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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity

-      Due to technical issues in vitro mutagenicity testing with the multi-constituent substance is not possible.

-      The available Ames test with the stabilizer is negative without metabolic activation and ambiguous with metabolic activation, the overall conclusion for the stabilizer is negative for mutagenicity.

-      The Ames test with SBP is negative and an MLA is ongoing.

-      IPP is positive with metabolic activation in the Ames test and the MLA. A comet assay is proposed to further investigate this endpoint.

-      QSAR data did not indicate the possibility of the multi-constituent substance to be positive in the Ames test (see QSAR doc section 13 Appendix 2). This has limited reliability since the molecular structure is not well presented in the models. In other words there is no alert but there is also no information on this chemistry in the data base.

-      Based on the available information (see QSAR doc section 13 Appendix 1), the multi-constituent substance is concluded not to be a mutagen pending the outcome of the proposed Comet assay. 

 

Clastogenicity

- SBP has a negative In vivo micronucleus assy.

- IPP and the stabilizer have negative In vitro micronucleus assays.

- Based on the structural similarities of IBP the same outcome is expected, see QSAR document section 13.

- Therefore, it can be concluded that the multi-constituent substance is not a clastogen and no further testing is required.

The robust summaries for the in vitro studies are unfortunately not available to the registrant at the time of dossier submission.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

See above.

Link to relevant study records

Referenceopen allclose all

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 2012.
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-clastogenic 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-clastogenic.under the conditions of the test.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned (based on read-across)
Study period:
2019
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out bisisopropyl peroxydicarbonate (IPP) CAS 105-64-6
- Name of the substance for which the testing proposal will be used [if different from tested substance] Reaction mass of IBP and IPP and SBP

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies: No OECD 489 GLP study is available
- Available non-GLP studies: No OECD 489 non-GLP study is available
- Historical human data: Not available
- (Q)SAR: Not suitable for this endpoint
- In vitro methods: No in vitro test to address this endpoint is available
- Weight of evidence: No available data for weight of evidence approach
- Grouping and read-across: 105-64-6 is a relevant structural analog (and constituent of the multi-constituent substance) and read across is proposed
- Substance-tailored exposure driven testing [if applicable]: Not applicable
- Approaches in addition to above [if applicable]: None
- Other reasons [if applicable]: required due to positive in vitro study results

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Adaptation is not possible

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed [if relevant] OECD 489
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes
Type of assay:
mammalian comet assay
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Justification for classification or non-classification

Not classified based on the available information.