Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1998-03-09 (start of experiment) to 1999-09-16 (end of experiment)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
Deviations to the Protocol:
- In deviation to the protocol the weight of eleven females was lower than 160 g (lowest weight was 150 .4 g).
- In deviation to the protocol animals of both sexes were treated with 15 mg/kg bw CPA instead of 10 mg/kg bw for the males.
- For short periods of time (hours) the lower limit of the relative humidity (30 %) was not reached and the upper limit (70 %) was exceeded. The lowest value was 23 %, the highest value was 91 % .
- No additional dose levels were tested at the sampling time 24 h after treatment.
These deviations had no influence on the quality or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
chromosome aberration assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-tert-butylbenzoic acid
EC Number:
202-696-3
EC Name:
4-tert-butylbenzoic acid
Cas Number:
98-73-7
Molecular formula:
C11H14O2
IUPAC Name:
4-tert-butylbenzoic acid
Constituent 2
Reference substance name:
Reference substance 001
Details on test material:
ANALOGOUS COMPOUND to p-tert butyltoluene

- Name of test material (as cited in study report): p-tert-Butylbenzoic acid (ptBBA); supplier: Clariant Chimie S.A.
- Molecular formula (if other than submission substance): C11 H14 O2
- Molecular weight (if other than submission substance): 178.23
- Smiles notation (if other than submission substance): c1(ccc(C(O)=O)cc1)C(C)(C)C
- InChl (if other than submission substance): InChI=1/C11H14O2/c1-11(2,3)9-6-4-8(5-7-9)10(12)13/h4-7H,1-3H3,(H,12,13)
- Physical state: solid, white
- Analytical purity: GC: 99.8 area-% (with no single impurity > 0.1 %), according to analytical certificate no. 339
- Impurities (identity and concentrations): no data
- Purity test date: 1997-08-28
- Lot/batch No.: 970715
- Expiration date of the lot/batch: storable for at least 12 months (August 1998 )
- Stability under test conditions: Stability in vehicle: > 1 week in DMSO, ethanol, and acetone
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
24 male and 24 female Wistar rats
- Source: RCC Ltd., Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation: males: 179.1 ± 6 .76 g; females: 163 .3 ± 8 .98 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes: approximately 18 hours before treatment with the test item the animals received no food but water was available ad libitum
- Housing: single housing; cages: Makrolon, Type II, with wire mesh top
- Diet (ad libitum): pelleted standard diet (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24 °C
- Humidity (%): 23 - 91%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil (SIGMA-Aldrich)
- Justification for choice of solvent/vehicle: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was formulated in corn oil. All animals received a single standard volume of 10 ml/kg bw orally.
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Post exposure period:
sacrifice at 24 or 48 hours post dose
Doses / concentrations
Remarks:
Doses / Concentrations:
300, 600 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
groups of 6 males and/or 6 females, see table below
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
cyclophosphamide (purity at least 98%), dissolved in 0.9% NaCl solution (solution prepared on day of administration)
- Justification for choice of positive control(s): no data
- Route of administration: oral (gavage)
- Doses / concentrations: 15 mg/kg bw, dosing volume: 10 ml/kg bw

Examinations

Tissues and cell types examined:
femoral bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose levels were determined to be the doses that caused toxic reactions without having effects on survival within 48 hours. Different maximum doses were determined : males = 600 mg/kg bw, females = 300 mg/kg bw. These doses were applied for the sampling intervals 24 h and 48 h after treatment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Approximately 18 hours before treatment with the test item the animals received no food but water ad libitum. Before the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test item, CPA, or vehicle control once. Twelve animals, six males and six females, were treated per dose group.
Prior (2.5 hours) to sacrifice, animals were injected intraperitoneally with the spindle inhibitor colcemid (2.0 mg/kg bw ) to arrest cells in the metaphase.

DETAILS OF SLIDE PREPARATION:
After anesthesia with CO2 the animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with approximately 5 ml hypotonic potassium chloride solution (0.56 % w/v, prewarmed to 37°C). The hypotonic cell suspension was then incubated for 20 min at 37°C. The cells were sedimented by a brief centrifugation (1000 rpm), the hypotonic supernatant was discarded and the cell pellet was fixed with 3+1 absolute methanol+glacial acetic acid fixative for 60 min. Then, the cell pellet was gently resuspended with fixative and stored overnight at 4°C. Prior to preparing the slides the fixative was changed and enough fixative was added to make a relatively thin cell suspension. The fixative-cell suspension was spread by flame-drying and stained with Giemsa. Cover slips were mounted with EUKITT. One or more slides were made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. 100 well spread metaphases per animal were scored for cytogenetic damage on coded slides. The number of chromosome aberrations per metaphase was determined. Only metaphases with the species-characteristic chromosome number of 42 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis; 1000 cells were scored) was determined.
The cells of five animals per sex and group were evaluated as described.
Evaluation criteria:
A test item is classified as mutagenic if it induces a dose-related increase in the number of structural chromosomal aberrations and a statistically significant positive response for at least one of the test points.
A test item producing no dose-related increase in the number of structural chromosomal aberrations and no statistically significant positive response at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the non-parametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.
Statistics:
non-parametric Mann-Whitney test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 400, 500, 600, 800, 900, 1000 mg/kg bw; dosing volume: 10 ml/kg bw
- Clinical signs of toxicity in test animals: death, reduction of spontaneous activity, eyelid closure, apathy, abdominal position
- Harvest times: 1, 6, 24, 48 h

Any other information on results incl. tables

The doses for the cytogenetic assay were determined in pre-experiments for toxicity. The doses of 300 and 600 mg/kg bw, respectively, were estimated as to be close to the maximum tolerated doses. In the cytogenetic assay 2 males died after treatment with 600 mg/kg bw of the test item.

A weak reduction (females only) of the mitotic indices after treatment with the test item was observed at preparation interval 24 hours indicating a cytotoxic effectiveness of the test item.

No statistically significant increase in the frequency of aberrant cells occurred after treatment with the test item as compared to the vehicle control.

An appropriate reference mutagen (cyclophosphamide) was used as positive control and showed a distinct and statistically significant increase of induced aberration frequency.

 

For details, see attached files.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce chromosome mutations as determined by the chromosome aberration test with bone marrow cells of the rat.
Therefore, p-tert Butyl benzoic acid (ptBBA) is considered to be non-clastogenic in this chromosome aberration assay in vivo.