Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation in bacteria: Key study. Method according to OECD 471, OECD 472, and Guidelines for Screening Mutagenicity Testing Of Chemicals (Japan). The test item was not mutagenic with or without metabolic activation. Supporting study: Method according to Zeiger (1992), similar to OECD 471 (GLP study). The test item was not mutagenic with or without metabolic activation.

In-vitro cytogenicity study in mammalian cells or in-vitro micronucleus study: Key study. Method according to OECD 473 and Guidelines for Screening Mutagenicity Testing Of Chemicals (Japan). The substance was not mutagenic with or without metabolic activation.

In-vitro gene mutation study in mammalian cells: Data waiving (study scientifically not necessary / other information available). In accordance with Column 2 of REACH Annex VIII, the study does not need to be conducted since there is adequate data from reliable in vivo cytogenicity studies.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
(NPP No. 237, No. 306, No. 302, No. 303 on Mar. 31, 1984)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot number: 10705, manufactured by Tama Chemical Industry Co., Ltd.
- Purity: 99.7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature shielded from light


Target gene:
Histidine-requiring gene in four strains of S. typhimurium, and tryptophan requiring gene in E. coli WP2.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
Escherichia coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (liver rat)
Test concentrations with justification for top dose:
- Each strain was tested at six concentrations within the following: 0, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500, 5000 µg/plate.
- Justification: A preliminary test was carried out in the range of 50 to 5000 µg/plate with only one replicate per strain. Antibacterial activity was observed at 5000 µg/plate in all strains except for Escherichia coli WP2 with and without metabolic activation, at 1500 µg/plate without metabolic activation in TA100, TA 1535 and TA1537 and with metabolic activation in TA100 and TA1537. On this basis, the highest dose in the first study was 2500 µg/plate for Salmonella typhimurium TA1535, T98 and T100 and 5000 µg/plate in Escherichia coli WP2 with and without metabolic activation. For 1537 the highest dose was set at 1250 µg/plate without metabolic activation and 2500 µg/plate with it. A second test was performed at the same doses used in the first test except the maximum dose of the metabolic activation test in TA1537 which was changed to1250 μg / plate as in the non-activation test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO) from Wako Pure Chemical Industries, Ltd, lot number: DSL5887
- Justification for choice of solvent/vehicle: a stability test in DMSO solution was conducted. Dicyclohexylcarbodiimide was prepared to be 50 mg / ml in DMSO and further diluted with the same solvent in a ratio of 2 to 3. The product was immediately used for the test. Two concentrations at the highest concentration (50 mg / ml) and at the lowest concentration (0.25 mg / ml) used in this test were carried out. The average content of the samples after 3 hours of preparation was 99.7 and 100%, respectively, with respect to the average (0 hour) of the initial value. These values satisfied the laboratory criteria (over 90% of the initial measurement average value).
The stability of the substance at the concentrations prepared for this test was also checked. For the 50 mg / ml solution were obtained values between 97.4 to 101%, 106 to 107% for the 0.781 mg / ml solution and 91.1-103% for the 0.391 mg / ml solution. These values also met the criteria of the standard laboratory operating procedure (the mean content is more than 85% of the aggregate quantity).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Plate incorporation method, with and without metabolic activation. 2 ml of top agar, 0.1 ml of test solution, 0.5 ml of phosphate buffer solution (0.5 ml of S9 mixture solution in metabolic activation test) and 0.1 ml of assay bacterial solution were mixed in a small test tube and then placed on a synthetic medium flat plate. After solidification, plates were incubated at 37ºC for 48 hours. As a control group, DMSO or positive control substance solutions were used instead of the test substance preparation solution. After the incubation period, the number of revertant mutant colonies generated was calculated and their average value and standard deviation were obtained, respectively.

DURATION
- Preincubation period: no preincubation period.
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays):
No specific selection agent was used. The lack of amino-acid in the medium allowed only mutants to grow because of their ability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS:
Three replicates per strain, dose and substance (vehicle, positive control substances and test substance).

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.
Evaluation criteria:
The substance was considered to be mutagenic if:
- the number of revertant colonies counted in the plate containing the test substance was increased more than twice as compared with the counts of the negative control
- a dose dependence was recognized in the increase in counts.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 and 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 μg/plate.
Vehicle controls validity:
valid
Untreated 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:
cytotoxicity
Remarks:
at 2500 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1250 and 2500 μg/plate.
Vehicle controls validity:
valid
Untreated 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:
cytotoxicity
Remarks:
at 625 and 1250 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: due to the antibacterial activity observed in the preliminary study, the highest dose selected for each strain in both tests was known to have antibacterial activity. This was confirmed in both tests: the highest dose for each strain showed inhibition against growth of the bacteria, in some cases in the two high doses, but although this antibacterial activity was observed, no increase in the number of mutant colonies dose-dependent was detected.

HISTORICAL CONTROL DATA: not specified.

Table 1. Number of revertants in the preliminary test.

Group

dose

(μg/plate)

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

Solvent control

-

-

138 ± 7.2

12 ± 2.1

11 ± 8.1

26 ± 2.6

8 ± 1.7

Test substance

50

-

76

9

8

21

8

150

-

82

5

14

13

8

500

-

62

11

10

10

7

1500

-

59*

5*

9

7

0*

5000

-

2*

0*

9

6*

0*

Positive controls

Substance and dose

 

AF2

0.01

SA

0.5

AF2

0.01

AF2

0.1

9AA

80

 

 

-

669 ± 30.1

319 ± 16.8

139 ± 15.9

561 ± 15.5

2329 ± 590.8.

Solvent control

-

+

139 ± 7.0

15 ± 3.8

15 ± 2.1

39 ± 3.1

11 ± 2.1

Test substance

50

+

115

15

17

21

7

150

+

138

13

22

22

12

500

+

88

8

24

28

6

1500

+

55*

8

13

8

5*

5000

+

22*

2*

8

3*

0*

Positive controls

Substance and dose

 

2AA

1

2AA

2

2AA

10

2AA

0.5

2AA

2

 

 

+

798 ± 52.2

245 ± 6.1

498 ± 24.7

189 ± 17.2

182 ± 18.2

* inhibition was observed against growth of the bacteria

Table 2. Number of revertants (mean ± SD) in the first test.

