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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse Mutation Study: Salmonella typhimurium (TA1535, TA1537, TA100 & TA98) and Escherichia coli (WP2uvrA) negative with and without metabolic activation.

Chromosome aberration: Negative with and without metabolic activation (cultured peripheral human lymphocytes).

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:
13 to 27 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Test System Salmonella typhimurium bacteria and Escherichia coli bacteria Rationale Recommended test system in international guidelines (e.g. EPA, OECD, EEC).
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD30521R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cell coat)
gg : mutation in the galactose metabolism
chi : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicilin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196.C).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
In the dose range finding test, Hatcol 2352 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Hatcol 2352 emulsified on the plates at dose levels of 1000 µg/plate and upwards.
In the first and in the second mutation assay, Hatcol 2352 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix.
Vehicle / solvent:
Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycin (DM), 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
Test substance preparation: The test substance was dissolved in ethanol (Lichrosolv, Merck). Test substance concentrations were prepared directly prior to use and used within 4 hours after preparation.

Cell culture
Preparation of bacterial cultures: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.
Permeabilization of the Escherichia coli strain: WP2uvrA bacteria were washed twice in 0.25 the original volume of ice-cold 0.12 M Tris-HCL buffer pH 8.0, then gently resuspended in 0.2 vol. 0.12 M Tris-HCL, 0.5 mM EDTA pH 8.0, and shaken for 2.5 min at 37°C. MgCl2 was then added to a final concentration of 10 mM. The cells were centrifuged and resuspended in the original volume of nutrient broth.
Agar plates: Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purifed agar (Oxoid, code L28) in Vogel-Bonner Medium E, 20 g glucose.
N.B. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin and 15 µg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan.
Top agar: Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid, code L 11) and 0.5% (w/v)
Sodium Chloride was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 1°C.
Environmental conditions: All incubations were carried out in the dark at 37 ± 1°C. The temperature was monitored during the experiment.

Metabolic activation system: Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany.

Study design
Dose range finding test: Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of Hatcol 2352 used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.
Mutation assay: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1°C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Colony counting: The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
No formal hypothesis testing was done.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose range finding test
Hatcol 2352 was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
This dose range finding test is reported as a part of the first experiment of the mutation test.
Precipitate: The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards.
Precipitation of Hatcol 2352 on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 ¡.g/plate and upwards. This precipitate was judged as an emulsion of Hatcol 2352 at the end of the incubation period.
Toxicity: To determine the toxicity of Hatcol 2352, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutation assay
Based on the results of the dose range finding test, Hatcol 2352 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitate: Hatcol 2352 precipitated in the top agar at the concentrations of 333 and 1000 µg/plate.
Precipitation of Hatcol 2352 on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate. This precipitate was judged as an emulsion of Hatcol 2352 at the end of the incubation period.
Toxicity: The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
Number of revertants: All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiment 1: Mutagenic response of Hatcol 2352 in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

324± 26

340± 81

460± 98

938± 42

596± 16

Solvent control

7± 2

8± 4

20± 3

118± 5

10± 5

3

 

 

 

141± 13

9± 3

10

9± 1

15± 5

16± 5

127± 11

9± 4

33

7± 4

8± 2

15± 5

123± 6

12± 2

100

7± 2

11± 2

18± 3

130± 2

8± 3

333

7± 1

10± 2

14± 1

149± 7

11± 2

1000E

8± 8

11± 4

13± 3

142± 7

11± 4

3330E

 

 

 

132± 13

9± 2

5000E

 

 

 

130± 15

14± 4

With S9-mix1

Positive control

195± 26

202± 29

679± 36

942± 42

94± 13

Solvent control

8± 2

9± 4

18± 4

138± 2

12± 2

3

 

 

 

159 V 9

11± 4

10

7± 2

8± 3

21± 2

147± 13

12± 12

33

6± 1

11± 5

22± 1

142± 3

11± 3

100

8± 3

7± 2

22± 7

153± 10

13± 4

333

8± 2

11± 2

22± 2

131± 7

13± 3

1000E

11± 1

6± 3

20± 1

149± 13

15± 1

3330E

 

 

 

146± 7

11± 1

5000E

 

 

 

141± 9

11± 3

Solvent control: 0.1ml ethanol

1 The S9-mix contained 5% (v/v) S9 fraction

E Hatcol 2352 emuslified on the plates

 

Experiment 2: Mutagenic response of Hatcol 2352 in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

231± 16

244± 31

588± 29

546± 56

763± 75

Solvent control

10± 3

8± 1

19± 1

127± 6

10± 3

10

14± 1

5± 1

19± 2

123± 6

11± 2

33

12± 2

5± 0

21± 4

132± 13

8± 3

100

11± 2

5± 1

14± 2

124± 9

10± 2

333

12± 5

5± 1

20± 3

119± 15

9± 2

1000E

7± 1

6± 1

13± 3

128± 9

9± 2

With S9-mix1

Positive control

114± 13

148± 18

340± 55

492± 15

65± 8

Solvent control

11± 2

6± 2

19± 2

122± 5

8± 2

10

12± 1

5± 2

20± 2

102± 4

10± 3

33

12± 3

6± 3

24± 1

108± 6

11± 2

100

10± 2

7± 2

21± 2

109± 4

14± 5

333

10± 2

6± 2

22± 3

113± 13

9± 3

1000E

12± 3

7± 2

22± 3

108± 5

9± 1

Solvent control: 0.1ml ethanol

1 The S9-mix contained 5% (v/v) S9 fraction

E Hatcol 2352 emuslified on the plates

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Based on the results of this study it is concluded that Hatcol 2352 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of Hatcol 2352 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

Hatcol 2352 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

The study procedures described in this report were based on the following guidelines:

- OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (Adopted July 21, 1997).

- European Economic Community (EEC). Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity; B.13/14: "Mutagenicity: "Reverse Mutation Test using bacteria". EEC Publication Commission Directive (Published June 8, 2000).

Batch K16156 of Hatcol 2352 was a clear pale yellow liquid. The test substance was soluble in ethanol.

In the dose range finding test, Hatcol 2352 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Hatcol 2352 emulsified on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first and in the second mutation assay, Hatcol 2352 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. Hatcol 2352 emulsified on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings. Hatcol 2352 did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that Hatcol 2352 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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:
28 February 2003 to 14 March 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Dose range finding test
Eight concentrations of HATCOL 3344: 3, 10, 33, 100,333, 1000, 3330 and 5000 µg/plate were tested in triplicate.
Mutation assay
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Vehicle / solvent:
The test substance was dissolved in ethanol (Lichrosolv, Merck). Test substance concentrations were prepared directly prior to use and used within 4 hours after preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycin, 2-aminoanthracene
Details on test system and experimental conditions:
CELL CULTURE
Preparation of Bacterial cultures: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.
Permeabilization of the Escherichia coli strain: WP2uvrA bacteria were washed twice in 0.25 the original volume of ice-cold 0.12 M Tris-HCL buffer pH 8.0, then gently resuspended in 0.2 vol. 0.12 M Tris-HCL, 0.5 mM EDTA pH 6.0, and shaken for 2.5 min at 37°C. MgCl2 was then added to a final concentration of 10 mM. The cells were centrifuged and resuspended in the original volume of nutrient broth.
Agar plates: Agar plates (ø 9 em) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18g purified agar (Oxoid, code l28) in Vogel-Bonner Medium E, 20 g glucose.
N.B. The agar plates for the test with the Salmonella typhimunum strains also contained 12.5 µg/plate biotin and 15 µg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan.
Top agar: Top agar medium, containing 0.6% (w/v) agar and 0.5% (w/v) NaCl, was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 1°C.
Environmental conditions: All incubations were carried out in the dark at 37 ± 1°C. The temperature was monitored during the experiment.

TREATMENT OF THE TEST SUBSTANCE: The test substance was dissolved in ethanol (Lichrosolv, Merck). Test substance concentrations were prepared directly prior to use and used within 4 hours after preparation.

EXPERIMENTAL PROCEDURE
Dose range finding test: Selection of an adequate range of doses was based on a dose range finding test with the tester strain TA100 in the absence and presence of S9-mix. Eight concentrations of HATCOL 3344, 3, 10, 33, 100,333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of HATCOL 3344 used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.
Mutation assay: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1°C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Colony counting: The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.
Evaluation criteria:
DATA EVALUATION AND STATISTICAL PROCEDURES
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
No formal hypothesis testing was done.

Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE RANGE FINDING TEST
HATCOL 3344 was tested in the tester strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
Precipitate: The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation of HATCOL 3344 on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards.
Toxicity: To determine the toxicity of HATCOL 3344 the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

MUTATION ASSAY
Based on the results of the dose range finding test, HATCOL 3344 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537, TA98 and WP2uvrA and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitate: HATCOL 3344 precipitated in the top agar at concentrations of 333 and 1000 µg/plate. Precipitation of HATCOL 3344 on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate.
Toxicity: The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
Number of revertants: All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

MUTAGENIC RESPONSE OF HATCOL 3344 IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

Experiment 1

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

718±49

446± 65

351± 40

978± 78

577± 24

Solvent control

8± 1

5± 2

17± 3

132± 14

21± 5

3

 

 

 

105± 13

 

10

9± 1

7± 3

17± 2

98± 12

14± 2

33

7± 5

5± 2

19± 3

101± 11

15± 3

100

6± 3

4± 2

15± 2

112± 14

13± 3

333

6± 1

6± 1

18± 1

113± 8

16± 4

1000SP

7± 4

5± 2

17± 3

111± 1

18± 7

3330SP

 

 

 

104± 2

 

5000SP

 

 

 

121± 7

 

With S9-mix1

Positive control

213± 9

851± 55

1154± 58

1403± 125

86± 12

Solvent control

7± 4

8± 3

22± 2

115± 20

20± 4

3

 

 

 

109± 12

 

10

8± 2

5± 2

24± 3

112± 14

21± 3

33

11± 3

4± 2

18± 4

114± 8

16± 9

100

5± 2

7± 2

22± 5

110± 6

14± 1

333

7± 1

6± 2

15± 2

102± 6

17± 6

1000SP

10± 3

7± 3

22± 4

113± 8

17± 2

3330SP

 

 

 

116± 6

 

5000SP

 

 

 

120± 10

 

Solvent control: 0.1ml ethanol;1The S9-mix contained 5% (v/v) S9 fraction;SPSlight Precipitate

 

MUTAGENIC RESPONSE OF HATCOL 3344 IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

Experiment 2

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

259± 8

242± 16

346± 15

1002± 30

585± 7

Solvent control

8± 2

5± 2

17± 3

128± 10

8± 3

10

11± 4

6± 1

18± 4

117± 14

10± 3

33

10± 3

6± 3

19± 4

124± 8

8± 3

100

10± 5

6± 2

16± 6

119± 8

9± 2

333

12± 1

9± 4

15± 1

138± 7

8± 4

1000SP

11± 4

6± 2

15± 3

126± 5

12± 2

With S9-mix1

Positive control

99± 3

251± 3

469± 18

463± 34

240± 3

Solvent control

8± 3

5± 1

25± 3

115± 7

13± 4

10

10± 2

7± 4

25± 9

112± 5

13± 3

33

8± 2

5± 3

27± 10

123± 7

12± 3

100

9± 2

5± 1

31± 4

115± 5

11± 3

333

9± 2

6± 3

23± 2

125± 6

14± 3

1000SP

11± 3

4± 1

25± 5

124± 9

12± 1

Solvent control: 0.1ml ethanol;1The S9-mix contained 5% (v/v) S9 fraction;SPSlight Precipitate

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Based on the results of this study it is concluded that HATCOL 3344 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

HATCOL 3344 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

In the dose range finding test, HATCOL 3344 was tested up to concentrations of 5000 µg/platein the absence and presence of S9-mix in the tester strain TA100 HATCOL 3344 precipitatedon the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn wasnot reduced at any of the concentrations tested and no biologically relevant decrease in thenumber of revertants was observed.

In the first and in the second mutation assay, HATCOL 3344 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. HATCOL 3344 precipitated on the platesat this dose level. The bacterial background lawn was not reduced at any ofthe concentrationstested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

HATCOL 3344 did not induce a dose-related, two-fold increase in the number of revertant (His+)colonies in each of the fourtester strains (TA1535, TA1537, TA9S and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that HATCOL 3344 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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:
22 November 2002 to 06 December 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and trytophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
HATCOL 5236 was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
Based on the results of the dose range finding test, HATCOL 5236 was tested with concentrations of 10, 33, 100, 333, 1000 µg/plate In the absence and presence of S9-mix in two mutation assays.
Vehicle / solvent:
The test substance was dissolved in ethanol (Lichrosolv, Merck). Test substance concentrations were prepared directly prior to use and used within 4 hours after preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycin (DM); 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
Dose range finding test: Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of HATCOL 5236 used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.

Mutation assay: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1 °C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Colony counting: The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
No formal hypothesis testing was done.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE RANGE FINDING TEST: HATCOL 5236 was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.

Precipitate: The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards.
Precipitation of HATCOL 5236 on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards.

Toxicity: To determine the toxicity of HATCOL 5236, the reduction of the bacterial background lawn, the increase in the size of the micro-colonies and the reduction of the revertant colonies were examined.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

MUTATION ASSAY: Based on the results of the dose range finding test, HATCOL 5236 was tested up to concentrations of 1000 µg/plate In the absence and presence of S9-mix in two mutation assays.
The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Precipitate: HATCOL 5236 precipitated in the top agar at concentrations of 333 and 1000 µg/plate.
Precipitation of HATCOL 5236 on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate.

