Dibutyl peroxydicarbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance dibutyl peroxydicarbonate was tested in an in vitro genotoxicity testing battery (OECD 471, 476, 487 and 490, GLP) as required by Annex VIII of the REACH regulation (EC) 1907/2006.

Dibutyl peroxydicarbonate was tested negative in a bacterial reverse gene mutation test conducted according to OECD 471 and in an in vitro micronucleus assay conducted according to OECD 487.

In a mammalian cell HPRT mutation assay conducted according to OECD 476, the substance showed equivocal results for inducing mutagenic effects. However, in a follow up mammalian cell gene mutation assay according to OECD 490, dibutyl peroxydicarbonate did not induce mutagenic effects in mouse lymphoma L5178Y cells. Based on the weight of evidence, the test item was considered to not induce genetic toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-03-27 to 2017-11-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. Due to the nature of the test item it was not possible to prepare a solution of the test item with cell culture medium. Therefore the test item was dissolved in ethanol at a 200fold increased concentration and rediluted in cell culture medium at a ratio 1:200 to reach the final test item concentrations and a final concentration of 0.5% v/v ethanol in the samples. The test item solution were used within 1 hour. The solvent was compatible with the survival of the cells and the S9 activity.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections were carried out before freezing.
For the experiments thawed cultures were set up in 75 cm² cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 10^5 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made every 3-4 days.

MEDIA USED:
MEM medium supplemented with:
- 10% fetal bovine serum (FBS)
- 100 U/100 µg/mL penicillin/streptomycin solution
- 2 mM L-glutamine
- 2.5 µg/mL amphotericin
- 25 mM HEPES

Treatment Medium (short-term exposure):
- Complete culture medium with 0% FBS.

After Treatment Medium / Treatment Medium (long-term exposure):
- Complete culture medium with 10% FBS and 1.5 µg/mL cytochalasin B.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment:

Experiment 1:
- Without metabolic activation: 0.10, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, 25 and 50 µg/mL; 0.50, 1.0, 2.0 and 4.0 µg/mL was selected as highest concentration for the microscopic analysis of micronuclei.
- With metabolic activation: 50, 100, 200, 300, 400, 500, 750, 1000, 1250, 1500 and 2000 µg/mL; 50, 100 and 300 µg/mL was selected as highest concentration for the microscopic analysis of micronuclei.

Experiment 2:
- Without metabolic activation: 0.5, 1.0, 2.5, 5.0, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50, 75 and 100 µg/mL; 12.5, 17.5 and 20 µg/mL was selected as highest concentration for the microscopic analysis of micronuclei.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (0.5% v/v in culture medium)

Untreated negative controls:

yes

Remarks:

Cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Cell culture medium with 0.5% ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

clastogenic control; without metabolic activation, final concentration: 20 µg/mL

Untreated negative controls:

yes

Remarks:

Cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Cell culture medium with 0.5% ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

clastogenic control, with metabolic activation, final concentration: 2.5 µg/mL

Untreated negative controls:

yes

Remarks:

Cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Cell culture medium with 0.5% ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Colchicine

Remarks:

aneugenic control, without metabolic activation, final concentration: 1.5 and 0.08 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 50000 cells were seeded per cell culture flask, containing 5 mL complete culture medium (minimum essential medium supplemented with 10% FBS).
Seeding of the cultures: Three or four-day-old stock cultures (in exponential growth), more than 50% confluent, were rinsed with Ca-Mg-free PBS solution prior to the trypsin treatment. Cells subsequently were trypsinised with a solution of 0.2% trypsin in Ca-Mg-free PBS at 37 °C for 5 min. By adding complete culture medium the detachment was stopped and a single cell suspension was prepared.

Experiment 1:
Exponentially growing V79 cells were seeded into 25 cm² cell culture flasks (two flasks per test group). Approx. 50000 cells were seeded per cell culture flask, containing 5 mL complete culture medium (minimum essential medium supplemented with 10% FBS). After an attachment period of approx. 48 h, the complete culture medium was removed and subsequently the test item was added to the treatment medium in appropriate concentrations. The cells were incubated with the test item for 4 h in presence or absence of metabolic activation. At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in complete culture medium + 1.5 µg/mL cytochalasin B for 20 h at 37 °C.
Experiment 2:
If negative or equivocal results are obtained, they should be confirmed using continuous treatment (long-term treatment) without metabolic activation. Approx. 50 000 exponentially growing V79 cells were seeded in 25 cm² cell culture flasks in absence of metabolic activation. After an attachment period of approx. 48 h the test item was added in complete culture medium. 1 h later 1.5 µg/mL cytochalasin B were added and the cells were incubated for 23 h at 37 °C. At the end of the treatment the cell culture medium was removed and the cells were prepared for microscopic analysis.

Number of Cultures:
Duplicate cultures were performed at each concentration level except for the pre-experiment.

Preparation of the Cultures:
At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in about 9 mL complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature. Prior to this an aliquot of each culture was removed to determine the cell count by a cell counter (AL-Systems). After the treatment with the hypotonic solution the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.

Analysis of Micronuclei:
At least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei according to criteria of Fenech, i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmatic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.

As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells according to the following formula:
CBPI= (c1 x 1) + (c2 x 2) + (cx x 3)/n

c1: mononucleate cells
c2: binucleate cells
cx: multinucleate cells
n: total number of cells

The CBPI can be used to calculate the % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures: % Cytostasis= 100 – 100 x ((CBPIT – 1) / (CBPIC – 1))

CBPIT: Cytokinesis Block proliferation index of treated cultures
CBPIC: Cytokinesis Block proliferation index of control cultures

Evaluation criteria:

A mutation assay is considered acceptable if it meets the following criteria:
- The concurrent negative/solvent control is considered acceptable for addition to the laboratory historical negative/solvent control database.
- Concurrent positive controls should induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative/solvent control.
- Cell proliferation criteria in the negative/solvent control according to OECD 487 [4] should be fulfilled.
- All experimental conditions are tested unless one resulted in positive results.
- Adequate number of cells and concentrations are analysable.
- Criteria for the selection of top concentration are fulfilled.

A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).

When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system. A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.

Statistics:

The nonparametric Chi-Quadrat Test was performed to verify the results of the experiment. No statistically significant enhancement (p< 0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment I and II.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:The pH-value detected with the test item was within the physiological range (pH 7.4).
- Precipitation: No precipitate of the test item was noted in any concentration group evaluated in experiment I and II.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 1.0 µg/mL. At a concentration of 2.0 µg/mL a cytostasis of 51% and at a concentration of 4.0 µg/mL a cytostasis of 65% was noted.
In experiment I with metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 50 µg/mL. At a concentration of 100 µg/mL a cytostasis of 33% and at a concentration of 300 µg/mL a cytostasis of 61% was noted.
In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 17.5 µg/mL. At a concentration 20 µg/mL a cytostasis of 57% was noted.