Group

dose

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

Solvent control

-

-

124 ± 7.5

15 ± 2.1

13 ± 3.8

22 ± 3.0

10 ± 0.6

Test substance

39.1

-

 

 

 

 

8 ± 1.5

78.1

-

118 ± 6.5

11 ± 3.2

 

19 ± 6.1

7 ± 2.6

156.3

-

108 ± 11.5

8 ± 3.6

13 ± 0.6

20 ± 4.5

10 ± 1.0

312.5

-

103 ± 8.0

10 ± 1.7

15 ± 4.6

20 ± 4.0

7 ± 1.5

625

-

105 ± 6.1

11 ± 2.9

13 ±3.1

18 ± 2.5

6 ± 1.0

1250

-

96 ± 11.5

9 ± 1.2 *

11 ± 3.1

16 ± 5.0

2 ± 2.9*

2500

-

85 ± 8.1 *

6 ± 1.2 *

11 ± 4.4

11 ± 2.1*

 

5000

-

 

 

6 ± 3.8*

 

 

Positive controls

Substance and dose

 

AF2

0.01

SA

0.5

AF2

0.01

AF2

0.1

9AA

80

 

 

-

626 ± 20.2

228 ± 15.2

152 ± 8.9

560 ± 54.0

2965 ± 56.0

Solvent control

-

+

157 ± 26.3

13 ± 2.6

17 ± 6.2

41 ± 3.8

14 ± 4.2

Test substance

78.1

+

128 ± 10.7

17 ± 7.0

 

35 ± 1.0

10 ± 4.5

156.3

+

125 ± 12.7

13 ± 0.6

12 ± 2.0

27 ± 2.1

8 ± 1.5

312.5

+

124 ± 5.0

8 ± 4.5

13 ± 0.00

30 ± 1.5

8 ± 1.5

625

+

117 ± 16.8

11 ± 5.7

14 ± 2.9

41 ± 2.1

6 ± 1.0

1250

+

96 ± 16.9*

7 ± 1.2

11 ± 3.2

28 ± 3.1

3 ± 0.0*

2500

+

101 ± 2.6*

8 ± 1.2*

15 ± 3.2*

20 ± 8.7*

2 ± 2.1*

5000

+

 

 

9 ± 0.6*

 

 

Positive controls

Substance and dose

 

2AA

1

2AA

2

2AA

10

2AA

0.5

2AA

2

 

 

+

750 ± 26.1

212 ± 4.6

273 ± 22.9

226 ± 9.7

164 ± 10.7

* inhibition was observed against growth of the bacteria

Table 3. Number of revertants (mean ± SD) in the second test.

Group

dose

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

Solvent control

-

-

150 ± 15.3

16 ± 2.1

15 ± 4.0

21 ± 1.0

8 ± 1.0

Test substance

39.1

-

 

 

 

 

10 ± 4.9

78.1

-

123 ± 27.2

11 ± 5.5

 

21 ± 5.6

6 ± 2.9

156.3

-

125 ± 7.0

11 ± 2.1

16 ± 8.7

21 ± 6.1

5 ± 3.5

312.5

-

122 ± 19.2

9 ± 1.2

14 ± 4.4

24 ± 1.0

4 ± 0.6

625

-

115 ± 13.5

10 ± 2.3

13 ± 1.5

21 ± 4.0

5 ± 1.5*

1250

-

127 ± 3.0

7 ± 0.6*

12 ± 2.9

19 ± 1.2

4 ± 2.5*

2500

-

101 ± 7.3*

5 ± 2.3*

7 ± 4.47*

17 ± 4.5*

 

5000

-

 

 

3 ± 2.6*

 

 

Positive controls

Substance and dose

 

AF2

0.01

SA

0.5

AF2

0.01

AF2

0.1

9AA

80

 

 

-

709 ± 19.5

175 ± 11.2

162 ± 10.8

759 ± 15.3

4064 ± 478.1

Solvent control

-

+

160 ± 16.5

16 ± 4.5

17 ± 0.6

50 ± 5.3

12 ± 2.6

Test substance

39.1

+

 

 

 

 

7 ± 1.5

78.1

+

167 ± 20.6

17 ± 4.6

 

33 ± 5.1

10 ± 1.2

156.3

+

147 ± 5.5

12 ± 3.1

15 ± 1.7

34 ± 6.8

7 ± 1.5

312.5

+

138 ± 10.5

14 ± 3.1

15 ± 3.5

34 ± 7.5

5 ± 2.0

625

+

147 ± 11.8

13 ± 2.0

16 ± 4.0

31 ± 3.6

5 ± 1.5

1250

+

110 ± 7.5

10 ± 1.5

17 ± 2.9

24 ± 2.6

3 ± 2.6*

2500

+

126 ± 1.0*

8 ± 1.5*

 15 ± 1.5

24 ± 4.2*

 

5000

+

 

 

6 ± 5.3*

 

 

Positive controls

Substance and dose

 

2AA

1

2AA

2

2AA

10

2AA

0.5

2AA

2

 

 

+

922 ± 19.1

228 ± 14.0

711 ± 51.6

304 ± 26.3

203 ± 6.1

* inhibition was observed against growth of the bacteria

Conclusions:
The test item was not mutagenic under the test conditions, with or without metabolic activation.
Executive summary:

The ability of the test item to induce mutation was assessed by the bacterial reverse mutation test (Ames test) according to OECD Guidelines No. 471 and 472 and the Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) (GLP study). Four histidine requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and tryptophan requiring Escherichia coli WP2 uvrA were tested in triplicate at six concentrations of test item in the range 40 - 5000 μg/plate, with and without metabolic activation (S9 mix), based on the results of a preliminary range-finding test. The plate incorporation method was used; vehicle (DMSO) and positive controls were run in parallel. Although inhibitory activity was observed in all strains tested in one or two of the higher dose groups, no increase in the number of the relevant colonies related with the dose were observed under the experimental conditions applied. Based on the above results, the test item was considered non-mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
four bacterial strains instead of five
Principles of method if other than guideline:
Testing was performed as reported by Zeiger et al. (1992).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Source: Radian Corporation (Austin, TX).
Target gene:
Histidine gene.
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
other: 1535
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Five doses of the test substance for each strain within these concentrations 0.1, 0.3, 1, 3, 10, 33, 100, 333 and 666 were tested. The high dose was limited by toxicity, the other four concentrations were selected accordingly.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: no data.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-Nitro-o-phenylenediamine: TA98 without S9; 2-aminoanthracene with S9 with all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation method.
- The test substance was incubated with the Salmonella typhimurium strains either in buffer or S9 mix for 20 minutes at 37ºC. Top agar supplemented with L-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were counted following 48 hours incubation at 37° C.
- Each trial consisted of triplicate plates of concurrent positive and negative controls and five doses of test item. The S9 mix was obtained from Aroclor 1254-induced male Sprague Dawley liver (at 10 and 30%) or Syrian hamster liver (at 10 and 30%). All trials were repeated at the same or a higher S9 fraction.

