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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 487 (adopted 22. Jul 2010)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): PEROXAN EPC S
- Physical state: liquid
- Analytical purity: 98.1%
- Lot/batch No.: 1081430
- Expiration date of the lot/batch: 11 Nov 2012
- Storage condition of test material: -20 to -30 C degrees

Method

Species / strain
Species / strain / cell type:
lymphocytes: human periheral blood obtained by a donor
Details on mammalian cell type (if applicable):
Human peripheral blood lymphocytes were obtained from an adequate donor (healthy, non-smoking, no known recent exposures to genotoxic chemicals or radiation). Blood samples were drawn by venous puncture and collected in heparinized tubes. Blood cultures were set up within 24 hours after sample collection.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from the liver of male rats
Test concentrations with justification for top dose:
Pre-exp (Exp. I): 3472, 2951, 2508, 2258, 2032, 1524, 990, 495 µg/mL
Exp. II: 3465, 2945, 2503, 2253, 2028, 1521, 989, 494 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetra hydrofuran (THF) for the test material and 0.9% NaCl for the positive controls.
- Justification for choice of solvent/vehicle: based on the pre-experiment (exp. I) the test material was sufficiently soluble in THF.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
tetra hydrofurane (THF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
With S9-Exp. I & II
-Exposure duration: 4 ± 1 h; the medium was removed, fresh medium and cytoB were added. The cells were harvested 1.5-2 cell cycles later.

Without S9-Exp. I
-Exposure duration: 4 ± 1 h; the medium was removed, fresh medium and cytoB were added. The cells were harvested 1.5-2 cell cycles later.

Without S9, extended exposure-Exp. II
-Exposure duration: 20 ± 2 h (1.5 - 2 cell cycles) in the presence of cytoB. The cells were harvested at the end of the exposure period.

SPINDLE INHIBITOR (cytogenetic assays): cytoB
STAIN (for cytogenetic assays): 10% solution of Giemsa

NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: at least 500 cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (cytokinesis-block proliferation index)

Evaluation criteria:
The test item is considered to have no genotoxic effects if:
-the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory’s historical control data of the solvent control
-no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

The test item is considered to have genotoxic effects if:
-the number of micronucleated cells in all evaluated dose groups is above the range of the historical laboratory control data
-either a concentration-related increase of micronucleated cells or a statistically significant increase in the number of cells containing micronuclei is observed.
Statistics:
Statistical significance when p<0.01

Results and discussion

Test results
Species / strain:
lymphocytes: human, peripheral blood
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
Additional information on results:
Solubility of the test substance was determined in a seperate test.The test item was sufficiently soluble in tetra hydrofurane (THF).

Exp.I: no cytotoxicity or precipitation in any of the concentrations tested. The highest concentration showed haemolysis. No micronuclei were detected at all concentrations tested.

Exp.II: no cytotoxicity or precipitation in any of the concentrations tested. Sigificant increases in the number of binucleated cells with miconuclei when compared to the THF, were not considered a genotoxic effect, since the increase was lower than that observed in the solvent control NaCl 0.9%.

The study is considered acceptable, since micronucleus induction of the controls was in the range of historical data.
For detailed results see "any other informaion on results incl. tables".
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 8.1-a     ResultsofCytotoxicityTestExperiment l in theabsenceofS9

 

Treatment

Precipitation

Haemolysis

CBPI

%Cytostasis

Solvent control THF

No

No

1.86

 

Solvent control 0.9 % NaCI

No

No

1.97

 

Positive control MMC 0.3 µg/mL

No

No

1.86

4.0%

Test Item

 

 

 

 

3472 µg/mL

No

Yes

1.56

16.1%

2951 µg/mL

No

No

1.61

13.4%

2508 µg/mL

No

No

1.82

2.1%

2258 µg/mL

No

No

1.73

6.9%

2032 µg/mL

No

No

1,74

6.5%

1524 µg/mL

No

No

1.65

11.3%

990 µg/mL

No

No

1.86

0.3%

495 µg/mL

No

No

1.78

4.2%

 

 

Table8.1-b      ResultsofCytotoxicityTestExperimentlinthepresenceofS9

 

Treatment

Precipitation

Haemolysis

CBP

% Cytostasis

SolventcontrolTHF

no

no

1.79

 

Solventcontrol0.9%NaCI

no

no

1.79

 

PositivecontrolCPA

151- µg/mL

 

no

 

no

 

1.47

 

17.7%

Test Item

 

 

 

 

3472µg/mL

no

yes

1.60

10.2%

2951µg/mL

no

no

1.69

5.3%

2508µg/mL

no

no

1.61

10.1%

2258µg/mL

no

no

1.64

8.2%

2032µg/mL

no

no

1.69

5.3%

 1524µg/mL

no

no

1.77

0.9%

990µg/mL

no

no

1.83

-2.7%

495µg/mL

no

no

1.86

-3.9%


 

Table 8.1-c      Genotoxicity Results Experiment I

Treatment

Average CBPI

Cytostasis (%)

