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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:
07.05.-18-07.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: granules
Details on test material:
- Name of test material (as cited in study report): 2,2'-azobis(2-amidinopropane)-dihydrochloride
- Substance type: organic
- Physical state: solid
- Analytical purity: 99.1 %
- Lot/batch No.: I59596
- Expiration date of the lot/batch: 31. Dec. 2012
- Storage condition of test material: ambient temperature, dark

Method

Species / strain
Species / strain / cell type:
lymphocytes: peripheral blood lymphocytes, obtained from adequate donors.
Metabolic activation:
with and without
Metabolic activation system:
S9-liver enzyme mixture
Test concentrations with justification for top dose:
Experiment I: 21.3; 42.6; 85.3; 170.6; 341.1; 682.3; 1364.5; 2729 µg/mL
Experiment II: 21.2; 42.4; 84.8; 169.5; 339.1; 678.2; 1356.4; 2712.7 µg/mL
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Human peripheral blood lymphocytes were obtained from adequate donors (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.
Blood donors:
Experimental part Evaluated as Exposure date Donor Examined for micronuclei ratio
Pre-experiment (Experiment I)Exp. I 07. May 2012 female, 20 years old yes
Experiment II Exp. II 18. Jun. 2012 female, 26 years old yes
Primary cultures of human peripheral lymphocytes are preferred for this type of study be-cause of their low and stable background rate of micronuclei. In addition, human cells are generally the most relevant for risk assessment.

Cell Cultivation

The blood cultures were set up in defined time intervals within 24 hours after collection in 25 cm2 cell culture flasks for cell proliferation. The following volumes were added to the flask per 10 mL:
• 9 mL complete culture medium RPMI 1640
• 1 mL heparinised whole blood
The cultures were then incubated at 37 ± 1 °C in a humidified atmosphere with 5 % CO2.

Cell Treatment

In experiments with short exposure, about 72 ± 2 hrs after seeding, the culture medium was replaced with serum-free medium RPMI 1640 and solvent control resp. positive con-trol resp. test item were added. In the case of metabolic activation, 50 µl S9 mix per ml medium was used. The cell cultures were incubated at 37 ± 1 °C in a humidified atmos-phere with 5 % CO2 for 4 ± 1 hrs (exposure period). After exposure, the treatment medium was removed, cells were washed twice with saline G and reincubated for 1.5 - 2.0 cell cy-cles in complete culture medium RPMI 1640, with addition of cytochalasin B.
In the experiment with extended exposure, the cultures were supplemented with solvent control resp. positive control resp. test item and complete culture medium RPMI 1640. Cy-tochalasin B was added, and the cells were incubated at 37 ± 1 °C in a humidified atmosphere with 5 % CO2 until preparation (exposure time 20 ± 2 hrs.).
Concentration of Cytochalasin B was always 6 ± 1 µg/ml.

Harvesting Procedure

Each cell culture was harvested and processed separately. 20 ± 2 hrs after end of treat-ment (experiment I with and without metabolic activation, experiment II with metabolic acti-vation), resp. immediately after treatment in the extended exposure experiment (experi-ment II without metabolic activation), the cell cultures were transferred in vials and the cells were spun down by gentle centrifugation (1750 rpm, 10 minutes). The supernatant was discarded and the cells were resuspended in approximately 10 ml hypotonic solution (0.075M KCl). The cell suspension was allowed to stand at 37 ± 1 °C for 15 to 20 minutes. After removal of the hypotonic solution by centrifugation (1750 rpm, 10 minutes), the cell pellet was fixated with fixans (mixture of methanol and glacial acetic acid 3 : 1). After fixa-tion at 2 – 8 °C, minimum 30 minutes; the cell suspension was spun down by gentle cen-trifugation (1750 rpm, 10 minutes), the supernatant was discarded and the cell pellet was resuspended in fixans again. The washing procedures were repeated until the cell pellet was white.
The slides were prepared by dropping the cell suspension onto a clean microscope slide. The cells were then stained with a 10 % solution of Giemsa (MERCK, 64293 Darmstadt, Germany). All slides, including those of positive and solvent controls, were independently coded before microscopic analysis.
Evaluation criteria:
Evaluation of the slides was performed using Zeiss microscopes with 100 x oil immersion objectives. The generated data were recorded on raw data sheets.
In all replicates, the cytokinesis-block proliferation index (CBPI) was determined to assess cell proliferation using at least 500 cells per culture. From these determinations, the test item concentrations, which were evaluated for micronuclei, were defined.

