O,O-tert-butyl isopropyl monoperoxycarbonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start: 05 March 2001 Completed: 12 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: Micronucleus Test in mice

Test material

Test material information

Constituent 1

Reference substance name:

O,O-tert-butyl isopropyl monoperoxycarbonate

EC Number:

219-143-7

EC Name:

O,O-tert-butyl isopropyl monoperoxycarbonate

Cas Number:

2372-21-6

Molecular formula:

C8H16O4

IUPAC Name:

tert-butoxy propan-2-yl carbonate

additive 1

Reference substance name:

Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated

EC Number:

297-629-8

EC Name:

Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated

Cas Number:

93685-81-5

Molecular formula:

not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

isododecane

Test material form:

liquid

Details on test material:

Trigonox BPIC-C75

Specific details on test material used for the study:

Identification: Trigonox BPIC-C75
Chemical name: Tert-Butylperoxy isopropyl carbonate
CAS-Number: 2372-21-6
Description: Colourless liquid
Batch: 0007130467
Test substance storage: At room temperature in the dark
Stability under storage conditions: Not indicated
Expiry date: 11 January 2002

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Recommended test system in international guidelines (e.g. EPA, FDA, OECD, EEC). The NMRl BR mouse is used as test system because it is a readily available rodent species, which is commonly used for this purpose, with documented susceptibility to a wide range of toxic substances.

Sex:

male

Details on test animals or test system and environmental conditions:

Source: Charles River, Sulzfeld, Germany.
Age at Start of Treatment: Young adult animals were selected (6-8 weeks old}.
Identification: By unique number on the tail.
Allocation: Allocated to treatment groups as they came to hand from delivery boxes.
On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.
Mean body weights immediately prior to dosing (g) (mean ± S.D.):
A 32.6 ± 2.3
B 32.8 ± 1.3
C 33.0 ± 1.0
D 31.8 ± 0.4

ANIMAL HUSBANDRY
Conditions
The animals were housed in an air-conditioned room with approximately 15 air changes per hour and a controlled environment with a temperature of a temperature of 21 ± 3°C and a relative humidity of 30-70%. Due to cleaning procedures or performance of functional observations in the room, temporary deviations from the maximum level for humidity (with max. 20) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity. The room was illuminated with 12
hours artificial fluorescent light and 12 hours dark per day.

Accommodation
Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material (Sawi, Jelu Werk, Rosenberg, Germany). Certificates of analysis were examined and then retained in the NOTOX archives. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

Diet
Free access to standard pelleted laboratory animal diet (Altromin (code VRF 1), Lage, Germany). Certificates of analysis were examined and then retained in the NOTOX archives.

Water
Free access to tap-water. Certificates of analysis (performed quarterly) were examined and then retained in the NOTOX archives.

Administration / exposure

Route of administration:

other: oral intubation

Vehicle:

TRIGONOX BPIC-C75 was dissolved in corn oil (OPG, Utrecht, The Netherlands). TRIGONOX BPIC-C75 concentrations were treated with ultra-sonic waves until the test substance had completely dissolved.

Details on exposure:

TRIGONOX BPIC-C75 concentrations were dosed within 4 hours after preparation.
The route of administration was chosen to maximize the chance of the test article reaching the target tissue.
Feed was withheld 3- 4 h prior to dosing until administration of TRIGONOX BPIC-C75. The dosing volume was 10 ml/kg body weight. The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test article.

Duration of treatment / exposure:

Single dose

Post exposure period:

Dose range finding study: 3 days
Main study: 24 or 48 hours

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

recommended maximum dose

No. of animals per sex per dose:

In the dose range findng study 2 male and 2 female mice were treated at a single dose
In the main study 5 male mice per sampling time in each treatment group.

Control animals:

yes, concurrent vehicle

other: yes, concurrent positive

Positive control(s):

The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, F.R.G.) dissolved in physiological saline (Fresenius B.V., 's-Hertogenbosch, The Netherlands) dosed at a single oral intubation of 50 mg salt/kg body weight.

Examinations

Tissues and cell types examined:

erythrocytes in the bone marrow of the femora

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation 24 or 48 h after dosing of TRIGONOX BPIC-C75, 24 h after dosing of the vehicle and 48 h after dosing of the positive control. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1 :1 mixture of 9 6 % (v/v) ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with roundwhetted sides at an angle of approximately 45 ' over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.

Staining of the bone marrow smears
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

Analysis of the bone marrow smears for micronuclei
All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. Al first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same lime. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range finding study
In a dose range finding study 4 animals (2 males and 2 females per group) were dosed orally with 2000 mg/kg body weight.
All treated animals showed no abnormalities during an observation period of 3 days.

Micronucleus Test
Since no substantial differences between the sexes in toxicity or any observable toxic effects were observed, the micronuclues test was performed with male animals only and one dose level: 2000 mg/kg body weight. Five male animals were used in each treatment group.

Mortalitv and systemic toxic signs
All animals of the groups treated with TRIGONOX BPIC-C75 and the animals of the vehicle treated group and the positive control group showed no abnormalities.

Micronucleated polychromatic ervthrocytes
The mean number of micronucleated polychromatic erythrocytes scored in TRIGONOX BPIC-C75 treated groups were compared with the corresponding solvent control group. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of TRIGONOX BPIC-C75 treated animals compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical solvent control data range.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

Ratio polychromatic to normochromatic erythrocytes
The animals of the groups which were treated with TRIGONOX BPIC-C75 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups which were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes.

Applicant's summary and conclusion

Conclusions:

II is concluded that this test is valid and that TRIGONOX BPIC-C75 is not mutagenic in the micronucleus test under the in vivo experimental conditions described in this report.

Executive summary:

TRIGONOX BPIC-C75 was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Two groups each comprising 5 males received a single oral intubation of 2000 mg/kg body weight. After dosing all animals of the treatment group showed no abnormalities.

A vehicle treated group (corn oil) served as negative control, a group treated with a single oral intubation of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow of the groups treated with TRIGONOX BPIC-C75 was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone

marrow from the positive control group was harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. The dose range finding study at 2000 mg/kg/d had clinical findings suggesting that the substance was absorbed.

No increase in the frequency of micronuclealed polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with TRIGONOX BPIC-C75.

The groups that were treated with TRIGONOX BPIC-C75 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle

controls.

It is concluded that TRIGONOX BPIC-C75 is not mutagenic in the micronucleus test under the in vivo experimental conditions described in this report.