Toxicity: The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Number of revertants: All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

MUTAGENIC RESPONSE OF HATCOL 5236 IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

966± 106

631± 188

465± 50

1066± 51

611± 49

Solvent control

15± 3

8± 5

23± 7

157± 9

14± 4

3

 

 

 

156± 12

15± 3

10

12± 3

7± 2

24± 4

142± 19

14± 2

33

9± 1

7± 3

13± 1

160± 7

13± 2

100

8± 1

10± 2

24± 2

158± 18

9± 2

333

8± 1

7± 3

19± 6

156± 20

12± 4

1000SP

8± 3

5± 1

21± 3

149± 11

14± 5

3330SP

 

 

 

157± 18

13± 6

5000SP

 

 

 

149± 9

12±2

With S9-mix1

Positive control

138± 24

183± 81

591± 25

1003± 137

145± 16

Solvent control

12± 2

8± 4

19± 2

159± 15

14± 4

3

 

 

 

155± 17

15± 3

10

11± 4

12± 2

16± 1

153± 2

10± 1

33

12± 4

8± 1

21± 6

161± 8

15± 3

100

12± 4

8± 3

22± 5

149± 13

15± 2

333

10± 2

7± 4

19± 6

144± 18

15± 5

1000SP

11± 2

7± 3

16± 1

146± 9

13± 5

3330SP

 

 

 

148± 14

12± 3

5000SP

 

 

 

143± 17

12± 5

Solvent control: 0.1ml ethanol;1: The S9-mix contained 5% (v/v) S9 fraction;SP: Slight Precipitate

 

MUTAGENIC RESPONSE OF HATCOL 5236 IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

499± 14

370± 35

344± 35

676± 23

414± 100

Solvent control

6± 2

6± 1

14± 3

141± 27

20± 3

10

7± 2

7± 2

16± 3

120± 4

19± 4

33

9± 3

6± 2

19± 7

121± 15

19± 2

100

7± 2

6± 2

19± 3

125± 14

21± 4

333

7± 2

6± 1

19± 4

104± 9

21± 6

1000SP

8± 4

6± 4

14± 1

121± 12

25± 3

With S9-mix1

Positive control

103± 7

67± 5

350± 51

276± 20

199± 22

Solvent control

8± 2

30± 2

15± 2

117± 15

19± 5

10

8± 1

8± 4

27± 3

104± 9

26± 5

33

7± 2

9± 2

25± 2

98± 3

21± 6

100

10± 3

7± 3

27± 10

109± 15

18± 7

333

9± 3

6± 3

22± 1

96± 6

16± 7

1000SP

10± 5

5± 1

21± 3

101± 11

16± 5

Solvent control: 0.1ml ethanol;1: The S9-mix contained 5% (v/v) S9 fraction;SP: Slight Precipitate

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

HATCOL 5236 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

HATCOL 5236 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a trptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

In the dose range finding test, HATCOL 5236 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. HATCOL 5236 precipitated on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first and in the second mutation assay, HATCOL 5236 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. HATCOL 5236 precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

HATCOL 5236 did not induce a dose-related, two-fold increase in the number of revertant (His+)colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that HATCOL 5236 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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
Study period:
12 June 2003 to 16 August 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
metaphase cells
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes.
Details on mammalian cell type (if applicable):
Whole blood treated with an anti-coagulant (heparin) obtained from healthy male subjects was cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemicals.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
In the dose range finding test: 1, 3, 10, 33 and 100 µg Hatcol 3331/ml culture medium with and without S9-mix.
First cytogenetic assay:
Without and with S9-mix: 10, 33 and 100 µg Hatcol3331/ml culture medium (3 h exposure time, 24 h fixation time).
Second cytogenetic assay:
Without S9-mix 10, 33 and 100 µg Hatcol 3331/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time)
With S9-mix 10, 33 and 100 µg Hatcol3331/ml culture medium (3 h exposure time, 48 h fixation time)
Vehicle / solvent:
Halcol 3331 was dissolved in ethanol absolute (Lichrosolv, Merck).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Test System
Test System: Cultured peripheral human lymphocytes.
Rationale: Recommended test system in international guidelines (e.g. EPA, OECD, EEC).
Source: Healthy adult male volunteers.
Dose range finding study: 24 h fixation: age 37, AGT = 16.5 h (Dec. 2002); 48 h fixation: age 37, AGT = 17.5 h (Dec. 2002)
First cytogenetic assay: age 29, AGT = 16.4 h (Dec. 2002)
Second cytogenetic assay: age 39, AGT = 16.6 h (Dec. 2002)
(AGT= Average Generation Time of the cells).

Cell culture
Blood samples: Blood samples were taken from healthy adult male volunteers by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
F10 complete culture medium: F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/I) and 30 U/ml heparin.
Lymphocyte cultures: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml F10 complete culture medium (in the absence and presence of S9-mix respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Murex) was added.
Environmental conditions: All Incubations were carried out in a humid atmosphere (80-100%) containing 5 ± 0.5% CO2 in air in the dark, at 37 ± 1 °C. The temperature, humidity and CO2-percentage-were monitored throughout the experiment.

Metabolic activation system: Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats (6), which were obtained from Charles River (Sulzfeld, Germany).

Study design
Dose range finding test: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Hatcol 3331 was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
Lymphocyte cultures (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of Hatcol 3331 for 3 h, 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
The highest tested concentration was determined by the solubility of Hatcol 3331 in the culture medium.
After 3 h exposure to Hatcol 3331 in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time). The cells that were exposed for 24 hand 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 hand 48 h fixation time).
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assay considering the highest dose level was determined by the solubility.

Cytogenetic assay: The cytogenetic assay was carried out with minor modifications as described by Evans, 1984. Hatcol 3331 was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.
First cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 3331 for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time).
Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modifications of experimental conditions.
Second cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 3331 for 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
After 3 h exposure, the cells exposed to Hatcol 3331 in the presence of S9-mix were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44-46 h (48 h fixation time).
The cells that were treated for 24 h and 48 h in the absence of S9-mixwere not rinsed after exposure but were fixed immediately after 24 and 48h (24 h-and 48 h fixation time).

Chromosome preparation: During the last 2.5 h of the culture period, cell division was arrested by addition of the spindle inhibitor colchicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).

Preparation of slides: Fixed cells were dropped onto cleaned slides, which were immersed for 24 hours in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. Two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa solution in tap water.
Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.

Mitotic index/dose selection for scoring of the cytogenetic assay: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of slides for chromosome aberrations: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases no more metaphases were examined. Only meta phases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.
Evaluation criteria:
A chromosome aberration test was considered acceptable if it met the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances-should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate-cultures is observed.

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically and biologically signifcant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Chi-square test.
Key result
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Dose-range-finding test
At a concentration of 100 µg/ml Hatcol 3331 precipitated in the culture medium. Therefore, a concentration of 100 µg/ml was used as the highest concentration of Hatco13331.
In the dose range finding test blood cultures were treated with 1, 3, 10, 33 and 100 µg Hatcol 3331/ml culture medium with and without S9-mix.

First cytogenetic assay
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Without and with S9-mix: 10, 33 and 100 µg Hatcol3331/ml culture medium (3 h exposure time, 24 h fixation time).
All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Hatcol 3331 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Second cytogenetic assay
To obtain more information about the possible clastogenicity of Hatcol 3331, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Hatcol 3331 in the absence of S9 mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Hatcol 3331. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix 10, 33 and 100 µg Hatcol 3331/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time)
With S9-mix 10, 33 and 100 µg Hatcol3331/ml culture medium (3 h exposure time, 48 h fixation time)
All doses were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Hatcol 3331 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mitotic index of donor cultures treated with Hatcol 3331 in the dose range finding test

HATCOL 3331 concentration (µg/l)

Number of metaphases per 1000 cells

Absolute

Percentage of control

Without metabolic activation (-S9-mix)

3h exposure time, 24h fixation time

Controla)

79

100

1

71

90

3

75

95

10

79

100

33

70

89

100b)

81

103

24h exposure time, 24h fixation time

Controla)

25

100

1

23

92

3

24

96

10

27

108

33

30

120

100b)

24

96

48h exposure time, 48h fixation time

Controla)

26

100

1

22

85

3

20

77

10

19

73

33

20

77

100b)

22

85

With metabolic activation (+S9-mix)

3h exposure time, 24h fixation time

Controla)

85

100

1

90

106

3

83

98

10

76

89

33

79

93

100b)

93

109

a)Ethanol;b)Hatcol 3331 precipitated in the culture medium.

 

Mitotic index of donor cultures treated with Hatcol 3331 in the first cytognetic assay

HATCOL 3331 concentration (µg/l)

Number of metaphases per 1000 cellsa)

Absolute

Percentage of control

Without metabolic activation (-S9-mix)

3h exposure time, 24h fixation time

Controlb)

68 – 63

100

10

71 – 70

108

33

70 – 65

103

100c)

73 – 69

108

MMC-C; 0.5µg/ml

50 – 49

76

With metabolic activation (+S9-mix)

3h exposure time, 24h fixation time

Controlb)

63 – 66

100

10

78 – 63

109

33

81 – 75

121

100c)

66 – 65

102

CP; 15µg/ml

56 – 42

76

a)Duplicate cultures;b)Ethanol;c)Hatcol 3331 precipitated in the culture medium.

 

Mitotic index of donor cultures treated with Hatcol 3331 in the second cytogenetic assay

HATCOL 3331 concentration (µg/l)

Number of metaphases per 1000 cellsa)

Absolute

Percentage of control

Without metabolic activation (-S9-mix)

24h exposure time, 24h fixation time

Controlb)

33 – 28

100

10

27 – 32

97

33

31 – 29

98

100c)

27 - 31

95

MMC-C; 0.2µg/ml

13 – 16

48

48h exposure time, 48h fixation time

Controlb)

28 – 30

100

10

31 – 25

97

33

30 – 28

100

100c)

18 – 18

62

MMC-C; 0.1µg/ml

21 – 24

78

With metabolic activation (+S9-mix)

3h exposure time, 48h fixation time

Controlb)

50 – 66

100

10

62 – 66

110

33

69 – 78

127

100c)

70 – 56

109

CP; 15µg/ml

27 – 15

-d)

a)Duplicate cultures;b)Ethanol;c)Hatcol 3331 precipitated in the culture medium;d)CP was fixed after 24 hours. Thereafter, the mitotic index could not be calculated as percentage of control.

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Hatcol 3331 is not clastogenic in human lymphocytes.
Executive summary:

Evaluation of the ability of Hatcol 3331 to induce chromosome aberrations in cultured peripheral human lymphocytes.

This report describes the effect of Hatcol 3331 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Hatcol 3331 was tested in two independent experiments.

The study procedures described in this report were based on the following guidelines: OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted 21st July 1997). European Economic Community (EEC). Directive 2000/321 EC, Part B: Methods for the Determination of Toxicity; B.10: "Mutagenicity: In Vitro Mammalian Chromosome Aberration Test".

Batch D21287 of Hatcol 3331 was a clear colourless liquid with a purity of 97.3%. The test substance was soluble in ethanol

.

In the first cytogenetic assay, Hatcol 3331 was tested up to 100 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. Hatcol 3331 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Hatcol 3331 was tested up to 100 µg/ml for a 24 hand 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. In the presence of 1.8% (v/v) S9-fraction, Hatcol 3331 was also tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Hatcol 3331 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, intwo independently repeated experiments.

Finally, it is concluded that this test is valid and that Hatcol 3331 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

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
Study period:
18 June 2003 to 26 August 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Whole blood treated with an anti-coagulant (heparin) obtained from healthy male subjects was cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemicals.
Species / strain / cell type:
mammalian cell line, other: Cultured peripheral human lymphocytes.
Details on mammalian cell type (if applicable):
Healthy adult male volunteers.
Dose range finding study:
age 29, AGT = 16.4 h (Dec. 2002)
First cytogenetic assay:
age 39, AGT = 16.6 h (Dec. 2002)
Second cytogenetic assay:
Without S9-mix (24 h fixation time) and with 59-mix:
age 33, AGT = 16.8 h (Dec. 2002)
Without 59-mix (48 h fixation time):
age 29, AGT = 16.4 h (Dec. 2002)
(AGT= Average Generation Time of the cells).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
In the dose range finding test blood cultures were treated with 1, 3, 10, 33 and 100 µg Hatcol 3344/ml culture medium with and without S9-mix.

First cytogenetic assay
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Without and with S9-mix: 10, 33 and 100 µg HatcoI 3344/ml culture medium (3 h exposure time, 24 h fixation time).

Second cytogenetic assay
Without S9-mix 10, 33 and 100 µg Hatcol 3344/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time)
With S9-mix10, 33 and 100 µg Hatcol 3334/ml culture medium (3 h exposure time, 48 h fixation time).
Vehicle / solvent:
Halcol 3344 was dissolved in ethanol absolute (Lichrosolv, Merck). Hatcol 3344 concentrations were prepared directy prior to use. The final concentrtion of the solvent in the culture medium amounted 1.0% (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Study design
Dose range finding test: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Hatcol 3344 was tested in the absence and in the presence of 1.8% (v/v) S9-frction.
Lymphocyte cultures (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of Hatcol 3344 for 3 h, 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
The highest tested concentration was determined by the solubiilty of Hatcol 3344 in the culture medium.
After 3 h exposure to Hatcol 3344 in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HB55 was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time). The cells that were exposed for 24 hand 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 hand 48 h fixation time).
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assay considering the highest dose level was determined by the solubility.

Cytogenetic assay: The cytogenetic assay was carried out with minor modifications as described by Evans, 1984. Hatcol 3344 was tested in the absence and presence of 1.8% (v/v) S9wfrction in duplicate in two independent experiments.

First cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 3344 for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time).
Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modifications of experimental conditions.

Second cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 3344 for 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
After 3 h exposure, the cells exposed to Hatcol334 in the presence of S9-mix were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44-46 h (48 h fixation time).
The cells that were treated for 24 hand 48 h in the absence of 59-mix were not rinsed after exposure but were fixed immediately after 24 hand 48 h (24 hand 48 h fixation time).

Chromosome preparation: During the last 2.5 h of the culture period cell division was arrested by addition of the spindle inhibitor colchicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C.
After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1
v/v).

