Bis-sec-butyl peroxydicarbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (Ames) - Negative

Gene mutation in mammalian cells - Negative (QSAR Prediction)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2018 - 07 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Density: 1.06 g/mL at 5°C
- No correction factor was applied

Target gene:

Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor™ 1254-induced rat liver S9 fraction.

Test concentrations with justification for top dose:

Dose range finding experiment (all strains, with and without S9): 25, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate
First experiment (TA1537, TA98, TA100 and TA1535 without S9): 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10, 25, 50, and 100 μg/plate
First experiment (TA1537, TA 98, TA100 and TA1535 with S9 and WP2uvrA with and without S9): 0.5, 1.0, 2.5, 5.0, 10, 25, 50, 100, 250, and 500 μg/plate
Second experiment (TA1537, TA98, TA100 and TA1535 without S9): 0.5, 1.0, 2.5, 5.0, 10, 25, 50, and 100 μg/plate
Second experiment (TA1537, TA 98, TA100 and TA1535 with S9 and WP2uvrA with and without S9): 2.5, 5.0, 10, 25, 50, 100, 250, and 500 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: according to OECD test guideline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: ICR-191; 2-aminoanthracene

Remarks:

For more details on the positive control substances, see table 1

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

PERFORMANCE OF THE ASSAY: Sterile 12 × 75 mm test tubes were placed in heating blocks at 45°C to 47°C and the following items were added in a stepwise manner for each concentration of test or control substance: 2.00 mL of top agar, supplemented with 10% of a 0.5 mM histidine/biotin/tryptophan solution, 0.10 mL of indicator organisms (overnight culture), 0.10 mL of vehicle control or test substance, or 0.05 mL of positive control substance and 0.50 mL of metabolic activation mixture or PBS, for tests with or without S9, respectively. The tube contents were mixed gently and then poured onto minimal glucose plates. The top agar was allowed to set and the plates were incubated at 36°C to 38°C for 2 days.

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn

COLONY COUNTING:
The number of revertant colonies were counted by hand or with an automatic colony counter and then recorded. Plates that had a cytotoxic reduction in background lawn growth (> 50% reduction in the background lawn) were counted.

Evaluation criteria:

POSITIVE RESPONSE:
The test substance was considered positive for mutagenicity if it induced an increase of revertants per plate with increasing concentration. The increases should be at least 2 times the vehicle control background frequency for strains with high spontaneous levels (i.e., TA100) and three times for those with low spontaneous levels (TA1537, TA98, TA1535, and WP2 uvrA).
These increases should be seen in at least 2 or more successive concentrations or the response should be repeatable at a single concentration.

NEGATIVE RESPONSE:
The test substance was considered to be negative for inducing mutagenicity if it did not induce a response which fulfills the criteria for a positive response.

EQUIVOCAL RESPONSE:
Cases which did not clearly fit into the positive or negative criteria may be judged equivocal. In these cases the Study Director, based on sound scientific judgment, may take additional factors into consideration in evaluating the test results.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 250 μg/plate and ≥ 50 μg/plate (DRF study, with and without S9, resp.); ≥ 100 μg/plate and ≥ 50 μg/plate (first experiment, with and without S9, resp.); ≥ 250 μg/plate and ≥ 100 μg/plate (second experiment, with and without S9, resp.)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 100 μg/plate and ≥ 50 μg/plate (DRF study, with and without S9, resp.); ≥ 100 μg/plate and ≥ 25 μg/plate (first experiment, with and without S9, resp.); ≥ 100 μg/plate and ≥ 50 μg/plate (second experiment, with and without S9, resp.)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 250 μg/plate and ≥ 50 μg/plate (DRF study, with and without S9, resp.); ≥100 μg/plate (first experiment, with and without S9); ≥ 100 μg/plate (second experiment, with and without S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 250 μg/plate and ≥ 25 μg/plate (DRF study, with and without S9, resp.); ≥ 100 μg/plate and ≥ 25 μg/plate (first experiment, with and without S9, resp.); ≥ 100 μg/plate and ≥ 25 μg/plate (second experiment, with and without S9, resp.)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 250 μg/plate and ≥ 500 μg/plate (DRF study, with and without S9, resp.); ≥ 250 μg/plate (first and second experiment, with and without S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- No precipitation was observed in any of the experiments.
- There were no increases in the number of revertant colonies indicating a positive repsonse for inducing mutagenicity. Therefore, the test item is considered to be not mutagenic.
- The means of all positive control data were at least 3-fold greater than the means of the vehicle control data and comparable to the historical data.
- In both the first and second assay, criteria for a negative response were met for all tester strains with and without metabolic activation.

