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Genetic toxicity in vitro

Description of key information
In order to evaluate the genetic toxicity potential of the test substance one in vitro test according to OECD guideline 471 (i.e. Ames test) and one in vivo test according to OECD guideline 474 (i.e. MNT) were performed. Both tests were negative in result.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
199-06-01 to 1999-06-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline and GLP conform well documented scientific study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA 97a, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
The genotypes of the tested strains were checked at each study:
Histidine auxotrophy, ampicillin resistance, tetracycline resistance, UV-sensitivity and growth inhibition by crystal violet.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
metabolic activation system: (S9) fraction from livers of Wistar male rats, which were induced with Phenobarbital intraperitoneal and beta-Naphtoflavone orally.
Test concentrations with justification for top dose:
1. Study (+/- S9):
All strains: 0.005, 0.05, 0.5 (mg/plate)
Based on the reults of the 1. study concentrations of the second study were selected.

2. Study (+/- S9):
TA97a, TA98, TA 100: 0.5, 0.16, 0.5, 1.6, 5 (mg/plate)
TA102: 0.016, 0.05, 0.16, 0.5, 1.6 (mg/plate)

3. Study:
TA102 (- S9): 0.5, 1.6, 5 (mg/plate)
TA102 (+ S9): 5 (mg/plate)
Vehicle / solvent:
Dimethy sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
other: ICR 191; 4-Nitro-o-phenylen-diamine; Nitrofurantoine; 2-Aminoanthracen, Danthron
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

First study was conducted with 3 concentration levels of the test substance from 0.005 – 0.5 mg/plate in geometrical series of factor 10.
Based on the results of the first study concentrations in a logarithmic series with a factor of √ 10 were tested in the second and third study as specified. Plates for confirmation of genotypes were incubated for 24 h and plates of mutagenicity test for 48 h, respectively, at 37 ± 1 °C. Genotypes were evaluated for each study.
Plates of the mutagenicity test were inspected for present and reduced background lawn after an incubation time of 48 h, respectively. Colonies per plate (revertants) were counted if no reduced background lawn was observed.
Evaluation criteria:
Evaluation:
Since the reduced background lawn is regarded to be a cytotoxic effect, plates with reduced background lawn were not included into evaluation procedures. Arithmetic mean values and standard deviations were calculated out of colonies per plate of three replicates.

The test substance is to be interpreted mutagenic if there is a concentration effect relationship and the induction rate is ≥2.

Validity criteria:
Spontaneous revertants (negative controls) had to be within the following ranges:
- TA 97a ± S9: 60- 300 revertants/plate
- TA98 ±S9: 15- 50 revertants/plate
- TA 100 ± S9: 60 - 200 revertants/plate
- TA 102 ± S9: 240 - 460 revertants/plate

The induction rates of the positive controls had to be ≥2
Species / strain:
other: TA 97a, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Summary of Mutagenic and cytotoxic effects of test substance

S. typhimurium strain

S9

Tested concentration

[mg/plate]

Lowest mutagenic concentration[mg/plate]

Lowest cytotoxic concentration[mg/plate]

TA97a

0.005 - 5.0

none

5

+

0.005 - 5.0

none

none

TA98

0.005 - 5.0

none

none

+

0.005 - 5.0

none

none

TA100

0.005 - 5.0

none

5

+

0.005 - 5.0

none

none

TA102

0.005 - 5.0

none

none

+

0.005 - 5.0

none

none

 

Table 2: Mutagenicity test (1. Study)

 

No. Revertants (mean of 3 plates)

Concentration

TA97a

TA98

TA100

TA102

[mg/plate]

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Negative Control

189

226

22

23

100

88

379

418

Test substance

0.5

158

141

13

27

71

87

cytotoxic

390

0.05

208

175

18

20

99

76

349

401

0.005

172

193

19

23

102

79

349

364

 

Table 3: Mutagenicity test (2. Study)

 

No. Revertants (mean of 3 plates)

Concentration

TA97a

TA98

TA100

TA102

[mg/plate]