Preparation of slides: Fixed cells were dropped onto cleaned slides, which were immersed for 24 hours in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. Two slides were prepared per culture.
Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa solution in tap water.
Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.

Mitotic index/dose selection for scoring of the cytogenetic assay: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of slides for chromosome aberrations: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.
Evaluation criteria:
Acceptability of the assay
A chromosome aberration test was considered acceptable if it met the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
Data evaluation and statistical procedures
A test substance was considered positive (clastogenic) in the chromosome aberration test if
a) It induced a dose-related statistically significant (Chi-square test, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not c1astogenlc) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Chi-square test.
Key result
Species / strain:
mammalian cell line, other: Cultured peripheral human lymphocytes.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
Dose range finding test: At a concentration of 100 µg/ml Hatcol 3344 precipitated in the culture medium. Therefore, a concentration of 100 µg/ml was used as the highest concentration of Hatcol 3344.
In the dose range finding test blood cultures were treated with 1, 3, 10, 33 and 100 µg Hatcol 3344/ml culture medium with and without S9-mix.

First cytogenetic assay: Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Without and with S9-mix: 10, 33 and 100 µg HatcoI 3344/ml culture medium (3 h exposure time, 24 h fixation time).
All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Hatcol 3344 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Second cytogenetic assay: To obtain more information about the possible clastogenicity of Hatcol 3344, a second cytogenetic assay was performed In which human lymphocytes were continuously exposed to Hatcol 3344 in the absence of S9 mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Hatcol 3344. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 10, 33 and 100 µg Hatcol 3344/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time)
With S9-mix: 10, 33 and 100 µg Hatcol 3344/ml culture medium (3 h exposure time, 48 h fixation time)
All doses were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Hatcol 3344 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Evaluation of the results: The ability of Hatcol 3344 to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments.
The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Both in the absence and presence of S9-mix Hatcol3344 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mitotic index of donor cultures treated with Hatcol 3344 in the dose range finding test

Hatcol 3344 concentration (µg/ml)

Number of metaphases per 1000 cells

Absolute

Percentage of control

Without metabolic activation (-S9-mix)

3h exposure time, 24h fixation time

Controla)

78

100

1

69

88

3

72

92

10

83

106

33

84

108

100b)

64

82

24h exposure time, 24h fixation time

Controla)

34

100

1

34

100

3

31

91

10

32

94

33

43

126

100b)

38

112

48h exposure time, 48h fixation time

Controla)

25

100

1

28

112

3

29

116

10

22

88

33

23

92

100b)

27

108

With metabolic activation (+S9-mix)

3h exposure time, 24h hour fixation time

Controla)

69

100

1

75

109

3

75

109

10

71

103

33

82

119

100b)

75

109

a)Ethanol

b)Hatcol 3344 precipitated in the culture medium

 

Mitotic index of donor cultures treated with Hatcol 3344 in the first cytogenetic assay

Hatcol 3344 concentration (µg/ml)

Number of metaphases per 1000 cellsa)

Absolute

Percentage of control

Without metabolic activation (-S9-mix)

3h exposure time, 24h fixation time

Controlb)

63 – 58

100

10

61 – 57

98

33

60 – 39

82

100c)

48 – 54

84

MMC-C; 0.5µg/ml

39 – 40

65

With metabolic activation (+S9-mix)

3h exposure time, 24h hour fixation time

Controlb)

61 – 57

100

10

55 -54

92

33

49 – 58

91

100c)

56 – 48

88

CP; 15µg/ml

24 – 26

42

a)Duplicate cultures

b)Ethanol

c)Hatcol 3344 precipitated in the culture medium

 

Mitotic index of donor cultures treated with Hatcol 3344 in the second cytogenetic assay

Hatcol 3344 concentration (µg/ml)

Number of metaphases per 1000 cellsa)

Absolute

Percentage of control

Without metabolic activation (-S9-mix)

24h exposure time, 24h fixation time

Controlb)

33 – 31

100

10

28 – 28

88

33

31 -34

102

100c)

31 – 24

86

MMC-C; 0.2µg/ml

23 – 28

80

48h exposure time, 48h fixation time

Controlb)

28 – 36

100

10

30 – 32

97

33

23 – 28

80

100c)

25 – 29

84

MMC-C; 0.1µg/ml

24 – 30

84

With metabolic activation (+S9-mix)

3h exposure time, 48h hour fixation time

Controlb)

36 – 54

100

10

37 – 48

94

33

44 – 55

110

100c)

60 – 34

104

CP; 15µg/ml

35 – 23

-d)

a)Duplicate cultures

b)Ethanol

c)Hatcol 3344 precipitated in the culture medium

d)CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Hatcol 3344 is not clastogenic in human lymphocytes under the experimental conditons described in this report.
Executive summary:

Evaluation of the ability of Hatcol 3344 to induce chromosome aberrations in cultured peripheral human lymphocytes.

This report describes the effect of Hatcol 3344 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9 -mix). The possible clastogenicity of Hatcol 3344 was tested in two independent experiments.

The study procedures described in this report were based on the following guidelines: OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted 21st July 1997). European Economic Community (EEC). Directive 2000/321EC, Part B: Methods for theDetermination ofToxicity; B.10: "Mutagenicity: In Vitro Mammalian Chromosome Aberration Test".

Batch H102-01-28 of Hatcol 3344 was a clear colourless liquid with a purity of 96.9%. The test substance was soluble in ethanol.

In the first cytogenetic assay, Hatcol 3344 was tested up to 100 µg/ml for a 3 h exposure timewith a 24 h fixation time in the absence and presence of S9-mix. Hatcol 3344 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Hatcol 3344 was tested up to 100 µg/ml for a 24 hand 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9 -mix. In the presence of 1.8% (v/v) S9 fraction Hatcol 3344 was also tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Hatcol 3344 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, in two independently repeated experiments.

Finally, it is concluded that this test is valid andthat Hatcol 334 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

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
Study period:
18 June 2003 to 06 September 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromosome aberrtions
Species / strain / cell type:
mammalian cell line, other: cultured peripheral human lymphocyes.
Details on mammalian cell type (if applicable):
Test System: Cultured peripheral human lymphocytes.
Rationale: Recommended test system in international guidelines (e.g. EPA, OECD, EEC).
Source: Healthy adult male volunteers.
Dose range finding study: age 37, AGT = 17.5 h (Dec. 2002)
First cytogenetic assay: age 36, AGT= 17.0 h (Dec. 2002)
Second cytogenetic assay: age 28. AGT = 16.2 h (Dec. 2002)
(AGT= Average Generation Time of the cells).

Cell culture
Blood samples: Blood samples were taken from healthy adult male volunteers by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
F10 complete culture medium: F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/l) and 30 U/ml heparin.
Lvmphocyte cultures: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml F10 complete culture medium (in the absence and presence of S9-mix respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Murex) was added.
Environmental conditions: All incubations were carried out in a humid atmosphere (80-100%) containing 5 ± 0.5% CO2in air in the dark at 37 ± 1 °C. The temperature, humidity and CO2 percentage were monitored throughout the experiment.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
In the dose range finding test blood cultures were treated with 1, 3, 10, 33 and 100 µg Hatcol 5236/ml culture medium with and without S9-mix.
First cytogenetic assay:
Without and with S9-mix: 10, 33 and 100 µg Hatcol 5236/ml culture medium (3 h exposure time, 24 h fixation time).
Second cytogenetic assay:
Without S9-mix: 10, 33 and 100 µg Hatcol 5236/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time)
With S9-mix: 10, 33 and 100 µg Hatcol 5236/ml culture medium (3 h exposure time, 48 h fixation time)
Vehicle / solvent:
Hatcol 5236 was dissolved in ethanol absolute. Hatcol 5236 concentrations were prepared directly prior to use. The final concentration of the solvent in the culture medium amounted 1.0% (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Dose range finding test
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Hatcol 5236 was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
Lymphocyte cultures (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/mI) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of Hatcol 5236 for 3 h, 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
The highest tested concentration was determined by the solubility of Hatcol 5236 in the culture
medium.
After 3 h exposure to Hatcol 5236 in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time). The cells that were exposed for 24 hand 48 h in the absence of S9-mix were not rinsed after exposure but were fied immediately (24 hand 48 h fixation time).
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assay considering the highest dose level was determined by the solubility.

Cytogenetic assay
The cytogenetic assay was carried out with minor modifications as described by Evans, 1984. Hatcol 5236 was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.

First cytogenetic assay
Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 5236 for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min. 150 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrgation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time).
Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modifications of experimental conditions.

Second cvoaenetic assay
Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 5236 for 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
After 3 h exposure, the cells exposed to Hatcol 5236 in the presence of 59-mix were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44-46 h (48 h fixation time).
The cells that were treated for 24 hand 48 h in the absence of 59-mix were not rinsed after exposure but were fixed immediately after 24 hand 48 h (24 hand 48 h fixation time).

Chromosome preparation
During the last 2.5 h of the culture period, cell division was arrested by addition of the spindle inhibitor colchicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining ceil pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).

Preparation of slides
Fixed cells were dropped onto cleaned slides, which were immersed for 24 hours in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. Two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa solution in tap water.
Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.

Mitotic index/dose selection for scoring of the cytogenetic assay
The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of slides for chromosome aberrations
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was > 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.
Evaluation criteria:
Acceptability of the assay
A chromosome aberration test was considered acceptable if it met the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.

Data evaluation and statistical procedures
A test substance was considered positive (clastogenic) in the chromosome aberration test if
a) it induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Chi-square test
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocyes.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose range finding test
At a concentration of 100 µg/ml Hatcol 5236 precipitated in the culture medium. Therefore, a concentration of 100 µg/ml was used as the highest concentration of Hatcol 5236.
In the dose range finding test blood cultures were treated with 1, 3, 10, 33 and 100 µg Hatcol 5236/ml culture medium with and without S9-mix.

First cytogenetic assay
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Without and with S9-mix: 10, 33 and 100 µg Hatcol 5236/ml culture medium (3 h exposure time, 24 h fixation time).
All dose levels were selected for scoring of chromosome aberrations. In the absence of S9-mix, Hatcol 5236 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
In the presence of S9-mix, Hatcol 5236 induced a statistically significant increase in the number of cells with chromosome aberrations at the intermediate concentration of 33 µg/ml, only when gaps were included.

Second cytogenetic assay
To obtain more information about the possible clastogenicity of Hatcol 5236, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Hatcol 5236 in the absence of S9 mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Hatcol 5236. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 10, 33 and 100 µg Hatcol 5236/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time)
With S9-mix: 10, 33 and 100 µg Hatcol 5236/ml culture medium (3 h exposure time, 48 h fixation time)
All doses were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Hatcol 5236 did not Induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mitotic index of donor cultures treated with Hatcol 5236 in the dose range finding test

Hatcol 5236 concentration (µg/ml)

Number of metaphases per 1000 cells

Absolute

Percentage of control

Without metabolic activation (-S9-mix)

3h exposure time, 24h fixation time

Controla)

53

100

1

53

100

3

55

104

10

52

98

33

48

91

100b)

56

106

24h exposure time, 24h fixation time

Controla)

25

100

1

21

84

3

22

88

10

26

104

33

26

104

100b)

21

84

48h exposure time, 48h fixation time

Controla)

22

100

1

23

105

3

21

95

10

16

73

33

18

82

100b)

14

64

With metabolic activation (+S9-mix)

3h exposure time, 24h fixation time

Controla)

48

100

1

46

96

3

43

90

10

46

96

33

48

100

100b)

45

94

a)Ethanol;b)Hatcol 5236 precipitated in the culture medium

 

Mitotic index of donor cultures treated with Hatcol 5236 in the first cytogenetic assay

Hatcol 5236 concentration (µg/ml)

Number of metaphases per 1000 cellsa)

Absolute

Percentage of control

Without metabolic activation (-S9-mix)

3h exposure time, 24h fixation time

Controlb)

50 – 45

100

10

55 – 46

106

33

47 – 49

101

100c)

43 – 47

95

MMC-C; 0.5µg/ml

30 – 31

64

With metabolic activation (+S9-mix)

3h exposure time, 24h fixation time

Controlb)

44 – 46

100

10

46 – 500

107

33

50 – 48

109

100c)

48 – 44

102

CP; 15µg/ml

18 – 24

47

a)Duplicate cultures;b)Ethanol;c)Hatcol 5236 precipitated in the culture medium.

 

Mitotic index of donor cultures treated with Hatcol 5236 in the second cytogenetic assay

Hatcol 5236 concentration (µg/ml)

Number of metaphases per 1000 cellsa)

Absolute

Percentage of control

Without metabolic activation (-S9-mix)

25h exposure time, 24h fixation time

Controlb)

56 – 49

100

10

54 – 45

94

33

49 – 48

92

100c)

42 – 48

86

MMC-C; 0.2µg/ml

28 – 28

53

48h exposure time, 48h fixation time

Controlb)

25 – 24

100

10

26 – 19

92

33

19 – 22

84

100c)

27 – 32

120

MMC-C; 0.1µg/ml

19 – 24

88

With metabolic activation (+S9-mix)

3h exposure time, 48h fixation time

Controlb)

32 – 43

100

10

39 – 37

101

33

29 – 35

85

100c)

41 – 36

103

CP; 15µg/ml

19 – 27

-d)

a)Duplicate cultures;b)Ethanol;c)Hatcol 5236 precipitated in the culture medium;d)CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

It is concluded that this test is valid and that Hatcol 5236 is not clastogenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

Evaluation of the ability of Hatcol 5236 to induce chromosome aberrations in cultured peripheral human lymphocytes.

This report describes the effect of Hatcol 5236 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Hatcol 5236 was tested in two independent experiments.

The study procedures described in this report were based on the following guidelines:

-OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vito MammalianChromosomeAberration Test (adopted 21st July 1997).

-European Economic Community (EEC). Directive 2000132/EC, Part B: Methods for theDetermination ofToxicity; B.10: "Mutagenicity: In Vitro Mammalian Chromosome Aberration Test".

