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REACH

A mixture of: esters of C14-C15 branched alcohols with 3,5-di-t-butyl-4-hydroxyphenyl propionic acid; C15 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate; C13 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate

EC number: 413-750-2 | CAS number: 171090-93-0 ANOX 1315; ANOX BF; DURAD AX 38

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro - Ames assay

ANOX BF did not induce any significant increase in the number of reversions, either in the absence or in the presence of metabolic activation.

ANOX® 1315 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Genetic toxicity in vitro - Mouse Lymphoma Mutagenesis Assay

Anox 1315 was concluded to be negative in the L5178Y/TK^+/-Mouse Lymphoma Mutagenesis Assay.

Genetic toxicity in vitro - Chromosome aberration test

ANOX BF up to the concentration of 150 µg/ml did not induce statistically significant increase of cells with chromosome aberrations in CHO cells.

Link to relevant study records

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2014 to 15 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (adopted July 21, 1997).

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

No further details specified in the study report.

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. OECD, EC and MITI).
Source Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames) (TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006) and (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) (WP2uvrA, 2008)

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Not specified

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation system
Rat liver microsomal enzymes (S9 homogenate) was obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that had been injected intraperitoneal with Aroclor 1254 (500 mg/kg).

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per 10 ml: 30 mg NADP (Randox) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Test concentrations with justification for top dose:

Range finding study: 5.4, 17, 52, 164, 512, 1600 & 5000 Source of S9 µg/plate
Main study: 52, 164, 512, 1600 & 5000 µg/plate
No justification detailed in the study report.

Vehicle / solvent:

Solvent used: Ethanol

Untreated negative controls:

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 6-Chloro-9-(3-N-(2-Chloroethyl-amino)propylamino-2-methoxyacridine dichloride [ICR-191]; 2-aminoanthracene [2-AA]

Details on test system and experimental conditions:

First experiment
Seven concentrations of the test substance, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Second experiment
Based on the results of the first mutation assay, five doses (increasing with approximately half-log steps) of the test substance were selected and tested in triplicate in each strain in the second experiment. The highest concentration of ANOX® 1315 used in the second mutation assay was 5 mg/plate.

Experimental procedure
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
The vehicle control and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating 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 inverted and incubated in the dark at 37.0 ± 1.0 °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 were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually and evidence of test article precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

Acceptability of the assay
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Data evaluation and statistical procedures
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
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) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a positive response will be repeated, 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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

First mutation experiment
ANOX® 1315 was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix.

Precipitate
Precipitation of ANOX® 1315 on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 and 5000 μg/plate in all tester strains, except in tester strain TA1535 where the test substance already precipitated on the plates at 512 μg/plate in the absence of S9-mix.

Toxicity
To determine the toxicity of ANOX® 1315, 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.

Mutagenicity
No biologically increase in the number of revertants was observed upon treatment with ANOX® 1315 under all conditions tested.
In strain TA1535, fluctuations in the number of revertant colonies above the laboratory historical control data range were observed in the absence and presence of S9-mix at several dose levels. However, since the increases were not three-fold (a maximum of 1.4-fold was reached), these increases were not considered to be relevant.
In strain TA100, fluctuations in the number of revertant colonies above the laboratory historical control data range were observed in the presence of S9-mix at several dose levels. However, since the increases were not two-fold (a maximum of 1.1-fold was reached), these increases were not considered to be relevant.

Second mutation assay
To obtain more information about the possible mutagenicity of ANOX® 1315, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation experiment, ANOX® 1315 was tested up to concentrations of 5000 μg/plate.

Precipitate
Precipitation of ANOX® 1315 on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate and at 1600 and 5000 μg/plate 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.

Mutagenicity
No biologically relevant increase in the number of revertants was observed upon treatment with ANOX® 1315 under all conditions tested.
In strain TA1535, fluctuations in the number of revertant colonies above the laboratory historical control data range were observed in the presence of S9-mix at several dose levels. However, since no increase above the concurrent vehicle control was seen, these increases were not considered to be relevant.

TABLE OF RESULTS

Range Finding Study

Name of test substance: ANOX® 1315

Date of experiment: from 24 April 2014 to 28 April 2014

S9-mix (-)

Dose level per plate (µg/plate)

Number of Revertants, (Mean), +/- SD

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Solvent control (ethanol)

29 (30)

30 0.6

4 (6)

4 3.5

20 (18)

19 3.2

124 (130)

121 12.5

144

38 (28)

15 11.8

5.4

34 (31)

38 9.5

12 (6)

3 5.2

20 (18)

16 2.1

136 (131)

141 12.7

117

23 (30)

23 12.1

46 (32)

31 13.5

8 (9)

14 5.0

27 (23)

26 6.1

131 (124)

122 6.7

118

31 (34)

30 6.1

23 (24)

26 1.7

4 (6)

5 3.2

19 (19)

16 3.0

131 (125)

131 10.4

113

22 (25)

19 8.5

164

30 NP (35)

39 NP 4.5

35 NP

10 (7)

3 3.6

14 (19)

24 5.0

148 (139)

147 14.7

122

33 (26)

18 7.5

512

34 SP (34)

25 SP 1.2

33 SP

11 NP (8)

7 NP 3.1

5 NP

20 NP (16)

12 NP 4.0

16 NP

150 NP (143)

131 NP 10.4

148 NP

26 NP (36)

48 NP 11.2

33 NP

1600

23 SP (31)

37 SP 7.2

33 SP

7 SP (4)

1 SP 3.1

3 SP

26 SP (23)

19 SP 3.6

24 SP

148 SP (134)

137 SP 15.7

117 SP

24 SP (35)

44 SP 10.1

37 SP

5000

31 n SP (35)

41 n SP 5.1

34 n SP

10 n SP (10)

8 n SP 1.5

11 n SP

33 n SP (26)

19 n SP 7.0

26 n SP

150 n SP (137)

140 n SP 14.7

121 n SP

37 n SP (36)

31 n SP 4.2

39 n SP

S9 mix (-)

Name

Dose level

No. of revertants

ICR-191

MMS

4-NQO

2.5

650

628 (633)

641 7.0

630

324 (306)

313 21.8

282

905 (883)

887 23.7

858

1008 (999)

986 11.5

1003

1355 (1294)

1329 84.7

1197

MMS methylmethanesulphonate

SA sodium azide

4-NQO 4-nitroquinoline N-oxide

NF 2-nitrofluorene

ICR-191 6-Chloro-9-(3-N-(2-Chloroethyl-amino)propylamino-2-methoxyacridine dihydrochloride

NP No precipitate

SP Slight precipitate

n Normal bacterial background lawn

Date of experiment: from 24 April 2014 to 28 April 2014

S9-mix (+)

Dose level per plate (µg/plate)

Number of Revertants, (Mean), +/- SD

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Solvent control (ethanol)

31 (33)

35 2.1

7 (9)

7 2.9

24 (31)

31 7.0

140 (138)

117 20.6

158

46 (38)

30 8.0

5.4

26 (29)

30 3.1

5 (9)

10 3.2

22 (25)

27 2.6

125 (125)

124 0.6

125

30 (30)

38 8.0

35 (36)

37 1.2

4 (8)

12 4.0

24 (28)

33 4.6

146 (141)

139 4.7

137

30 (30)

34 3.5

31 (37)

35 6.7

11 (10)

8 1.7

34 (35)

41 6.0

137 (130)

125 6.2

128

38 (33)

26 6.1

164

42 (41)

33 8.0

7 (8)

4 4.0

33 (28)

16 10.4

140 (133)

121 10.2

137

39 (35)

35 4.0

512

29 NP (32)

33 NP 2.3

33 NP

7 NP (11)

12 NP 3.6

14 NP

27 NP (26)

19 NP 6.1

31 NP

137 NP (133)

146 NP 26.4

151 NP

37 NP (37)

39 NP 2.0

35 NP

1600

35 SP (43)

48 SP 6.7

45 SP

7 SP (4)

1 SP 3.1

3 SP

27 SP (26)

19 SP 6.1

31 SP

137 SP (150)

161 SP 12.1

152 SP

31 SP (39)

50 SP 10.0

35 SP

5000

39 n SP (47)

50 n SP 7.0

52 n SP

19 n SP (22)

18 n SP 5.5

28 n SP

30 n SP (39)

49 n SP 9.5

38 n SP

133 n SP (141)

144 n SP 7.0

146 n SP

38 n SP (43)

46 n SP 4.2

44 n SP

S9 mix (+)

Name

Dose level

No. of revertants

2AA

2.5

256 (256)

265 8.5

248

381 (389)

414 22.5

371

705 (667)

624 40.7

672

1189 (1203)

1262 53.0

1159

180 (176)

163 11.9

186

2AA 2-aminoanthracene

NP No precipitate

SP Slight precipitate

n Normal bacterial background lawn

TABLE OF RESULTS

Main Study

Name of test substance: ANOX® 1315

Date of experiment: from 01 May 2014 to 05 May 2014

S9-mix (-)

Dose level per plate (µg/plate)

Number of Revertants, (Mean), +/- SD

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Solvent control (ethanol)

30 (32)

29 4.9

12 (10)

11 2.6

20 (19)

20 1.2

129 (119)

109 10.0

118

27 (32)

24 10.8

24 (18)