Total No. of BNC examined

Total No. of MBNC

% MBNC

Experiment I: exposure period 4 hrs without S9

SolventcontrolTHF

1.86

--

2035

11

0.54%

SolventcontrolNaCl0.9%

1.94

--

2276

8

0.35%

PositivecontrolMMC0.3µg/mL

1.86

4.0%

2100

79

**3.76%

Test item3472µg/mL

1.56

16.1%

2067

9

0.44%

Test item2951µg/mL

1.61

13.4%

2111

3

0.14%

Test item2508µg/mL

1.82

2.1%

2093

1

0.05%

Experiment I: exposure period 4 hrs with S9

SolventcontrolTHF

1.79

--

2025

10

0.49%

SolventcontrolNaCI0.9%

1.79

--

2055

9

0.44%

PositivecontrolCPA15µg/mL

1.47

17.7%

2139

70

**3.27%

Testitem3472µg/mL

1.60

10.2%

2060

5

0.24%

Testitem2951µg/mL

1.69

5.3%

2083

7

0.34%

Testitem2508µg/mL

1.61

10.1%

2074

4

0.19%

 

 

 

 

Table8.2-a      ResultsofCytotoxicity TestExperiment IIin theabsenceofS9

Treatment

Precipitation

Haemolysis

CBPI

%Cytostasis²

Solvent control THF

No

No

1.84

 

Solvent control 0.9 % NaCI

No

No

1.93

 

Positive control MMC 0.3 µg/mL

No

No

1.81

6.4%

Test Item

 

 

 

 

3465 µg/mL

No

No

1.71

7.2%

2945 µg/mL

No

No

1.71

7.5%

2503 µg/mL

No

No

1.76

4.7%

2253 µg/mL

No

No

1.68

9.0%

2028 µg/mL

No

No

1,70

7.9%

1521 µg/mL

No

No

1.67

9.3%

989 µg/mL

No

No

1.76

4.6%

494 µg/mL

No

No

1.75

4.9%

 

 


 

 

 

 

 

 

 

 

 

Table 8.2-b     Results of Cytotoxicity Test Experiment II in the presence of S9

Treatment

Precipitation

Haemolysis

CBPI

%Cytostasis²

Solvent control

No

No

1.80

 

Solvent control 0.9 % NaCI

No

No

1.73

 

Positive control CPA 15 µg/mL

No

No

1.53

11.5%

Test Item

 

 

 

 

3465 µg/mL

No

No

1.77

1.6%

2945 µg/mL

No

No

1.86

-3.4%

2503 µg/mL

No

No

1.81

-0.1%

2253 µg/mL

No

No

1.85

-2.5%

2028 µg/mL

No

No

1,76

2.3%

1521 µg/mL

No

No

1.81

-0.1%

989 µg/mL

No

No

1.79

0.5%

494 µg/mL

No

No

1.82

-1.0%

 

 

Table 8.2-c      Results Experiment II

Concentrations (µg/mL)

Average CBPI

Cytostasis (%)

Total No. of BNC examined

Total No. of MBNC

% MBNC

Experiment II: exposure period 22 ± 2hrs without S9

SolventcontrolTHF

1.84

--

2046

5

0.24%

SolventcontrolNaCl0.9%

1.93

--

2041

15

*0.73%

PositivecontrolMMC0.3µg/mL

1.81

6.4%

2107

59

**2.80%

Test item3465µg/mL

1.71

7.2%

2059

14

**0.68%

Test item2945µg/mL

1.71

7.5%

1849

13

*0.70%

Test item2503µg/mL

1.76

4.7%

2040

13

*0.64%

Experiment I: exposure period 4 hrs with S9

SolventcontrolTHF

1.80

--

2040

5

0.25%

SolventcontrolNaCI0.9%

1.73

--

2032

7

0.34%

PositivecontrolCPA15µg/mL

1.53

11.5%

1825

33

**1.81%

Test item3465µg/mL

1.77

1.6%

2110

6

0.28%

Test item2945µg/mL

1.86

-3.4%

2044

7

0.34%

Test item2503µg/mL

1.81

0.1%

2026

7

0.35%


 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

On the basis of this result, the test item does not induce chromosomal aberrations in human lymphocytes.
Executive summary:

In a mammalian cell cytogenicity test (micronucleus test) human peripheral blood lymphocytes (obtained by a donor) were exposed to PEROXAN EPC S (analytical purity: 98.1%) in tetradyrofuran (THF) in the presence and absence of mammalian metabolic activation (liver S9 mix, male rats, treated with Aroclor 1254). Prior to exposure the cells were properly cultured in the presence of phytohaemagglutinin.

Three experiments were run. The first experiment was deemed invalid and therefore, it is not reported here. The rest two were as follows:

Experiment I

With and without S9 mix / 4 hrs exposure: 3472, 2951, 2508, 2258, 2032, 1524, 990, 495 µg/mL

Micronuclei were scored at 3472, 2951, 2508 µg/mL

Experiment II

With and without S9 mix / 20 ± 2 hrs exposure: 3465, 2945, 2503, 2253, 2028, 1521, 989, 494 µg/mL

Micronuclei were scored at 3465, 2945, 2503 µg/mL

 

Cytotoxicity was not seen at any concentration level. There was no evidence of a biologically relevant increase in the micronucleated cells at all concentrations tested. The positive controls did induce the appropriate response and the negative controls were within the normal range according to historical data. 

 

This study is classified as acceptable.This study satisfies the requirement for Test Guideline OECD 487 for in vitro cytogenicity (mammalian cell micronucleus test) data.