 

Batch H20139 of Hatcol 5236 was a clear pale yellow liquid with a purity of 97.6%. The test substance was soluble in ethanol.

 

In the first cytogenetic assay, Hatcol 5236 was tested up to 100 µg/ml for a 3 h exposure timewith a 24 h fixation time in the absence and presence of S9-mix. Hatcol 5236 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Hatcol 5236 was tested up to 100 µg/ml for a 24 hand 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. In the presence of 1.8% (v/v) S9 -frction Hatcol 5236 was also tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

First cytogenetic assay

In the absence of S9-mix, Hatcol 5236 did not induce a statistically significant or biologicallyrelevant increase in the number of cells with chromosome aberrations.

In the presence of S9-mix, Hatcol 5236 induced a statistically significant increase in the numberof cells with chromosome aberrations at the intermediate concentration of 33 µg/ml, only whengaps were included. Since the type of aberrations observed were only breaks and gaps, theincrease was not dose related, only observed at the intermediate concentration of 33 µg/ml, only when gaps were included and the number of cells with chromosome aberrations was well within our historical control data range, the Increase was considered not to be biologically relevant.

 

Second cytogenetic assay

Hatcol 5236 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix.

 

Finally, it is concluded that this test is valid and that Hatcol 5236 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
06. Oct. - 16. Feb. 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD). (Purity of test substance not given, evalation criteria not given.) For read-across, maximum reliability score is 2.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
purity of test substance is not given (responsibility of the sponsor)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagles essential medium with HEPES buffer (MEM), supplemented with:L-glutamine, penicillin/streptomycin, amphotericin B, 15% foetel calf serum- Properly maintained: yes- Periodically checked for Mycoplasma contamination: no data- Periodically checked for karyotype stability: no data- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats pretreated with phenobarbitone (80 mg/kg) and ß-naphtoflavone (100 mg/kg)
Test concentrations with justification for top dose:
Experiment I:4 hour (with and without): 240, 320, 400 µg/mLExperiment II4 hour (with): 240, 320, 400 µg/mL24 hour (without): 240, 320, 400 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C (MMC; 0.2 and 0.4 µg/mL; -S9), cyclophosphamide (CP; 10 µg/mL; +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Exposure duration: 4 h (with and without S9), 24 h (without)- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 20 h; 24 h treatment: 0 hSPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL (demecolcine)STAIN (for cytogenetic assays): 5% Gurrs Giemsa for 5 minutesNUMBER OF REPLICATIONS: 2NUMBER OF CELLS EVALUATED: 200 per cultureDETERMINATION OF CYTOTOXICITY- Method: mitotic index of 2000 cells OTHER EXAMINATIONS:- Determination of polyploidy: yes
Evaluation criteria:
no data
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's exact test.
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: no- Effects of osmolality: no- Precipitation: cloudy precipitates were observed at and above 40 and 80 µg/mL in the 24-hour continuous and 4-hour pulse treatment groups, respectivelyRANGE-FINDING/SCREENING STUDIES:The dose range tested was 10-320 µg/mL. the test material produced some weak toxicity in the 4-hour treatment group but not the 24-hour treatment group. Toxicity could not be reproduced in the main experiment (scorable metaphases at every dose level).COMPARISON WITH HISTORICAL CONTROL DATA:The results are in range with historical control data.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 3 + 4: Test results of experiment I.

Test item

Concentration

Mitotic Index

Aberrant cells in %

 Experiment I

in µg/mL

in %

with gaps

without gaps

Exposure period 4h, fixation time 20h, without S9 mix

control

0

100

1

0

MMC

0.4

35

53

37

Test substance

240

98P

0.5

0

320

110P

0

0

400

91P

0.5

0.5

Exposure period 4h, fixation time 20h, with S9 mix

control

0

100

1

0.5

CP

10

20

35.5

28.5

Test substance

240

89P

1.5

0

320

89P

0.5

0

400

108P

3.5

1

Test item

Concentration

Mitotic Index

Aberrant cells in %

 Experiment II

in µg/mL

in %

with gaps

without gaps

Exposure period + fixation time 24h, without S9 mix

control

0

100

1.5

0

MMC

0.4

41

70

67

Test substance

240

61

0.5

0.5

320

53

0.5

0

400

83

0

0

Exposure period 4h, fixation time 20h, with S9 mix

control

0

100

0.5

0

CP

10

30

67

56

Test substance

240

103

1

0

320

76

1

0

400

110

1.5

0.5

Table5 +6: Mean Frequency of Polyploid Cells (%)

Experiment I

dose level µg/mL

harvest time 24 hours

4 hours without S9

4 hours with S9

0

0.0

0.0

240

0.0

0.0

320

0.0

0.0

400

0.0

0.0

MMC 0.4

0.0

NA

CP 10

NA

0.0

dose level µg/mL

harvest time 24 hours

24 hours without S9

4 hours with S9

0

0.5

0.0

240

0.0

0.0

320

0.0

0.0

400

0.0

0.5

MMC 0.4

0.0

NA

CP 10

NA

0.0

Conclusions:
Interpretation of results (migrated information):negative
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
Study period:
26 Mar - 08 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted in 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 supplemented with 5% (v/v) heat-inactivated horse serum- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9-mix), prepared from rats pretreated with phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
First experiment: 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (8%, v/v))Second experiment: 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (12%, v/v))
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
+ S9: cyclophosphamide, 15 and 5 µg/mL for 3 and 24 h treatment, respectively; - S9: methylmethanesulfonate, 7.5 µg/mL
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspensionDURATION- Preincubation period: No- Exposure duration: cells were exposed to the test material for 3 h and 24 h in the presence and absence of S9-mix, respectively. - Expression time (cells in growth medium): For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. For determination of the mutation frequency cells were plated and incubated for 11-12 days. After that, cells were stained for 2 h by adding 0.5 mg/mL MTT (Sigma) to each well. The plates were scored for cloning efficiency and mutation frequency with the naked eye or with the microscope.SELECTION AGENT (mutation assays): RPMI 1640 supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).DETERMINATION OF CYTOTOXICITY- Method: relative total growth
Evaluation criteria:
Several criteria including a concentration-related, or a reproducible increase in mutation frequencies determined a positive result.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at and above 333 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: at and above 333 µg/mL RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity data was obtained by treating cells for 3 h and 24 h respectively with a number of increasing test substance concentrations. The highest concentration tested was 750 µg/ml due to poor solubility of the test substance. No toxicity was observed with and without metabolic activation up to and at the maximum dose level tested with 3 h or 24 h incubation.COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all controls were in the range of the historical controls
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Results of experiment 1

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10E6

 

 

 

 

 

total

Without metabolic activation, 3 h treatment

SC1

100

104

100

100

74

SC2

85

97

0.3

99

98

104

102

74

1

101

102

108

109

71

3

100

101

107

107

94

10

93

98

104

97

67

33

120

94

100

120

63

100

113

101

107

121

61

333*

104

113

120

124

64

750*

405

101

107

112

74

MMS

71

68

72

51

835

With 8% (v/v) metabolic activation, 3 h treatment

SC1

100

70

100

100

65

SC2

69

64

0.3

96

60

86

83

74

1

115

68

98

113

60

3

109

40

57

62

84

10

127

72

104

132

52

33

114

46

66

75

84

100

122

76

108

133

63

333*

115

62

89

102

72

750*

104

58

84

87

53

CP

50

32

45

22

1617

 

Table 2: Results of experiment 2

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10E6

 

 

 

 

 

total

Without metabolic activation, 24 h treatment

SC1

100

66

100

100

90

SC2

79

75

0.3

112

77

106

119

88

1

116

80

110

128

82

3

117

72

100

117

79

10

120

85

117

140

66

33

114

74

101

116

83

100

121

69

95

115

83

333*

116

70

97

112

70

750*

116

66

91

106

71

MMS

101

49

67

68

1502

With 12% (v/v) metabolic activation, 3 h treatment

SC1

100

93

100

100

80

SC2

93

76

0.3

103

84

90

93

74

1

113

83

89

101

81

3

107

97

104

112

60

10

105

94

101

107

80

33

103

93

100

103

67

100

102

105

114

116

57

333*

106

91

99

104

74

750*

103

93

100

103

73

CP

72

75

81

58

1082

 

RSG: Relative Suspension Growth; CE: Cloning efficiency; RS: Relative Survival; RTG: Relative Total Growth; SC: Solvent Control (DMSO); MMS: Methylmethansulfonate; CP: Cyclophosphamide

*: Precipitation of test substance

Conclusions:
Interpretation of results (migrated information):negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
06 May - 29 Aug 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. For read-across, maximum reliability score is 2.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
September 1995
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
September 1995
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S. typhimurium strains)trp operon ( E. coli strains)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (Spraque Dawley rats, male, Aroclor 1254 induced)
Test concentrations with justification for top dose:
0, 10, 33, 100, 333 and 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide (SA; 1 μg/plate, TA1535 and TA100); 9-Aminoacridine (9AA; 75 µg/plate, TA 1537); 2-Nitrofluorene (2NF; 1 µg/plate, TA98 and TA 1538); Methylmethanesulfonate (MMS; 1000 µg/plate, WP2 uvrA) with S9: 2-Amino-anthracene (2-AA; 1 μg/pl
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)DURATION- Exposure duration: 48 to 72 h- Expression time (cells in growth medium): 48 to 72 hDETERMINATION OF CYTOTOXICITY - Method: Inspection of the bacterial backgroung lawn wit a dissecting microscope
Evaluation criteria:
Revertant colonies were counted and the mean and standard deviation were calculated and compared to the controls.All Salmonella tester strains must demonstrate the presence of the deep rough mutation and the deletion of the uvrA gene. Cultures of the TA98 and TA100 strains must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion of the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls. Tester strain titers must be above 30.000.000 cells/ml. The mean of each positive control must be at least three-fold increased to the controlls. A minimum of three non-toxic dose levels are recquired to evaluate assay data.
Statistics:
Mean and standard deviation were calculated
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
other: An unacceptable vehicle control value with the tester strain TA1537, contamination of tester strain TA98, experiments were repeated
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle 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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: >100 µg/plateRANGE-FINDING/SCREENING STUDIES: YesCOMPARISON WITH HISTORICAL CONTROL DATA: Yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiment 1:

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 1535

TA1537

TA98

TA100

 

TA 1538

WP2uvrA

-

Vehicle

12

4

17

111

5

21

 

10

3

4

15

115

6

16

 

33

5

5

19

113

6

11

-

100

9

4

19

98

7

12

-

333

7

5

11

116

4

10

-

1000

8

6

21

120

7

13

Positive

controls

- S9

Name

SA

9AA

2NF

SA

2NF

MMS

Concentrations

(μg/plate)

1.0

75

1.0

1.0

1.0

1000

Number of colonies/plate

241

40

105

377

179

143

+

Vehicle

14

5

18

133

7

11

 

10

9

5

27

114

12

16

+

33

9

3

21

113

8

13

+

100

8

7

26

108

9

13

+

333

9

4

27

115

6

8

+

1000

10

5

17

111

9

13

Positive

controls

+ S9

Name

2AA

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1.0

1.0

1.0

1.0

1.0

10

Number of colonies/plate

72

127

888

904

783

57

SA: Sodium azide

9AA : 9-Aminoacridine

MMS: Methylmethanesulfonate

2-AA: 2-Aminoanthracene

2NF: 2-Nitrofluorene

 

 

Experiment 2/3:

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 1535

TA1537

TA98

TA100

 

TA 1538

WP2uvrA

-

Vehicle

14

10

23

126

11

27

 

10

7

13

16

117

7

27

 

33

9

15

23

124

8

19

-

100

5

13

22

120

6

18

-

333

12

9

17

110

11

16

-

1000

8

14

17

125

5

21

Positive

controls

- S9

Name

SA

9AA

2NF

SA

2NF

MMS

Concentrations

(μg/plate)

1.0

75

1.0

1.0

1.0

1000

Number of colonies/plate

429

757

125

601

221

195

+

Vehicle

8

5

19

147

14

24

 

10

9

7

17

142

16

30

+

33

10

5

19

136

18

25

+

100

10

5

20

132

13

31

+

333

10

4

17

138

12

19

+

1000

10

7

19

125

12

19

Positive

controls

+ S9

Name

2AA

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1.0

1.0

1.0

1.0

1.0

10

Number of colonies/plate

85

97

530

647

1041

88

SA: Sodium azide

9AA : 9-Aminoacridine

MMS: Methylmethanesulfonate

2-AA: 2-Aminoanthracene

2NF: 2-Nitrofluorene

Conclusions:
Interpretation of results (migrated information):negative Under the tested experimental conditions the test substance did not induce gene mutations in S. typhimurium strains and in an E. coli strain up to the maximum of solubility. Therefore it is not considered to be mutagenic in this bacterial mutagenicity tes
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
22 May - 28 Oct 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. For read-across, maximum reliability score is 2.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adpoted in 1995
Deviations:
yes
Remarks:
Only basic data on test substance given
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy´s 5A medium supplemented with 10% FBS, 100 units penicillin and 100 µg streptomycin/ml and 2 mM L-glutamine- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9-mix (Spraque-Dawley)
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/mLChromosomal abberation assay: (157, 313), 625, 1250, 2500, 5000 µg/mL. Concentrations 157 and 313 µg/mL were not evaluated for CA
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, 0.08 and 0.15 µg/mL, -S9; mitomycin C 10 µg/mL, +S9
Remarks:
0.08 and 0.15 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Exposure duration: 4 and 20 h without S9 activation and 4 h with activation.STAIN (for cytogenetic assays): 5% GiemsaNUMBER OF CELLS EVALUATED: A minimum of 200 metaphase spreads (100 per duplicate flask) at each concentration used.
Evaluation criteria:
Criteria for cytotoxicity of the test substance: (1) cell growth inhibition relative to the solvent controlCriteria for chromosomal damage: (1) Number and types of aberrations found, the percentage of structurally and numerically damaged cells in the total population were counted.
Statistics:
The frequency of structural aberrations per cell was calculated. The statistical analysis of the percent aberrant cells was performed with Fisher´s Exact Test. It was used to compare pair wise the percent aberrant cells of each treatment group with that of the solvent control. In the event of positive control, the Cochran-Armitage test was used to measure dose- responsiveness. The dose response was estimated by linear regression curves. Difference in the sensitivity of mutagenic compounds was established by comparing the slopes of corresponding dose-response regression curves. For a comparison of mean values, Student's t test was used.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
48% in the highest dose for treatment of 4 h without metabolic activation, 33% with metabolic activation and 3% for 20 h exposure without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: 8 in the highest dose- Effects of osmolality: 285 in the highest doseRANGE-FINDING/SCREENING STUDIES: Yes, cell growth inhibition of 83% without and 3% with metabolic activation at the highest dose tested (5000 µg/mL).COMPARISON WITH HISTORICAL CONTROL DATA: Yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Chromosomal aberration – Summary

