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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
Remarks:
Read across data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
The study contains experimental data of a read-across analogue. This data was used as a basis for the SIDS dossier. All data were checked and validated by BUA. A final evaluation of the human health part has been performed by the Federal Institute for Risk Assessment (BfR).
Qualified BUA personnel performed quality control on the full SIDS dossier submitted by the industry. This quality control process followed internal BUA guidelines/instructions for the OECD/ICCA peer-review process. This included: i) A full (or update) literature search to verify completeness of data provided by industry in the IUCLID/HEDSET, ii) Review of data and assessment of the quality of data, iii) Review of data evaluation. iv) Check the adequacy of the selection process for key studies for OECD endpoints and, where relevant, for non-OECD endpoints by checking original reports/publications. v) Review of key study description according to robust summaries requirements; completeness and correctness are checked against original reports/publications (if original reports are missing: reliability (4), i.e. reliability not assignable). vi) Review of validity of structure-activity relationships, vii) Review of full SIDS dossier (including SIAR, SIAP and proposal for the conclusion and recommendation for further work), viii) In case of data gaps, review of testing plan or rationale for not testing.

Data source

Reference
Reference Type:
review article or handbook
Title:
In vivo micronucleus assay of a read-across analogue.
Author:
UNEP Publications
Year:
2003
Bibliographic source:
OECD SIDS Initial Assessment Report

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
July 21, 1997
GLP compliance:
no
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
(E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one
EC Number:
201-224-3
EC Name:
(E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one
Cas Number:
79-77-6
Molecular formula:
C13H20O
IUPAC Name:
trans-beta ionone
Details on test material:
- Name of the test chemical: β-Ionone
- Molecular formula: C13H20O
- Molecular weight: 192.3 g/mol
- SUbstance type: Organic

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
No data
Sex:
male
Details on test animals or test system and environmental conditions:
Healthy male Crl: NMRI mice (breeder: Charles River, Deutschland GmbH, GER) with a mean weight of about 29g (with an age range of about 5-8 weeks) were used in the test.

5 males/dose were received a single ip injection.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Olive oil was used as a vehicle of the test substance.
Details on exposure:
Frequency of dosing: single injection (ip)
Dosing volume: 10 ml/kg bw
Control groups:
negative: 1 x vehicle control (10 ml/kg bw olive oil)
positive: 1 x 20 mg/kg bw cyclophosphamide (CPP) for
clastogenic effects (10 ml/kg bw), 1 x 0.15 mg/kg bw
vincristine (VCR) for aneugenic effcts (10 ml/kg bw)
5 males/dose were received a single ip injection.
Duration of treatment / exposure:
24 hours for treatment groups.
48 hours for satelite groups.
Frequency of treatment:
A single ip injection was applied in all control and treatment groups.
Post exposure period:
24 hours for treatment groups
48 hours for satelite groups
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day
Dose / conc.:
0 mg/kg bw/day
Remarks:
Satelite control
Dose / conc.:
750 mg/kg bw/day
Remarks:
Satelite top dose
No. of animals per sex per dose:
Treatment groups: 5 males/dose
Satelite groups: 5 males/ groups
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPP) for clastogenic effects (10 ml/kg bw)
Vincristine (VCR) for aneugenic effcts (10 ml/kg bw)

Examinations

Tissues and cell types examined:
Bone marrow PCEs and NMEs.
Details of tissue and slide preparation:
Samples of bone marrow of the 2 femurs were collected. Preparation of the bone marrow was performed according to the method of Schmidt (1976 and 1977) and Salamone et al. (1980).
Evaluation criteria:
Microscopic evaluation: 2000 polychromatic erythrocytes (PCEs) from each animal of every test group were investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) were also scored. The ratio of polychromatic to normochromatic erythrocytes was determined.
Statistics:
U-test according to Mann-Whitney (modified rank test according to Wilcoxon) test was used to demonstrate differences between control and dose groups.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Remarks:
No statistically significant increase in the number of polychromatic erythrocytes containing either small or large micronuclei was noted at any doses tested.
Toxicity:
yes
Remarks:
Clinical signs of toxicity was observed at 500 and 750 mg/kg bw.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 1. Effect on PCE/NCE ratio - Mean numbers of PCEs and NCEs

Interval

 

24 hrs

48 hrs

 

PCEs

NCEs

NCEs

Vehicle

10000

4594

3439

250 mg/kg bw

10000

3755

 

500 mg/kg bw

10000

4646

 

750 mg/kg bw

10000

2476

2804

CPP (20 mg/kg bw)

10000

3920

 

VCR (0.15 mg/kg bw)

10000

5374

 

Table 2. Mean number of PCEs containing MN per 1,000 PCE at 24 hrs (differentiation between small and large micronuclei)

 