DURATION
- Preincubation period: 20 minutes at 37ºC
- Exposure duration: 48 hours at 37ºC

SELECTION AGENT (mutation assays):
No specific selection agent was used. The lack of histidine in the medium allowed only mutants to grow because of their ability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS:
Three replicates for positive control, solvent control and each dose of the test item..

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.
Evaluation criteria:
A positive response was defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. There was no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
A negative response was obtained when no increase in revertant colonies was observed following chemical treatment.
An equivocal response was defined as an increase in revertants that was not dose related, was not reproducible, or was not of sufficient magnitude to support a determination of mutagenicity.
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 33 µg/plate without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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
Positive controls validity:
valid
Additional information on results:
The test item was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535, with or without induced rat or hamster liver S9 activation enzymes.

Revertants/plate (presented as mean ± SD from three plates)

 

 

-S9 mix

+ hamster S9

+ rat S9 mix

Strain

Dose

(µg/plate)

Trial 1

Trial 2

10%

30 %

10 %

30 %

TA98

0

13 ± 1.2

12 ± 2.9

20 ± 1.2

17 ± 3.6

13 ± 1.0

17± 1.5

0.1

 

13 ± 1.5

 

 

 

 

0.3

13 ± 1.8

15 ± 3.3

 

 

 

 

1

16 ± 4.2

11 ± 0.6

 

 

 

 

3

13 ± 2.2

9 ± 1.2

16 ± 1.0

 

16 ± 0.9

 

10

11 ± 1.2

11 ± 2.8

11 ± 1.0

15 ± 2.4

12 ± 1.0

14 ± 0.6

33

9 ± 0.3*

 

13 ± 1.3

14 ± 1.2

13 ± 3.2

13 ± 1.5

100

 

 

10 ± 2.5

13 ± 2.6

15 ± 2.3

14 ± 0.3

333

 

 

8 ± 3.3*

12 ± 2.1

9 ± 0.7*

11 ± 1.5

666

 

 

 

13 ± 1.5*

 

7 ± 0.9*

Trial summary

 

Negative

Negative

Negative

Negative

Negative

Negative

Positive controls

 

4-nitro-o-phenylenediaminne

2-AA

 

 

287 ± 25.2

298 ± 21.1

349 ± 4.7

265 ± 17.2

226 ± 5.0

223 ± 13.1

TA100

0

84 ± 1.0

97 ± 5.5

105 ± 8.1

92 ± 1.9

98 ± 7.5

107 ± 5.4

0.1

 

87 ± 3.8

 

 

 

 

0.3

86 ± 3.7

97 ± 6.7

 

 

 

 

1

85 ± 2.3

100 ± 3.8

 

 

 

 

3

88 ± 6.8

92 ± 6.1

95 ± 1.5

 

103 ± 2.9

 

10

85 ± 0.0

62 ± 3.8*

89 ± 1.2

101 ± 0.3

98 ± 4.3

102 ± 5.2

33

toxic

 

88 ± 7.9

90 ± 0.3

94 ± 4.3

112 ± 6.7

100

 

 

94 ± 3.2

88 ± 5.8

83 ± 2.5

97 ± 2.8

333

 

 

57 ± 5.4*

87 ± 0.9

22 ± 11.7*

86 ± 1.9

666

 

 

 

8 ± 5.7*

 

39 ± 5.8*

Trial summary

 

Negative

Negative

Negative

Negative

Negative

Negative

Positive controls

 

SA

2-AA

 

 

829 ± 11.4

792 ± 19.7

522 ± 26.4

612 ± 11.8

428 ± 13.5

492 ± 35.4

TA1535

0

8 ± 2.3

12 ± 2.3

11 ± 1.5

10 ± 0.9

15 ± 1.5

12 ± 1.8

0.1

10 ± 0.9

11 ± 1.8

 

 

 

 

0.3

8 ± 0.9

11 ± 1.5

 

 

 

 

1

10 ± 0.3

10 ± 3.3

 

 

 

 

3

11 ± 2.5

13 ± 2.1

10 ± 1.9

9 ± 2.6

12 ± 2.7

10 ± 1.0

10

8 ± 1.2

6 ± 0.9

9 ± 0.3

9 ± 1.5

10 ± 1.9

11 ± 2.2

33

 

 

9 ± 1.8

10 ± 1.9

12 ± 2.0

10 ± 2.3

100

 

 

7 ± 0.6

8 ± 2.2

7 ± 0.6

15 ± 2.3

333

 

 

4 ± 0.9*

6 ± 0.6

9 ± 1.5

9 ± 1.5

Trial summary

 

Negative

Negative

Negative

Negative

Negative

Negative

Positive controls

 

SA

2-AA

 

 

651 ± 20.4

443 ± 6.4

101 ± 7.3

263 ± 18.0

100 ± 6.1

119 ± 3.4

TA97

0

98 ± 9.0

115 ± 9.8

128 ± 6.3

151 ± 4.3

132 ± 15.3

162 ± 4.9

0.1

112 ± 5.4

122 ± 6.7

 

 

 

 

0.3

115 ± 4.8

133 ± 5.4

 

 

 

 

1

119 ± 4.3

123 ± 10.7

 

 

 

 