Conclusions:

Based on the results of an Ames test, performed according to OECD guideline 471 and GLP principles, Di-sec-butyl peroxydicarbonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
2. MODEL

DEREK Nexus v.6.0.1; 2018
Biovia Discovery Studio (TOPKAT); 2018
VEGA-QSAR v.1.1.4; 2017
OECD QSAR Toolbox v.4.2; 2018
Danish QSAR database (which includes Leadscope, CASE Ultra and SciQSAR models); 2016
ACD / ToxSuit v2.95

With respect to the prediction of Mammalian Cell Gene Mutation, the Danish QSAR database and OECD Toolbox were used.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CCC(C)OC(=O)OOC(=O)OC(C)CC

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
5. APPLICABILITY DOMAIN
6. ADEQUACY OF THE RESULT

QMRF and QPRF are included in the attached QSAR report.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Software tool(s) used including version:
OECD QSAR Toolbox v.4.2; 2018
Danish QSAR database (which includes Leadscope, CASE Ultra and SciQSAR models); 2016

- Model description: see field 'Justification for non-standard information', 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'

Key result

Species / strain:

not specified

Metabolic activation:

not specified

Genotoxicity:

other: Negative or inconclusive (out of domain)

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Predictions by the Danish QSAR database and the OECD toolbox were negative or inconclusive (out of domain), either in vitro or in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Chromosome aberration in mammalian cells (Mice) - Negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 19 April 2012 and 03 January 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Qualifier:

according to guideline

Guideline:

other: Japanese METl/MHLW/MAFF guidelines for testing of new chemical substances.

GLP compliance:

not specified

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Specific details on test material used for the study:

Identification: Di-sec-butyl-peroxydicarbonate (CAS# 19910-65-7)
Description: clear colourless liquid
Purity: 97.1% w/w
Batch number: 0808181901
Date received: 21 December 2011
Expiry date: 12 January 2013
Storage conditions: -20°C (approximately) in the dark

Species:

mouse

Strain:

ICR

Details on species / strain selection:

The test system was chosen because the mouse has been shown to be a suitable model for this type of study and is the recommended species in the test method.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sufficient albino Hsd: IC R (CD-1®) strain mice were obtained from Harlan Laboratories UK Ltd., Oxon, UK. At the start of the main test the mice weighed 24 to 30 g and were approximately six to ten weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding. Free access to mains drinking water and food (Harlan Teklad 2014C Global Certified Rodent Diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

Due to the nature of the test item an initial investigation was performed to determine the compatibility of the vehicle (dried corn oil) with the test item as available information indicated that the break down of the test item when added to some vehicles may cause rapid hydrolysis and the release of gases, which may cause adverse effects in the live animal.
Dried corn oil was considered to be suitable as a vehicle for the test item with no evidence of a release of gases when mixed together.

Details on exposure:

The test item was formulated as a solution in dried corn oil. The test item was removed from cold storage (approximately -20°C) and formulations were prepared at the required testing concentrations of 1250, 625, and 312.5 mg/kg. Due to the instability of the test item formulations, the animals were dosed within 35 minutes of formulation to ensure the temperature remained as low as possible.