Micronuclei Analysis:
In experiment I without metabolic activation the micronucleated cell frequencies of the negative control (0.60%) and the solvent control (1.00%) were within the historical control limits of the negative control (0.39% – 1.40%,) and the solvent control (0.45% - 1.55%), respectively. The mean values of micronucleated cell frequencies found after treatment with the test item were 0.70% (0.5 µg/mL), 0.70% (1.0 µg/mL), 0.90% (2.0 µg/mL) and 0.85% (4.0 µg/mL). The number of micronucleated cells found in the groups treated with the test item was within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.
In experiment I with metabolic activation the micronucleated cell frequencies of the negative control (1.55%) and the solvent control (1.28%) were within the historical control limits of the negative control (0.37% – 1.68%) and the solvent control (0.23%-1.88%), respectively. The mean values of micronucleated cell frequencies found after treatment with the test item were 0.85% (50 µg/mL), 1.55% (100 µg/mL) and 1.20% (300 µg/mL). The number of micronucleated cells found in the groups treated with the test item was within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control. In experiment II without metabolic activation the micronucleated cell frequencies of the negative control (0.95%) and the solvent control (0.70%) were within the historical control limits of the negative control (0.39% – 1.40 %) and the solvent control (0.45% - 1.55%), respectively. The mean values of micronucleated cell frequencies found after treatment with the test item were 0.65% (12.5 µg/mL), 0.80% (17.5 µg/mL) and 1.05% (20 µg/mL). The number of micronucleated cells found in the groups treated with the test item was within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.
The nonparametric x² Test was performed to verify the results in both experiments. No statistically significant enhancement (p< 0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment I and II


For individual results please refer to Table 3 to 5 in box "Any other information on results incl. tables".

Remarks on result:

other:

Remarks:

Experiment 1

Table 3: Test for cytotoxicity

Dose Group	Concentration [µg/mL]	CBPI	Relative Cell Growth [%]	Cytostasis [%]	Precipitate +/-
without metabolic activation
C	0	1.53	101	0	-
S	0	1.53	100	0	-
1	7.8	1.31	60	40	-
2	15.6	1.23	43	57	-
3	31.3	1.36	68	32	-
4	62.5	1.31	60	40	-
5	125	1.26	50	50	-
6	250	1.03	5	95	-
7	500	1.02	5	95	+
8	1000	1.01	2	98	+
with metabolic activation
C	0	1.44	123	0	-
S	0	1.36	100	0	-
1	7.8	1.49	134	0	-
2	15.6	1.37	102	0	-
3	31.3	1.38	105	0	-
4	62.5	1.40	110	0	-
5	125	1.36	98	2	-
6	250	1.35	97	3	-
7	500	1.25	70	30	-
8	1000	1.18	50	50	+
9	1500	1.21	57	43	+
10	2000	1.16	45	55	+

C: Negative Control (Culture medium)

CBPI: Cytokinesis Block Proliferation Index, CBPI = ((c₁x 1) + (c₂x 2) + (c_xx 3))/n

Relative Cell Growth:100 x ((CBPI_{Test conc}– 1) / (CBPI_control-1))

c₁:mononucleate cells

c₂:binucleate cells

c_x:multinucleate cells

n:total number of cells

 

Cytostasis [%] = 100- Relative Cell Growth [%]

the cytostasis is defined 0, when the relative cell growth exceeds 100%

Table 4: Experiment 1 and 2, without metabolic activation

	Dose Group	Concentration [µg/mL]	Number of cells evaluated	Cytostasis [%]	Relative Cell Growth [%]	Micro-nucleated Cells Frequency [%]	Historical Control Limits Negative Control	Precipitation	Statistical Significant Increase^a
Exp. I 4 h treatment, 24 h fixation interval	C	0	2000	2	98	0.60	0.39% - 1.40%	/	/
	S	0	2000	0	100	1.00		/	/
	3	0.50	2000	12	88	0.70		-	-
	4	1.0	2000	20	80	0.70		-	-
	5	2.0	2000	51	49	0.90		-	-
	7	4.0	2000	65	35	0.85		-	-
	MMS	20	2000	18	82	2.85		-	+
	Colc	1.5	2000	18	82	2.55		-	+
Exp. II 24 h treatment, 24 h fixation interval	C	0	2000	0*	128	0.95	0.39% - 1.40%	/	/
	S	0	2000	0	100	0.70		/	(
	7	12.5	2000	30	70	0.65		-	-
	9	17.5	2000	16	84	0.80		-	-
	10	20	2000	57	43	1.05		-	-
	MMS	20	2000	0*	108	5.85		-	+
	Colc	0.08	1906	1	99	8.07		-	+

Table 5: Experiment 1, with metabolic activation

	Dose Group	Concentration [µg/mL]	Number  of cells evaluated	Cytostasis [%]	Relative Cell Growth [%]	Micro- Nucleated Cells Frequency [%]	Historical Control Limits Negative Control	Precipitation	Statistical Significant Increase^a
Exp. I 4 h treatment, 24 h fixation interval	C	0	2000	5	95	1.55	0.37% - 1.68%	/	/
	S	0	4000	0	100	1.28		/	/
	1	50	2000	21	79	0.85		-	-
	2	100	4000	33	67	1.55		-	-
	4	300	4000	61	39	1.20		-	-
	CPA	2.5	2000	30	70	5.75		-	+

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In an in vitro mammalian micronucleus assay (OECD 487), V79 cells cultured in vitro were exposed to Dibutyl peroxydicarbonate (50.6% peroxide) in cell culture medium with 0.5% ethanol in experiment 1 (short-term exposure, 4 h) at concentrations of 0.50, 1.0, 2.0 and 4.0 µg/mL (without metabolic activation) and at 50, 100 and 300 µg/mL (with metabolic activation). In experiment 2 (long-term exposure 24 h, without metabolic activation), concentration of 12.5, 17.5 and 20 µg/mL were used.

In experiment 1 without metabolic activation the mean values of micronucleated cell frequencies found after treatment with the test item were 0.70% (0.5 µg/mL), 0.70% (1.0 µg/mL), 0.90% (2.0 µg/mL) and 0.85% (4.0 µg/mL). In experiment I with metabolic activation the mean values of micronucleated cell frequencies found after treatment with the test item were 0.85% (50 µg/mL), 1.55% (100 µg/mL) and 1.20% (300 µg/mL). In experiment 2 without metabolic activation the mean values of micronucleated cell frequencies found after treatment with the test item were 0.65% (12.5 µg/mL), 0.80% (17.5 µg/mL) and 1.05% (20 µg/mL). No statistically significant enhancement (p< 0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment 1 and 2.