3

112 ± 9.4

141 ± 10.5

124 ± 3.6

166 ± 6.0

141 ± 8.1

154± 3.6

10

118 ± 10.7

105 ± 7.8

144 ± 7.4

172 ± 0.9

154 ± 4.4

159 ± 3.8

33

 

 

157 ± 3.8

165 ± 3.5

153 ± 8.2

165 ± 3.2

100

 

 

158 ± 6.6

153 ± 3.5

152 ± 13.5

150 ± 2.8

333

 

 

118 ± 9.7

149 ± 7.2

116 ± 7.4

155 ± 6.0

Trial summary

 

Negative

Negative

Negative

Negative

Negative

Negative

Positive controls

 

9-AA

2-AA

 

 

341 ± 22.0

370 ± 32.7

447 ± 13.1

439 ± 22.2

325 ± 11.0

323 ± 20.0

* slight toxicity

Conclusions:
The test item was not mutagenic with and without metabolic activation.
Executive summary:

A bacterial reverse mutation assay (Ames test) was performed on the test item, according to the method reported by Zeiger (1992), similar to OECD 471 (GLP study). Four histidine requiring strains of Salmonella typhimurium (TA 97, TA98, TA100 and TA1535) were tested in triplicate at five concentrations of the test item within the following: 0.1, 0.3, 1, 3, 10, 33, 100, 333 and 666 μg/plate, with and without metabolic activation (S9 mix, prepared from the livers of Aroclor 1254-induced male Sprague Dawley rats or Syrian hamsters, at 10 and 30%), using the pre-incubation method. Vehicle and positive controls were run in parallel. No increase in revertant colonies was observed following chemical treatment in any of the conditions tested. Based on these results, the item was considered not mutagenic.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot No.:10705, manufactured by Tama Chemical Industry Co., Ltd., provided by Japan Chemical Industry Association.
- Purity >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: an acetone stability test was performed in the Hadano Laboratory Analytical Chemistry Laboratory. The test sunstance was stable in acetone for 3 hours at a concentration range of 0.0245 to 6.20 mg / ml. The concentration of the test substance was also measured in the highest and lowest concentration solutions and all were within the allowable range (average content of 85% or more) (data not shown)

FORM AS APPLIED IN THE TEST (if different from that of starting material): the test substance was a slightly pale yellow crystalline mass. It was dissolved in acetone for performing the test. A stock solution was prepared each time it was used.
Species / strain / cell type:
other: CHL cells (Chinese Hamster cells)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Research - Resource Bank (JCRB)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle MEM culture medium supplemented with 10% fetal bovine serum (FCS; Bocknek, lot number SF 70521)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver, induced with phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
To determine the treatment concentration used in the chromosomal aberration test, its influence on cell proliferation was investigated in a preliminary test. Based on the preliminary test, the maximum dose was the dose capable of inhibiting 50% of growth. In the long term assay, it was calculated to be 1.90 μg / ml and in the short term assay with metabolic activation was 31.0 μg / ml. The concentrations selected for the long term assay were 0.48, 0.95 and 1.90 μg / ml and the concentrations for the short term assay were 7.8, 15.5 and 31.0 μg / ml (the second concentration was half the top dose and the third concentration was half the second).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone.
- Justification for choice of solvent/vehicle: acetone was used as a solvent because the test substance was soluble in acetone. Information on the stability was not obtained, but in the stability test in acetone carried out at the Hadano Laboratory Analytical Chemistry Laboratory, it was stable for 3 hours at a concentration range of 0.0245 to 6.20 mg/ml.
Untreated negative controls:
yes
Remarks:
untreated control
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 2 × 10^4 CHL cells were seeded in a Petri dish (diameter 6 cm) containing 5 ml of the culture solution and cultured in a 37° C CO 2 incubator (5% CO 2).
- Direct method (24 and 48h): cells were cultured for 3 days, then, the culture solution was discarded, and culture broth with the test item was added, and cultured for 48h.
- Metabolic activation method (6h treatment, 18h post-exposure): cells were cultured for 4 days, then, the culture solution was discarded, and exposed to medium with test item for 6h. After processing, fresh culture medium was added and further cultured for 18h. Two petri dishes were used for each concentration.
- Two hours before the end of the culture, colcemid was added to the culture medium so that the final concentration is about 0.1 μg/ml, and after completion of the culture, each group of cells was diluted with phosphate buffer solution. Then, the culture solution was discarded, and the cells were washed with phoshpate buffer solution, fixed with 10% neutral formalin solution, and stained with Giemsa stain. Six slide specimens were prepared for each petri dish.

DURATION
- Preincubation period: 3 - 4 days
- Exposure duration: for the direct method (long treatment assay): 24 and 48 hours; for the metabolic activation method (short treatment assay, with and without metabolic activation): 6h.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid was added at 0.1 µg/ml two hours before the end of the incubation.

STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: three different assays were performed without replicates.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 200 cells were analysed per group for detecting structural aberrations, 800 cells were analysed per group for detecting polyploidy.

DETERMINATION OF CYTOTOXICITY: mitotic index: inhibition of mitosis (except in the second short term assay without metabolic activation, where was also examined the mitotic index MI).
- Tests with metabolic activation: inhibition of mitosis and determination of mitotic index.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
According to the criteria of Ishidate et al (1987), it was considered that :
- the test substance induced chromosomal abnormality if at least 10 % of cells presented chromosomal aberrations (positive result)
- the test substance did not induce chromosomal aberrations if less than 5 % presented chromosomal aberrations (negative result)
Results between 5 to 10% were considered as false positives.
Key result
Species / strain:
other: Chinese Hamster cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the highest dose caused 50% inhibition.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
To determine the concentrations to be used in the chromosomal aberration test, the inhibitory effect of the test substance on the proliferation of CHL cells was measured. The proliferation degree was measured using a monolayer cultured cell densitometer (Monocellater -Olympus). The ratio between the degree of proliferation for each concentration of the test substance and the degree of proliferation in the control group (only solvent) was determined. As a result, the growth inhibitory concentration of about 50% was calculated to be 1.90 μg / ml for the long term treatment and 31.0 μg / ml fot the short term treatment with metabolic activation. These concentrations were set to be the highest concentrations to be tested. The second concentration was half of the highest and the third concentration half of the second.