 

Treatment

[µg/ml]

S9 activation

Treatment/

harvest time [h]

Mitotic index

Cells scored

Aberrations/cell (mean±SD)

Cells with aberrations [%]

numerical

structural

Vehicle (Ethanol)

-

4/20

6.9

200

0.005±0.071

2.5

0.5

625

-

4/20

5.9

200

0.000±0.000

2.0

0.0

1250

-

4/20

5.9

200

0.025±0.186

4.0

2.0

2500

-

4/20

6.5

200

0.025±0.186

2.5

2.0

5000

-

4/20

4.4

200

0.000±0.000

0.5

0.0

MMC (0.08)

-

4/20

6.1

200

0.150±0.788

3.5

9.0*

Vehicle (Ethanol)

+

4/20

7.8

200

0.025±0.157

2.0

2.5

625

+

4/20

7.0

200

0.020±0.140

1.0

2.0

1250

+

4/20

6.9

200

0.035±0.184

3.0

3.5

2500

+

4/20

7.4

200

0.030±0.222

2.0

2.0

5000

+

4/20

7.7

200

0.010±0.100

2.5

1.0

CP (10)

+

4/20

2.3

200

0.950±1.591

2.5

45.5*

Vehicle (Ethanol)

-

20/20

7.6

200

0.020±0.140

1.0

2.0

625

-

20/20

6.4

200

0.015±0.158

1.5

1.0

1250

-

20/20

5.5

200

0.025±0.186

2.0

2.0

2500

-

20/20

6.0

200

0.025±0.157

2.5

2.5

5000

-

20/20

6.2

200

0.020±0.172

2.0

1.5

MMC (0.08)

-

20/20

5.8

200

0.220±0.513

1.0

18.0*

MMC = Mitomycine C

CP = Cyclophosphamide

* = p≤0.01; Fisher´s exact test

Conclusions:
Interpretation of results (migrated information):negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23 Feb - 26 Jul 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (no details on analytical purity of the test substance given).
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no details on analytical purity of the test substance given
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no details on analytical purity of the test substance given
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
yes
Remarks:
no details on analytical purity of the test substance given
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: in the absence of S9 mix: McCoy's 5A Medium containing 10% foetal bovine serum and 2 mM L-glutamine; in the presence of S9 mix: serum-free McCoy's 5A Medium containing 2 mM L-glutamine - Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague Dawley rats treated with Aroclor
Test concentrations with justification for top dose:
Range-finder toxicity test: 0, 20, 39, 78, 156, 313, 625, 1250 and 2500 µg/mL (3 h treatment), with and without S9Initial and repeat experiment (main assay): 25, 75, 250, 750 and 2500 µg/mL (3 h treatment), with and without S9Chromosome aberration analysis: 75, 750, and 2500 µg/mL (without S9); 25, 250 and 2500 µg/mL (with S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone- Justification for choice of solvent/vehicle: a vehicle solubility test was previously performed to assess the solubility of the test substance in DMSO, water and acetone. The results of this test showed that the test substance was soluble in acetone at the concentrations required for this study. The non-cytotoxic dose volume for acetone is 50 µL/flask, therefore based on solubility, 2500 µg/mL was the highest dose that could be achieved in this assay.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
- S): 9,10-dimethyl- 1,2-benzanthracene (DMBA, 10 µg/mL in acetone); + S9: 1-methyl-3-nitro-1-nitrosoguanidine (MNNG, 0.6 µg/mL in acetone
Positive control substance:
9,10-dimethylbenzanthracene
other: 1-methyl-3-nitro-1-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Exposure duration: 3 h- Fixation time (start of exposure up to fixation or harvest of cells): A) Range-finder toxicity test: 19 h B) Initial experiment (main assay): 19 hC) Repeat experiment (main assay): 19 and 43 hSPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL Colcemid®STAIN (for cytogenetic assays): 5% GiemsaNUMBER OF REPLICATIONS: 2NUMBER OF CELLS EVALUATED: 100 per cultureDETERMINATION OF CYTOTOXICITY- Method: mitotic index and cell confluency (range-finder toxicity test and main assay); other: cell count (range-finder toxicity test)
Evaluation criteria:
For a test substance to be considered as positive, one of the following conditions must be met:- a statistically significant dose-related increase in the mean percentage of aberrant cells and, in at least one of the treatment groups, the mean percentage of aberrant cells exceeds 5%.OR- a reproducible and statistically significant response for at least one of the treatment groups is observed. In addition, the mean percentage of aberrant cells exceeds 5%.A positive result indicates that the test substance induces chromosomal aberrations in cultured mammalian somatic cells. If neither of the above conditions are met, the test substance is considered as non-mutagenic in this system.
Statistics:
The number of cells with at least one aberrant chromosome and the number of cells examined in each replicate were used for statistical analysis. The number of aberrant individual chromosomes per cell was not statistically analysed. The results of the positive control group were compared to the appropriate vehicle control group by the Fisher Exact Test to assure that the assay was performed in an appropriate manner. To test for homogeneity of the replicates, each pair of replicates was compared by Fisher Exact Test. Two times the sum of the log of the individual two-sided significance levels was compared to chi-square distribution with 2k degrees of freedom (k is the number of replicate pairs). If the test failed, further investigation would be pursued and the remaining analyses would not be performed. To test for differences among the control and the treated groups, a 2x2-Fisher Exact Test was performed. If differences were shown to exist at the 0.05 level or less, individual 2x2-Fisher Tests were performed to determine which of the treated groups differed from the control group. A permutation test (Hoeffding, 1952) was performed to test for dose-related trends. Statistical analysis included the calculation of means for percent confluency, percent mitotic cells, percent aberrant cells, percent frequency of aberrations, and cell counts. Significance was reported when p < 0.05.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: in the range-finder toxicity test, visual solubility observations were made 3 h after treatment. Cloudiness was evident at all concentrations with S9. Complete solubility was observed at concentrations ranging from 10 to 39 µg/mL without S9. Cloudiness was evident at concentrations of 78 µg/mL through 625 µg/mL without S9. Oil droplets were noted without metabolic activation at concentrations ≥ 1250 µg/mL. A notable decrease in confluency (≥ 50% reduction compared with vehicle controls) were observed at concentrations of 625 µg/mL and 1250 µg/mL with S9 and 2500 µg/mL without S9. Decreases in cell survival and mitotic index (MI) of ≥ 50% from the vehicle control were not observed in either the activated or non-activated series. Cell morphology was normal at all concentrations tested, however debris of undetermined origin was noted in activated flasks at concentrations ≥ 156 µg/mL and non-activated flasks ≥ 313 µg/mL. Based on these results, 25, 75, 250, 750 and 2500 µg/mL were selected as concentrations for the main chromosomal aberration assay.ADDITIONAL INFORMATION ON CYTOTOXICITY: in both experiments of the main study, there was no notable decrease in the percent confluency (≥ 50% reduction compared to the vehicle control) at any concentration with or without S9. Cell morphology was normal at all concentrations tested. Similarly, no notable decrease (≥ 50%) in MI values was noted for the test substance concentrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

CHROMOSOME ANALYSIS

A) INITIAL ASSAY

There were no statistically significant differences or dose-related trends in the percentage of cells with chromosomal aberrations between the treated and the control groups either with or without metabolic activation. The percentage of aberrant cells in the vehicle control groups, with and without metabolic activation, was 2.5 and 1.0%, respectively. Both values fell within the 0 to 5% range of acceptance for the vehicle control. The percentage of aberrant cells in the treatment groups ranged from 2.0 to 3.0% for the metabolically activated series and from 0.5 to 3.0% in the non-activated series.

Table 1. Test results of the initial experiment

Test item

Concentration in µg/mL

Mitotix index in % of vehicle control

Aberrant cells in % per 200 cells scored

Frequency of abberations in % per 200 cells scored

with gaps

without gaps

Exposure period 3 h, fixation time 19 h, without S9 mix

Acetone

a

100

1.0

1.5

1

MNNG

0.6

26

12.0**

5.0

12.5

Test substance

75

77

0.5

2.5

0.5

750

80

3.0

1.0

3

2500

54

2.0

0.0

2

Exposure period 3h, fixation time 19 h, with S9 mix

Acetone

a

100

2.5

2.5

3.0

DMBA

10

59

19.0**

5.0

26.0

Test substance

25

112

3.0

3.0

3.0

250

100

2.5

1.5

2.5

2500

104

2.0

3.0

2.0

MNNG = 1-methyl-3-nitro-1-nitrosoguanidine; DMBA = 9,10-dimethyl-1,2-benzanthracene

a = concentration: 50 µL/flask; b = positive controls were not required for the 43 h harvest

*p < 0.05; **p < 0.01

B) REPEAT ASSAY

There was a statistically significant difference (p < 0.05) in the percentage of cells with chromosomal aberrations between the vehicle and the highest concentration (2500 µg/mL) in the 19 h harvest with metabolic activation. The permutation test for the 19 h harvest with metabolic activation also appears to be significant (p<0.01) indicating a dose-related trend in the percentage of cells with chromosomal aberrations. This increase in the percentage of aberrant cells does not appear to be biologically significant due to the fact that the percentage of aberrant cells fell within the acceptable range for the vehicle control (0-5%).

The percentage of aberrant cells in the vehicle control groups at both harvest intervals (with and without metabolic activation) was 2.5% or less. These values fell within the 0 to 5% range of acceptance for the vehicle control. The percentage of aberrant cells observed in the 19 h treated groups ranged from 1 to 3.0%. The percentage of aberrant cells observed in the 43 h treated groups ranged from 0.5% to 2.5%.

Table 2. Test results of the repeat experiment

Test item

Concentration in µg/mL

Mitotix index in % of vehicle control

Aberrant cells in % per 200 cells scored

Frequency of abberations in % per 200 cells scored

with gaps

without gaps

Exposure period 3 h, fixation time 19 h, without S9 mix

Acetone

a

100

1.0

1.0

1.0

MNNG

0.6

42

8.0**

2.0

13.5

Test substance

75

89

2.0

2.5

2.5

750

65

1.5

1.0

1.5

2500

82

1.5

1.0

2.0

Exposure period 3 h, fixation time 43 h, without S9 mix

Acetone

a

100

2.5

2.5

2.5

MNNG

0.6

b

b

b

b

Test substance

75

105

0.5

1.0

0.5

750

95

0.5

1.5

0.5

2500

68

1.5

2.0

1.5

Exposure period 3 h, fixation time 19 h, with S9 mix

Acetone

a

100

0.0

1.5

0.0

DMBA

10

66

10.5**

7.0

11.5

Test substance

25

107

1.0

1.0

1.0

250

105

1.0

1.0

1.0

2500

60

3.0* #

1.0

3.5

Exposure period 3 h, fixation time 43 h, with S9 mix

Acetone

a

100

1.0

1.0

1.0

DMBA

0.6

b

b

b

b

Test substance

25

89

2.5

2.5

2.5

250

70

1.5

1.0

1.5

2500

68

2.0

0.0

2.0

MNNG = 1-methyl-3-nitro-1-nitrosoguanidine; DMBA = 9,10-dimethyl-1,2-benzanthracene

a = concentration: 50 µL/flask; b = positive controls were not required for the 43 h harvest