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Negative Control

196

204

18

16

177

131

365

335

Test substance

5.0

cytotoxic

144

13

10

cytotoxic

115

317

405

1.6

126

170

9

10

94

96

302

450

0.5

181

201

15

18

111

125

298

415

0.16

198

208

13

29

145

104

392

404

0.05

179

177

18

19

154

111

424

443

 

  Table 4: Mutagenicity test (3. Study)

 

No. Revertants

(mean of 3 plates)

Concentration

TA102

[mg/plate]

- S9

+ S9

Negative Control

330

339

Test substance

5

225

293

1.6

309

Nt

0.5

311

Nt

Nt = Not tested

Table 5: Induction rates of positive controls

strain

Reference substance

(µg/plate)

 (S9)

Induction rate/study

1.

2.

3.

TA 97a

ICR 191 acridine mutagen dihydrochloride

0.5

>5.3

>5.1

Nt

2-Aminoanthracene

2.0

+

>4.4

>4.9

Nt

TA 98

4-nitro-1,2-phenylenediamine

0.5

3.2

5.5

Nt

2-Aminoanthracene

2.0

+

>43.5

>62.5

Nt

TA 100

Nitrofrantoine

0.2

4.4

2.7

Nt

2-Aminoanthracene

2.0

+

11.3

>7.7

Nt

TA 102

Cumene hydroperoxide

100

>2.6

>2.7

>3.0

Danthron

30

+

>2.4

>3.0

>3.0

Nt = Not tested

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

Based on the results of this test, the test substance is regarded to be not mutagenic.
Executive summary:

In order to assess the mutagenic effects of the test substance it were determined in a reverse mutation assay according to OECD test guideline 471 in three independent studies. The test system were the Salmonella typhimurium strains TA97a, TA 98, TA100 and TA102 with and without metabolic activation system S9. Positive and negative controls were included in each study. Duration of each study was 48 hours. The test substance was applied once at test intitiation for the 1. study for all strains at the concentrations (with and without S9) of 0.005, 0.05 and 0.5 (mg/plate) dissolved in vehicle DMSO. Based on the results of the 1. study, concentrations of 0.5., 0.16, 0.5 and 5 mg/plate (with and wihtout S9) were selected for the second study for strains TA97a, TA98 and TA 100. For the strain TA102 concentrations of 0.016, 0.05, 0.16, 0.5, 1.6 (mg/plate) with and without S9 were selected. For the third study the strain TA102 was tested without S9 at concentrations of 0.5, 1.6 and 5 (mg/plate) and with S9 at concentration of 5 mg/plate. The validity of the test system was approved with sufficient positive controls.

Based on the results of this test the test substance is regarded to be not mutagenic.

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

Additional information

Additional information from genetic toxicity in vitro:

The mutagenic effects of the test item were determined in a reverse mutation assay with Salmonella typhimurium. Test systems were the strains TA97a, TA 98, TA 100 and TA 102 with (+) and without (-) the metabolic activation system S9 (from male Wistar rats). Concentrations tested were 0.005 - 0.05 - 0.16 - 0.5 - 1.6 and 5 mg/plate. Three replicates per concentration level and control were performed. Based on the results of this study the test item was found to have no mutagenic effects on Salmonella typhimurium strains TA 97a, TA 100, TA 98, and TA 102 with (+) and without (-) the metabolic activation system S9 from Wistar rats at concentrations up to 5 mg/plate.

In order to study the induction of micronuclei in bone marrow cells Hostacor 4323 was tested in an in vivo micronucleus test in erythrocytes according to OECD 474 guideline performed in NMRI mice. The test substance was administered to ten mice (5/ sex) twice at an interval of 24 hours orally by gavage to the test animals at a dose of 2000 mg/kg bw. The vehicle, sesame oil, was administered in the same way to the negative control groups. Cyclophosphamide was used as positive substance and was administered once orally at a dose of 50 mg/kg bw.The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with Hostacor 4323. Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system.

Justification for classification or non-classification

Based on the available data no classification is warranted according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).

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