The positive controls did induce the appropriate response. In conclusion, it can be stated that during the study described and under the experimental conditions reported, Dibutyl peroxydicarbonate did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, Dibutyl peroxydicarbonate is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

 

This study is classified as acceptable and satisfies the requirements for Test Guideline OECD 487 for in vitro mutagenicity data.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-04-03 to 2017-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name: Dibutyl peroxydicarbonate
- Common name: Peroxan NBC-50
- CAS No: 13215-49-9
- Batch No: 1230518-02
- Molecular weight: not provided by the sponsor
- Physical state: liquid
- Colour: clear
- Density: 0.88 g/cm³
- Active components: 50.6% Peroxide
- Expiry date: not provided by the sponsor
- Storage conditions: =< -20°C
- Safety precautions: routine hygienic procedures will be sufficient to assure personnel helath and safety

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED:
Sources of cells:
TA98, TA1535 and TA102: MOLTOX, INC., NC 28607, USA
TA100 and TA1537: Xenometrix AG, Switzerland

MEDIA USED:
Nutrient medium (per litre):
- 8 g Nutrient Borth
- 5 g NaCl
(solution of 125 µL ampicillin (10 mg/mL) (TA98, TA100, TA102) was added to retain phenotypic characteristics of strain)

Vogel-Bonner-salts (per litre)
- 10 g MgSO4 x 7 H2O
- 100 g citric acid
- 175 g NaNH4HPO4 x 4 H2O
- 500 g K2HPO4

Vogel-Bonner Medium E agar plates (per litre)
- 15 g Agar Agar
- 20 mL Vogel-Bonner salta
- 50 mL glucose-solution (40%)

Overlay agar (per litre)
- 7 g Agar Agar
- 6 g NaCl
- 10.5 mg L-histidine x HCl x H2O
- 12.2 mg biotin

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The test item concentrations in the main experiments were chosen according to the results of the pre-experiment:
Experiment 1: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
Experiment 2: 0.00158, 0.0050, 0.0158, 0.050, 0.158, 0.50 and 1.58 µL/plate, all tester strains (with metabolic activation) and TA102 (with and without metabolic activation); 0.05, 0.158, 0.50, 1.58, 5.0, 15.8 and 50 nL/plate, all tester strains (without metabolic activation), except TA102

Vehicle / solvent:

- Vehicle: ethanol

Untreated negative controls:

yes

Remarks:

A. dest.

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

yes

Remarks:

A. dest.

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Untreated negative controls:

yes

Remarks:

A. dest.

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

yes

Remarks:

A. dest.

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
- in agar (plate incorporation)

EXPERIMENTAL PERFORMANCE
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
- 100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
- 500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
- 100 µL Bacteria suspension (cf. Preparation of bacteria, pre-culture of the strain),
- 2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of background lawn or reduction in number of revertants down to mutation factor of approximately < 0.5 in relation to solvent control

Evaluation criteria:

The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of solvent control.

Statistics:

not applicable

Key result

Species / strain:

S. typhimurium, other: TA98, TA102, TA1535, TA1537

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:

Precipitation of the test item was observed in all tester strains used in experiment I at concentrations of 2.5 µL/plate and higher (with and without metabolic activation).
Toxic effects of the test item were noted in most tester strains evaluated in experiment I and II. In experiment I, toxic effects were observed in tester strain TA98 at concentrations of 1.0 µL/plate and higher (without S9 mix). In tester strains TA1535 and TA102 toxic effects of the test item were noted at concentrations of 1.0 µL/plate and higher (without S9 mix) and at concentrations of 2.5 µL/plate and higher (with S9 mix). In tester strain TA1537 toxic effects were observed at concentrations of 1.0 µL/plate and higher (with and without S9 mix). In experiment II toxic effects of the test item were noted in tester strain TA98 at concentrations at 15.8 nL/plate and higher (without metabolic activation) and at a concentration of 1.58 µL/plate (with S9 mix). In tester strains TA100, TA1535 and TA1537 toxic effects of the test item were observed at concentrations of 15.8 nL/plate and higher (without metabolic activation) and at concentrations of 0.50 µL/plate and higher (with metabolic activation). In tester strain TA102 toxic effects of the test item were noted at concentrations of 0.050 µL/plate and higher (without S9 mix) and at a concentration of 1.58 µL/plate (with S9 mix).

Remarks on result:

other:

Remarks:

Experiment 1

Pre-experiment for toxicity and Main experiments I and II:

Result tables are attched in box "Attached background material"

Conclusions:

In conclusion, the test item is not genotoxic in the bacterial reverse gene mutation assay in the presence and absence of mammalian metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471) strains of S. typhimurium (TA1537, TA1535, TA102, TA100 and TA98) were exposed to Dibutyl peroxydicarbonate in ethanol at concentrations of 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µl/plate (experiment I) in the presence and absence of metabolic activation. In experiment II all tester strains (with metabolic activation) and TA 102 (with and without metabolic activation) were exposed to concentrations of 0.00158, 0.0050, 0.0158, 0.050, 0.158, 0.50 and 1.58 µL/plate. Tester strain TA102 (without metabolic activation) was tested at concentrations of 0.050, 0.158, 0.50, 1.58, 5.0, 15.8 and 50 nL/plate. The positive controls did induce the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-04-24 to 2017-04-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 29 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT (hypoxanthine-guanine-phosphoribosyl-transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins BioPharma Product Testing Munich GmbH
- Doubling time: 12 - 14 h
- Cloning efficiency: > 50%
- Modal number of chromosomes: Yes

MEDIA USED
- Type and identity of media: Complete culture media

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

Pre-test for toxicity:
0.025, 0.05, 0.1, 0.25, 0.5, 1, 1.5 and 2 mg/mL without/with S9 mix
Main test:
Experiment I: 0.005, 0.01, 0.03, 0.04, 0.06, 0.08, 0.1, 0.3 and 0.5 mg/mL without S9 mix and 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2 mg/mL with S9 mix
Experiment II: 0.0002, 0.0005, 0.001, 0.002, 0.005, 0.007, 0.01, 0.02, 0.03 and 0.04 mg/mL without S9 mix

The test item was investigated at the following concentrations:
Experiment I: 0.005, 0.01, 0.03, 0.04, 0.06 mg/mL without S9 mix and 0.025, 0.05, 0.1, 0.25, 0.5 mg/mL with S9 mix
Experiment II: 0.007, 0.01, 0.02, 0.03 and 0.04 mg/mL without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol

Untreated negative controls:

yes

Remarks:

MEM medium

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (1 % v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

300 µg/mL, without S9

Untreated negative controls:

yes

Remarks:

MEM medium

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (1 % v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

1.0 µg/mL, with S9

Details on test system and experimental conditions:

Method of application: in medium
DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours (short time exposure, Experiment I with and without metabolic activation)
20 hours (long time exposure, Experiment II without metabolic activation)
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 7-12 days

SELECTION AGENT (mutation assays): 11 µg/mL thioguanine (TG)

NUMBER OF REPLICATIONS: one culture per test group (expression period)

DETERMINATION OF CYTOTOXICITY: Cytotoxicity (relative survival) was calculated based on the cloning efficiency of cells plated immediately after treatment adjusted by any loss of cells during treatment

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation:
No precipitation of the test item was noted in any of the experiments.

