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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-12 to 2012-08-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: micronucleus assay in vivo

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butyl 3,5,5-trimethylperoxyhexanoate
EC Number:
236-050-7
EC Name:
tert-butyl 3,5,5-trimethylperoxyhexanoate
Cas Number:
13122-18-4
Molecular formula:
C13H26O3
IUPAC Name:
tert-butyl 3,5,5-tris(methylperoxy)hexanoate

Test animals

Species:
mouse
Strain:
other: CRL: NMRI BR
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: TOXI COOP Zrt. 1103 Budapest, Cserkesz u. 90.
- Age at study initiation: Young adult mice (7 -8 weeks)
- Weight at study initiation: 28.0g - 32.4 g
- Assigned to test groups randomly: Yes
- Fasting period before study: no
- Housing: Group caging (up to 2 and 5 animals/cage)
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes (per hr): 12 air exchanges per hour by central air-condition system
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 2012-04-11 To: 2012-04-13

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: sunflower oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- positive control: Cyclophosphamide (positive control) was dissolved in aqua ad iniectabilia for treatment
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The necessary amount of the test item was weighed into a calibrated volumetric flask and vehicle added and stirred to obtain homogenous formulations. It was then diluted to the final volume with vehicle. The concentration of the test item solutions were chosen to assure the same dosing volumes in mice for all dose levels (10 mL/kg).

CRITERIA FOR DOSE SELECTION:
A preliminary toxicity test was performed to identify the appropriate maximum dose level for the main test. The preliminary toxicity test determined whether there are differences in toxicity between the sexes or not. Groups of two male and female mice were treated on one occasion by oral gavage at dose levels of 2000 mg/kg bw. The treatment volume was 10 mL/kg bw. Animals were examined regularly for toxic signs and mortalities. On the basis of results of preliminary toxicity test, doses for the Mouse Micronucleus Test were: 500, 1000 and 2000 mg/kg bw.
Duration of treatment / exposure:
single dosage
Frequency of treatment:
single dosage
Post exposure period:
- untreated control, low and mid dose groups the sampling from bone marrow was performed once at 24 hours after treatment;
- high dose and the negative control groups the sampling from bone marrow was performed twice at 24 and 48 hours after treatment
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
untreated control, low and mid dose group, positive control group: 5 male rats
negative control group and hogh dose group: 10 male rats (5 rate for the 24h sampling time, 5 rats for the 48h sampling time)
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- Route of administration: intraperitoneally
- Doses / concentrations: 10 mL/kg bw (60 mg/kg bw)

Examinations

Tissues and cell types examined:
Please refer to details of tissue and slide preparation
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
The animals from each group were weighed and bone marrow was obtained from two exposed femurs of the mice from every dose- time point immediately after sacrificing. The bone marrow was flushed with foetal bovine serum (5 mL). After vortex mixing, the cell suspension was concentrated by centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides. Slides were then dried at room temperature.

DETAILS OF SLIDE PREPARATION:
Subsequently the slides were stained as follow:
Fixed for a minimum of 5 minutes in methanol and allowed to air-dry.
1. Stained with Giemsa solution for 25 minutes.
2. Rinsing in distilled water.
3. Drying at room temperature (at least 12 hours).
4. Coating with EZ-mounting

METHOD OF ANALYSIS:
Prior to microscopic analysis, one slide from each animal was given a code number for blind microscopic analysis. The code labels were covering the original animal numbers to ensure that the slides scored without bias. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells were expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field.
The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 200 immature erythrocytes.
Evaluation criteria:
A micronucleus is defined in following way:
– A bluish mauve strongly coloured uniform circular particle in the cell.
– The particle should have a certain size and it should be located inside the cells.
– During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval.
– Cells with two or more micronuclei were counted as single micronucleated cells.

The Micronucleus Test is considered acceptable if it meets the following criteria:
– The proportion of polychromatic erythrocytes among total erythrocytes in treated groups is not less than 20 % of the control value.
– the frequencies of micronucleated polychromatic erythrocytes found in the negative and /or solvent controls are consistent with the range of historical laboratory control data.
– the positive control item should produce biologically relevant increases in the number of micronucleated polychromatic erythrocytes.
– Each treated and control group should include at least 5 analysable animals

The test item would have been considered to have shown genotoxic activity in this study if the following criteria are met:
– increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals of a single dose group compared to the corresponding negative controls
– the increases are dose-related
– the increases are statistically significant.
– the increases exceeded historical control range for this laboratory

The test item was considered to have given a negative response because no reproducible, statistically significant increases were observed
Statistics:
The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative control group. Statistically analysis was performed using Kruskal Wallis Non Parametric ANOVA test (Kruskal-Wallis one-way analysis of variance).