In the short term treatment study without metabolic activation, inhibition of mitosis was observed in all groups, so chromosome analysis was impossible. Additional test were conducted at lower concentrations: 0.12, 0.25, 0.49 and 0.1 µg/ml.

Table 1. Results of chromosome analysis of Chinese Hamster cells (CHL) by direct method (long term treatment)

Group

Concentration

(µg/ml)

Time of exposure

No of cells analysed

No. of structural aberrations

gap ctb cte csb cse f  mu total

Others (1)

N. of cells with aberrations (%)

TAG        TA

Polyploid (%) (2)

Judgement (3)

SA      NA

Solvent

0

24

200

1    0    0    0    0    0    0    1 

0

0.5         0

0.25

 

DCC

0.48

24

200

3    0    0    0    0    0    0    3 

0

1.5         0

0.75

-       -

DCC

0.95

24

200

0    0    0    0    0    0    0    0 

0

0            0

0.38

-       -

DCC

1.90

24

200

1    0    0    0    0    0    0    1 

0

0.5         0

0.13

-       -

MC

0.05

24

200

21  42   59    0    0     1   10 133

0

40.5      71

0.38

+       -

Solvent

 0

48

200

0    0    0    0    0    0    0    0 

0

1.0         0

0.25

 

DCC

0.48

48

200

0    0    0    0    0    0    0    0 

0

2.0         0

0.38

-       -

DCC

0.95

48

200

0    0    0    0    0    0    0    0 

0

0.5         0

0.25

-       -

DCC

1.90

48

0

 

 

 

 

Tox  Tox

MC

0.05

48

200

0    0    0    0    0    0    0    0 

1

39.0     36.5

0.25

+       -

 

Abbreviations. Gap: chromatid gap and chromosome gap; ctb: chromatid break; cte: chromatid exchange; csb: chromosome break; cse: chromosome exchange; f: fragment (deletion); mul: multiple aberrations; TAG: total number of cells with aberrations; TA: total number of cells with aberrations except gap; SA: structural aberration; NA: numerical aberration; MC: mitomycin C; Tox: toxicity.

(1)  Others such attenuation and premature chromosome condensation, were excluded from the number of structural aberrations.

(2)  Eight hundred cells were analyzed in each group.

(3)  Judgment was done on the basis of the criteria of Ishidate (1987)

High concentration group treated for 48 hours could not be analyzed due to mitosis suppression.

Table 2. Results of chromosome analysis of Chinese Hamster cells (CHL) by metabolic activation method (short term treatment)

Group

Concentration

(µg/ml)

 

 

S9 mix

Time of exposure

No of cells analysed

No. of structural aberrations

Gap   ctb   cte   csb   cse  f  mu total

Others (1)

N. of cells with aberrations (%)

TAG    TA

Polyploid (%) (2)

Judgement (3)

SA      NA

Solvent

0

-

6

200

 0     0     0     0     0     0     0      0

0

0.5      0

0.13

 

DCC

7.8

-

6

0

 

 

 

 

Tox Tox

DCC

15.5

-

6

0

 

 

 

 

Tox Tox

DCC

31.0

-

6

0

 

 

 

 

Tox Tox

CPA

5

-

6

200

1     4     3     0     0      0     0     0 

0

0.5     0.5

0.50

-      -

Solvent

 

+

6

200

0     0     0     0     0      0     0     0

0

0       0

0.63

 

DCC

7.8

+

6

200

4     0     2     0     0      0     0     0

6

 2.0     0.5

0.38

-       -

DCC

15.5

+

6

200

3     0     0     0     0      0     0      3

3

1.5      0

0

-       -

DCC

31.0

+

6

0

3     0     0     0     0      0     0      3

3

1.5      0

0

-       -

CPA

5

+

6

200

32   66  136   0    0      3   20  25 

1

56.5   54.5

0.50

+       -

 

Abbreviations. Gap: chromatid gap and chromosome gap; ctb: chromatid break; cte: chromatid exchange; csb: chromosome break; cse: chromosome exchange; f: fragment (deletion); mul: multiple aberrations; TAG: total number of cells with aberrations; TA: total number of cells with aberrations except gap; SA: structural aberration; NA: numerical aberration; CPA: cyclophosphamide; Tox: toxicity.

(1)  Others such attenuation and premature chromosome condensation, were excluded from the number of structural aberrations.

(2)  Eight hundred cells were analyzed in each group.

(3)  Judgment was done on the basis of the criteria of Ishidate (1987)

Dividing cells were not obtained in the groups without S9 mix. 

Table 3. Results of chromosome analysis of Chinese Hamster cells (CHL) by metabolic activation method (short term treatment)

Since dividing cells were not obtained in the treated groups without metabolic activation, an addittional test was performed. The test item concentration was reduced until analyzable dividing cells were obtained.

Group

Concentration

(µg/ml)

 

 

S9 mix

Time of exposure

No of cells analysed

No. of structural aberrations

Gap   ctb  cte  csb  cse  f  mu total

Others (1)

N. of cells with aberrations (%)

TAG    TA

Polyploid (%) (2)

Judgement (3)

SA      NA

Solvent

0

-

6

200

 2 0     0     0     0     0     0     2

0

1.0      0

0.38

 

DCC

0.12

-

6

200

 3    0     0     0     0     0     0     3

0

1.5  0

0.13 

-      -

DCC

0.25

-

6

200

 4    1     1     0     0     0     0     6

0

3.0 1 

0.13

-      -

DCC

0.49

-

6

200

 3    0     0     0     0     0     0     3

0

1.5 0 

0.25 

 -      -

MC

0.1

-

6

200

10  37   46    0     1      1    0    95

0

34.0   32.5

0.13

-      -

Abbreviations. Gap: chromatid gap and chromosome gap; ctb: chromatid break; cte: chromatid exchange; csb: chromosome break; cse: chromosome exchange; f: fragment (deletion); mul: multiple aberrations; TAG: total number of cells with aberrations; TA: total number of cells with aberrations except gap; SA: structural aberration; NA: numerical aberration; MC: mitomycin C; Tox: toxicity.

(1)  Others such attenuation and premature chromosome condensation, were excluded from the number of structural aberrations.

(2)  Eight hundred cells were analyzed in each group.