Toxicity:
A biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in experiment I and II with and without metabolic activation. In experiment I without metabolic activation, the relative survival was 1% for the highest concentration (0.3 mg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2.0 mg/mL with a relative survival of 0%. In experiment II without metabolic activation, the relative survival was 15% for the highest concentration (0.04 mg/mL) evaluated.

Mutagenicity:
In the experiment without and with metabolic activation all validity criteria were met. The mutant values of the negative and solvent controls were within or even lower than the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls were > 50%. The positive controls, DMBA (1 µg/mL) and EMS (300 µg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems. In the experiment I without metabolic activation, the mutant values of the negative controls, the solvent controls and two of five mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 7.8-39.7 mutants per 10^6 cells). The positive control EMS induced a distinct increase in mutant frequency with 273.2 mutants/10^6 cells.

The mutant frequencies of the negative controls were 39.5 and 39.2 mutants per 10^6 cells, of the solvent controls 35.2 and 23.9 mutants per 10^6 cells, respectively.

Remarks on result:

other: equivocal results obtained in short-term and long-term experiments

Table1:  Experiment I – Toxicity, without metabolic activation

Dose Group	Concen-tration	Number of cells at the		Number of colonies per flask			CE [%]	Adjusted CE [%]	Relative Survival (RS) [%]
	[mg/mL]	beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	12019000	151	142	147	73	88	96
NC2		10000000	13804000	176	104	140	70	97	106
S1	0	10000000	11815000	158	158	158	79	93	100
S2		10000000	11237000	160	159	160	80	90
1	0.005	10000000	8925000	128	111	120	60	53	58
2	0.01	10000000	13821000	107	110	109	54	75	82
3	0.03	10000000	7786000	52	75	64	32	25	27
4	0.04	20000000	17000000	46	37	42	21	18	19
5	0.06	20000000	10778000	24	22	23	12	6	7
6	0.08	20000000	7174000	21	19	20	10	4	4
7	0.1	20000000	5168000	25	31	28	14	4	4
8	0.3	20000000	2815200	12	5	9	4	1	1
EMS	300 µg/mL	10000000	11645000	178	145	162	81	94	103

NC:     negative control

S:        solvent control (0.5% v/v ethanol)

CE:     cloning efficiency

EMS:  Ethylmethanesulfonate [300 µg/mL]

Table 2:  Experiment I – Mutagenicity, without metabolic activation

		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration	Number of colonies per flask			CE [%]	Number of colonies per flask							CE [%]	Mutant Frequency per 10^6cells
	[mg/mL]	I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	148	171	160	80	8	12	18	11	14	12.6	3.3	0.0032	39.5
NC2		155	146	151	75	15	12	8	8	16	11.8	3.4	0.0030	39.2
S1	0	154	153	154	77	7	16	9	10	12	10.8	3.1	0.0027	35.2
S2		180	163	172	86	9	9	5	9	9	8.2	1.6	0.0021	23.9
1	0.005	147	148	148	74	11	11	12	9	8	10.2	1.5	0.0026	34.6
2	0.01	160	140	150	75	19	15	20	17	16	17.4	1.9	0.0044	58.0
3	0.03	135	135	135	68	9	9	9	7	6	8.0	1.3	0.0020	29.6
4	0.04	141	142	142	71	11	13	11	12	12	11.8	0.7	0.0030	41.7
5	0.06	151	148	150	75	8	18	14	16	15	14.2	3.4	0.0036	47.5
EMS	300 µg/mL	176	141	159	79	81	88	90	82	92	86.6	4.4	0.0217	273.2

NC:     negative control

S:        solvent control (0.5% v/v ethanol)

CE:     cloning efficiency

EMS:  Ethylmethanesulfonate

 Table 3:  Experiment I – Toxicity, with metabolic activation

Dose Group	Concen-tration	Number of cells at the		Number of colonies per flask			CE [%]	Adjusted CE [%]	Relative Sur-vival (RS) [%]
	[mg/mL]	beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	13464000	128	157	143	71	96	88
NC2		10000000	12274000	135	151	143	72	88	81
S1	0	10000000	13362000	149	175	162	81	108	100
S2		10000000	12648000	177	168	173	86	109
1	0.025	10000000	12274000	122	128	125	63	77	71
2	0.05	10000000	11373000	111	81	96	48	55	50
3	0.1	10000000	10047000	88	126	107	54	54	49
4	0.25	10000000	7854000	44	39	42	21	16	15
5	0.5	20000000	14110000	36	38	37	19	13	12
6	0.75	20000000	8602000	43	50	47	23	10	9
7	1	20000000	8806000	14	25	20	10	4	4
8	1.5	20000000	6528000	14	24	19	10	3	3
9	2	20000000	3434000	6	6	6	3	1	0
DMBA	1 µg/mL	10000000	13413000	151	158	155	77	104	95

NC:        negative control

S:           solvent control (0.5% v/v ethanol)

CE:        cloning efficiency

DMBA:  7,12-dimethylbenz(a)anthracene

Table 4:  Experiment I – Mutagenicity, with metabolic activation

		CE in non-selective medium				CE in selective medium
Dose Group	Concentration	Number of colonies per flask			CE [%]	Number of colonies per flask							CE [%]	Mutant Frequency per 10^6cells
	[mg/mL]	I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	145	149	147	74	10	11	12	6	9	9.6	2.1	0.0024	32.7
NC2		164	165	165	82	9	2	2	6	4	4.6	2.7	0.0012	14.0
S1	0	143	142	143	71	7	7	6	11	9	8.0	1.8	0.0020	28.1
S2		141	132	137	68	10	10	6	6	9	8.2	1.8	0.0021	30.0
1	0.03	155	154	155	77	8	7	4	7	5	6.2	1.5	0.0016	20.1
2	0.05	145	131	138	69	5	6	8	4	5	5.6	1.4	0.0014	20.3
3	0.1	169	160	165	82	10	13	4	6	6	7.8	3.2	0.0020	23.7
4	0.25	142	124	133	67	6	13	15	13	10	11.4	3.1	0.0029	42.9
5	0.5	154	148	151	76	13	14	13	11	16	13.4	1.6	0.0034	44.4
DMBA	1 µg/mL	147	171	159	80	81	60	68	66	54	65.8	9.0	0.0165	206.9

