Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
- Name of test material (as cited in study report): TK 12891/1 (also known as 2,4-di-tert.-butylphenol)
- Physical state: yellow solid
- Analytical purity: 99.1%
- Supplier: Sigma-Aldrich
- Lot/batch No.: S43419-228
- Expiration date of the lot/batch: 24 July 2009
- Storage condition of test material: stored at ambient temperature in the dark when not in use

Test animals

Species:
rat
Strain:
other: CD rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK
- Weight at study initiation: mean(males): 266 - 312 g; mean(females): 160 - 193 g
- Housing: in sets of twos and threes
- Diet: SDS Rat and Mouse Maintenance Diet No. 1 which was obtained from Special Diet Services Limited, England. (ad libitum)
- Water: tap water (ad libitum)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 46 - 87
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose volume used for both the control and test item treated animals was a constant 10 mL/kg body weight.

Duration of treatment / exposure:
Bone marrow samples were taken 48 h after the initial 0 h dose.
Frequency of treatment:
Twice (Rats were dosed at 0 h and 24 h.)
Post exposure period:
Bone marrow samples were taken 48 h after the initial 0 h dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
males: 1000 mg/kg bw/day; females: 200, 400 and 800 mg/kg bw/day
Basis:
analytical conc.
No. of animals per sex per dose:
- vehicle control: 5 males and 5 females;
- low/mid dose: 5 females each;
- high dose: 10 males and 10 females (The high dose group of rats consisted of an increased group size of 10 males and 10 females of which 5 males and 5 females provided the regular assessment base. The additional rats were processed in normal fashion and the slides labelled with the original animal number. The slides from this spare group were kept as a contingency in case of unscheduled deaths or potential sex differences. In the event of death, the first available animal in the relevant contingency group replaced the missing animal. Preparations were made from remaining contingency group animals and the slides were kept as spares.);
- positive control: 5 males;
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Doses / concentrations: 50 mg/kg bw/day (Cyclophosphamide was prepared fresh as a 5 mg/mL solution in distilled water. It was administered to the positive control animals in dose volumes of 10 mL/kg to give the required target dose of 50 mg/kg)

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
A drop of the suspension including bone marrow cells was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube/animal. The smear was left to air dry, fixed in methanol for ca 5 min and then immersed for 15 min in 15% Giemsa stain, prepared in tap water, to give optimum erythrocyte discrimination. The stained smears were finally rinsed in distilled water for ca 1 min and left to air dry overnight. Permanent slide preparations were made by sealing coverslips onto the glass slides using DPX mounting medium.

METHOD OF ANALYSIS:
The better of the 2 prepared slides was selected for examination and the coded slides assessed blind by the same operator. At least two thousand (2000) polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micronucleated cells (MN-PCE) determined. As a control against inclusion of artefacts, or action of a mutagen on the G2 and/or mitotic phase of the cell cycle, the numbers of micronucleated normochromatic erythrocytes (MN-NCE) in mature red blood corpuscles were also recorded (Maier and Schmid, 1976; Hamoud et al, 1989). In addition, scored micronuclei were assigned on the basis of size into small or large categories, historically defined as micronuclei occupying less or more than 25% of the visible cellular area. This classification provided a non-specific measure of compound induced spindle dysfunction, as large micronuclei appear to derive from lagging chromosomes caused by damage to the mitotic apparatus during bone marrow erythropoiesis (Yamamoto and Kikuchi, 1980; Vanderkerken et al, 1989). The PCE/NCE ratio, a measure of any induced systemic toxicity, was determined by counting a minimum total of 1000 erythrocytes (PCE + NCE) per marrow preparation.

- Positive Response:

The test would be judged positive if an increase in the number of micronucleated polychromatic erythrocytes (MN-PCE) was obtained for one or more of the test item treated dose groups. That is, an increase greater than 10% over the expected historical control ranges for a group of animals. The increase observed should be biologically relevant and statistically significant relative to concurrent and historical control frequencies for MN-PCE and/or MN-NCE induction.