*p < 0.05; **p < 0.01

Conclusions:
Interpretation of results (migrated information):negative
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:
03 April 2014 to 02 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S. typhimurium strains)trp operon ( E. coli strain)
Species / strain / cell type:
S. typhimurium, other: TA97a, TA1535, TA98 and TA100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
A cytotoxicity screen was conducted in the Salmonella typhimurium TA-100 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 µI/plate) of the test article, two plates per dose.Based on the cytotoxicity results, five concentrations (0.05, 0.1, 0.5, 1 and 5 µI/plate) of the test article were tested in each of five bacterial tester strains (E. coliWP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100).
Vehicle / solvent:
Identity: Acetone, Lot#124017Supplied by: Fisher ScientificDate Received: 15 Feb 2013Expiration Date: Jul 2017Storage: Room temperature.Description: Clear colorless liquidSample Preparation: Used as received.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-Aminoanthracene (2AA), Acridine, 6-chloro-9-(3-((2-chloroethyl)amino)propyl)amino-2-methoxy, dihydrochloride (ICR-191), Daunomycin (DM),
Details on test system and experimental conditions:
Sample PreparationScreen: 50 µI of the test article were brought to a volume of 1 ml with Acetone to yield a 50 µI/ml solution (stock for a dose of 5 µl/plate) (clear colorless liquid). Additional dilutions were made with Acetone (clear colorless liquids).Main Assay: 500 µI of the test article were brought to a volume of 10 ml with Acetone to yield a 50 µI/ml solution (stock for a dose of 5 µl/plate) (clear colorless liquid). Additional dilutions were made with Acetone (clear colorless liquids).Independent Repeat Assay: 500 µI of the test article were brought to a volume of 10 ml with Acetone to yield a 50 µl/ml solution (stock for a dose of 5 µI/plate) (clear colorless liquid). Additional dilutions were made with Acetone (clear colorless liquids).Preparation of the Tester Strains: Bacterial cultures were inoculated by the addition of a lyophilized disk of each tester strain to Oxoid No.2 nutrient broth (Molecular Toxicology, Inc. (Moltox) Boone, NC, cat. #26-555). Ampicillin was added to the nutrient broth to ensure the retention of R-factor plasmid in tester strains TA-97a, TA-98 and TA-100. The cultures were incubated at 37°C ± 2°C with agitation. The cultures were used after they reached the late exponential growth phase as determined by absorbance readings at 600 nm.Exogenous Metabolic Activation: An activation buffer containing 10% S9 obtained from the livers of Aroclor 1254-treated adult Sprague Dawley rats was prepared according to the manufacturer's (Moltox) instructions. Each vial of Regensys B (Moltox cat# 60-201) was reconstituted with approximately 1 ml of Regensys A (Moltox cat# 60-200). The solution was transferred back into the Regensys A bottle S9 (Moltox cat# 11-101) was then mixed with Regensys A+B to yield a 10% S9 buffer stock solution. The stock was separated into aliquots in sterile 15 ml conical tubes and refrigerated at 2-8°C until used.The exogenous metabolic activation mixture was added to one set of all doses - each test article concentration, vehicle control and positive control for each of the bacterial tester strains.Treatment of the Test System: Top agar supplemented with appropriate amino acids were prepared, as 2 ml aliquots, and maintained at 45-50°C in sterile culture tubes. Dulbecco's Phosphate Buffered Saline (DPBS) was added to the tubes not undergoing S9 activation (i.e. without S9, or -S9) to maintain equal dosing volumes. 0.1 ml of bacteria was added to the top agar, followed by 0.1 ml of the test article, vehicle control or positive control. For the activation portion of the test, 0.5 ml of S9 mixture was added last. The contents were gently vortexed and overiaid onto minimal glucose agar plates. After the mixture had solidified, the plates were incubated at 37°C ± 2°C for 48-72 hours. Plates that were not scored immediately following the incubation period were stored at 2-8°C until scoring.An independent repeat (confirmatory) test was dosed using a preincubation method. One-hundred microliters of the test article concentration or Control was preincubated with 0.1 ml of the bacteria tester strain, along with 0.5 ml of Phosphate Buffered Saline or the metabolic activation (S9) system. These mixtures were incubated for 20 minutes or more at approximately 3rC prior to being mixed with the overlay agar and poured onto the surface of minimal agar plates. At the request of the Sponsor, the independent repeat (confirmatory) test was aborted after the plates were dosed.Screen: Prior to the cytotoxicity screen, solubility of the test article was checked in tissue culture water (TCH2O), Dimethyl sulfoxide (DMSO), and Acetone. The test article was freely soluble at a concentration of 50 µI/ml only in Acetone and the Study Director chose Acetone as the vehicle for the study A cytotoxicity screen was conducted in the TA-100 tester strain using eight concentrations (0.001,0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 µI/plate) of the test article, two plates per concentration, with and without S9. A vehicle control (Acetone) was run concurrently, with and without S9. The plates were incubated at 37°C ± 2°C for 48-72 HoursMain Assay: Five concentrations (0.05, 0.1, 0.5, 1 and 5 µI/plate) of the test article were tested in each of five bacterial tester strains (Escherichia coli WP2 uvrA, and Salmonella typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Two sets of culture plates were dosed per concentration (+S9 and -S9). A vehicle control and positive controls specific to each bacterial strain were treated in a similar manner as the test article concentrations. The plates were incubated at 37°C ± 2°C for 48-72 hours.Revertant Colony Count: Counting of the revertants per plate was performed using an Alphalmager™ 2200 (Alpha Innotech Corporation, San Leandro, CA) fluorescence imager. Proper function of the imager was verified against a standard template (e.g. high (1000), medium (100) and low (10) counts) prior to each daily use. The number of revertants was recorded, along with observations of cytotoxicity. Routine examination (under a light microscope) of the bacterial background lawn was used to determine cytotoxicity of the test article.The plates were also examined visually for test article precipitate.Independent Repeat Assay: The guidelines recommend that equivocal results be clarified by further testing, preferably using a modification of experimental conditions (e.g. concentrations tested), and that negative results need to be confirmed on a case-by-case basis. Justification should be provided when confirmation of negative results is not considered necessary. There is no recommendation of an independent repeat assay when the assay results are clearly positive.At the request of the Sponsor, an independent repeat assay (confirmatory test) was aborted after all tester strains had been dosed with test material and plated but before the plates were scored.Quality Check of the Assay:Sterility Test: Sterile technique is required throughout the course of the study. To ascertain each component's sterility, a small amount (1 ml) was placed on agar plates and incubated to encourage growth of any contaminating microorganisms. The plates were incubated for 48-72 hours at 37°C ± 2°C and visually observed for microbial growth.Vehicle Control: The spontaneous reversion rate, as represented by the mean colony forming units (CFU), for each strain of bacteria was calculated and compared to in-house historical ranges.Positive Control: Positive control treatment for each tester strain of bacteria must result in at least a 2-fold increase of revertants over the mean vehicle control value. The effectiveness of the exogenous metabolic activation mixture was demonstrated by the positive response of the control.
Evaluation criteria:
Plates were scored based on the number of revertant colony-forming units present per plate. The number of revertants of each test article plate were averaged and plotted versus concentration of the test article. The mean number of revertants of each dose was divided by the mean for the vehicle control value to obtain a ratio to vehicle. In evaluating the data, cytotoxicity of the test article as well as quality checks of the assay were taken into account.In general, a 2-fold increase with or without metabolic activation is considered a positive response. Dose-related increases approaching a 2-fold increase are deemed equivocal.A negative result is determined by the absence of a dose-related increase in all five tester strains, again taking into account cytotoxicity of the test article as well as the quality checks of the assay.Positive results from the bacterial reverse mutation test indicate that the substance induces point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested strains.
Statistics:
Not specified in the study report.
Key result
Species / strain:
S. typhimurium, other: TA97a, TA1535, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Screen: There was no diminution or clearing of the background lawn observed at any dose level, indicating that the test article was not cytotoxic to tester strain TA-100 at 0.001 to 5 µI/plate. The Study Director chose 5 µI/plate as the top test article concentration for the main test.Main Assay: The assay was run in all five strains on triplicate plates, Positive and vehicle controls were run concurrently for all five strains, on six plates per strain. All plating was with and without exogenous metabolic activation, S9. No reduction or clearing of the bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test article under test conditions. There was no significant increase or dose-dependent increase of the number of revertants in any tester strain treated with the test article in the presence or absence of S9, All Positive and Negative Control values were within acceptable ranges, and all criteria for a valid study were met. However, TA-98 (with and without S9) and WP2 uvrA (with S9) initially did not pass QC parameters and had to be repeated. TA-98 with S9 did not pass the negative control QC parameters (average revertant colonies higher than historical range) but passed positive control parameter (greater than 2-fold increase over negative control), TA-98 without S9 did not pass either QC parameter and WP2 uvrA with S9 did not pass the positive control QC parameter, Upon repeating dosing for these two strains (TA-98 with and without S9, and WP2 uvrA with S9) all QC parameters were within acceptable ranges.Independent Repeat Assay: An independent repeat assay was dosed in all five tester strains using test article concentrations of 0.05, 0.1, 0.5, 1, and 5 µI/plate and the pre-incubation dosing method. However, at the request of the Sponsor the assay was terminated before the plates were scored.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1

Test Article: H2925 (CAS No. 68130-53-0), Lot# 2013090401

Tester Strain: E.coli EP2 uvrA

Treatment

S9

CFU

Mean

Std. Dev.

Std. Error of Mean

Old Increase over Vehicle

Acetone (Vehicle Control)

+

63

50

51

48

43

52

51.3

6.5

2.7

 

5µl/plate

+

49

61

61

 

57.0

6.9

4.0

1.1

1µl/plate

+

47

65

54

55.32

9.1

5.2

1.1

0.5µl/plate

+

57

55

60

57.3

2.5

1.5

1.1

0.1µl/plate

+

47

54

61

54.0

7.0

4.0

1.1

0.05µl/plate

+

47

53

53

51.0

3.5

2.0

1.0

10µg 2AA (Positive Control)

+

132

131

166

152

121

134

141.0

14.7

6.0

2.7*

Acetone (Vehicle control)

-

73

41

98

98

119

83

85.3

26.7

10.9

 

5µl/plate

-

49

107

38

 

64.7

37.1

21.4

0.8

1µl/plate

-

95

94

75

88.0

11.3

6.5

1.0

0.5µl/plate

-

62

76

94

77.3

16.0

9.3

0.9

0.1µl/plate

-

45

59

68

57.3

11.6

6.7

0.7

0.05µl/plate

-

69

50

37

52.0

16.1

9.3

0.6

2.5µl MMS (Positive Control)

-

238

440

421

394

368

338

366.5

72.7

29.7

4.3*

*= 2-fold or more increase over Vehicle Control

 

Table 2

Test Article: H2925 (CAS No. 68130-53-0), Lot# 2013090401

Tester Strain: S. typh. TA-97a

Treatment

S9

CFU

Mean

Std. Dev.

Std. Error of Mean

Old Increase over Vehicle

Acetone (Vehicle Control)

+

63

54

68

61

81

88

69.2

12.9

5.3

 

5µl/plate

+

57

78

73

 

69.3

11.0

6.3

1.0

1µl/plate

+

75

80

80

78.3

2.9

1.7

1.1

0.5µl/plate

+

72

71

77

73.3

3.2

1.9

1.1

0.1µl/plate

+

77

75

59

70.3

9.9

5.7

1.0

0.05µl/plate

+

75

65

53

64.3

11.0

6.4

0.9

10µg 2AA (Positive Control)

+

611

564

541

497

496

468

529.5

52.8

21.5

7.7*

Acetone (Vehicle control)

-

40

52

46

58

63

56

52.5

8.4

3.4

 

5µl/plate

-

54

69

59

 

60.7

7.6

4.4

1.2

1µl/plate

-

57

59

65

60.3

4.2

2.4

1.1

0.5µl/plate

-

56

57

82

68.3

13.1

7.5

1.3

0.1µl/plate

-

69

58

65

64.0

5.6

3.2

1.2

0.05µl/plate

-

66

69

47

60.7

11.9

6.9

1.2

1µg ICR191 (Positive control)

-

1044

984

936

930

1091

1023

986.3

77.3

31.5

18.8*

*= 2-fold or more increase over Vehicle Control

 

Table 3

Test Article: H2925 (CAS No. 68130-53-0), Lot# 2013090401

Tester Strain: S. typh. TA-1535

Treatment

S9

CFU

Mean

Std. Dev.

Std. Error of Mean

Old Increase over Vehicle

Acetone (Vehicle Control)

+

10

8

10

13

12

9

10.3

1.9

0.8

 

5µl/plate

+

9

16

11

 

12.0

3.6

2.1

1.2

1µl/plate

+

5

10

9

8.0

2.6

1.5

0.8

0.5µl/plate

+

8

10

11

9.7

1.5

0.9

0.9

0.1µl/plate

+

10

11

14

11.7

2.1

1.2

1.1

0.05µl/plate

+

12

8

9

9.7

2.1

1.2

0.9

10µg 2AA (Positive Control)

+

47

44

36

44

48

52

45.2

5.4

2.2

4.4*

Acetone (Vehicle control)

-

11

12

7

7

16

8

10.2

3.5

1.4

 

5µl/plate

-

17

14

13

 

14.7

2.1

1.2

1.4

1µl/plate

-

8

13

6

9.7

2.9

1.7

1.0

0.5µl/plate

-

9

14

12

11.7

2.5

1.5

1.1

0.1µl/plate

-

11

5

12

9.3

3.6

2.2

0.9

0.05µl/plate

-

11

5

10

8.7

3.2

1.9

0.9

1.5µg NaN3(Positive Control)

-

545

548

534

552

515

482

529.3

26.7

10.9

51.9*

*= 2-fold or more increase over Vehicle Control

Conclusions:
Interpretation of results (migrated information):negative with and without metabolic activationUnder test conditions, test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, did not have mutagenicity potential.
Executive summary:

Objective: The purpose of this study is to evaluate the mutagenic potential of a test article based on the reversion of selective growth mutations in several strains of Salmonella typhimurium bacteria and in Escherichia coli WP2 uvrA bacteria, in the presence and absence of S9 activation. This protocol is based on OECD Guideline for Testing of Chemicals: No. 471 - Bacterial Reverse Mutation Test and U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 - Bacterial Reverse Mutation Test.

 

Method Synopsis: Prior to the cytotoxicity screen, solubility of the test article was checked in tissue culture water (TCH2O), Dimethyl sulfoxide (DMSO), and Acetone. The test article was freely soluble at a concentration of 50µI/mlonly in Acetone and the Study Director chose Acetone as the vehicle for the study. A cytotoxicity screen was conducted in the Salmonella typhimurium TA-100 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5µI/plate)of the test article, two plates per dose. The test article was combined with the bacteria and top agar in the presence and absence of a metabolic activation mixture (S9) and overlaid onto minimal glucose agar plates. An Acetone vehicle control was run concurrently, with and without S9.

Based on the cytotoxicity results, five concentrations (0.05, 0.1, 0.5, 1 and 5µI/plate)of the test article were tested in each of five bacterial tester strains(E.coliWP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Vehicle controls and positive controls specific to each bacterial strain were treated in a similar manner as the test article concentrations. The plates were incubated at 37°C ± 2°C for 48-72 hours. Revertant colony growth was determined by counting the colonies per plate using an Alphalmager™ imaging system. The number of revertants of the test article treatment plates and positive control plates was divided by the number of revertants of the vehicle plates. In general, a positive result is determined by a 2-fold increase above the vehicle control.