NC:       negative control

S:          solvent control (0.5% v/v ethanol)

CE:       cloning efficiency

DMBA:  7,12-dimethylbenz(a)anthracene

 

Table 5:  Experiment II – Toxicity, without metabolic activation

Dose Group	Concen-tration	Number of cells at the		Number of colonies per flask			CE [%]	Adjusted CE [%]	Relative Survival (RS) [%]
	[mg/mL]	beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	38250000	182	209	196	98	374	107
NC2		10000000	39610000	207	240	224	112	443	126
S1	0	10000000	36040000	188	196	192	96	346	100
S2		10000000	35870000	201	196	199	99	356
1	0.0002	10000000	34170000	198	221	210	105	358	102
2	0.0005	10000000	33830000	202	206	204	102	345	98
3	0.001	10000000	34680000	230	189	210	105	363	103
4	0.002	10000000	33830000	184	193	189	94	319	91
5	0.005	10000000	36550000	204	183	194	97	354	101
6	0.007	10000000	37230000	187	180	184	92	342	97
7	0.01	20000000	64260000	232	259	246	123	394	112
8	0.02	20000000	71060000	257	269	263	132	467	133
9	0.03	20000000	48960000	223	214	219	109	267	76
10	0.04	20000000	13906000	166	141	154	77	53	15
EMS	300 µg/mL	10000000	32130000	176	172	174	87	280	80

NC:     negative control

S:        solvent control (0.5% v/v ethanol)

CE:     cloning efficiency

EMS:  Ethylmethanesulfonate

Table 6:  Experiment II – Mutagenicity, without metabolic activation

		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration	Number of colonies per flask			CE              [%]	Number of colonies per flask							CE              [%]	Mutant Frequency per 10^6cells
	[mg/mL]	I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	175	189	182	91	2	8	0	5	3	3.6	2.7	0.0009	9.9
NC2		196	181	189	94	4	7	9	12	7	7.8	2.6	0.0020	20.7
S1	0	161	169	165	83	9	7	7	9	10	8.4	1.2	0.0021	25.5
S2		186	202	194	97	7	5	5	9	4	6.0	1.8	0.0015	15.5
6	0.007	183	194	189	94	7	3	6	5	5	5.2	1.3	0.0013	13.8
7	0.01	183	210	197	98	7	6	5	4	10	6.4	2.1	0.0016	16.3
8	0.02	213	218	216	108	14	6	8	9	9	9.2	2.6	0.0023	21.3
9	0.03	191	220	206	103	5	5	7	15	9	8.2	3.7	0.0021	20.0
10	0.04	178	205	192	96	14	11	14	12	11	12.4	1.4	0.0031	32.4
EMS	300 µg/mL	156	168	162	81	202	203	210	240	211	213.2	13.9	0.0533	658.0

 NC:     negative control

S:        solvent control (0.5% v/v ethanol)

CE:     cloning efficiency
EMS:  Ethylmethanesulfonate

Conclusions:

In this study, under the given conditions, the results of the mutagenic potential of the test item dibutyl peroxydicarbonate, were found to be equivocal.

Executive summary:

In a mammalian cell HPRT gene mutation assay conducted in accordance with OECD 476, V79 cells cultured in vitro were exposed to dibutyl peroxydicarbonate (50.6% peroxide) in 1% ethanol investigated at concentrations of 0.005, 0.01, 0.03, 0.04, 0.06, 0.08, 0.1 and 0.3 mg/mL in the absence of mammalian metabolic activation (experiment I, 4-hour exposure) and at concentrations of 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5 and 2 mg/mL in the presence of mammalian metabolic activation (experiment I, 4-hour exposure). For experiment II (20-hour exposure), concentrations tested were 0.0002, 0.0005, 0.001, 0.002, 0.005, 0.007, 0.01, 0.02, 0.03 and 0.04 mg/mL in the absence of metabolic activation. An experiment II with metabolic activation could not be run for technical reasons. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency, showing the validity of the experiments.

 

In experiment I without metabolic activation, a biologically relevant increase of mutants was found after treatment with the test item at cytotoxic concentrations. The concentration 0.04 mg/mL induced a slight increase above the historic control range. A statistical analysis displayed that these mutant frequencies were significantly increased over those of the solvent controls, but no dose-response relationship was determined. Therefore, no clearly positive response is observed.

 

In experiment II without metabolic activation, the mutant values were all within or even lower than the historical control data. The highest mutant frequency was observed at the highest evaluated concentration of 0.04 mg/mL with a relative survival of 15%. This increase was also statistically significant compared to the solvent control and a dose-response relationship was determined. However, as the mutant values of the test item were within the historical control data, these significances were considered as not biologically relevant. Thus, the 20 h exposure without S9 was clearly negative.

 

In experiment I with metabolic activation, a dose related increase in both cytotoxicity and mutant frequency was observed. The top two concentrations induced appropriate maximum levels of cytotoxicity and very slight, but significantly increased mutant frequencies compared to the control, which minimally meet the criteria for a positive response.

 

In summary, as the short-term and long-term treatments without metabolic activation were contrary to each other and the short-term incubation with metabolic activation was slightly positive, the all over result was regarded as equivocal.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS OECD 476 for in vitro mutagenicity data.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-07-19 to 2018-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Name: Dibutyl peroxydicarbonate
Common name: Peroxan NBC-50
CAS No.: 16215-49-9
Physical state: liquid
Colour: clear
Density: 0.88 g/cm3
Active components: 50.5% peroxide

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source: Pergan GmbH (Sponsor), batch no.: 1262684-01
- Purity test date: 2018-06-26

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ≤ -20 °C

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was diluted in ethanol (0.5% v/v)

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins Munich stock
- Cell cycle length, doubling time or proliferation index: 10-12 hours doubling time, cloning efficiency >50%
- Methods for maintenance in cell culture if applicable: Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Roswell Park Memorial Institute (RPMI) 1640, supplemented with 9.0 µg/mL hypoxanthine; 15.0 µg/mL thymidine, 22.5 µg/mL glycine and 0.1 µg/mL methotrexate
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: yes, by culturing in RPMI 1640 supplemented with 9.0 µg/mL hypoxanthine, 15.0 µg/mL thymidine, 22.5 µg/mL glycine, 0.1 µg/mL methotrexate

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

without metabolic activation: 50, 60, 70, 72, 74, 76, 78, and 80 µg/mL
with metabolic activation: 25, 50, 75, 100, 150, 300, 500, 750 µg/mL

The selection of the concentrations used in the main experiment was based on data from the pre-experiment. Due to the unusual toxicity 76 µg/mL (without metabolic activation) and 300 µg/mL (with metabolic activation) were selected as the highest concentrations. Since there was first an increase of toxicity followed by a decrease, two upper concentrations (78 and 80 µg/mL, without metabolic activation; 500 and 750 µg/mL, with metabolic activation) were evaluated in the main experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 0.5% v/v ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL.
Based on the results of the solubility test ethanol (0.5% v/v; Supplier: AppliChem; Lot: 7A012351) was used as solvent.