22 9.3

19 (11)

1 9.1

30 (24)

23 5.1

113 (105)

90 13.0

112

33 (40)

39 7.5

164

16 (22)

23 5.6

10 (7)

5 2.9

29 (21)

26 11.9

106 (105)

94 10.1

114

27 (30)

33 3.1

512

22 NP (23)

20 NP 3.6

27 NP

7 NP (9)

10 NP 1.7

10 NP

34 NP (25)

22 NP 7.9

19 NP

118 NP (117)

112 NP 4.2

120 NP

29 NP (36)

48 NP 10.4

31 NP

1600

20 SP (31)

42 SP 11.0

30 SP

14 SP (9)

10 SP 5.6

3 SP

16 SP (18)

27 SP 8.6

10 SP

109 SP (97)

93 SP 11.0

88 SP

33 SP (38)

48 SP 8.4

34 SP

5000

31 n SP (30)

33 n SP 3.1

27 n SP

18 n SP (13)

12 n SP 5.0

8 n SP

24 n SP (22)

20 n SP 2.1

23 n SP

116 n SP (115)

127 n SP 13.1

101 n SP

42 n SP (36)

29 n SP 6.7

38 n SP

S9 mix (-)

Name

Dose level

No. of revertants

ICR-191

MMS

4-NQO

2.5

650

804 (812)

793 23.5

838

517 (553)

584 33.8

558

834 (813)

842 43.5

763

926 (940)

932 19.3

962

1534 (1473)

1291 160.4

1594

MMS methylmethanesulphonate

SA sodium azide

4-NQO 4-nitroquinoline N-oxide

NF 2-nitrofluorene

ICR-191 6-Chloro-9-(3-N-(2-Chloroethyl-amino)propylamino-2-methoxyacridine dihydrochloride

NP No precipitate

SP Slight precipitate

n Normal bacterial background lawn

Date of experiment: from 01 May 2014 to 05 May 2014

S9-mix (+)

Dose level per plate (µg/plate)

Number of Revertants, (Mean), +/- SD

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Solvent control (ethanol)

38 (36)

45 10.7

7 (12)

14 4.4

23 (28)

33 5.0

143 (142)

144 2.6

139

46 (46)

41 4.5

30 (22)

18 6.7

14 (11)

14 5.2

41 (38)

41 4.6

103 (107)

103 11.5

116

37 (42)

44 4.0

164

30 (34)

30 6.9

3 (6)

10 3.6

38 (40)

50 9.6

109 (110)

99 11.5

122

34 (38)

34 6.9

512

34 NP (27)

16 NP 9.5

30 NP

10 NP (9)

11 NP 2.1

7 NP

20 NP (30)

26 NP 12.5

44 NP

141 NP (122)

116 NP 16.8

109 NP

44 NP (43)

39 NP 3.6

46 NP

1600

24 SP (27)

35 SP 6.7

23 SP

5 SP (8)

15 SP 5.8

5 SP

38 SP (35)

31 SP 3.5

35 SP

98 SP (101)

113 SP 11.2

91 SP

39 SP (46)

38 SP 12.4

60 SP

5000

35 n SP (36)

39 n SP 2.3

35 n SP

10 n SP (16)

22 n SP 6.0

15 n SP

44 n SP (32)

22 n SP 11.1

30 n SP

113 n SP (106)

107 n SP 8.1

97 n SP

50 n SP (54)

46 n SP 11.2

67 n SP

S9 mix (+)

Name

Dose level

No. of revertants

2AA

2.5

220 (194)

192 25.1

170

612 (490)

484 118.6

375

718 (693)

679 21.7

682

1726 (1545)

1490 160.3

1420

288 (277)

249 24.4

294

2AA 2-aminoanthracene

NP No precipitate

SP Slight precipitate

n Normal bacterial background lawn

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative control values were within the laboratory historical control data ranges, except for TA1535 in the presence of S9-mix (first and second experiment) and TA100 in the presence of S9-mix (first experiment).
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that ANOX® 1315 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 ANOX® 1315 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

ANOX® 1315 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 (rat liver S9-mix induced by Aroclor 1254).

The study procedures described in this report were based on the most recent OECD, EC and Japanese guidelines.

Batch WBF3F0004 of ANOX® 1315 was a clear yellow viscous liquid with a purity of 93.5% by GC. The test substance was dissolved in ethanol.

In the first mutation assay, ANOX® 1315 was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. ANOX® 1315 precipitated on the plates at dose levels of 1600 and 5000 μg/plate in all tester strains, except in tester strain TA1535 where the test substance already precipitated on the plates at 512 μg/plate in the absence of S9-mix. 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 second mutation assay, ANOX® 1315 was tested up to concentrations of 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix. ANOX® 1315 precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

ANOX® 1315 did not induce a biologically significant dose-related 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.

The negative control values were within the laboratory historical control data ranges, except for TA1535 in the presence of S9-mix (first and second experiment) and TA100 in the presence of S9-mix (first experiment). However, since these values were just outside the limit of the range, the validity of the test was considered to be not affected.

The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that ANOX® 1315 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 25, 1991 to March 8, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Organization for Economic Cooperation and Development Guidelines, Section 4, Subpart 471, Paris 1981 and subsequent revisions.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No specified

Metabolic activation:

with and without

Metabolic activation system:

Adult male Sprague Dawley rats purchased from Charles River Company (Calco) weighing 150-230 g, received a single intraperitoneal dose of 500 mg/kg (2.5 ml/kg) of Aroclor 1254 dissolved in corn oil (200 mg/ml). Five days thereafter, the rats were killed and their livers removed. The livers were homogenized for 30 seconds at 4 deg. C in three 10-seconds bursts with 0.15M KCI (solution to tissue ratio 3:1). The homogenate was centrifuged at 8,000xg for 10’ in a Servall refrigerated supercentrifuge. The supernatant was divided into aliquots, deep frozen, and stored at -70 deg. C. Protein concentration in the supernatant was assayed by the Biuret method. The capacity of the supernatant to metabolically activate an indirect mutagen (2-Aminofluorene) was assayed by the Ames test.
An adequate amount of S9 Mix was prepared in an ice cold bath immediately before use. The composition was the following:
0.1 ml of Liver microsomal suspension (S9)
0.9 ml of phosphate buffer pH 7.4 0.01M
20.0 µl of MgCl2 0.5M and KCI 2M
1.3 mg of glucose-6-phospahte
2.97 mg of NADP

Test concentrations with justification for top dose:

The test article was assayed undiluted and at four serial 1:10 dilutions: t.q., 1:10, 1:100, 1:1000. 1:10000 corresponding to (on the basis of d=0.93) 9.3, 93, 930, 9300 93000 µg/plate.

Vehicle / solvent:

Test article assayed undiluted.

Untreated negative controls:

Negative solvent / vehicle controls:

True negative controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: Hydrazine sulphate;Doxorubicine HCI; 2-Aminofluorene

Details on test system and experimental conditions:

Preparation of the test article solutions
The test article was used undiluted. Furthermore, it was diluted with water for injection to obtain t.q., 1:10, 1:100, 1:1000, 1:10000 dilutions.

Plate test without and with metabolic activation
Of the test article solutions according to the dosage level or negative or positive controls 0.1 ml were introduced into sterile test tubes containing 2.5 ml (test without) or 2 ml (test with) of soft agar kept liquid in a thermostatic bath at 45 deg. C.
Of suspension of Salmonella strains in a stationary growth phase, 0.1 ml were rapidly added.
Of S9 Mix 0.5 ml were also added only for the test with metabolic activation.
The test tubes were shaken rapidly and the contents poured onto plates containing solid selective growth medium. The plates were incubated at 37 deg. C for 72 hours.
Three plates per dose per Salmonella strain were prepared both for the test without and with metabolic activation.
The following positive control were used: Hyd for strain TA 1535, 9-AA for strain TA 1537, and Doxo for strains TA 1538, TA 98 and TA 100 for the test without metabolic activation and 2-AF for strains TA 1538, TA 98 and TA 100 for the test with metabolic activation.
The tubes and plates were adequately identified. The reverted colonies per plate were counted following incubation.

Rationale for test conditions:

Performed in accordance with recognised test guidelines.

Evaluation criteria:

The test can be considered to have been conducted correctly if compliance with the following conditions has been achieved:
a) The sterility check must prove negative for bacterial growth.
b) The growth of all the strains must be inhibited by crystal violet; the growth of the strains TA 1535, TA 1537, TA 1538 must be inhibited by ampicillin, while the growth of strains TA 98 and TA 100 must not.
c) The frequency of spontaneous reversion for each strain must fall within the range reported by literature and by the laboratory data.
d) The activity of the microsomal preparation must be confirmed by its capability to activate the positive control which requires a metabolic transformation in order to explicate its mutagenic effect.
e) The number of colonies reverted owing to the mutagenic activity of the positive controls must be statistically greater than (Student’s t test) and at least double the manner of spontaneously reverted colonies.

The test article is considered positive:
-if the number of reverted colonies is statistically significant increased in comparison to the number of control reversions (Student’s t test);
-if a dose-dependent can be verified, that is, a positive correlation between the number of reversions in the dose in an interval of at least 3 doses (linear regression test).