Tester strains WP2 uvrA (with S9) and TA-98 (with and without S9) failed the quality checks. The main test was repeated for these strains and passed the quality checks. The results of the repeat main test are reported; the original data is maintained in the study file.

At the request of the Sponsor, an independent repeat assay (confirmatory test) was aborted after all tester strains had been dosed with test material and plated but before the plates were scored.

 

Summary: Test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, in the vehicle, Acetone, was tested in a Bacterial Reverse Mutation Assay. In the screen, the test article did not show obvious cytotoxicity to tester strain TA-100 at any concentration, with or without S9. In the main test, the test article at 0.05 to 5µI/plate,with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of any bacterial tester strain, indicating that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay.

 

Conclusion: Under test conditions, test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, did not have mutagenicity potential.

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
Study period:
26 Mar - 11 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 supplemented with 5% (v/v) heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
First experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with and without metabolic activation (8%, v/v))
Second experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with metabolic activation (12%, v/v)); 0.1, 1, 3, 10, 33, 100, 200, 250 µg/mL (without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
in the absence of S9-mix Migrated to IUCLID6: 15 and 5 µg/mL for 3 and 24 h treatment period
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in the presence of S9-mix Migrated to IUCLID6: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: cells were exposed to the test material for 3 h and 24 h
- Expression time (cells in growth medium): Cells in the final suspension after treatment were counted with the coulter particle counter. For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. Cells were plated for the determination of the cloning efficiency and mutation frequency. For the determination of the mutation frequency cells were plated and incubated for 11-12 d. After that, cells were stained for 2 h by adding 0.5 mg/mL MTT (Sigma) to each well. The plates were scored for cloning efficiency and mutation frequency with the naked eye or with the microscope.

SELECTION AGENT (mutation assays): RPMI 1640 supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth
Evaluation criteria:
Measurement of cytotoxicity by determining the relative cloning efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period.
There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency.
Statistics:
The cloning efficiency (CE) was determined as follows:
P(0)= Number of empty wells divided by the total number of wells
CE= P(0)/number of cells plated per well

Relative survival rate (RS): RS= [CE(test)/CE(control)] x 100
Relative total growth (RTG): RTG= RSG x RSday2 / 100
Suspension growth (SG): [Day 0 cell count/1.25x10E005] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count]
Relative suspension growth (RSG): SG(test)/SG(control) x 100

RSday2= CEday2(test) / CEday2(control) x 100

The growth rate (GR) was calculated for the solvent control cultures:
- 3 h treatment: [Day 1 cell count/1.25x105] x [Day 2 cell count/1.25x10E005]
- 24 h treatment: [Day 0 cell count/1.25x105] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count/1.25x10E005]


The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:

MF= {-ln P(0)/number of cells plated per well)/CE day2 x 10E-006

Small and large colony mutation frequencies were calculated in an identical manner.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
and above (precipitating concentration: 100 µg/mL, tested up to 250 µg/mL)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 100 µg/mL

RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity data was obtained by treating cells for 3 h and 24 h, respectively, with a number of increasing test substance concentrations. The highest concentration tested was 200 µg/ml due to poor solubility of the test substance. No toxicity was observed with and without metabolic activation up to and at the maximum dose level tested with 3 h incubation. 24 h incubation resulted in 64% relative suspension growth in the absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all controls were in the range of the historical controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Experiment 1 - 3 hours with and without S9 mix

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10-6

 

 

 

 

 

total

Without metabolic activation, 3 h treatment

SC1

100

94

100

100

89

SC2

108

73

0.03

98

101

100

98

63

0.1

92

99

98

90

83

0.3

111

102

101

112

58

1

107

98

97

104

64

3

110

101

100

110

83

10

98

99

98

96

83

33

98

110

109

106

90

100*

74

94

93

69

97

MMS

70

63

63

44

1022

With 8% (v/v) metabolic activation, 3 h treatment

SC1

100

77

100

100

82

SC2

84

87

0.03

96

90

112

107

71

0.1

92

104

129

119

60

0.3

80

108

135

108

55

1

93

105

131

121

69

3

97

90

112

109

65

10

95

84

104

99

71

33

93

81

101

94

91

100*

42

83

103

43

98

CP

20

37

47

9

1107

 

Table 2: Experiment 2 - 3 hours with and 24 hours without S9 mix

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10-6

 

 

 

 

 

total

Without metabolic activation, 24 h treatment

SC1

100

102

100

100

62

SC2

104

57

0.1

97

83

80

78

87

1

94

105

102

96

68

3

102

90

87

89

65

10

104

115

111

115

54

33

105

83

80

84

53

100*

102

98

95

97

55

200*

116

104

101

116

52

250*

112

108

105

118

51

MMS

80

81

79

63

631

With 12% (v/v) metabolic activation, 3 h treatment

SC1

100

77

100

100

60

SC2

91

84

0.03

116

58

69

81

108

0.1

97

80

95

93

86

0.3

94

80

95

90

76

1

99

81

97

96

88

3

102

89

106

108

71

10

104

86

103

106

73

33

119

86

103

122

83

100*

105

77

91

96

72

CP

31

54

64

20

814

 

RSG: Relative Suspension Growth; CE: Cloning efficiency; RS: Relative Survival; RTG: Relative Total Growth; SC: Solvent Control (DMSO); MMS: Methylmethansulfonate; CP: Cyclophosphamide

*: Precipitation of test substance

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (shorter exposure period. Lack of data on test substance, no positive controls for 40 h time point)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 1997
Deviations:
yes
Remarks:
(in both experiments, cultures without metabolic activation were exposed to the test substance for about 16 h, no positive control for the 40 h time point)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A Medium containing 10% (v/v) fetal bovine serum and 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
other: WBL clone
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague-Dawley rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
40, 80 and 160 µg/mL with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on results of a solubility test, acetone was selected as the vehicle. The test substance was not soluble in water or dimethyl sulfoxide at any of the concentrations (10, 25, 50% (v/v)) tested. The test substance was soluble as a 50% mixture in acetone.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 0.6 µg/mL (v/v) in acetone; + S9: 7,12-Dimethylbenz[a]anthracene (DMBA), 10 µg/mL (v/v) in acetone
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
+S9: ca. 3 h (± 0.5 h)
- S9: ca. 16 h (± 0.5 h)
- Fixation time (start of exposure up to fixation or harvest of cells): ca. 16 h (± 0.5 h); second experiment - ca. 16 and 40 h (± 0.5 h)

SPINDLE INHIBITOR (cytogenetic assays): 0.2 mL Colcemid® (10 mg/mL (v/v) in cell culture medium)
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2 replications (16 h) and 1 replication (40 h), respectively

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
Evaluation criteria:
A test substance was considered positive in the chromosome aberration test if:
1. A statistically significant dose-related increase in the percentage of aberrant cells and in at least one of the treatment groups, the percentage of aberrant cells exceeds 5%. OR
2. A reproducible and statistically significant response for at least one of the treatment groups is observed. In addition, the mean percentage of aberrant cells exceeds 5%.
A positive result indicates that under the test conditions the test substance induces chromosomal aberrations in cultured mammalian somatic cells.
If neither of the above conditions exist, the test substance is considered nonmutagenic or negative for inducing chromosomal aberrations in this system.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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 substance is not soluble in water, therefore it was dissolved in acetone.
- Precipitation: Final concentrations of the test substance in medium of 10, 20, 39, 78, 156, 313, 625, 1250 and 2500 µg/mL were tested by visual and microscopic methods for precipitation immediately, 30 minutes and 3 h after dosing. Traces of the test substance were observed microscopically at all test concentrations equal to or greater than 78 µg/mL. Therefore, the upper limit of the culture medium solubility of the test substance was considered to be between 39 and 78 µg/mL. Based on these results, the study director selected the following concentrations for the toxicity pretest: 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL.
In the main experiments, slight precipitation was observed in the second experiment after 16 h at 160 µg/mL without metabolic activation. Precipitation was not noted at any other harvest of a 160 µg/mL culture.

RANGE-FINDING/SCREENING STUDIES: To determine a concentration selection for the aberration assay, a toxicity pretest was conducted with concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL of the test substance with and without metabolic activation. The concentrations tested were based on the results of a culture medium solubility test. The cultures with metabolic activation were treated for 3 h (± 0.5 h). The cultures without metabolic activation were treated until 2-3 h prior to harvest. All cultures were harvested about 16 h from the beginning of treatment. After harvest, the number of cells that survived treatment were counted using a hemacytometer to evaluate cytotoxicity and the mitotic indices (number of mitotic cells per 1000 total cells) were determined to evaluate cell cycle suppression. The selected concentrations for the aberration assay were based on the results of the cell count data and mitotic index data. The highest reduction in cell survival was observed at 160 µg/mL without metabolic acvtivation, where reduction in viability of 37% was noted. Other less notable reductions in cell survival were noted (see table 3), but were not indicative of a concentration-related trend. Based on these results, the concentrations selected for the aberration assay were 40, 80 and 160 µg/mL.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Test results of experiment 1

Test item

Concentration

Mitotic Index

Aberrant cells

Aberration frequency

 

 in µg/mL

in %

in %

in %

Exposure period 16 h, fixation time 16 h, without S9 mix

vehicle

0.5% (v/v)

6.8

0.5

0.5

MNNG

0.6

6.2

22.5**

24.5

Test substance

40

5.6

0.5

0.5

80

6.5

0.5

0.5

160

7.2

1.0

1.0

Exposure period 3 h, fixation time 16 h, with S9 mix

Acetone

0.5% (v/v)

5.5

1.0

1.0

DMBA

10

2.6

33.5**

42.0

Test substance

40

5.3

1.5

1.5

80

6.3

0.5

0.5

160

4.6

2.0

2.0

 

**statistically significantly higher than vehicle control (p<0.001)

MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)

 

Table 2. Test results of experiment 2

Test item

Concentration

Mitotic Index

Aberrant cells

Aberration frequency

 

 in µg/mL

in %

in %

in %

Exposure period 16 h, fixation time 16 h, without S9 mix

vehicle

0.5% (v/v)

7.2

1.0

1.0

MNNG

0.6

5.4

17.5**

17.5

Test substance

40

7.1

1.0

1.0

80

5.4

0.5

0.5

160

7.1

2.5

2.5

Exposure period 3 h, fixation time 16 h, with S9 mix

Acetone

0.5% (v/v)

2.2

0.0

0.0

DMBA

10

4.5

33.0**

43.0

Test substance

40

2.2

1.0

1.0

80

2.4

2.0

2.0

160

2.0

1.5

1.5

Exposure period 16 h, fixation time 40 h, without S9 mix

Acetone

0.5% (v/v)

3.4

2.5

2.0

MNNG #

0.6

---

---

---

Test substance

40

3.0

4.0

4.5

80

2.2

3.0

3.0

160

3.4

2.0

2.0

Exposure period 3 h, fixation time 40 h, with S9 mix

Acetone

0.5% (v/v)

4.8

2.5

2.5

DMBA #

10

---

---

---

Test substance

40

5.4

2.0

2.0

80

4.8

2.0

2.0

160

5.0

0.5

0.5

 

**statistically significantly higher than vehicle control (p<0.001)

MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)

# According to the study report, positive controls were not required for the 40 h harvest.

Table 3. Toxicity pretest results

Treatment Group
in µg/mL

Cell Survival in %*

+ S9

- S9

non-treated

107

102

vehicle

100

100

0.625

122

114

1.25

95

72

2.5

121

88

5

108

68

10

111

108

20

89

102

40

99

93

80

78

93

160

120

63

  * % cell survival as compared to vehicle

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (no details on analytical purity given)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no analytical purity reported
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S. typhimurium) and trp operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague Dawley rats treated i.p. with a single dose of 500 mg/kg bw Arochlor 1254
Test concentrations with justification for top dose:
Range-finding toxicity study (in TA 100 and WP2 uvrA): 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate, with and without metabolic activation
Main study (all strains): 33.3, 100, 333, 1000, 3330 and 5000 µg/plate, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
-S9: 2-NF (1 µg/plate, TA 98); SA (2 µg/plate, TA 100 and TA 1535); ICR-191 (2 µg/plate, TA 1537); 4-NQO (1 µg/plate, WP2 uvrA); +S9: BP (2.5 µg/plate, TA 98); 2-AA (2.5-25 µg/plate, TA 100, TA 1535, TA 1537 and WP2 uvrA)
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene; ICR-191
Remarks:
2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 52 ± 4 h

NUMBER OF REPLICATIONS: triplicates each in one experiment

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn
Evaluation criteria:
The results of the test were considered positive, if the following criteria were met:
- tester strains TA 98, TA 100 and WP2 uvrA: for a test article to be considered positive, it must produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
- tester strains TA 1535 and TA 1537: for a test article to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
Mean values and standard deviations of revertants per plate were calculated.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the absence of S9 mix, slight precipitation of the test substance was observed in all experiments at concentrations ≥ 100 µg/plate. In the absence of S9 mix, slight precipitates were noted at ≥ 333 µg/plate in the preliminary cytotoxicity study and at ≥ 1000 µg/plate in the main study.