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

0.5% v/v ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

0.5% v/v ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

0.5% v/v ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1x10^7 cells suspended in 11 mL RPMI medium with 5% horse serum

DURATION
- Preincubation period: none
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth


SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

Rationale for test conditions:

The mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT:
- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable
- The cloning efficiency of the negative and/or solvent controls is in the range 65% - 120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50 - 170 mutants per 10^6 cells
- The cell number of the negative/solvent controls should undergo 8 - 32-fold increase during a 2-day growth period (short-term treatment)
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 10^6 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10^6 cells
- The relative total growth must be greater than 10%.

Evaluation criteria:

Test item is considered mutagenic if the following criteria are met:
- the induced mutant frequency meets or exceeds the Global Evaluation factor of 126 mutants per 1x10^6 cells
- a dose-dependent increase in mutant frequency is detected

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference. Statistical significance at the 5% level (p < 0.05) was evaluated.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The relative total growth was 13.1% at 76 µg/mL without S9 and 13.7% at 300 µg/mL with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test item was within the physiological range.
- Effects of osmolality: The osmolality was within the physiological range.
- Precipitation: In the main experiment no precipitation was observed.

RANGE-FINDING/SCREENING STUDIES: The selection of the concentrations used in the main experiment was based on data from the pre-experiment according to the OECD guideline 490. Due to the nature of the test item an explicit determination of the toxicity in the pre-experiment was not clearly evaluable. Thus for the relative suspension growth (RSG) values a first increase in toxicity could be observed, followed by a further decrease. Therefore the selection of appropriate concentration was adapted to cover the toxicity range, containing the first time toxicity occurs and two upper evaluated concentrations.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): All mutant frequencies for negative, solvent and positive controls were found within the historical range of the test facility Eurofins Munich.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative suspension growth

For individual results see tables 1 to 10 in box "Any other information on results incl. tables".

Table 1: Pre-Experiment for Toxicity, without metabolic activation

Test Group	Concentration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]
C1	0	339000	963000	1320000	12.7	98.8
C2		363000	996000	1280000	12.7	99.1
S1	0	319000	870000	1390000	12.1	100.0
S2		325000	927000	1470000	13.6
1	25	325000	983000	1130000	11.1	86.4
2	50	356000	1020000	1380000	14.1	109.5
3	150	77500	44200	54800	0.2	1.3
4	500	98700	78700	269000	0.8	6.3
5 P	1000	51700	21200	24900	0.1	0.6
6 P	2000	61900	17400	13300	0.0	0.3

C:        Negative control

S:        Solvent control (0.5% ethanol; v/v)

P:        Precipitation

a:        Suspension Growth, SG = [((value 24 h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h*x20))]

* : for value 24 h > 3x10⁵ then value 24 h = 3x10⁵

b:         Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

Table 2: Pre-Experiment for Toxicity, with metabolic activation

Test Group	Concentration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]
C1	0	321000	873000	1360000	11.9	103.7
C2		313000	936000	1270000	11.9	103.8
S1	0	307000	879000	1190000	10.5	100.0
S2		325000	882000	1410000	12.4
1	25	311000	816000	1420000	11.6	101.2
2	50	327000	894000	1330000	11.9	103.9
3	150	258000	232000	737000	2.2	19.3
4 P	500	263000	416000	1430000	5.9	52.0
5 P	1000	175000	350000	1430000	5.0	43.7
6 P	2000	200000	219000	879000	2.6	23.0

C:        Negative control

S:        Solvent control (0.5% ethanol; v/v)

P:        Precipitation

a:        Suspension Growth, SG = [((value 24 h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h*x20))]

*: for value 24 h > 3x10⁵ then value 24 h = 3x10⁵

b:         Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

Table 3: Main Experiment - Toxicity data, without metabolic activation

Test Group	Concentration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]	RCE^c[%]	RTG^d[%]
C1	0	365000	773000	1600000	12.4	107.0	107.4	114.9
C2		397000	891000	1420000	12.7	109.5	100.4	109.9
S1	0	371000	726000	1510000	11.0	100.0	100.0	100.0
S2		374000	799000	1520000	12.1
1	50	358000	917000	1430000	13.1	113.5	89.8	101.9
3	60	325000	631000	1440000	9.1	78.6	92.6	72.9
5	70	304000	465000	1420000	6.6	57.2	111.2	63.5
6	72	302000	329000	970000	3.2	27.6	109.2	30.2
7	74	275000	303000	833000	2.5	21.8	85.8	18.7
8	76	266000	274000	553000	1.7	14.4	91.2	13.1
9	78	253000	310000	992000	3.1	26.6	98.8	26.3
10	80	192000	186000	352000	1.1	9.1	94.1	8.6
EMS	300	364000	693000	1500000	10.4	90.0	75.1	67.6
MMS	10	369000	729000	1260000	9.2	79.5	59.0	46.9

C:        Negative control

S:        Solvent control (0.5% ethanol; v/v)

P:        Precipitation

a:        Suspension Growth, SG = [((value 24h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h*x20))]

*: for value 24 h > 3x10⁵ then value 24 h = 3x10⁵

b:         Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

Table 4: Main Experiment - Mutagenicity data, without metabolic activation

		Cloning Efficiency (CE)			Mutagenicity Data
Test Group	Concen-tration [µg/mL]	Plate 1^e	Plate 2^e	CE^f[%]	Number of cultures / 96 wells					MF^g     [mutants / 10^6cells]	IMF^h      [mutants / 10^6cells]
					Plate 1^e	Plate 2^e	Plate 3^e	Plate 4^e	Mean
C1	0	76	83	110.1	25	26	26	32	27.3	152.1	/
C2		73	82	102.9	24	26	19	30	24.8	145.6	/
S1	0	70	80	95.0	16	27	17	23	20.8	129.1	/
S2		82	77	110.1	28	24	21	21	23.5	127.9	/
1	50	67	81	92.1	26	16	11	21	18.5	117.7	-10.9
3	60	73	77	95.0	21	25	23	25	23.5	147.9	19.4
5	70	78	83	114.0	22	22	31	23	24.5	129.9	1.4
6	72	82	78	112.0	36	36	31	19	30.5	173.1	44.5
7	74	72	73	88.0	31	33	39	33	34.0	249.2	120.7
8	76	80	69	93.5	31	30	29	36	31.5	213.1	84.6
9	78	78	76	101.2	27	35	31	25	29.5	182.2	53.6
10	80	78	73	96.5	39	32	35	26	33.0	219.7	91.2
EMS	300	68	68	77.0	75	63	77	70	71.3	895.0	766.5
MMS	10	63	56	60.4	68	66	68	68	67.5	1005.0	876.5