(3)  Judgment was done on the basis of the criteria of Ishidate (1987)

Conclusions:
The test item did not induce chromosomal abnormalities in CHL cells under the test conditions. Therefore, it is not mutagenic.
Executive summary:

The ability of the test item to induce chromosomal aberrations in cultured Chinese hamster cells (CHL) was examined according to the Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD Guideline 473 (GLP study). Two different types of assays were carried out: a long-term treatment (direct method) of 24 and 48 hours of continuous treatment with the test substance and a short-term treatment consisting of culture for 18 hours after treatment for 6 hours (metabolic activation method), with and without metabolic activation. Two hours before the end of the culture, colcemide was added and metaphase cells were collected, fixed and stained with Giemsa. For structural abnormalities, 200 cells were analyzed per group, and for ploidy, 800 cells were analyzed. Negative, solvent, and positive controls were included. The maximum dose for each assay was selected based on a cytotoxicity preliminary study: the dose capable of inhibiting 50% growth was selected as the maximum dose (1.90 µg/ml in the long-term assay, and 31.0 µg/ml in the short-term assay). In the test without metabolic activation, no dividing cells were obtained, so an additional test was performed at lower doses (0.12, 0.25 and 0.49 µg / ml). No chromosome structural abnormality or inducing effect of polyploid cells was observed in any of the tests at any concentration. Therefore, the test item is not mutagenic under test conditions.

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

Genetic toxicity in vivo

Description of key information

In vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus: Key study. Method similar to OECD 474, GLP study: An acute three-injection micronucleus study in bone marrow of male F344/N rats gave a negative result for genetic toxicity.

Key study. Method similar to OECD 474, GLP study: A subchronic peripheral blood micronucleus study in male and female B6C3F1 mice by dermal application gave a positive result for genetic toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- dicyclohexylcarbodiimide (CASRN 538-75-0; MW 206.32)
- Source and lot/batch No.of test material: Aldrich Chemical Co. (Milwaukee, WI)
- Purity: 99+%
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6 weeks
- Assigned to test groups randomly: yes
- Housing: individually housed
- Diet: NIH-07 Open Formula Diet, pelleted form (Zeigler Bros., Inc., Gardener, PA), ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3ºC
- Humidity (%): 50 ± 15%
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle.
Route of administration:
dermal
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the test item is moisture sensitive, so ethanol was the preferred solvent.
- Concentration of test material in vehicle: 1.5, 3.0, 6.0, or 12.0 mg/kg
- Dosing volume: 2.0 ml/kg bw.
Details on exposure:
TEST SITE: skin, not specified.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0 ml/kg bw.
- Concentration (if solution): 1.5, 3.0, 6.0, or 12.0 mg/kg.
- Constant volume or concentration used: yes
- For solids, paste formed: no
Duration of treatment / exposure:
90 days (13 weeks).
Frequency of treatment:
5 days per week.
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
6 mg/kg bw/day (nominal)
Dose / conc.:
12 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Because the blood samples were taken from animals on test in a subchronic toxicity study, there was no positive control group.
Tissues and cell types examined:
Normochromatic erythrocytes (PCE) from peripheral blood.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Doses were selected based on preliminary toxicity tests (no further data).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
- Treatment: 10 mice per dose group per sex, were administered the test item dissolved in ethanol via skin painting, five times per week for 13 weeks.
- At termination, the animals were examined for gross pathology and histopathology; blood chemistry and hematology parameters were also measured.

DETAILS OF SLIDE PREPARATION:
- At the end of the exposure period, blood samples were obtained from the retro-orbital sinus under anesthesia prior to euthanasia, and smears were immediately prepared and fixed in absolute methanol. Coded slides from each animal were stained with acridine orange [Tice et al. (1989)].

METHOD OF ANALYSIS:
- All slides were evaluated at 1,000x magnification using epi-illuminated fluorescence microscopy (450–490 nm excitation, 520 emission).
- the frequency of MN-NCE among 1,000 NCE per animal and the percentage of PCE among 1,000 erythrocytes was determined in each of 10 mice per sex per dose group.
Evaluation criteria:
In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.
Statistics:
The frequency of micronucleated cells among PCEs was analyzed by a statistical software package (Micronucleus Assay Data Management and Statistical software package (ver. 1.4) [Margolin and Risko, 1988; ILS, 1990]) that tested for increasing trend over dose groups with a one-tailed CochranArmitage trend test, followed by pairwise comparisons between each dosed group and the control group (ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. The %PCE data were analyzed by an analysis of variance (ANOVA) test based on individual animal data; pairwise comparisons between the exposure and control groups were made using a two-tailed Student’s t-test.
Key result
Sex:
male
Genotoxicity:
positive
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Key result
Sex:
female
Genotoxicity:
positive
Remarks:
weak
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): positive results were seen in male mice based on a trend test analysis (P 5 0.003), although none of the treated groups showed MN-NCE frequencies that were significantly higher than the corresponding control group. In female mice, increased frequencies of MN-NCE were seen in all four treatment groups; however, only the mice receiving 3.0 mg DCC/kg/day showed significantly elevated MN-NCE frequencies, and the trend P value was not significant. The results in females were judged weakly positive.
- Ratio of PCE/NCE (for Micronucleus assay): Neither sex showed chemical-induced effects on the %PCE in blood.

Table 2. Frequency of MN-PCE and %PCE in Peripheral Blood of Male and Female B6C3F1 Mice.

Dose

(mg/kg)

N

MN-PCE/

1000 PCE

(mean ± SE)

P value*

% PCE

(mean ± SE)

P value*

Males

0

10

3.6 ± 0.43

 

4.8 ± 0.25

 

1.5

10

3.1 ± 0.48

0.720

4.3 ± 0.08

0.114

3.0

10

5.3 ± 0.50

0.036

4.4 ± 0.11

0.264

6.0

10

5.7 ± 0.70

0.015

4.1 ± 0.14

0.048

12.0

10

5.7 ± 0.68

0.015

4.3 ± 0.14

0.107

 

 

P=0.003**

 

P = 0.071***

 

Females

0

10

2.7 ± 0.63

 

4.3 ± 0.19

 

1.5

10

3.7 ± 0.58

0.105

4.9 ± 0.14

0.009

3.0

10

5.1 ± 0.67

0.003

4.6 ± 0.16

0.085

6.0

10

4.3 ± 0.68

0.027

4.9 ± 0.22

0.015

12.0

10

4.4 ± 0.50

0.022

4.5 ± 0.22

0.191

 

 

P = 0.078**

 

P = 0.096

 

*: Comparison of individual dose groups to the concurrent control; significant at P 0.006. **: Trend test, one-tailed. Significant at P0.025. ***: ANOVA, significant at P ≤ 0.025.