- Inconclusive Response:

The test would be considered inconclusive if the levels of MN-PCE within any one dose group were increased above the established historical control frequencies for MN-PCE induction, but not high enough to meet the criteria for a positive response. That is an increase up to 10% over the maximum negative control frequency for a group of animals.
Evaluation criteria:
Acceptance Criteria:
- The prepared slides had uniform staining properties and sufficient number of PCE cells present to allow accurate micronucleus determination.
- The assay was considered acceptable as the MN-PCE frequencies for the vehicle control dosed rats were within the expected historical range. The ranges are defined in accordance with Charles River Laboratories experience of the bone marrow micronucleus test using CD rats.
- An adequate positive control response for at least 2 animals and the dose group as a whole.

Evaluation Criteria:

The average micronucleus incidence in vehicle control dosed and untreated CD rats, has in this laboratory been determined as 0.06±0.05% , a range of 0.01-0.13% per group of 5-6 animals and 0.03-0.11% per group of 10-12 animals. This frequency is in agreement with published data for micronucleus tests with CD rats (Tamura et al, 1990; Salamone and Mavournin, 1994). These historical data have been used in the evaluation of response in this test.
- Negative Response:
The test would be judged negative if no biologically relevant increases in the numbers of MN-PCE were observed, relative to the concurrent and established historical control frequencies for MN-PCE induction. No statistical analysis will be performed if the levels of MN-PCE induction fell within the determined historical control frequencies. A similar biological approach to the data, which avoids the need for statistical evaluations, has recently been described (Ashby and Tinwell, 1995). Variations in the MN-NCE frequencies and PCE/NCE ratios will also not be analysed statistically, unless clearly different from concurrent control values.
Statistics:
Not required

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
There were indications of bone marrow toxicity in the high dose males (PCE/NCE ratio of 0.43 compared to 0.56 in vehicle control group).
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
As toxicity information on the test item was available from the Sponsor, a limit toxicity test (3 males/3 females) was conducted prior to the micronucleus test to establish a suitable dose range for the micronucleus experiment. The limit toxicity study used a starting dose of 1600 mg/kg. Mortality occured at the ranger-finder doses of 1000, 1200, 1400 and 1600 mg/kg bw. At 1600 mg/kg bw each one of three males and females died on day 3 after dosing. Based on the findings of the toxicity study, the maximum tolerated doses were judged to be in the region of 1000 mg/kg/day for males and 800 mg/kg/day for females.



RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay): in the range of the negative control
- Ratio of PCE/NCE (for Micronucleus assay): There were indications of bone marrow toxicity in the high dose males (PCE/NCE ratio of 0.43 compared to 0.56 in vehicle control group).

Any other information on results incl. tables

Test group Sex No. of rats scored erythrocytes
Normochromatic cells (NCE) Polychromatic cells (PCE) PCE/NCE Mean +/- S.D.
No. of MN-NCE PCE analysed No. of MN-PCE % MN-PCE
vehicle (20 ml corn oil kg/day) male 5 8 10004 7 0.07 0.53 +/- 0.06
female 5 4 10008 4 0.04 0.58 +/- 0.05
male/female 10 12 20012 11 0.05 0.56 +/- 0.06
200 mg /kg/day female 5 1 10009 6 0.06 0.67 +/- 0.12
400 mg /kg/day female 5 3 10012 5 0.05 0.58 +/- 0.13
1000 mg /kg/day male 5 9 10011 3 0.03 0.43 +/-0.08
800 mg /kg/day female 5 2 10008 5 0.05 0.59 +/- 0.12
50 mg Cyclophosphamide /kg/day male 5 53¿ 10002 189¿ 1.89 0.34 +/- 0.07
PCE = polychromatic erythrocyts 
MN-PCE = micronucleated PCE
NCE = normochromatic erythrocyts
MN-NCE = micronucleated NCE
¿= positive response in PCE
¿ = evident response in NCE

No animal deaths occurred following dosing. Clinical signs of hunched, subdued behaviour, wet around anus, wet staining pergenital, wet faeces, piloerection, salivation red discharge (nose), laboured breathing and staggering were observed. There was no indication that the test item induced bone marrow micronuclei in the treated rats. The highest MN-PCE frequency recorded for the test item was in the low dose females where an incidence of 0.06% was observed. There were indications of bone marrow toxicity in the high dose males (PCE/NCE ratio of 0.43 compared to 0.56 in vehicle control group).

Applicant's summary and conclusion