CBPI = (MONC*1+BNC*2+MUNC*3)/n

CBPI Cytokinesis-block proliferation index
n Total number of cells
MONC Mononucleate cells
BNC Binucleate cells
MUNC Multinucleate cells

Cytotoxicity was calculated as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
Cytostasis % = 100 – 100 [(CBPIT – 1) / (CBPIC – 1)]
CBPIT Cytokinesis-block proliferation index of test item
CBPIC Cytokinesis-block proliferation index of solvent control

The number of binucleated cells with and without micronuclei in each treatment group was compared with the solvent control value.
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results
Statistics:
Statistical significance was tested using Fisher’s exact Test with the following equation:

rho(a) = [(a+b)!(c+d)!(a+c)!(b+d)!]/(n!a!b!c!d!)

a number of binucleated cells with micronuclei of the solvent control
b number of binucleated cells with micronuclei of the test item of the respective concentration
c number of binucleated cells without miconuclei of the solvent control
d number of binucleated cells without micronuclei of the test item of the respective concentration

This equation represents a hypergeometric distribution.
The resulting probability of the respective distribution was halved and the probabilities of the more extreme distributions (down to a value of 0 in the controls) were added to give the cumulated p-value of the tail of the distribution.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Remarks:
for more details check test system
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

1.1     Cytotoxicity Test (Pre-ExperimentàExp. I)

The results of the pre-experiment are presented in the following tables:

Table 8.1‑a      Results of Cytotoxicity Test Experiment I in the absence of S9

Treatment

Precipitation

Haemolysis

CBPI

% Cytostasis

Solvent control medium without FCS

no

no

1.962

--

Solvent control
0.9 % NaCl

no

no

2.011

--

Positive control MMC
0.15 µg/mL

no

no

1.916

4.7%

Test Item

 

 

 

 

2729 µg/mL

no

no

1.934

1.4%

1364.5 µg/mL

no

no

1.758

10.4%

682.3 µg/mL

no

no

2.009

- 2.4%

341.1 µg/mL

no

no

1.944

1.0 %

170.6 µg/mL

no

no

1.984

- 1.1%

85.3 µg/mL

no

no

1.975

-0.7%

42.6 µg/mL

no

no

2.010

- 2.5%

21.3 µg/mL

no

no

1.995

-1.7%

Table 8.1‑b      Results of Cytotoxicity Test Experiment I in the presence of S9

Treatment

Precipitation

Haemolysis

CBPI

% Cytostasis

Solvent control medium without FCS

no

no

1.997

--

Solvent control
0.9 % NaCl

no

no

1.983

--

Positive control CPA
15 µg/mL

no

no

1.683

15.2%

Test Item

 

 

 

 

2729 µg/mL

no

no

1.840

7.8%

1364.5 µg/mL

no

no

1.941

2.8%

682.3 µg/mL

no

no

1.954

2.1%

341.1 µg/mL

no

no

2.003

-0.3%

170.6 µg/mL

no

no

1.984

0.6%

85.3 µg/mL

no

no

1.993

0.2%

42.6 µg/mL

no

no

1.926

3.5%

21.3 µg/mL

no

no

2.022

- 1.3%

 

The pre-experiment showed no cytotoxicity in all concentrations. Therefore, the pre-experiment was evaluated as genotoxicity experiment I.

The concentrations to be evaluated for genotoxicity were selected on this base.

1.1.1    Genotoxicity Test

The results of the evaluated concentrations in Experiment I are presented in the following table:

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±1 hrs without S9

Solvent controlmedium without FCS

1.962

--

2060

11

0.53%

Solvent control NaCl 0.9 %

2.011

--

2103

7

0.33%

Positive control MMC
0.15 µg/mL

1.916

4.7%

2127

92

4.33%

Test item 2729mg/mL

1.934

1.4%

2049

10

0.49%

Test item 1364.5mg/mL

1.758

10.4%

2020

4

0.20%

Test item 682.3mg/mL

2.009

- 2.4%

2037

7

0.34%

Experiment I: exposure period 4±1 hrs with S9

Solvent controlmedium without FCS

1.997

--

2041

7

0.34%

Solvent control NaCl 0.9 %

1.983

--

2020

3

0.15%

Positive control CPA
15 µg/mL

1.683

15.2%

2058

42

2.04%

Test item 2729mg/mL

1.840

7.8%

2051

5

0.24%

Test item 1364.5mg/mL

1.941

2.8%

2041

10

0.49%

Test item 682.3mg/mL

1.954

2.1%

2032

4

0.20%

For abbreviations see Annex 5 – Glossary

 

1.1.2    Conclusion Experiment I

In experiment I with and without metabolic activation, no cytotoxicity, precipitation or haemolysis was observed in all concentrations.