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs at the highest dose tested.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:
Groups of two male and female mice were treated on one occasion by oral at dose levels of 2000 mg/kg bw.
- Clinical signs of toxicity in test animals:
The male mice dosed at 2000 mg/kg body weight showed medium, slight to medium decrease in activity and piloerection between 2 and 5 hours after the treatment. The female mice dosed at 2000 mg/kg body weight showed slight decrease in activity and piloerection between 2 and 4 hours after the treatment.
- Other:
No adverse reactions to treatment were observed in the male and female mice dosed 2000 mg/kg body weight between 5 and 48 hours after the treatment.

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei:
The frequencies of MPCEs for the untreated, negative (solvent) controls mice were within an acceptable range and compatible with the historical control data for this laboratory. Cyclophosphamide treated mice showed a large, statistically significant increase in the MPCEs number compared to the negative control, demonstrating an acceptable sensitivity of the test. The mean number of MPCEs in the Cyclophosphamide treated mice was above the the corresponding historical control data range, however within the assay acceptance range.

The single oral administration of 500 mg/kg bw, 1000 mg/kg bw and 2000 mg/kg body weight of tert. butylperoxy-3,5,5-trimethylhexanoate (TBPIN) did not induce biologically and statistically significant increases in the frequency of MPCEs in male mice at either 24 or 48 hours after the treatment compared to the concurrent negative (solvent) control group.

- Ratio of PCE/NCE:
The ratio of polychromatic to normochromatic cells was calculated on the basis of the number of mature cells encountered while accumulating 200 PCEs. At the 24-hour sampling the number of PCEs was decreased in the 1000 and 2000 mg/kg bw dose groups and at the 48-hour sampling in the 2000 mg/kg bw compared to the negative control group value. This effect demonstrated exposure of the bone marrow to the test item.


Any other information on results incl. tables

RESULTS OF DEFINITIVE STUDY

Clinical Signs and Mortality:

No animals died during the study. No adverse reactions to treatment were observed in the mice of the untreated, negative and positive control groups. The mice dosed at the 500 and 1000 mg/kg bw dose levels were symptom-free during the study. The male animals dosed 2000 mg/kg bw showed slight decreased in activity and piloerection at two and at five hours after the treatment and moderate decreased in activity and piloerection were observed between 3-4 and four hours after the treatment.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this assay the test item tert. butylperoxy-3,5,5-trimethylhexanoate (TBPIN) did not induce any statistically and biologically relevant increase in the number of micronucleated polychromatic erythrocytes at dose levels of 500, 1000 and 2000 mg/kg body weight after oral administration on one occasion in NMRI BR mice.
Executive summary:

The potential mutagenic activity of tert. butylperoxy-3,5,5-trimethylhexanoate (TBPIN) was examined in bone marrow of male NMRI BR mice. Based on a preliminary oral toxicity study doses of 500, 1000 and 2000 mg/kg bw were selected. Untreated negative (solvent) control and a positive control groups were included. Treatment was carried out with TBPIN by an oral single dosage. The test item was dissolved Sunflower oil with a constant treatment volume (10 mL/kg body weight). Cyclophosphamide dissolved in 0.9% NaCl solution (positive control) was administered once, intraperitoneally with a treatment volume of 10 mL/kg body weight. In the untreated control, low and mid dose groups the sampling from bone marrow was performed once at 24 hours after treatment and twice, at 24 and 48 hours after treatment in the high dose and the negative control groups. In animals treated with Cyclophosphamide (60 mg/kg bw.), the sampling was performed only at 24 hours post-treatment. Five animals per dose group were used on each occasion. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells.

The frequencies of micronucleated polychromatic erythrocytes (MPCEs) for the untreated, negative (solvent) and positive control mice were within acceptable ranges and compatible with the historical control data for this laboratory. The positive control values showed a large, statistically significant increase over the negative control values, demonstrating the sensitivity of the test. At the 24-hour sampling the number of PCEs was decreased in the 1000 and 2000 mg/kg bw dose groups and at the 48-hour sampling in the 2000 mg/kg bw compared to the negative control group value. This effect demonstrated exposure of the bone marrow to the test item.

The single oral administration of 500 mg/kg bw, 1000 mg/kg bw and 2000 mg/kg body weight of TBPIN did not induce biologically or statistically significant increase in the frequency of MPCEs in male mice at either 24 or 48 hours after the treatment compared to the concurrent control group.

In conclusion, no biologically or statistically significant increases in the frequency of MPCEs were seen in the groups of mice treated with TBPIN to the negative control group.Thus TBPIN did not show genotoxic activity in this mouse micronucleus test.