Statistics:

The mean and standard deviation were calculated for reversions read in each dosage group.
Comparison of the spontaneous reversions (in the negative control) with the ones in the test article plates and in the positive control plates were done by Student’s “t” test.
Significances were expressed in the following manner:
* p < 0.05
** p < 0.1
*** p < 0.001

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No appreciable increase of the number of reversions in comparison with the negative control was evident in either experiments, at any of the doses of [ANOX BF] for any strain, whether in the presence or in the absence of metabolic activation, while the reference mutagens induced a number of reverted colonies statistically greater than (Student’s t test) and at least double the mean number of spontaneous reverted colonies.

Mutagenicity test with Salmonella typhimurium TA 1535 strain without metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		24 – 26 – 29	26.33	2.52
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	22 – 20 – 18 34 – 20 – 23 32 – 28 – 30 24 – 26 – 26 30 – 27 – 24	20.00 25.68 30.00 25.33 27.00	2.00 7.37 2.00 1.15 3.00
Hydrazine	500 µg/p	116 – 131 – 118	121.67***	8.14

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 1537 strain without metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		10 – 7 – 16	11.00	4.58
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	10 – 13 – 9 8 – 5 – 13 12 – 10 – 10 9 – 8 – 12 11 – 12 – 14	10.67 8.67 10.68 9.67 12.33	2.08 4.04 1.15 2.08 1.53
9-AA	40 µ/p	51 – 37 – 49	45.67**	7.57

** p < 0.01

Mutagenicity test with Salmonella typhimurium TA 1538 strain without metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		17 – 28 – 21	22.00	5.37
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	19 – 14 – 20 20 – 13 – 15 19 – 23 – 20 21 – 17 – 15 18 – 14 – 19	17.67 16.00 29.67 17.67 17.00	3.21 3.61 2.08 3.06 2.65
Doxorubicine	4 µg/p	145 – 106 – 129	126.67***	19.60

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 98 strain without metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		36 – 35 – 41	37.33	3.21
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	37 – 35 – 38 31 – 40 – 35 28 – 36 – 43 30 – 31 – 42 40 – 33 – 30	36.67 35.33 35.67 34.33 34.33	1.53 4.51 7.51 6.66 5.13
Doxorubicine	4 µg/p	396 – 415 – 471	427.33***	38.99

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 100 strain without metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		210 – 174 – 213	199.00	21.70
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	170 – 193 – 217 203 – 226 – 209 204 – 212 – 187 212 – 200 – 218 205 – 208 – 191	194.00 212.67 201.00 210.00 201.33	23.52 11.93 12.77 9.17 9.07
Doxorubicine	4 µg/p	492 – 570 – 496	519.33***	43.92

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 1535 strain with metabolic activation of the compound [ANOX BF]

Compound

Conc.

Reversion/plate

Mean

Control

27 – 33 – 31

30.33

3.06

ANOX BF

1:10000

1:1000

1:100

1:10

Undiluted

27 – 32 – 38

39 – 34 – 23

28 – 36 – 34

37 – 29 – 29

31 – 28 – 33

32.33

32.00

32.67

31.67

30.67

5.51

8.19

4.16

4.62

2.52

Mutagenicity test with Salmonella typhimurium TA 1537 strain with metabolic activation of the compound [ANOX BF]

Compound

Conc.

Reversion/plate

Mean

Control

16 – 15 – 9

13.33

3.79

ANOX BF

1:10000

1:1000

1:100

1:10

Undiluted

16 – 9 – 12

19 – 13 – 15

13 – 18 – 16

16 – 12 – 9

8 – 13 – 19

12.33

15.67

12.33

13.33

3.51

3.06

2.52

3.51

5.51

Mutagenicity test with Salmonella typhimurium TA 1538 strain with metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		25 – 28 – 30	27.67	2.52
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	27 – 34 – 36 22 – 16 – 30 29 – 25 – 31 35 – 25 – 22 25 – 19 – 23	32.33 22.67 28.33 27.33 22.33	4.73 7.02 3.06 6.81 3.06
2-AF	5 µg/p	940 – 1320 – 1175	1145.00***	191.77

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 98 strain with metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		54 – 48 – 43	48.33	5.51
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	51 – 56 – 47 61 – 59 – 47 51 – 54 – 56 53 – 46 – 49 58 – 38 – 40	51.33 55.67 53.67 49.33 45.33	4.51 7.57 2.52 3.51 11.02
2-AF	5 µg/p	1300 – 1245 – 1117	1220.67***	93.90

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 100 strain with metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		241 – 236 – 225	234.00	8.19
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	227 – 256 – 251 216 – 239 – 221 208 – 261 – 233 224 – 250 – 239 244 – 254 – 219	244.67 225.33 234.00 237.67 239.00	15.50 12.10 26.51 13.05 18.03
2-AF	5 µg/p	530 – 666 – 621	605.67***	69.28

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 1535 strain without metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		20 – 24 – 28	24.00	4.00
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	30 – 24 – 28 20 – 26 – 23 20 – 29 – 25 24 – 19 – 21 17 – 28 – 19	27.33 23.00 24.67 21.33 21.33	3.06 3.00 4.51 2.52 5.86
Hydrazine	500 µg/p	97 – 110 – 104	103.67***	6.51

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 1537 strain without metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		12 – 9 – 14	11.67	2.52
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	7 – 12 – 6 11 – 8 – 14 7 – 8 – 8 9 – 10 – 16 5 – 6 – 8	8.33 11.00 7.67 11.67 6.33	3.21 3.00 0.58 3.79 1.53
9-AA	40 µg/p	87 – 95 – 80	87.33***	7.51

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 1538 strain without metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		19 – 17 – 17	17.67	1.15
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	10 – 17 – 13 19 – 18 – 10 13 – 12 – 16 10 – 21 – 19 17 – 16 – 13	13.33 15.67 13.67 16.67 15.33	3.51 4.93 2.08 5.86 2.08
Doxorubicine	4 µg/p	122 – 115 – 103	113.33***	9.61

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 98 strain without metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		45 – 36 – 37	39.33	4.93
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	35 – 30 – 32 37 – 40 – 42 39 – 35 – 38 34 – 30 – 36 31 – 33 – 41	32.33 39.67 37.33 33.33 35.00	2.52 2.52 2.08 3.06 5.29
Doxorubicine	4 µg/p	551 – 704 – 712	655.67***	90.73

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 100 strain without metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		236 – 191 – 215	214.00	22.52
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	236 – 238 – 221 224 – 226 – 216 221 – 218 – 219 197 – 181 -208 214 – 194 – 227	231.67 222.00 219.33 195.333 211.67	9.29 5.29 1.53 13.58 16.62
Doxorubicine	4 µg/p	579 – 622 – 657	519.33***	39.07

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 1535 strain with metabolic activation of the compound [ANOX BF]

Compound

Conc.

Reversion/plate

Mean

Control

26 – 25 – 29

26.67

2.08

ANOX BF

1:10000

1:1000

1:100

1:10

Undiluted

28 – 24 – 18

20 – 23 – 24

30 – 33 – 26

31 – 25 – 27

24 – 29 – 26

23.33

22.33

29.67

27.67

26.33

5.03

2.08

3.51

3.06

2.52

Mutagenicity test with Salmonella typhimurium TA 1537 strain with metabolic activation of the compound [ANOX BF]

Compound

Conc.

Reversion/plate

Mean

Control

11 – 9 – 13

11.00

2.00

ANOX BF

1:10000

1:1000

1:100

1:10

Undiluted

11 – 10 – 9

19 – 6 – 10

10 – 12 – 13

12 – 9 – 11

11 – 10 – 9

10.00

11.67

10.67

10.00

1.00

6.66

1.53

1.00

Mutagenicity test with Salmonella typhimurium TA 1538 strain with metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		19 – 22 – 20	20.33	1.53
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	22 – 13 – 15 19 – 20 – 17 18 – 19 – 21 19 – 21 – 17 21 – 19 -22	16.67 18.67 19.33 19.00 20.67	4.73 1.53 1.53 2.00 1.53
2-AF	5 µg/p	1075 – 921 – 1152	1049.33***	117.62

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 98 strain with metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		52 – 48 – 40	46.67	6.11
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	38 – 41 – 47 53 – 48 – 51 49 – 52 – 47 41 – 57 – 46 49 – 50 – 53	42.00 50.67 49.33 48.00 50.67	4.58 2.52 2.52 8.19 2.08
2-AF	5 µg/p	1221 – 1315 – 1297	1277.67***	49.89

*** p < 0.001

Mutagenicity test with Salmonella typhimurium TA 100 strain with metabolic activation of the compound [ANOX BF]

Compound	Conc.	Reversion/plate	Mean	SD
Control		230 – 197 – 209	212.00	16.70
ANOX BF ANOX BF ANOX BF ANOX BF ANOX BF	1:10000 1:1000 1:100 1:10 Undiluted	215 – 217 – 216 214 – 225 – 226 216 – 237 – 240 215 – 208 – 175 210 – 223 – 215	216.00 221.67 231.00 199.33 216.00	1.00 6.66 1308 21.36 6.56
2-AF	5 µg/p	693 – 704 – 715	704.00***	11.00

*** p < 0.001

Conclusions:

The test article [ANOX BF] assayed undiluted as supplied by the sponsor, did not induce any significant increase in the number of reversions, either in the absence or in the presence of metabolic activation, in TA 1535, TA 1537, TA 1538, TA 98 and TA 100 Salmonella typhimurium strains, in a duplicate experiment.