No relevant increase of the number of binucleated cells with micronuclei was found at the evaluated concentrations. Therefore, a second experiment (Experiment II) was necessary.


1.2     Experiment II

1.2.1    Cytotoxicity Test

The results of experiment II are presented in the following tables:

Table 8.2‑a      Results of Cytotoxicity Test Experiment II in the absence of S9

Treatment

Precipitation

Haemolysis

CBPI

% Cytostasis

Solvent control medium without FCS

no

no

2.046

--

Solvent control
0.9 % NaCl

no

no

1.979

--

Positive control MMC
0.15 µg/mL

no

no

1.864

5.8%

Test Item

 

 

 

 

2712.7 µg/mL

no

no

1.657

19.0%

1356.4 µg/mL

no

no

1.928

5.8%

678.2 µg/mL

no

no

1.963

4.1%

339.1 µg/mL

no

no

1.971

3.6%

169.5 µg/mL

no

no

1.966

3.9%

84.8 µg/mL

no

no

1.969

3.8%

42.4 µg/mL

no

no

1.936

5.4%

21.2 µg/mL

no

no

1.872

5.5%

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

Treatment

Precipitation

Haemolysis

CBPI

% Cytostasis

Solvent control medium without FCS

no

no

1.972

--

Solvent control
0.9 % NaCl

no

no

1.978

--

Positive control CPA
15 µg/mL

no

no

1.613

18.5%

Test Item

 

 

 

 

2712.7 µg/mL

no

no

1.661

15.7%

1356.4 µg/mL

no

no

1.680

14.8%

678.2 µg/mL

no

no

1.882

4.6%

339.1 µg/mL

no

no

1.831

7.1%

169.5 µg/mL

no

no

1.931

2.1%

84.8 µg/mL

no

no

1.867

5.3%

42.4 µg/mL

no

no

1.941

1.6%

21.2 µg/mL

no

no

1.965

0.3%

 


1.2.2    Genotoxicity Test

The results of the evaluated concentrations in Experiment II are presented in the following table:

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 20± 2hrs without S9

Solvent control medium without FCS

2.046

--

2050

4

0.20%

Solvent control NaCl 0.9 %

1.979

--

2053

8

0.39%

Positive control MMC
0.15 µg/mL

1.864

5.8%

2096

59

2.81%

Test item 2712.7mg/mL

1.657

19.0%

2050

6

0.29%

Test item 1356.4mg/mL

1.928

5.8%

2095

12

0.57%

Test item 678.2mg/mL

1.963

4.1%

2059

14

0.68%

Experiment II: exposure period 4±1 hrs with S9

Solvent control medium without FCS

1.972

--

2055

13

0.63%

Solvent control NaCl 0.9 %

1.978

--

2043

6

0.29%

Positive control CPA
15 µg/mL

1.613

18.5%

2083

57

2.74%

Test item 2712.7mg/mL

1.661

15.7%

1797

11

0.61%

Test item 1356.4mg/mL

1.680

14.8%

2040

0

0.00%

Test item 678.2mg/mL

1.882

4.6%

2002

0

0.00%

For abbreviations see Annex 5 – Glossary

 

1.2.3    Conclusion Experiment II

In experiment II, no relevant cytotoxic effect was observed.

No relevant increase of the number of binucleated cells with micronuclei was detected in the evaluated concentrations.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test item 2,2' - azobis(2-amidinopropane)-dihydrochloride (=2,2'-azobis[2-methylpropion­amidine] dihydrochloride) did not show any evidence of genotoxic activity in this in vitro test for the induction of micronuclei.
Executive summary:

For the analysis of the genotoxic potential of the test item2,2' - azobis(2-amidinopropane)-dihydrochloride (=2,2'-azobis[2-methylpropionamidine] dihydrochloride), 1000 binucleated cells per evaluated culture were scored for the presence of micronuclei. The recorded values were compared with a negative control (solvent only) and with positive controls (known aneugens/clastogens).

Two concentrations of the positive controls were always prepared, but only the higher concentration of the positive controls (in the presence and the absence of S9) was evaluated and compared with the historical data. The raw data of all positive controls will be archived..

The study is considered acceptable: micronucleus induction of the negative controls was in the range of the literature data[. Therefore, the study is considered valid. In both experiments, Mitomycin C (0.15 mg/mL) and CPA (15 µg/mL) were used as positive controls and showed distinct increases in the number of binucleated cells with micronuclei.

In this study, in both independent experiments in the absence and presence of S9 mix, no biologically relevant increase in binucleated cells with micronuclei was observed. The range of binucleated cells with micronuclei after treatment with the test item was similar to the range of the solvent control values.