C:        Negative control

S:        Solvent control (0.5% ethanol; v/v)

a:        Suspension Growth, SG = [((value 24hx30)/1x10⁷) x ((value 48 h x 20)/(value 24 h*x20))]

* : for value 24h > 3x10⁵then value 24h = 3x10⁵

b:         Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

c:        Relative Cloning Efficiency, RCE = [(CE_{dose group}/ CE_{of corresponding controls}) x 100]

Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

d:        Relative Total Growth, RTG = (RSG x RCE)/100

EMS:   Ethylmethanesulfonate

MMS:  Methylmethanesulfonate

Table 5: Main Experiment - Colony sizing, without metabolic activation

Test Group*	Concentration [µg/mL]	Wells with at least 1 colony	Large colonies	Small colonies	% small colonies
C1	0	109	87	22	20.2
C2		99	75	24	24.2
S1	0	83	56	27	32.5
S2		94	74	20	21.3
EMS	300	285	241	44	15.4
MMS	10	270	138	132	48.9

C:        Negative control

S:        Solvent control (0.5% ethanol; v/v)

e:        Number of cultures with cell growth.

f:         Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

g:        Mutant frequency, MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

h:        Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

EMS:   Ethylmethanesulfonate

MMS:  Methylmethanesulfonate

Table 6: Main Experiment - Toxicity data, with metabolic activation

Test Group	Concen-tration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]	RCE^c[%]	RTG^d[%]
C1	0	351000	926000	1410000	13.1	92.9	103.2	95.9
C2		377000	1010000	1430000	14.4	102.8	96.9	99.6
S1	0	350000	954000	1420000	13.5	100.0	100.0	100.0
S2		369000	983000	1480000	14.5
1	25	342000	927000	1450000	13.4	95.7	94.0	89.9
2	50	364000	975000	1390000	13.6	96.5	89.8	86.7
3	75	336000	826000	1420000	11.7	83.5	100.0	83.5
4	100	364000	782000	1460000	11.4	81.3	98.4	80.0
5	150	266000	338000	885000	3.0	21.3	96.9	20.6
6	300	276000	269000	643000	1.9	13.7	100.0	13.7
7	500	256000	277000	879000	2.6	18.8	96.9	18.2
8	750	282000	366000	1320000	4.8	34.4	104.9	36.1
B[a]P	2.5	329000	514000	1380000	7.1	50.5	73.3	37.0

C:        Negative control

S:        Solvent control (0.5% ethanol; v/v)

EMS:   Ethylmethanesulfonate

MMS:  Methylmethanesulfonate

*: Based on the non-mutagenic effects of the test item, an assessment of clastogenicity was not feasible.

Table 7: Main Experiment - Mutagenicity data, with metabolic activation

		Cloning Efficiency (CE)			Mutagenicity Data
Test Group	Concen-tration [µg/mL]	Plate 1^e	Plate 2^e	CE^f[%]	Number of cultures / 96 wells					MF^g     [mutants / 10^6cells]	IMF^h      [mutants / 10^6cells]
					Plate 1^e	Plate 2^e	Plate 3^e	Plate 4^e	Mean
C1	0	78	74	98.0	11	11	16	10	12.0	68.3	/
C2		75	73	92.1	17	10	10	12	12.3	74.4	/
S1	0	76	75	96.5	16	14	24	14	17.0	101.7	/
S2		73	76	93.5	15	12	13	8	12.0	71.6	/
1	25	71	75	89.3	17	12	22	17	17.0	109.7	23.0
2	50	73	70	85.4	16	15	21	13	16.3	109.0	22.4
3	75	78	72	95.0	18	12	14	14	14.5	86.4	-0.3
4	100	76	73	93.5	13	15	10	16	13.5	81.2	-5.4
5	150	73	75	92.1	10	12	21	14	14.3	88.0	1.3
6	300	77	73	95.0	19	16	20	22	19.3	118.0	31.3
7	500	73	75	92.1	21	18	19	16	18.5	116.4	29.7
B[a]P	2.5	68	61	69.6	40	47	41	43	42.8	424.0	337.3

C:        Negative control

S:        Solvent control (0.5% ethanol; v/v)

a:        Suspension Growth, SG = [((value 24hx30)/1x10⁷) x ((value 48 h x 20)/(value 24 h*x20))]

   * : for value 24h > 3x10⁵then value 24h = 3x10⁵

b:         Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

c:        Relative Cloning Efficiency, RCE = [(CE_{dose group}/ CE_{of corresponding controls}) x 100]

Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

d:        Relative Total Growth, RTG = (RSG x RCE)/100

B[a]P:  Benzo[a]pyrene

Table 8: Main Experiment - Colony sizing, with metabolic activation

Test Group*	Concentration [µg/mL]	Wells with at least 1 colony	Large colonies	Small colonies	% small colonies
C1	0	48	38	10	20.8
C2		49	43	6	12.2
S1	0	68	53	15	22.1
S2		48	38	10	20.8
B[a]P	2.5	171	95	76	44.4

C:        Negative control

S:        Solvent control (0.5% ethanol; v/v)

e:        Number of cultures with cell growth.

f:         Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

g:        Mutant frequency,MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

h:        Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

B[a]P:  Benzo[a]pyrene

Table 9: Main Experiment - Biometry, without metabolic activation

Test Group	Concentration [µg/mL]	mean Mutant Frequency	mean induced Mutant Frequency	p-value	statistical Significance
C1	0	152.1	/	0.2236	-
C2		145.6	/
S1	0	129.1	/	/	/
S2		127.9	/	/	/
1	50	117.7	-10.9	0.6545	-
3	60	147.9	19.4	0.2404	-
5	70	129.9	1.4	0.6505	-
6	72	173.1	44.5	0.1394	-
7	74	249.2	120.7	0.0040	+
8	76	213.1	84.6	0.0040	+
9	78	182.2	53.6	0.0263	+
10	80	219.7	91.2	0.0061	+
EMS	300	895.0	766.5	0.0040	+
MMS	10	1005.0	876.5	0.0040	+

C:        Negative control

S:        Solvent control (0.5% ethanol; v/v)

B[a]P:  Benzo[a]pyrene

*:         Based on the non-mutagenic effects of the test item, an assessment of clastogenicity was not feasible.

