Bis(2-ethylhexyl) peroxydicarbonate

Genetic toxicity: in vitro

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

Guideline:

other: OECD 487 (adopted 22. Jul 2010)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) peroxydicarbonate

EC Number:

240-282-4

EC Name:

Bis(2-ethylhexyl) peroxydicarbonate

Cas Number:

16111-62-9

Molecular formula:

C18H34O6

IUPAC Name:

3-({[({[(2-ethylhexyl)oxy]carbonyl}peroxy)carbonyl]oxy}methyl)heptane

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.