Executive summary:

Appraisal of the possible genotoxic activity exercised by the test article [ANOX BF].

The present test was performed according to “Organization for Economic Cooperation and Development Guidelines”, Section 4, Subpart 471, Paris 1981 and subsequent revisions.

The test article was used undiluted. Furthermore, it was diluted with water for injection to obtain t.q., 1:10, 1:100, 1:1000, 1:10000 dilutions.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 27, 1997 to May 14, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

"Organization for Economic Cooperation and Development Guidelines", Section 4, Subpart 473, Paris 1981 subsequent revisions

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

"Annex to Commission Directive 92/69/EEC of July 3 1, 1992 adapting to technical progress for the seventeenth time Council Directive 67/548iEEC on approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (Test method B.10)".

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

chromatids

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary cell line (CHO), obtained from the European Collection of Animal cell Cultures (ECACC), Salisbury, U.K.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Not specified

Metabolic activation:

with and without

Metabolic activation system:

Adult male Sprague Dawley rats, weighing from 150 to 250 g, supplied by the Charles River Company of Calco, received a single intraperitoneal administration of an Aroclor 1254 solution in corn oil (200 mg/ml) at the dosage of 500 mg/kg (2.5 ml/kg solution in commercial corn oil).
On the fifth day thereafter, the animals were sacrificed and the liver of each removed.
The livers were homogenized for 30 seconds at 4OC in three 10- second bursts with 0.15M KC1 (3 ml per gram of liver).
The homogenate was centrifuged for 20 minutes at 9,000xg in a refrigerated supercentrifuge. The supernatant was divided into fractions and deep frozen at -80°C.

Test concentrations with justification for top dose:

Exp. No. 960696/1: 500, 150, 50, 15, 5, 1.5 and 0.5 mg/ml
Exp. No. 960696/2: 15, 5: 1.5,0.5,0.15 and 0.05 mg/ml
No justification specified in the study report.

Vehicle / solvent:

Acetone (Merck, batch No. K23088714 628, expiry date June 30, 2001)
When necessary, exact amounts of ANOX BF were weighed in suitable containers and solubilized with acetone and further diluted in the same solvent in order to obtain the test concentrations.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

4-5.10e5 cells were seeded in 5 ml medium (Ham's F12 - Sigma, batch No. 26H2340, expiry date December 1997, supplemented with FBS) in 25 cm2 flasks.
Two cultures per each experimental point were prepared and incubated at 37 °C for about 24 hours. Just before treatment the medium was replaced.
In the test without metabolic activation 4 ml of fresh medium were put into each flask and 1 ml of the test article solutions, according to the dosage levels, or of the vehicle control was added.
In the test with metabolic activation 4 ml of fresh medium containing the S9 metabolic activation system were put into each flask and then 1 ml of the test article solutions or of the vehicle control was added.
Of the positive control solutions, 0.05 ml of EMS were put into 4.95 ml of fresh medium (test without metabolic activation) or 0.05 ml of CP were put into 4.95 m of fresh medium containing the S9 metabolic system (test with metabolic activation).
After 3 hours of incubation at 37 ± 1 °C in a humidified atmosphere of 5% CO2 in air the cultures were washed twice with saline, refed with 5 ml of fresh medium and returned to the incubator for additional 17 hours.
The cells were collected by tripsinization 20 hours from the initiation of treatment (1st harvest at 1.5 times normal cell cycle).
Two hours prior to harvest colcemid (GIBCO, batches No.s 21N9162 and 11P4368, expiry dates September 30, 1997 and April 30, 1998, respectively) was added to the cultures at a final concentration of 0.2 µg/ml, in order to stop cell division in metaphase.
Then cells were centrifugated, treated with hypotonic solution (a 1:1 mixture of 0.5% sodium citrate and 0.5% KC1, pre-warmed at 37 °C) (Merck, batch No. 207A627248, expiry date February 2001) washed twice with fixative (methanol:glacial acetic acid, 3:1 v/v) (Methanol, Merck, batch No. K2333 1709641, expiry date September 30, 200 1) – Glacial acetic acid, Merck, batch No. K23575963, expiry date November 2001), dropped onto glass microscope slides and air-dried.
The slides were stained with Giemsa (Merck batch No. 540014479, expiry date January 31, 1998) and permanently mounted.

Repetition of the assay
The experiment was repeated in an independent assay.
As negative results were obtained in the first trial, in the repeat test the cultures in the absence of metabolic activation were incubated at 37 °C for 18 hours in the presence of drug before harvest, while in the test with metabolic activation they were incubated at 37 °C for 3 hours.
Since the positive controls are used to determine the sensitivity of CHO cells to mutagens and to confirm the appropriate metabolic activity of the liver homogenate fractions, they were incubated for 3 hours, both with and without metabolic activation, according to our laboratory standard.
Furthermore, the repeat test included an additional sample, both with and without metabolic activation at approximately 24 hours later (44th hour).
For the latter harvest the same exposure conditions of the second trial were used.
At the second fixation time only the highest not severely cytotoxic dose (150 µg/ml) was analyzed.
Vehicle control was also scored.

Glass slide reading
Two glass slides per each culture were prepared.
Toxicity of the test article ANOX BF on CHO cells was evaluated examining the uncoded slides for the number of metaphase cells per 1000 cells (mitotic index) and determining the mitotic index reduction (%) in the treated cultures as compared to the vehicle control.
For metaphase analysis all slides were randomly assigned code numbers and scored blind.
For each test article dose and for the untreated, vehicle and positive controls 200 metaphases/dose (100 metaphases/culture) underwent microscope analysis, at 1250 magnifications and scored for chromatid and chromosome type aberrations.
The XY coordinates for each cell with chromosomal aberrations were recorded.
The aberrations found, classified according to the general criteria, were registered on appropriate forms.

The aberrations found are classified as:
Fragment: Any piece of supernumerary chromatid material, free or displaced, in association or not with a parent chromatid. Minute fragment are classified as fragments.
Gap: Undisplaced, unstained area less than one chromatid wide. If the gap involves both chromatid arms in the same position it is defined as an isochromatid gap (isolocus gap). Both possibilities are classified as gaps.
Break: A lesion involving any separation or discontinuity of a chromatid piece with consequent derangement of the axial integrity with or without the chromatid fragment that originated.
Exchange: Exchanges can be of two types: intrachanges, when the damage occurs within one chromosome and involves the internal rearrangement within the same chromosome, and interchanges, when the damage concerns two different chromosoms and the rearrangements occur between the two chromosomes: these can be of asymmetrica type when the two chromosomes involved have not symmetrical plane and of symmetrical type when they do.

In qualitative evaluation of aberration tests it is well to keep in mind that the presence of any type of exchange is indication of possible hereditary genetic damage and therefore is particularly important, whereas breaks and chromatid gaps alone do not constitute clear indication of genetic damage.

It is generally held that gaps, in particular, are not valid indicators of true genetic damage, since they can in fact derive from methodologic artefacts.

Polyploid and endoreduplicated cells were recorded.

Rationale for test conditions:

By this test, damage to chromatids caused by a test substance can be identified as chromosome aberrations. These are microscopically examined and classified according to the type of damage caused to one or more chromosomes. In practice, after an appropriate time for treatment of the cultured CHO cells with the test substance at different concentrations, the cells in mitosis are blocked with colchicine at the metaphase. The cells are fixed, dropped on glass slides and stained. The metaphases are accurately examined for the presence of chromosome aberrations.

Evaluation criteria:

Criteria for determination of a valid test
- Negative controls
The frequency of cells with structural chromosome aberrations in the untreated or solvent control must be no greater than 5%.
- Positive controls
The percentage of cells with aberrations must be statistically increased (p ≤ 0.05, Fisher's exact test) relative to the vehicle control.

Criteria for determination of a positive response
In this assay, the test article is considered to have clastogenic properties if the following criteria are fulfilled:
i) statistically significant increases in the incidence of cells bearing aberrations are observed at one or more concentrations;
ii) the increases are reproduced in independent assays;
iii) the incidence of aberration-bearing cells in the treated cultures exceeds 5.0%.

The evaluation is based on the set of results which excludes gaps.
Where borderline results are obtained, the results including gaps, or the combined data from all of the treatment-levels, may be adduced as evidence for clastogenic activity.