Conclusions:
The test item gave a positive result in male and female B6C3F1 mice.
Executive summary:

A subchronic peripheral blood micronucleus study in male and female B6C3F1 mice was performed for the test item, by a method similar to OECD 407 (GLP study). Ten animals per sex per dose group were exposed to 0 (control), 1.5, 3, 6, 12 mg/kg bw of test item in ethanol by skin painting, five days a week for 13 weeks. At the end of the exposure period, blood samples were obtained from the retro-orbital sinus under anesthesia prior to euthanasia, and smears were immediately prepared, fixed in absolute methanol and stained with acridine orange. Peripheral blood analysis included assessment of the frequency of MN-PCE among 1000 PCE and the %PCE among 1000 erythrocytes. After 13 weeks of dermal administration, the test item induced significant increases in the frequency of micronucleated normochromatic erythrocytes in peripheral blood of mice, based on the results of a trend analysis, although none of the treated groups showed a significant increase in the frequency of micronucleated NCEs compared to the control. In females, the frequency of all treated groups was greater than the control, but only significant at 3 mg/kg bw, without a significant trend. No effect was observed on the percentage of polychromatic erythrocytes, suggesting no measurable induced toxicity. Based on the available information, the test item gave a positive result under test conditions.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to other assay used for intermediate effect derivation
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- dicyclohexylcarbodiimide (CASRN 538-75-0; MW 206.32)
- Source and lot/batch No.of test material: Aldrich Chemical Co. (Milwaukee, WI)
- Purity: 99+%
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
MN testing is typically carried out in rats using short-term exposures and bone marrow PCE analysis because of the efficiency with which the rat spleen removes damaged mature erythrocytes from circulation [Schlegel and MacGregor, 1984].
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 9 - 16 weeks
- Assigned to test groups randomly: yes
- Housing: individually housed
- Diet: Purina Laboratory Rodent Chow 5001, pelleted form (Zeigler Bros., Inc., Gardener, PA), ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: at least 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3ºC
- Humidity (%): 50 ± 15%
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle.
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: the test item is moisture sensitive, so corn oil was the preferred solvent.
- Concentration of test material in vehicle: 5.0, 10.0, 15.0, or 20.0 mg/kg
- Dosing volume: 0.4 ml.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: standard NTP 3-i.p. injection protocol [Shelby et al., 1993]: the chemical was suspended using a Tek-Mar Tissue-mizer (Tissumizer; Tekmar Co., Cincinnati, Ohio) at the appropiate concentration, and was administered within 30 min of preparation. All treatments were by intraperitoneal injection at a volume of 0.4 ml/mouse.
Duration of treatment / exposure:
72h
Frequency of treatment:
daily
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 male F344/N rats per dose group.
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide (CASRN 50-18-0; Sigma, St. Louis, MO).
- Route of administration: intraperitoneal.
- Doses / concentrations: 25 mg/kg.
- Dosing volume: 0.4 ml.
Tissues and cell types examined:
Polychromatic erythrocytes (PCE) from bone marrow.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: information from a previous 13-week study was used to select the range of doses in this test (see 'Cross-reference').

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Treatment: rats were injected intraperitoneally three times at 24-hour intervals, using the standard NTP 3-i.p. injection protocol [Shelby et al., 1993].

DETAILS OF SLIDE PREPARATION:
- 24 hours after the third injection, the animals were killed by CO2 asphyxiation and femoral bone marrow spreads were prepared, air-dried, and fixed using absolute methanol [Tice et al., 1989]: bone marrow cells from an extracted femur were flushed onto a microscope slide (numbered with the animal ear tag code) using two to three drops of fetal calf serum. The flushed material was mixed well and then spread using another microscope slide. After air drying, the bone marrow and peripheral blood samples were fixed by treating the slides with absolute methanol for 5 min. The coded slides were stained with acridine orange (Sigma, St. Louis, MO).

METHOD OF ANALYSIS:
- All slides were evaluated at 1,000x magnification using epi-illuminated fluorescence microscopy (450–490 nm excitation, 520 emission).
- 2000 polychromatic erythrocites (PCEs) were scored for the frequency of micronucleated cells in 2-5 rats per dose per group. The percentage among the total erythrocyte population (200) was scored for each dose group as a measure of toxicity.
Evaluation criteria:
In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.
Statistics:
The frequency of micronucleated cells among PCEs was analyzed by a statistical software package (Micronucleus Assay Data Management and Statistical software package (ver. 1.4) [Margolin and Risko, 1988; ILS, 1990]) that tested for increasing trend over dose groups with a one-tailed CochranArmitage trend test, followed by pairwise comparisons between each dosed group and the control group (ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. The %PCE data were analyzed by an analysis of variance (ANOVA) test based on individual animal data; pairwise comparisons between the exposure and control groups were made using a two-tailed Student’s t-test.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Information from a previous 13-week study was used to select the range of doses in this test (see 'Cross-reference').
- Dose range: 0, 0.75, 1.5, 3, 6 or 12 mg/kg bw of test item in ethanol (0.5 mL/kg).
- Clinical signs of toxicity in test animals: All 12 mg/kg male and female core study rats died or were found moribund and sacrificed prior to day 45. Final mean body weight and body weight gain of 6 mg/kg males were significantly less than those of the vehicle controls.

RESULTS OF DEFINITIVE STUDY
- Results of the rat MN tests, which used doses that induced mortality and bone marrow toxicity, were clearly negative.
- Induction of micronuclei (for Micronucleus assay): No significant increase observed.

Table 2. Frequency of MN-PCE and %PCE in Bone Marrow of Male F344 Rats.