RANGE-FINDING/SCREENING STUDIES: in a preliminary cytotoxicity study, the tester strains TA 100 and WP2 uvrA were treated with the test substance at concentrations ranging from 6.67 to 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). No cytotoxicity was observed in these strains up to the limit dose of 5000 µg/plate, neither with nor without addition of S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Test results of experiment (plate incorporation)

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)

EXPERIMENT

S9-Mix

Without

 

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

SC

25 ± 2

80 ± 9

14 ± 8

3 ± 1

26 ± 3

Test material

 

33.3 µg

19 ± 4

88 ± 5

12 ± 5

4 ± 4

19 ± 2

100 µg

21 ± 7

96 ± 12

11 ± 2

5 ± 4

19 ± 4

333 µg

22 ± 2

92 ± 9

13 ± 2

5 ± 5

23 ± 10

1000 µg

25 ± 6

97 ± 11

25 ± 2

5 ± 3

30 ± 3

3330 µg

22 ± 1

101 ± 3

10 ± 1

3 ± 2

32 ± 2

5000 µg

20 ± 6

85 ± 6

15 ± 3

4 ± 2

27 ± 3

PC

 

2-NF

206 ± 42

-

-

-

-

SA

-

549 ± 71

480 ± 3

-

-

ICR-191

-

-

-

277 ± 33

-

4-NQO

 -

 -

 -

260 ± 43

S9-Mix

 

With

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

SC

36 ± 2

102 ± 2

19 ± 1

7 ± 1

25 ± 5

Test material

 

33.3 µg

36 ± 7

93 ± 9

15 ± 2

8 ± 4

22 ± 5

100 µg

34 ± 7

97 ± 17

16 ± 4

9 ± 2

22 ± 6

333 µg

34 ± 6

87 ± 6

17 ± 2

9 ± 4

27 ± 3

1000 µg

39 ± 9

94 ± 17

17 ± 3

8 ± 2

26 ± 6

3330 µg

38 ± 8

92 ± 6

16 ± 4

6 ± 3

29 ± 11

5000 µg

34 ± 5

139 ± 5

20 ± 3

3 ± 2

24 ± 4

PC

 

 

 

 

 

BP

471 ± 14

-

-

-

-

2-AA

-

918 ± 296

124 ± 6

171 ± 32

278 ± 24

SC = Solvent control; PC = Positive control substances; SD = standard deviation;

2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene

Conclusions:
Interpretation of results (migrated information):
negative
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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 May - 08 July 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted in 1983
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK Limited), Margate, Kent, UK
- Age at study initiation: 5-9 weeks for phase I (determination of the maximum tolerated dose) and 7-9 weeks for phase II (Micronucleus test) of the study
- Assigned to test groups randomly: Yes
- Housing: 5 per cage in mobile mouse cage racks, housed per sex
- Diet: Porton Combined Diet, ad libitum
- Water: filtered tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): 25
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
The study consisted in two phases: in phase I the maximum tolerated dose (MTD) was determined, on the basis of lethalities or severe toxicity observed over a four-day observation period following a single intraperitoneal injection.
In phase II, male and female animals were weighed and given a single intraperitoneal injection of corn oil (vehicle control), cyclophosphamide (positive control) or test substance prepared in corn oil.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
24 h and 48 h
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 65 mg/kg bw in physiological saline
Tissues and cell types examined:
Monochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
No deaths or severe adverse effects occurred in Phase I of the study with doses up to 5000 mg/kg bw. This dose was selected as MTD.

TREATMENT AND SAMPLING TIMES: 24 h and 48 h after dosing

DETAILS OF SLIDE PREPARATION: Bone Marrow smears were stained with polychrome methylene blue and eosin

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were evaluated for micronuclei per slide. In addition, 1000 erythrocytes were counted to determine the percentage of polychromatic erythrocytes in the total erythrocyte population.
Evaluation criteria:
Increase in the incidence of micronucleated polychromatic erythrocytes in any sex or at any time point.
Percentage of polychromatic erythrocytes.
Statistics:
The incidence of micronucleated polychromatic erythrocytes and percentage of polychromatic erythrocytes in the erythrocyte sample were considered by analysis of variance regarding each combination of sampling time, dose level and sex as a separate group. Results were examined to determine wether any differences between vehicle control and test substance treated groups were consistent between sexes and across sampling times.
Each group mean was compared with the vehicle control group mean at the corresponding sampling time using a one-sided Student´s t-test based on the error mean square in the analysis.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times.
Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is, however, considered not to be statistically significant compared to the concurrent control values.
The positive control induced stastistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Mean incidence of micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5

 

Table 1: Males

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 mL/kg

0.8 ± 0.8

1.0 ± 1.2

12

Cyclophosphamide

65 mg/kg

24.4 ± 6.0**

 

13

Test substance

5000 mg/kg

0.6 ± 0.6

0.4 ± 0.6

 

Table 2: Females

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

0.2 ± 0.5

1.4 ± 1.1

12

Cyclophosphamide

65 mg/kg

18.4 ± 7.3**

 

13

 Test substance

5000 mg/kg

0.4 ± 0.9

0.4 ± 0.9

 

 

Mean percentage of polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5

 

Table 3: Males

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

48.0 ± 5.6

44.3 ± 7.5

12

Cyclophosphamide

65 mg/kg

41.4 ± 4.4*

 

13

 Test substance

5000 mg/kg

42.2 ± 7.0*

43.3 ± 1.9

 

Table 4: Female

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

41.9 ± 4.8

41.9 ± 1.7

12

Cyclophosphamide

65 mg/kg

45.9 ± 3.49

 

13

 Test substance

5000 mg/kg

46.5 ± 5.8

48.0 ± 5.2

Conclusions:
Interpretation of results (migrated information): negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

HATCOL 2352

Evaluation of the mutagenic activity of Hatcol 2352 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

Hatcol 2352 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

Batch K16156 of Hatcol 2352 was a clear pale yellow liquid. The test substance was soluble in ethanol.

In the dose range finding test, Hatcol 2352 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Hatcol 2352 emulsified on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first and in the second mutation assay, Hatcol 2352 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. Hatcol 2352 emulsified on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings. Hatcol 2352 did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that Hatcol 2352 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

HATCOL 3331

Evaluation of the ability of Hatcol 3331 to induce chromosome aberrations in cultured peripheral human lymphocytes.

This report describes the effect of Hatcol 3331 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Hatcol 3331 was tested in two independent experiments.

Batch D21287 of Hatcol 3331 was a clear colourless liquid with a purity of 97.3%. The test substance was soluble in ethanol.

In the first cytogenetic assay, Hatcol 3331 was tested up to 100 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. Hatcol 3331 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Hatcol 3331 was tested up to 100 µg/ml for a 24 hand 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. In the presence of 1.8% (v/v) S9-fraction, Hatcol 3331 was also tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Hatcol 3331 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, intwo independently repeated experiments.

Finally, it is concluded that this test is valid and that Hatcol 3331 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

 

HATCOL 3344

HATCOL 3344 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

In the dose range finding test, HATCOL 3344 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the tester strain TA100 HATCOL 3344 precipitated on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first and in the second mutation assay, HATCOL 3344 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. HATCOL 3344 precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

HATCOL 3344 did not induce a dose-related, two-fold increase in the number of revertant (His+)colonies in each of the four tester strains (TA1535, TA1537, TA9S and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that HATCOL 3344 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Evaluation of the ability of Hatcol 3344 to induce chromosome aberrations in cultured peripheral human lymphocytes.

This report describes the effect of Hatcol 3344 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9 -mix). The possible clastogenicity of Hatcol 3344 was tested in two independent experiments.

Batch H102-01-28 of Hatcol 3344 was a clear colourless liquid with a purity of 96.9%. The test substance was soluble in ethanol.

In the first cytogenetic assay, Hatcol 3344 was tested up to 100 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. Hatcol 3344 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Hatcol 3344 was tested up to 100 µg/ml for a 24 hand 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9 -mix. In the presence of 1.8% (v/v) S9 fraction Hatcol 3344 was also tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Hatcol 3344 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, in two independently repeated experiments.

Finally, it is concluded that this test is valid and that Hatcol 3344 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

 

HATCOL 5236

HATCOL 5236 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a trptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

In the dose range finding test, HATCOL 5236 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. HATCOL 5236 precipitated on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first and in the second mutation assay, HATCOL 5236 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. HATCOL 5236 precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

HATCOL 5236 did not induce a dose-related, two-fold increase in the number of revertant (His+)colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that HATCOL 5236 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Evaluation of the ability of Hatcol 5236 to induce chromosome aberrations in cultured peripheral human lymphocytes.

This report describes the effect of Hatcol 5236 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Hatcol 5236 was tested in two independent experiments.  

Batch H20139 of Hatcol 5236 was a clear pale yellow liquid with a purity of 97.6%. The test substance was soluble in ethanol.

In the first cytogenetic assay, Hatcol 5236 was tested up to 100 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. Hatcol 5236 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Hatcol 5236 was tested up to 100 µg/ml for a 24 hand 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. In the presence of 1.8% (v/v) S9 -frction Hatcol 5236 was also tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

First cytogenetic assay

In the absence of S9-mix, Hatcol 5236 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

In the presence of S9-mix, Hatcol 5236 induced a statistically significant increase in the number of cells with chromosome aberrations at the intermediate concentration of 33 µg/ml, only when gaps were included. Since the type of aberrations observed were only breaks and gaps, the increase was not dose related, only observed at the intermediate concentration of 33 µg/ml, only when gaps were included and the number of cells with chromosome aberrations was well within our historical control data range, the Increase was considered not to be biologically relevant.

Second cytogenetic assay

Hatcol 5236 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix.  

Finally, it is concluded that this test is valid and that Hatcol 5236 is not clastogenic in human lymphocytes under the experimental conditions described in the report.

HATCOL 1510

In an Ames test (OECD Guideline 471), under test conditions, test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, did not have mutagenicity potential.

 

CAS 11138-60-6

In an Ames Test with substance, decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS 11138-60-6), was not mutagenic with or without metabolic activation. In an in vitro chromosome aberration test, the same read-across substance was not clastogenic in the CHO cell culture test system, with or without metabolic activation. Regardless of dose level (from 625 g/ml to as high as 5000 g/ml) and dosing regimen, the test substance was concluded to be negative for structural and numerical chromosome aberrations, with or without metabolic activation.

 

CAS 189120-64-7

An in vitro chromosome aberration test wAS conducted with substances, CAS 189120-64-7. This substance provided negative results for structural and numerical chromosome aberrations, with or without metabolic activation.

 

CAS 403507-18-6

An in vitro chromosome aberration tests were conducted with substances, CAS 403507-18-6; this substance provided negative results for structural and numerical chromosome aberrations, with or without metabolic activation.

 

CAS 85186-89-6

An in vitro Mammalian Cell Gene Mutation Test was conducted with substance CAS No. 85186-89-6 (Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane) according to OECD Guideline 476. Mouse lymphoma L5178Y cells were dosed with 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/ml with and without metabolic activation. No signs of toxicity were observed, DMSO was used as vehicle, precipitation was observed at and above 100 µg/ml. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, indicated by the total number of colonies per plate.

 

CAS 67762-53-2

The mutagenic potential of Fatty acids, C5-9, tetraesters with pentaerythritol (CAS 67762-53-2) was tested in a reverse mutation assay according to OECD Guideline 471 and under GLP conditions (Mecchi, 1999). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA were used. Tester strains were incubated with test material dissolved in ethanol at concentrations of 33.3, 100, 333, 1000, 3330 and 5000 µg/plate with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9 mix). Vehicle and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. Thus, Fatty acids, C5-9, tetraesters with pentaerythritol did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

 

CAS 189200-42-8

The mutagenic potential of Fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS 189200-42-8) was tested in a reverse mutation assay comparable to OECD Guideline 471 and under GLP conditions (Przygoda, 1995). The following Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 were used. Tester strains were incubated with the test material dissolved in acetone at concentrations of 0.5, 5, 50, 500, 5000 µg/plate in the first experiment and 50, 100, 500, 1000 and 5000 µg/plate in the repeat experiment with and without the addition of a metabolic activation system (Arochlor 1254 induced rat liver S9 mix). Vehicle, negative and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. No cytotoxicity was observed but beading of the test substance occured in the initial assay and repeat assay at 500 µg/plate and above with and without metabolic activation in all strains. Thus, Fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritoland tripentaerythritoldid not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

An in vitro mammalian chromosome aberration test was performed with Fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS 189200-42-8) in Chinese hamster ovary cells (CHO cells) comparable to OECD Guideline 473 and under GLP conditions (Przygody, 1995). Duplicate cultures of CHO cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In the first experiment, cells were exposed to the test substance for 3 hours and for 16 hours followed by 16 hours expression time with and without metabolic activation, respectively. The test substance was dissolved in acetone and used at concentrations of 40, 80 and 160 µg/mL. In the second experiment cells were again exposed for 3 hours and for 16 hours followed by 16 hours expression time with and without metabolic activation, respectively. Additionally, cells were exposed for 3 and 16 hours followed by 40 hours expression time with and without metabolic activation, respectively. The same substance concentrations as in first experiment were used. The test substance did not induce cytotoxicity but a precipitate was visible in the second experiment at 160 µg/mL after 16 hours incubation without metabolic activation. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. N-Methyl-N-Nitro-N-Nitrosoguanidine and 7,12-Dimethylbenz[a]anthracene were used as positive control materials inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level tested in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to CHO cells in vitro.

 

CAS 15834-04-5

An in vitro Mammalian Cell Gene Mutation Assay according to OECD Guideline 476 and GLP was performed with 2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate (CAS 15834-04-5) in mouse lymphoma L5178Y cells (Verspeek-Rip, 2010). In the first experiment, the cells were treated for 3 hours with 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL in the presence or absence of S9-mix (8% (v/v)). In the second experiment, test concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL were applied with metabolic activation (12%, v/v) for 3 h and 0.1, 1, 3, 10, 33, 100, 200, 250 µg/mL without metabolic activation for 24 hours. The test substance was tested up to precipitating concentrations (100 µg/mL and above). Cyclophosphamide and methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. Positive and negative controls were valid and in range of historical control data. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. It was concluded that 2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

 

Additional information from genetic toxicity in vitro:

HATCOL 1765

Fatty acids, C5-10, esters with pentraerythritol (CAS No. 68424-31-7) were found to be not genotoxic in the micronucleus assay in vivo after intraperitoneal application. A single intraperitoneal injection was given to groups of 5 male and 5 female mice at a dose level of 5000 mg/kg bw. Bone marrow samples were taken 24 and 48 hours after dosing.

No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times.

Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is, however, considered not to be biologically significant compared to the concurrent control values.

The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen (Griffiths, 1992).

Justification for classification or non-classification