Statistics:

From the statistical viewpoint, only metaphases containing the modal number (+/-2) of centromeres were analyzed.
For the statistical analysis, Fisher's Exact Test was used to compare the number of cells with aberrations in control and treated cultures (test article or reference mutagen). The analysis was performed using sets of data either including or excluding gaps.
Significances were expressed as follows:
* p < 0.05
** p < 0.01
*** p < 0.001

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

500, 1500 and 5000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

From 500 µg/ml upward, when the test article was added to the incubation mixture, droplets visible to the naked eye formed on its surface.
From 150 µg/ml downward small droplets of the test article distributed throughout the incubation mixture were observed when it was examined using a microscope.
Toxicity of the test article on CHO cells was evaluated examining the slides for the number of metaphase cells per 1000 cells (mitotic index) in the treated cultures as compared to the vehicle control, the reduction of which is indicative of toxicity.
At the dosage levels of 500, 1500 and 5000 µg/ml the test substance proved to be cytotoxic on the test systems, both with and without metabolic activation and very few metaphases were seen on the slides at harvesting.
At 150 µg/ml in the test without metabolic activation the mitotic index reduction compared to the vehicle control was 38%.
At the other two doses 15 and 50 µg/ml, no significant cytotoxic effects were detected.
At 15, 50 and 150 µg/ml with metabolic activation the mitotic index reduction was about 25%.
At the 72 hour sampling time, at 150 µg/ml without metabolic activation, the mitotic index reduction compared to the control value was 33%.
At the other two concentrations assayed no significant cytotoxic effects were seen.
At 50 and 150 µg/ml with metabolic activation the mitotic index reduction was 20-30%.
At 15 µg/ml no significant cytotoxic effect was detected.
At 96 hour sampling time, at 150 µg/ml, the mitotic index reductions compared to the control values were 20 and 26% in the test without and with metabolic activation, respectively.
Sampling time: 20th and 44th hour: At neither sampling time, at none of the test article concentration assayed was there an incidence of cells with chromosome aberration statistically different from the control group, either in the presence or in the absence of metabolic activation.
As expected, the reference mutagens produced statistically significant increases in the percentage of cells with chromosome aberrations, both including and excluding gaps (p < 0.001).
Furthermore, at none of the test article concentrations assayed was observed an incidence of polyploid or endoreduplicated cells statistically different from the control group, either in the presence or in the absence of metabolic activation.

Mitotic index – First test

Sampling time: 20th hour

Test without metabolic activation

M.I. (°)

M.I. reduction %

Treatment

Vehicle control

ANOX BF

Dose µg/ml

150

48/1000

44/1000

46/1000

30/1000

Test with metabolic activation

M.I. (°)

M.I. reduction %

Treatment

Vehicle control

ANOX BF

Dose µg/ml

150

56//1000

43/1000

42/1000

41/1000

(°) M.I. = mitotic index (number of metaphases cells/1000 cells)

Mitotic index – Second test

Sampling time: 20th hour

Test without metabolic activation

M.I. (°)

M.I. reduction %

Treatment

Vehicle control

ANOX BF

Dose µg/ml

150

42/1000

41/1000

44/1000

28/1000

Test with metabolic activation

M.I. (°)

M.I. reduction %

Treatment

Vehicle control

ANOX BF

Dose µg/ml

150

44//1000

39/1000

32/1000

35/1000

(°) M.I. = mitotic index (number of metaphases cells/1000 cells)

Mitotic index – Second test

Sampling time: 44th hour

Test without metabolic activation

M.I. (°)

M.I. reduction %

Treatment

Vehicle control

ANOX BF

Dose µg/ml

150

49/1000

39/1000

Test with metabolic activation

M.I. (°)

M.I. reduction %

Treatment

Vehicle control

ANOX BF

Dose µg/ml

150

27/1000

20/1000

(°) M.I. = mitotic index (number of metaphases cells/1000 cells)

Conclusions:

The results of this study show that the ANOX BF up to the concentration of 150 µg/ml, both in the absence and in the presence of metabolic activation, did not induce statistically significant increase of cells with chromosome aberrations in cultured CHO cells, in two independent experiments.

Executive summary:

The clastogenic potential of the test article ANOX BF was investigated by identifying chromosome aberrations in cultured Chinese Hamster ovary cells (CHO) in two independent experiments.

The studies were performed with and without rat liver S9 fraction as metabolizing system.

The test article vehicle (acetone) was used as negative control.

In each experiment, Ethylmethane sulfonate (EMS) (605 µg/ml) and Cyclophospharnide (CP) (50 µg/ml) were used as positive controls to assess the sensitivity of the Chinese Hamster ovary cells (CHO) to mutagens as well as the activity of the metabolizing system.

The preliminary cytotoxicity test was performed as part of the main study.

In the first trial (Exp. No.960696/1), the test article was tested at the dosage levels of 5, 15, 50,150.500, 1500 and 5000 µg/ml.

The culture treatment, performed at 24 hours, was 3 hours long, both with and without metabolic activation.

The cell harvesting was carried out at the 20th hour.

The test article did not affect pH and osmolality of the treatment medium at any of the dosage levels assayed.

From 500 µg/ml upward, when the test article was added to the incubation mixture, droplets visible to the naked eye formed on its surface.

From 150 µg/ml downward small droplets of the test article distributed throughout the incubation mixture were observed when it was examined using a microscope.

Toxicity of the test article on CHO cells was evaluated examining the slides for the number of metaphase cells per 1000 cells (mitotic index) in the treated cultures as compared to the vehicle control, the reduction of which is indicative of toxicity.

At the dosage levels of 500, 1500 and 5000 µg/ml the test substance proved to be cytotoxic on the test systems, both with and without metabolic activation and very few metaphases were seen on the slides at harvesting.

At 150 µg/ml in the test without metabolic activation the mitotic index reduction compared to the vehicle control was 38%.

At the other two doses, 15 and 50 µg/ml, no significant cytotoxic effects were detected.

At 15, 50 and 150 µg/ml with metabolic activation the mitotic index reduction was about 25%.

Taking into account the above considerations 15, 50 and 150 µg/ml were selected for metaphase analysis, both in the presence and in the absence of metabolic activation.

The test was repeated in an independent assay (Exp. no. 960696/2).

In the second experiment the culture treatment was 18 and 3 hours long in the absence and in the presence of metabolic activation, respectively.

In this trial the same concentrations analyzed in the first experiment were scored.

At the 72 hour sampling time, at 150 µg/ml without metabolic activation, the mitotic index reduction compared to the control value was 33%.

At the other two concentrations assayed no significant cytotoxic effects were seen.

At 50 and 150 µg/ml with metabolic activation the mitotic index reduction was 20-30%.

At 15 µg/ml no significant cytotoxic effect was detected.

Since negative results were obtained in the first assay, the repeat test included an additional sample with and without metabolic activation at approximately 24 hours later (44^thhour).

At the second fixation time only the vehicle control and the highest dose of the test article (150 µg/ml) were scored, both in the presence and in the absence of S9 metabolizing system.

At this sampling time, the mitotic index reductions compared to the control values were 20 and 26% in the test without and with metabolic activation, respectively.

At neither sampling time, at none of the test article concentration assayed was there an incidence of cells with chromosome aberration statistically different from the control group, either in the presence or in the absence of metabolic activation.

As expected, the reference mutagens, Ethylmethane sulphonate (EMS) and Cyclophosphamide (CP), produced statistically significant increases in the percentage of cells with chromosome aberrations, both including and excluding gaps p < 0.001).

At none of the test article concentrations assayed was observed an incidence of polyploid cells or endoreduplications statistically different from the control group, either in the presence or in the absence of metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2003 to 13 October 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Guideline 476 (In Vitro Mammalian Cell Gene
Mutation Test), Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, publishes
by OECD, Paris, February 1998.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y cells, clone 3.7.2C, were obtained fkom Patricia Poorman-Allen, Glaxo Wellcome Inc.,
Research Triangle Park, NC on 15 August 1995. Each lot of cryopreserved cells was tested using the agar culture and Hoechst staining procedures and found to be free of mycoplasma
contamination. Prior to use in the assay, L5 178Y cells were cleansed of spontaneous TK-'- cells by culturing in a restrictive medium (Clive and Spector, 1975).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor-1254,500 mgkg, five days prior to sacrifice. The S9 was batch prepared and stored at I-70°C until used. Each bulk preparation of S9 was assayed for sterility and its ability to metabolize 2-arninoanthracene and 7,12-dimethyl-benz(a)anthracene to forms mutagenic to Salmonella typhimurium TA 100.
Immediately prior to use, the S9 was mixed with the cofactors and Fischer's Medium for Leukemic Cells of Mice with 0.1% Pluronics (Fa) to contain 250 pL S9, 6.0 mg nicotinamide adenine dinucleotide phosphate (NADP), 1 1.25 mg DL-isocitric acid, and 750 pL FOP per mJi S9-activation mixture and kept on ice until used. The cofactor/FoP mixture was adjusted to pH 7.0 and filter sterilized prior to the addition of S9.

Test concentrations with justification for top dose:

Based on the results of the preliminary toxicity assay, the doses chosen for treatment of the extended treatment assay ranged fiom 5 to 250 pg/mL for non-activated cultures with a 24-hour exposure.
The concentrations chosen for cloning were 3.75,7.5, 18.75,37.5 and 75 pg/mL.

Vehicle / solvent:

Ethanol was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in ethanol at 500 mg/mL, the maximum concentration tested.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

Solubility Test
A solubility test was conducted to select the vehicle. The test was conducted using one or more of the following solvents in the order of preference as listed: purified water, dimethyl sulfoxide, and ethanol. The test article was tested to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration, up to 50 mg/mL for aqueous vehicles or 500 mg/mL for organic vehicles.

Preliminary Toxicity Assay
The preliminary toxicity assay was used to establish the optimal dose levels for the mutagenesis assay. L5178Y cells were exposed to the solvent alone and nine concentrations of test article ranging fiom 0.5 to 5000 &rnL in both the absence and presence of S9-activation with a 4-hour exposure and fiom 0.25 to 2500 ,ug/mL without activation with a 24-hour exposure.
Cell population density was determined 24 and 48 hours after the initial exposure to the test article.
The cultures were adjusted to 3x10' cellslml after 24 hours only. Cultures with less than 3x10~ cells/mL were not adjusted. Toxicity was measured as suspension growth of the treated cultures relative to the growth of the solvent control cultures after 48 hours.