Dose

(mg/kg)

N

MN-PCE/

1000 PCE

(mean ± SE)

P value*

% PCE

(mean ± SE)

P value*

Trial 1

0

5

0.4 ± 0.19

 

52.1 ± 1.10

 

5

5

0.1 ± 0.10

0.910

34.5 ± 3.03

0.003

10

4

0.5 ± 0.20

0.376

33.9 ± 2.18

0.002

15

0.3 ± 0.25

-

30.3 ± 7.75

-

20

3

1.3 ± 0.67

0.018

33.0 ± 4.31

0.050

 

 

P = 0.006***

 

P < 0.001**

 

CP#

5

15.1 ± 1.51

< 0.001

2.3 ± 0.41

< 0.001

Trial 2

0

 

0.6 ± 0.37

 

55.2 ± 4.62

 

10

 

0.7 ± 0.20

0.391

51.0 ± 6.07

0.599

15

 

1.0 ± 0.45

0.159

34.2 ± 3.29

0.008

20

 

0.7 ± 0.20

0.391

44.4 ± 7.37

0.255

 

 

P = 0.289***

 

P < 0.079**

 

CP#

 

45.2 ± 8.47

< 0.001

2.8 ± 0.96

< 0.001

*Comparison of individual dose groups to the control; significant at P 0.008; **:ANOVA, significant at P 0.025; ***: Trend test, one-tailed. Significant at P  0.025; #:Cyclophosphamide, positive control; a: data not included in the overall statistical evaluation due to insufficient animals.

Conclusions:
Acute bone marrow MN studies with DCC in male F344 rats, using intraperitoneal (i.p.) injection, yielded negative results. Therefore, the test item was not mutagenic under test conditions.
Executive summary:

An acute three-injection micronucleus study in bone marrow of male F344/N rats was performed for the test item, by a method similar to OECD 407 (GLP study). Five animals per dose group were exposed to 5, 10, 15, 20 mg/kg bw of test item in corn oil by intraperitoneal injection daily for 3 days. Negative and positive (cyclophosphamide) controls were run in parallel. One day after the last injection, the animals were killed by CO2 asphyxiation and femoral bone marrow spreads were prepared, air-dried, fixed using absolute methanol, and stained with acridine orange. At termination, bone marrow analysis included assessment of the frequency of MN-PCE among 2,000 PCE and the %PCE among 200 erythrocytes. Results of the rat MN tests, which used doses that induced mortality and bone marrow toxicity, were clearly negative at all doses tested. Therefore, the test item was not mutagenic under test conditions.

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

Additional information

In-vitro gene mutation in bacteria:

- Key study: A bacterial reverse mutation test (Ames test) according to OECD 471 and 472 and the Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) was performed with the test item (GLP study). Four histidine requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and tryptophan requiring Escherichia coli WP2 uvrA were tested in triplicate at six concentrations of test item in the range 40 - 5000 μg/plate, with and without metabolic activation (S9 mix). Although inhibitory activity was observed in all strains tested in the higher dose groups, no increase in the number of the relevant colonies was observed. Therefore, the test item was not mutagenic.

- Key study: A bacterial reverse mutation assay (Ames test) was performed on the test item, according to the method reported by Zeiger (1992), similar to OECD 471 (GLP study). Four histidine requiring strains of Salmonella typhimurium (TA 97, TA98, TA100 and TA1535) were tested in triplicate at five concentrations in the range of 0.1- 6666 μg/plate of test item, with and without metabolic activation (S9 mix). No increase in revertant colonies was observed. Therefore, the item was considered not mutagenic.

In-vitro cytogenicity study in mammalian cells or in-vitro micronucleus study: Key study. A chromosomal aberration test in CHL cells was performed with the test item according to OECD 473 / Guidelines for Screening Mutagenicity Testing Of Chemicals (Japan). Two different types of assays were carried out: a long-term treatment (direct method) of 24 and 48 hours of continuous treatment with the test substance and a short-term treatment consisting of culture for 18 hours after treatment for 6 hours (metabolic activation method), with and without metabolic activation. The maximum dose for each assay was selected based on a cytotoxicity preliminary study: the dose capable of inhibiting 50% growth was selected as the maximum dose (1.90 µg/ml in the long-term assay, and 31.0 µg/ml in the short-term assay). No chromosome structural abnormality or inducing effect of polyploid cells was observed at any concentration. Therefore, the test item is not mutagenic.

In-vitro gene mutation study in mammalian cells: Data waiving (study scientifically not necessary / other information available). In accordance with Column 2 of REACH Annex VIII, the study does not need to be conducted since there is adequate data from reliable in vivo cytogenicity studies.

In vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus:

- Key study. Method similar to OECD 474, GLP study: An acute three-injection micronucleus study in bone marrow of male F344/N rats was performed. Five animals per dose group were exposed to 5, 10, 15, 20 mg/kg bw of test item in corn oil by intraperitoneal injection daily for 3 days. Bone marrow analysis included assessment of the frequency of MN-PCE among 2,000 PCE and the %PCE among 200 erythrocytes. The results, which used doses that induced mortality and bone marrow toxicity, were clearly negative at all doses tested. Therefore, the test item was not mutagenic.

- Key study. Method similar to OECD 474, GLP study: A subchronic peripheral blood micronucleus study in male and female B6C3F1 mice by dermal application was performed. Ten animals per sex per dose group were exposed to 0 (control), 1.5, 3, 6, 12 mg/kg bw of test item in ethanol by skin painting, five days a week for 13 weeks. Peripheral blood analysis included assessment of the frequency of MN-PCE among 1000 PCE and the %PCE among 1000 erythrocytes. After 13 weeks of dermal administration, the test item induced significant increases in the frequency of micronucleated normochromatic erythrocytes in peripheral blood of mice, based on the results of a trend analysis, although none of the treated groups showed a significant increase in the frequency of micronucleated NCEs compared to the control. In females, the frequency of all treated groups was greater than the control, but only significant at 3 mg/kg bw, without a significant trend. No effect was observed on the percentage of polychromatic erythrocytes, suggesting no measurable induced toxicity. Based on the available information, the test item gave a positive result under test conditions.

Justification for classification or non-classification

Based on the available information, the substance is not classified for mutagenicity according to CLP Regulation (EC) no. 1272/2008.