Small

Large

Total

Vehicle

1.3

0.1

1.4

250 mg/kg bw

1.6

0.0

1.6

500 mg/kg bw

1.7

0.1

1.8

750 mg/kg bw

1.2

0.0

1.2

CPP (20 mg/kg bw)

10.9

0.2

11.1 (p≤0.01)

VCR (0.15 mg/kg bw)

35.7

11.5

47.2 (p≤0.01)

Table 3. Mean number of PCEs containing MN per 1,000 PCE at 48 hrs (differentiation between small and large micronuclei) 

 

Small

Large

Total

Vehicle

0.7

0.0

0.7

750 mg/kg bw

1.0

0.0

1.0

Applicant's summary and conclusion

Conclusions:
The substance (E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one (CAS 79-77-6), was tested negative (non-clastogenic) in a bone marrow micronucleus assay using male NMRI mice. The study was performed according to OECD TG 474 (1997).
Executive summary:

The read across substance, i.e., (E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one (CAS 79-77-6), was tested for chromosomal damage (clastogenicity) and for the ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. The test was performed according to OECD TG 474 (1997). Test doses were selected based on an initial experiment. In this pretest for determination of the acute i.p. toxicity, deaths were observed at 2000 mg/kg bw. Mice treated with 750 and 1000 mg/kg bw showed evident signs of toxicity, and some animals were sacrificed moribund. At 500 mg/kg bw, all animals survived, but l signs of clinical toxicity were noted. There were no distinct differences in the symptoms between males and female mice. Thus, only males were used for the cytogenetic investigations. Doses of 750, 500 and 250 mg/kg bw were selected for the main test. The test substance was dissolved in olive oil and was administered once intraperitoneally to male NMRI mice (5 mice/dose) at 0 (VC), 250, 500 and 750 mg/kg bw. Additional 5 mice were injected with the vehicle or 750 mg/kg of test substance served as a satellite group. Samples of the bone marrow of the 2 femurs were taken 24 (treatment groups) and 48 hrs (satelite groups) after the last treatment. 2000 polychromatic erythrocytes (PCEs) from each animal of every test group were investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) were also scored. The ratio of polychromatic to normochromatic erythrocytes was determined. After administration of the vehicle, test substance and positive controls, the animals were examined for clinical signs of toxicity. U-test according to Mann-Whitney (modified rank test according to Wilcoxon) was performed to confirm differences between control and dose groups. Results: No mortality was observed at any dose level. Clinical signs of toxicity were observed at 500 and 750 mg/kg bw, which included poor general state, irregular respiration, squatting posture. These clinical signs were reversible after 2 days. At 250 mg/kg bw only minor signs of clinical toxicity were observed after 2 and 4 hours of administration of the test substance (squatting posture). No inhibition of erythropoiesis, determined from the PCE/NCE ratio, was detected. The vehicle and the positive control substances, Cyclophosphamide (CPP, 20 mg/kg bw) and vincristine (VCR, 0.15 mg/kg bw), caused no evident signs of toxicity. The following mean number of PCEs and NCEs were observed: vehicle (PCEs: 10000, NCEs: 4594 (24 hrs), 3439 (48 hrs)), at 250 mg/kg bw (PCEs: 10000, NCEs:3755), at 500 mg/kg bw (PCEs: 10000, NCEs: 4646), at 750 mg/kg bw (PCEs: 10000, NCEs: 2476 (24 hrs), 2804 (48 hrs), CPP (PCEs: 10000, NCEs: 3920), VCR (PCEs: 10000, NCEs: 5374). The administration of the test substance did not lead to any statistically significant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was nearly the range of the concurrent negative control in all dose groups and within the range of the historical control data (mean 1.6, min. 0.3, max. 3.3, SD 0.6; n=393). The positive controls led to the expected increases in micronuclei (either small or large). The mean number of PCEs containing MN per 1,000 PCE at 24 hrs (differentiation between small and large micronuclei) were the follows: vehicle (small: 1.3, large:0.1, total: 1.4), at 250 mg/kg bw (small: 1.6, large: 0.0, total: 1.6), at 500 mg/kg bw (small: 1.7, large: 0.1, total: 1.8), at 750 mg/kg bw (small: 1.2, large: 0.0, total: 1.2), CPP (small: 10.9, large: 0.2, total: 11.1; p ≤0.01), VCR (small: 35.7, large: 11.5 total: 47.2; p≤0.01). The mean number of PCEs containing MN per 1,000 PCE at 48 hrs (small and large and total number of micronuclei) were the follows: vehicle (small: 0.7, large: 0.0, total: 0.7) at 750 mg/kg bw (small: 1.0, large: 0.0, total: 1.0). Conclusion: The source substance, i.e., (CAS 79-77-6), did not have a chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.