Mutagenesis Assays
The initial mutagenesis assay (with and without S9 activation with a 4-hour exposure) and extended treatment assay (without activation with a 24-hour exposure) were used to evaluate the mutagenic potential of the test article. L5 178Y mouse lymphoma cells were exposed to the solvent alone and eight concentrations of test article in duplicate in both the absence and presence of S9. Positive controls, with and without S9-activation, were tested concurrently.

Treatment of the Target Cells
The mutagenesis assay was performed according to a protocol described by Clive and Spector (1975). Treatment was carried out in conical tubes by combining 6 x 1o 6 ~5 1~ xY/TK+'c- ells, FOP medium or S9 activation mixture, and 100 pL dosing solution of test or control article in solvent or
solvent alone in a total volume of 10 ml for the 4-hour exposure. Due to the inherent toxicity of ethanol, 50 pL aliquots of test article in solvent or solvent alone were used for the 24-how exposure. The positive controls were treated with MMS (at final concentrations in treatment medium of 10 and 20 pg/mL with a 4-hour exposure or 2.5 and 5.0 pg/ml with a 24-hour exposure) and 7,12-DMBA (at final concentrations in treatment medium of 2.5 and 4.0 pglml). Treatment tubes were gassed with 5*l% CO2 in air, capped tightly, and incubated with mechanical mixing for 4 or 24 hours at 37*1°C. The preparation and addition of the test article dosing solutions were carried out under amber lighting and the cells were incubated in the dark during the exposure period. After the treatment period, the cells were washed twice with FOP or FOP supplemented with 10% horse serum, 2 rnM Lglutamine, 100 U penicillinhL and 100 pg streptomycinhd, (Flop).
After the second wash, the cells were resuspended in Flop, gassed with 5*1% C02 in air and placed on the roller drum apparatus at 37hl "C.

Expression of the Mutant Phenotype
For expression of the mutant phenotype, the cultures were counted using an electronic cell counter and adjusted to 3x10~c ellslml at approximately 24 and 48 hours after treatment in 20 and 10 mL total volume, respectively. Cultures with less than 3x10~ce lls1mL were not adjusted.
For expression of the TK-'- cells, cells were placed in cloning medium (C.M.) containing 0.23% dissolved granulated agar in FOP plus 20% horse serum. Two flasks per culture to be cloned were labeled with the test article concentration, activation condition, and either TFT (trifluorothymidine,
the selective agent) or VC (viable count). Each flask was prewarmed to 37*1°C, filled with 100 mL C.M., and placed in an incubator shaker at 37*1°C until used. The cells were centrihged at 1000 rpm for 10 minutes and the supernatant was decanted. The cells were then diluted in C.M. to
concentrations of 3x10~c ells/100 mL C.M. for the TFT flask and 600 cells/100 rnL C.M. for the VC flask. After the dilution, 1.0 mL of stock solution of TFT was added to the TFT flask (final concentration of 3 pg/mL) and both thls flask and the VC flask were placed on the shaker at 125 rpm and 37*1°C. After 15 minutes, the flasks were removed and the cell suspension was divided equally into each of three appropriately labeled petri dishes. To accelerate the gelling process, the plates were placed in cold storage (approximately 4°C) for approximately 30 minutes. The plates were then incubated at 37*l "C in a humidified 5*l % COz atmosphere for 10- 14 days.

Scoring Procedures
After the incubation period, the VC plates were counted for the total number of colonies per plate and the total relative growth determined. The TFT-resistant colonies were then counted for each culture with 220% total relative growth (including at least one concentration with 110% but
QO% total growth.). The diameters of the TFT-resistant colonies for the positive and solvent controls and, in the case of a positive response, the test article-treated cultures were determined over a range of approximately 0.2 to 1.1 rnm. The rationale for this procedure is as follows: Mutant L5178Y TIC'- colonies exhibit a characteristic frequency distribution of colony sizes. The precise distribution of large and small TFT-resistant mutant colonies appears to be the characteristic mutagenic "finger-print" of carcinogens in the L5178Y TK+'- system (Clive et al., 1979; DeMarini et al., 1989). Clive et al. (1979) and Hozier et al. (1981) have presented evidence to substantiate the hypothesis that the small colony variants cany chromosome aberrations associated with chromosome 11, the chromosome on which the TK locus is located in the mouse (Kozak and Ruddle, 1977). They suggested that large colony mutants received very localized damage, possibly in the form of a point mutation or small deletion within the TK locus, whle small colony mutants received damage to collateral loci concordant with the loss of TK activity.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

A result was considered positive if a concentration-related increase in mutant frequency was observed and one or more dose levels with 10% or greater total growth exhibited mutant frequencies of 2100 mutants per lo6 clonable cells over the background level.
A result was considered equivocal if the mutant frequency in treated cultures was between 55 and 99 mutants per 10E6 clonable cells over the background level.
A result was considered negative if the mutant frequency in treated cultures was fewer than 55 mutants per 10E6 clonable cells over the background level.

Criteria for a Valid Test
The following criteria must be met for the mutagenesis assay to be considered valid:
Negative Controls:
The spontaneous mutant frequency of the solvent control cultures must be within 20 to 100 TFT-resistant mutants per lo6 surviving cells. Low spontaneous mutant frequencies, i.e., 20 to 40 mutants per lo6 surviving cells, are considered acceptable if small colony recovery is demonstrated (Mitchell et al., 1997). The cloning efficiency of the solvent control group must be greater than 50%.

Positive Controls:
At least one concentration of each positive control must exhibit mutant fi-equencies of 2100 mutants per lo6 clonable cells over the background level. The colony size distribution for the MMS positive control must show an increase in both small and large colonies (Moore et al., 1985; Aaron et al., 1994).

Test Article-Treated Cultures:
A minimum of four analyzable concentrations with mutant kequency data will be required. Ideally, the highest concentration should produce at least 80% toxicity (no more than 20% survival; ICH, 1996). In the case of a test article with a steep toxicity curve (no concentrations with 10-20% survival), the results may be considered acceptable if a dose spacing of I2-fold is used and the highest dose tested showed <20% survival or total kill (Sofimi et al., 1997).

Statistics:

Not specified

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:

>150 µg!mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility Test
Ethanol was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in ethanol at 500 mg/mL, the maximum concentration tested.

Preliminary Toxicity Assay
The maximum dose tested in the preliminary toxicity assay was 5000 µg!mL with a 4-hour exposure and 2500 µg/mL without activation with a 24-hour exposure. Visible precipitate was present at concentrations > 150 µg/mL in treatment medium with a 4-hour exposure and 2250 pg/mL in treatment medium with a 24-hour exposure. No visible precipitate was present at concentrations of < 50 µg/mL in treatment medium with a 4-hour exposure and < 75 µg/mL with a 24-hour exposure. The osmolality of the solvent control was 324 mmo/kg and the osmolality of the highest soluble dose at the beginning of treatment, 50 µg/mL, was 351 mmol/kg. Suspension growth relative to the solvent controls was 80% without activation with a 4-hour exposure, 70% with S9 activation with a 4-hour exposure, and 115% without activation with a 24-hour exposure at 5000 pg/mL. Based on the results of the toxicity test, the doses chosen for the mutagenesis assay ranged from 5 to 150 µg/mL for both the non-activated and S9-activated cultures with a 4-hour exposure and fiom 5 to 250 µg/mL without activation with a 24-hour exposure. However, due to a calculation error, the actual doses ranged from 3.75 to 187.5 µg/rnL for the 24-hour exposure without activation.

Mutagenesis Assays
Visible precipitate was present at concentrations > 150 µg/mL in treatment medium. No visible preciptate was present at concentrations of < 125 µg/mL in treatment medium. In the non-activated system, cultures treated with concentrations of 50, 75, 100, 125, and 150 µg/rnL were cloned and produced a range in suspension growth of 90% to 107%. In the S9-activated system, cultures treated with concentrations of 50, 75, 100, 125, and 150 µg/mL were cloned and produced a range in suspension growth of 88% to 97%.
One cloned non-activated culture exhibited a mutant frequency between 55 and 99 mutants per 10e6 clonable cells over that of the solvent control. A dose-response trend was not observed in the nonactivated or S9-activated systems. The total growth ranged kom 74% to 110% for the non-activated cultures at concentrations of 50 to 150 pg/mL and 89% to 124% for the S9-activated cultures at concentrations of 50 to 150 µg/mL.
The results of the initial assay were equivocal at one dose in the absence of S9 activation and negative presence of S9 activation. Because no unique metabolic requirements were known about the test article, an independent repeat assay was performed only in the absence of S9 for a 24-hour exposure period.
Visible precipitate was present at concentrations > 75 pg/rnL in treatment medium. No visible precipitate was present at concentrations of < 37.5 µg/mL in treatment medium. Cultures treated with concentrations of 3.75, 7.5, 18.75, 37.5, and 75 µg/mL were cloned and produced a range in suspension growth of 99% to 126%.
No cloned cultures exhibited mutant fiequencies that were between 55 and 99 mutants per 10e6 clonable cells over that of the solvent control. A dose-response trend was not observed. The total growth ranged fiom 110% to 147% at concentrations of 37.5 to 75 µg/mL.
The TFT-resistant colonies for the positive and solvent control cultures &om both assays were sized according to diameter over a range fiom approximately 0.2 to 1.1 rnm. The colony sizing for the MMS positive control yielded the expected increase in small colonies, verifying the adequacy of the methods used to detect small colony mutants.