Table 10: Main Experiment - Biometry, with metabolic activation

Test Group	Concentration [µg/mL]	mean Mutant Frequency	mean induced Mutant Frequency	p-value	statistical Significance
C1	0	68.3	/	0.2236	-
C2		74.4	/
S1	0	101.7	/	/	/
S2		71.6	/	/	/
1	25	109.7	23.0	0.2606	-
2	50	109.0	22.4	0.0990	-
3	75	86.4	-0.3	0.7798	-
4	100	81.2	-5.4	> 0.9999	-
5	150	88.0	1.3	0.9091	-
6	300	118.0	31.3	0.0424	+
7	500	116.4	29.7	0.0424	+
B[a]P	2.5	424.0	337.3	0.0020	+

C:        Negative control

S:        Solvent control (0.5% ethanol; v/v)

EMS:   Ethylmethanesulfonate

MMS:  Methylmethanesulfonate

+:        significant

-:         not significant

Conclusions:

The test item dibutyl peroxydicarbonate is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

In a mammalian cell gene mutation assay using the Thymidine Kinase Gene conducted according to OECD Guideline 490, mouse lymphoma L5178Y cells cultured in vitro were exposed to dibutyl peroxydicarbonate (50.5% active peroxide) at concentrations of 50, 60, 70, 72, 74, 76, 78, and 80 µg/mL without S9 metabolic activation and at concentrations 25, 50, 75, 100, 150, 300, 500, and 750 µg/mL with S9 metabolic activation. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. Due to the unusual toxicity 76 µg/mL (without metabolic activation) and 300 µg/mL (with metabolic activation) were selected as the highest concentrations. Since there was first an increase of toxicity followed by a decrease, two upper concentrations (78 and 80 µg/mL, without metabolic activation; 500 and 750 µg/mL, with metabolic activation) were evaluated in the main experiment. The experiment without and with metabolic activation was performed as 4 h short-term exposure assay.

The relative total growth (RTG) was 13.1% (at concentration 76 µg/mL without metabolic activation) and 13.7% (at concentration 300 µg/mL with metabolic activation) at time of first toxicity. In the experiment with metabolic activation for the highest evaluated concentrations 500 and 750 µg/mL, an increase in toxicity was observed. A relative total growth value of 18.2% at 500 µg/mL and 36.1% at 750 µg/mL was observed. In the experiment without metabolic activation, the increase in toxicity at concentrations 78 and 80 µg/mL had RTG values of 26.3% and 8.6%, respectively.

The positive controls EMS, MMS and B(a)P showed distinct effects in mutation frequency, thus proving the ability of the test system to detect potential mutagenic effects. In the main experiment without and with metabolic activation, all validity criteria were met. Furthermore, the negative and vehicle controls had valid mutant frequencies. In experiments with the test item, no biologically relevant increase was observed. In the experiment without metabolic activation a statistically significant concentration-response relationship was observed. Since the Global Evaluation Factor was not exceeded in any evaluated concentrations and a strong toxicity was observed in the highest tested concentrations, this effect was considered as not biologically relevant. In the experiment with metabolic activation no evidence for a concentration-response relationship was detected. Based on the non-mutagenic effects of the test item, an assessment of clastogenicity was not feasible. The positive controls proved the test system valid in indicating potential clastogenic effects.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 490 for in vitro mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Dibutyl peroxydicarbonate was evaluated for genotoxicity/mutagenicity in four of the standard genetic toxicology tests. It was evaluated in the Ames test, the in vitro mammalian HPRT gene mutation assay, the in vitro mammalian mouse lymphoma gene mutation assay using the thymidine kinase locus and in the in vitro mammalian micronucleus test. Negative results were obtained in the bacterial Ames test, the mouse lymphoma assay and the micronucleus test. The in vitro mammalian HPRT assay gave borderline results. The study director concluded that dibutyl peroxydicarbonate is considered to be mutagenic at cytotoxic concentrations in the first experiment without and with metabolic activation in the HPRT locus using V79 cells of the Chinese Hamster. As the mutagenic potential of the test item could not be verified in the long-term experiment without metabolic activation, the response was concluded to be equivocal in the HPRT mutagenicity test.

Because the results were different, it is important to compare the results observed in the HPRT and the mouse lymphoma assays. Three different test conditions were used for the HPRT assay, 4 h and 20 h exposure without exogeneous metabolic activation (S9) and a 4 h exposure with S9. The 4 h exposure without S9 had background mutant frequencies that were close to the maximum values shown in the laboratory historical controls. While there was an overall dose response both for cytotoxicity and for mutant frequency, there was substantial variability and the highest mutant frequency occurred at one of the lower concentrations (a concentration that demonstrated only a minor amount of cytotoxicity). Thus, the response without S9, while suggestive of a positive response, was not clearly positive. The 4 h exposure with S9 showed substantial variability in the concurrent negative controls. There was a dose related increase in both cytotoxicity and mutant frequency. The top two concentrations induced appropriate maximum levels of cytotoxicity and very small increases in mutant frequency. These increases were sufficient to minimally meet the criteria for a positive response. The 20 h exposure without S9 was clearly negative. A 20 h exposure with S9 cannot be run for technical reasons. Thus, the overall response in the HPRT assay was a weak positive (observed with S9).

For the mouse lymphoma assay, two test conditions, 4 h exposure with and without S9, were used. Without S9, there was a dose related increase in cytotoxicity. Ten concentrations were used to cover the dose range and several concentrations induced the appropriate maximum levels of cytotoxicity. Because none of these cultures showed an increase in mutant frequency that met the criteria for a positive response, the result was clearly negative. With S9 there was also a dose related increase in cytotoxicity. Three of the eight concentrations induced the appropriate maximum level of cytotoxicity. There was no dose related increase in the mutant frequency. The result with S9 was also clearly negative. Thus, the overall response for the mouse lymphoma assay was a clear negative.

When comparing the results obtained in the three gene mutation assays (Ames test, HPRT and the mouse lymphoma assay) that were conducted for dibutyl peroxydicarbonate, it is important to bear in mind that while all three assays respond to test materials that induce point mutations, the mouse lymphoma assays detects a much broader range of genetic damage (OECD TG490). If dibutyl peroxydicarbonate was capable of inducing point mutations, it would be expected that positive results would have been obtained in all three assays. Because the results seen in the HPRT assay were not robustly positive, it can be concluded that the clear negative results for the Ames test and the mouse lymphoma assay outweigh the results and that dibutyl peroxydicarbonate is not mutagenic. Because the mouse lymphoma assay can detect viable chromosome damage, the negative results in both that assay, and the in vitro micronucleus test indicate that dibutyl peroxydicarbonate does not induce chromosomal effects. Therefore, it can be concluded that dibutyl peroxydicarbonate is not genotoxic/mutagenic.

Justification for classification or non-classification

Based on the available data for mutagenicity used in a weight of evidence approach, dibutyl peroxydicarbonate is not considered to be genotoxic and no classification is warranted in accordance with CLP regulation (EC) 1272/2008.