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

Range-finding test - 2 days
Micronucleus test - 24 or 48 hours

Dose / conc.:

1 250 mg/kg bw (total dose)

Dose / conc.:

625 mg/kg bw (total dose)

Dose / conc.:

312.5 mg/kg bw (total dose)

No. of animals per sex per dose:

Range-finding test: 4 males and 2 females treated at 1250 mg/kg bodyweight
Micronucleus test: 7 males per dose

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Five mice were dosed orally with cyclophosphamide at 50 mg/kg bodyweight. Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test.

Tissues and cell types examined:

Erythrocytes from the bone marrow within both femurs.

Details of tissue and slide preparation:

Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Gronwald/Giemsa, allowed to air-dry and a
cover slip applied using mounting medium.

Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. Where possible, the incidence of micronucleated cells per 2000 polychromatic erythrocytes ( PCE-blue stained immature cells) per animal was scored.
Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.
A compound is considered mutagenic when a statistically significant, dose-responsive and toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to the vehicle control group.
If these criteria were not fulfilled, then the test item would be considered non-mutagenic under the conditions of the test.
A compound is considered toxic to the bone marrow when the mean polychromatic to normochromatic ratio in treated animals is statistically significantly lower than the vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing
Report, Part Ill (1989). The data set was analysed following a √(x + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Range-finding Toxicity Test
In the initial range finder two male animals were dosed with the test item at 1250 mg/kg via the oral route, the clinical signs observed were as follows: hunched posture, ptosis, piloerection and ataxia up to 4 hours after dosing with no clinical signs observed at 24 or 48 hours. The clinical signs observed were close to the severity limits and were considered to be at the maximum achievable dose. Therefore four additional animals (two male and 2 female) were dosed at 1250 mg/kg as a confirmatory test. The clinical signs observed were very similar to the first test and included: hunched posture, ptosis and ataxia, the animals recovered after two hours with no clinical signs observed at 4, 24 or 48 hours. The maximum achievable dose of 1250 mg/kg was selected as the top dose with 625 and 312.5 mg/kg as the intermediate and lower doses respectively in the main test using the oral route of administration.
The test item showed no marked difference in its toxicity to male or female mice; therefore the main test was performed using male mice only.

Micronucleus Test
Mortality Data and Clinical Observations
There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test item at and above 625 mg/kg in both the 24 and 48-hour groups where applicable, and included hunched posture, ptosis and ataxia. The observations were very similar to the range finding toxicity test in that no clinical signs were observed at the 24 and 48 hour time-points where applicable.

Evaluation of Bone Marrow Slides
No statistically significant decreases in the PCE/NCE ratio were observed at any test item dose level. Whilst no statistically significant decreases were recorded there was evidence of reductions in both the 24 and 48-hour dose groups, which accompanied with the clinical signs was taken to confirm exposure to the bone marrow had been achieved.
There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. The test item was found not to produce a toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.
The observation of clinical signs was taken to indicate that systemic absorption had occurred and the target tissue was exposed to the test item.

Conclusions:

The test item was considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction.

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Mammalian Erythrocyte Micronucleus Test", Method 812 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the USE PA (TSCA) O P PTS 870.5395, E PA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METl/MHLW/MAFF guidelines for testing of new chemical substances.

Methods.

A range-finding test was performed to confirm a suitable dose level of the test item, route of administration, and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum achievable dose of 1250 mg/kg with 625 and 312.5 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and

smear preparations made and stained. Polychromatic ( PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Additional groups of mice were given a single oral dose of corn oil (7 male mice) or dosed orally with cyclophosphamide (5 male mice), to serve as vehicle and positive controls respectively. Vehicle and positive control animals were killed after 24 hours.

Results.

There were no premature deaths at any dose level. Clinical signs were observed in animals dosed with the test item at and above 625 mg/kg and included hunched posture, ptosis and ataxia.

There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group. No statistically significant decreases in the PCE/NCE ratio were observed at any test item dose level.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusion.

The test item was considered to be non-mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)