PRELIMINARY TOXICITY ASSAY USING Anox 1315

Test Article Concentration

(µg/mL)

Cell Concentration

(X10⁶)^a

Suspension Growth

Day 1

Day 2

Total^b

% Control^c

WITHOUT ACTIVATION (4-hour)

Solvent 1

Solvent 2

1.5

150+

500+

1500+

5000+

1.308

1.210

1.275

1.163

1.183

1.258

1.154

1.124

1.261

1.205

1.257

1.488

1.465

1.478

1.594

1.552

1.535

1.549

1.585

1.427

1.514

1.181

21.6

19.7

20.9

20.6

20.4

21.5

19.9

19.8

20.0

20.3

16.5

101

100

104

WITH S9 ACTIVATION (4-hour)

Solvent 1

Solvent 2

1.5

150+

500+

1500+

5000+

0.839

0.871

0.872

0.897

0.865

0.869

0.867

0.891

0.685

0.849

0.656

1.485

1.462

1.501

1.416

1.474

1.425

1.551

1.424

1.486

1.529

1.346

13.8

14.1

14.5

14.1

14.2

13.8

14.9

14.1

11.3

14.4

9.8

104

101

107

101

103

WITHOUT ACTIVATION (24-hour)

Solvent 1

Solvent 2

.25

.75

2.5

7.5

250+

750+

2500+

0.921

0.923

0.961

0.984

0.977

0.956

0.967

0.887

0.759

1.035

1.199

1.176

1.281

1.110

1.256

1.238

1.316

1.028

1.262

1.009

1.130

1.084

12.0

13.1

11.9

13.7

13.4

14.0

11.0

12.4

8.5

13.0

14.4

109

107

111

103

115

Solvent = ethanol

1 and 2 are duplicate cultures

+ - precipitating dose

^a– Cultures containing <0.3x10⁶cells/mL on day 1 and 2 are considered to have 0% total suspension growth.

^b– Total suspension growth = Day 1 cell conc. x Day 2 cell conc.

-------------------------- -------------------------

0.3x10⁶cells/mL Day 1 adjusted cell conc.

^c- % of control suspension growth = total treatment suspension growth x 100

------------------------------------------------

average solvent control

total suspension growth

CLONING DATA FOR L5178Y/TK^+/-MOUSE LYMPHOMA CELLS TREATED WITH Anox 1315 IN THE ABSENCE EXOGENOUS METABOLIC ACTIVATION Initial Assay (4-hour exposure)

Test Article Concentration

(µg/mL)

TFT Colonies

VC Colonies

Mutant Freq.^a

Induced Mutant Freq.^b

% Total Growth^c

Counts

Mean

Counts

Mean

Solvent 1

Solvent 2

120

133

87 ± 4

97 ± 42

150

176

178

157

165

178

164 ± 11

170 ± 9

106

114

Mean Solvent Mutant Frequency = 110

50 A

50 B

109

107

98 ± 8

97 ± 7

132

120

152

142

157

137

147 ± 11

133 ± 9

133

146

75 A

75 B

103

120

101

103

94 ± 8

108 ± 9

158

166

146

158

185

144

163 ± 16

156 ± 9

115

138

100 A

100 B

101

116

60 ± 18

88 ± 30

143

162

126

149

142

137

137 ± 8

149 ± 10

117

-22

125 A

125 B

127

123

114

149

120

139

120 ± 5

137 ± 11

199

183

168

157

183 ± 13

137 ± 36

131

200

110

150+ A

150+ B

118

87 ± 4

75 ± 30

158

178

191

217

157

166

169 ± 16

187 ± 22

104

-6

-30

104

103

Positive Control – Methyl Methanesulfonate (µg/mL)

41 ± 6

38 ± 13

91 ± 3

27 ± 2

281

-20

172

Solvent = ethanol

A and B or 1 and 2 are duplicate cultures

+ - precipitating dose

^a– Mutant frequency (per 10⁶surviving cells = Average # TFT coloniesx 200

-------------------------------

Average # VC colonies

^b– Induced mutant frequency per 10⁶surviving cells) = mutant frequency – average mutant frequency of solvent controls

^c- % total growth = (% suspension growth x % cloning growth)

-------------------------------------------------------------

100

TOTAL COMPUND TOXICITY DATA FOR L5178Y/TK+/- MOUSE LYMPHOMA CELLS TREATED WITH Anox 1315 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION Initial Assay (4-hour exposure)

Test Article Concentration

(µg/mL)

Cell Concentration

(x 10⁶)^a

Susp Growth

Cloning Growth

% Total Growth^e

Day 1

Day 2

Total^b

%Cntl^c

Avg VC

%Cntl^d

Solvent 1

Solvent 2

1.188

1.159

1.511

1.440

2.0

18.5

164

170

50 A

50 B

1.111

1.044

1.548

1.587

19.1

18.4

147

133

75 A

75 B

1.089

1.058

1.546

1.567

18.7

18.4

163

156

100 A

100 B

1.207

1.052

1.541

1.488

20.7

17.4

107

137

149

125 A

125 B

1.186

1.068

1.466

1.464

19.3

17.4

100

1836

137

110

150+ A

150+ B

1.179

1.054

1.517

1.514

19.9

17.7

103

169

187

101

112

104

103

Positive Control – Methyl Methanesulfonate (µg/mL)

0.842

0.787

1.383

0.844

12.9

7.4

Solvent = ethanol

1 and 2 are duplicate cultures

+ - precipitating dose

^a– Cultures containing <0.3x10⁶cells/mL on day 1 and 2 are considered to have 0% total suspension growth.

^b– Total suspension growth = Day 1 cell conc. x Day 2 cell conc.

-------------------------- -------------------------

0.3x10⁶cells/mL Day 1 adjusted cell conc.

c - % of control suspension growth = total treatment suspension growth x 100

------------------------------------------------

average solvent control

total suspension growth

^d– Mutant frequency (per 10⁶surviving cells = Average # TFT coloniesx 200

-------------------------------

Average # VC colonies

^e- % total growth = (% suspension growth x % cloning growth)

-------------------------------------------------------------

100

CLONING DATA FOR L5178Y/TK^+/-MOUSE LYMPHOMA CELLS TREATED WITH Anox 1315 IN THE PRESENCE EXOGENOUS METABOLIC ACTIVATION Initial Assay (4-hour exposure)

Test Article Concentration

(µg/mL)

TFT Colonies

VC Colonies

Mutant Freq.^a

Induced Mutant Freq.^b

% Total Growth^c

Counts

Mean

Counts

Mean

Solvent 1

Solvent 2

37 ± 16

9 ± 3

123

157

173

130

107

133

134 ± 28

140 ± 12

Mean Solvent Mutant Frequency = 34

50 A

50 B

51 ± 10

48 ± 21

153

173

152

182

184

189

163 ± 15

181 ± 7

114

124

75 A

75 B

100

65 ± 2

70 ± 27

175

179

165

157

169

162

170 ± 4

166 ± 9

111

110

100 A

100 B

49 ± 21

60 ± 29

151

176

137

149

165

171

151 ± 11

165 ± 12

110

125 A

125 B

62 ± 14

32 ±5

150

173

153

116

145

123

149 ± 3

137 ±25

103

150+ A

150+ B

70 ± 4

55 ± 25

164

142

172

166

169

183

168 ± 3

164 ± 17

112

115

Positive Control – 7,12 Dimethylbenz(a)anthracene (µg/mL)

2.5

146

103

74 ±16

110± 27

166

133

127

142 ± 17

71 ± 12

104

311

278

Solvent = ethanol

A and B or 1 and 2 are duplicate cultures

+ - precipitating dose

^a– Mutant frequency (per 10⁶surviving cells = Average # TFT coloniesx 200

-------------------------------

Average # VC colonies

^b– Induced mutant frequency per 10⁶surviving cells) = mutant frequency – average mutant frequency of solvent controls

^c- % total growth = (% suspension growth x % cloning growth)

-------------------------------------------------------------

100

TOTAL COMPUND TOXICITY DATA FOR L5178Y/TK+/- MOUSE LYMPHOMA CELLS TREATED WITH Anox 1315 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION Initial Assay (4-hour exposure)

Test Article Concentration

(µg/mL)

Cell Concentration

(x 10⁶)^a

Susp Growth

Cloning Growth

% Total Growth^e

Day 1

Day 2

Total^b

%Cntl^c

Avg VC

%Cntl^d

Solvent 1

Solvent 2

1.069

0.914

1.412

1.496

16.8

15.2

134

140

50 A

50 B

0.966

0.925

1.429

1.460

15.3

15.0

163

181

119

132

114

124

75 A

75 B

0.981

0.916

1.310

1.434

14.3

14.6

170

166

124

121

111

110

100 A

100 B

0.897

0.928

1.420

1.421

14.1

14.6

151

165

110

121

110

125 A

125 B

0.923

0.902

1.469

1.412

15.1

14.1

149

137

109

100

103

150+ A

150+ B

0.897

0.938

1.464

1.484

14.6

15.5

168

164

123

119

112

135

Positive Control – 7,12 Dimethylben(a)anthracene (µg/mL)

2.5

0.781

0.571

1.314

0.872

11.4

505

142

104

Solvent = ethanol

1 and 2 are duplicate cultures

+ - precipitating dose

^a– Cultures containing <0.3x10⁶cells/mL on day 1 and 2 are considered to have 0% total suspension growth.

^b– Total suspension growth = Day 1 cell conc. x Day 2 cell conc.

-------------------------- -------------------------

0.3x10⁶cells/mL Day 1 adjusted cell conc.

c - % of control suspension growth = total treatment suspension growth x 100

------------------------------------------------

average solvent control

total suspension growth

^d– Mutant frequency (per 10⁶surviving cells = Average # TFT coloniesx 200

-------------------------------

Average # VC colonies

^e- % total growth = (% suspension growth x % cloning growth)

-------------------------------------------------------------

100

CLONING DATA FOR L5178Y/TK^+/-MOUSE LYMPHOMA CELLS TREATED WITH Anox 1315 IN THE ABSENCE EXOGENOUS METABOLIC ACTIVATION Extended Treatment Assay (24-hour exposure)

Test Article Concentration

(µg/mL)

TFT Colonies

VC Colonies

Mutant Freq.^a

Induced Mutant Freq.^b

% Total Growth^c

Counts

Mean

Counts

Mean

Solvent 1

Solvent 2

43 ± 5

25 ± 12

175

169

137

136

160 ± 17

108 ± 20

Mean Solvent Mutant Frequency = 50

3.75 A

3.75 B

44 ± 24

29 ± 7

162

138

150

125

146

148

153 ± 7

137± 9

-7

143

110

7.5 A

7.5 B

43 ± 8

19 ± 6

145

156

146

151

129

142

140 ±8

150±6

-25

114

128

18.75 A

18.75 B

27 ± 10

24 ± 4

158

123

146

143

113

160

139 ± 19

142 ± 15

-12

-16

115

37.5 A

37.5 B

54 ± 11

37± 26

163

190

173

150

173

166

170 ± 5

169± 16

-6

147

124

75+ A

75+ B

33 ± 18

51 ± 17

181

150

157

137

158

140

165 ± 11

142 ± 6

-10

133

118

Positive Control – Methyl Methanesulfonate (µg/mL)

2.5

201

100

217

111

138

100 ± 9

185± 34

109

116

85 ± 7

101 ± 16

236

366

186

316

Solvent = ethanol

A and B or 1 and 2 are duplicate cultures

+ - precipitating dose

^a– Mutant frequency (per 10⁶surviving cells = Average # TFT coloniesx 200

-------------------------------

Average # VC colonies

^b– Induced mutant frequency per 10⁶surviving cells) = mutant frequency – average mutant frequency of solvent controls

^c- % total growth = (% suspension growth x % cloning growth)

-------------------------------------------------------------

100

TOTAL COMPUND TOXICITY DATA FOR L5178Y/TK+/- MOUSE LYMPHOMA CELLS TREATED WITH Anox 1315 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION Extended Treatment Assay (24-hour exposure)

Test Article Concentration

(µg/mL)

Cell Concentration

(x 10⁶)^a

Susp Growth

Cloning Growth

% Total Growth^e

Day 1

Day 2

Total^b

%Cntl^c

Avg VC

%Cntl^d

Solvent 1

Solvent 2

1.163

1.134

1.288

1.343

16.6

16.9

160

108

3.75 A

3.75 B

1.408

1.186

1.349

1.379

21.1

18.2

126

108

153

137

114

102

143

110

7.5 A

7.5 B

1.256

1.334

1.318

1.298

18.4

19.2

110

115

140

150

104

111

114

128

18.75 A

18.75 B

1.289

1.261

1.302

1.304

18.7

18.3

111

109

139

142

103

106

115

37.5 A

37.5 B

1.286

1.113

1.369

1.345

19.6

16.6

117

170

169

126

147

124

75+ A

75+ B

1.186

1.230

1.374

1.369

18.1

18.7

108

111

165

142

123

106

133

118

Positive Control – Methyl Methanesulfonate (µg/mL)

2.5

1.459

1.313

1.340

1.080

21.7

15.8

129

101

Solvent = ethanol

1 and 2 are duplicate cultures

+ - precipitating dose

^a– Cultures containing <0.3x10⁶cells/mL on day 1 and 2 are considered to have 0% total suspension growth.

^b– Total suspension growth = Day 1 cell conc. x Day 2 cell conc.

-------------------------- -------------------------

0.3x10⁶cells/mL Day 1 adjusted cell conc.

c - % of control suspension growth = total treatment suspension growth x 100

-----------------------------------------------------------------------

average solvent control total suspension growth

^d– Mutant frequency (per 10⁶surviving cells = Average # TFT coloniesx 200

-------------------------------

Average # VC colonies

^e- % total growth = (% suspension growth x % cloning growth)

-------------------------------------------------------------

100

Conclusions:

All criteria for a valid study were met as described in the protocol, except as noted in the Deviations section. Under the conditions of this study, test article Anox 1315 was concluded to be negative in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.

Executive summary:

The test article, Anox 1315, was tested in the L5178Y/TK^+/-Mouse Lymphoma Mutagenesis Assay in the absence and presence of Aroclor-induced rat liver S9. The preliminary toxicity assay was used to establish the concentration range for the mutagenesis assays. The initial and extended treatment mutagenesis assays were used to evaluate the mutagenic potential of the test article.

In the preliminary toxicity assay, the maximum concentration of Anox 1315 in treatment medium was 5000 µg/mL. Visible precipitate was present at concentrations ≥150 µg/mL in treatment medium with a 4-hour exposure and ≥250 µg/mL in treatment medium with a 24-hour exposure.

No visible precipitate was present at concentrations of ≤50 µg/mL in treatment medium with a 4-hour exposure and ≤ 75 µg/mL with a 24-hour exposure. Selection of concentrations for the mutation assay was based on solubility profile. Substantial toxicity, i.e., suspension growth of ≤50% of the solvent control, was not observed at any concentration with or without S9 activation.

Based on the results of the preliminary toxicity assay, the doses chosen for treatment of the initial mutagenesis assay ranged from 5 to 150 µg/mL for both the non-activated and S9-activated cultures. Visible precipitate was present at concentrations ≥150 µg/mL in treatment medium. No visible precipitate was present at concentrations of ≤125 µg/mL in treatment medium. The concentrations chosen for cloning were 50, 75, 100, 125, and 150 µg/mL with and without S9 activation. One non-activated cloned culture exhibited a mutant frequency between 55 and 99 mutants per 10⁶clonable cells over that of the solvent control. There was no dose-response trend.

Toxicity in the cloned cultures, i.e., total growth of 150% of the solvent control, was not observed any concentration with or without S9 activation.

Based on the results of the preliminary toxicity assay, the doses chosen for treatment of the extended treatment assay ranged from 5 to 250 µg/mL for non-activated cultures with a 24-hour exposure. Due to a calculation error, the actual doses ranged fi-om 3.75 to 187.5 µg/mL. Visible precipitate was present at concentrations 275 µg/mL in treatment medium. No visible precipitate was present at concentrations of ≤37.5 µg/mL in treatment medium. The concentrations chosen for cloning were 3.75,7.5, 18.75,37.5 and 75 µg/mL. No cloned cultures exhibited mutant frequencies between 55 and 99 mutants per 10⁶clonable cells over that of the solvent control. There was no dose-response trend. Toxicity in the cloned cultures was not observed any concentration with or without S9 activation.

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

Under the conditions of this study, test article Anox 1315 was concluded to be negative in the L5178Y/TK^+/-Mouse Lymphoma Mutagenesis Assay.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo - Micronucleus test

ANOX BF is not mutagenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro - Ames Assay

The test article was used undiluted. Furthermore, it was diluted with water for injection to obtain t.q., 1:10, 1:100, 1:1000, 1:10000 dilutions.

Genetic toxicity in vitro - Ames Assay

Based on the results of this study it is concluded that ANOX® 1315 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Genetic toxicity in vitro - Mouse Lymphoma Mutagenesis Assay

Based on the results of the preliminary toxicity assay, the doses chosen for treatment of the extended treatment assay ranged from 5 to 250 µg/mL for non-activated cultures with a 24-hour exposure. Due to a calculation error, the actual doses ranged from 3.75 to 187.5 µg/mL. Visible precipitate was present at concentrations 275 µg/mL in treatment medium. No visible precipitate was present at concentrations of ≤37.5 µg/mL in treatment medium. The concentrations chosen for cloning were 3.75,7.5, 18.75,37.5 and 75 µg/mL. No cloned cultures exhibited mutant frequencies between 55 and 99 mutants per 10⁶clonable cells over that of the solvent control. There was no dose-response trend. Toxicity in the cloned cultures was not observed any concentration with or without S9 activation.

Under the conditions of this study, test article Anox 1315 was concluded to be negative in the L5178Y/TK^+/-Mouse Lymphoma Mutagenesis Assay.

Genetic toxicity in vitro - Chromosome Aberration Assay