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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Sep 2001 - 04 Nov 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002
Reference Type:
publication
Title:
A re-assessment of styrene-induced clastogenicity in mice in a subacute inhalation study.
Author:
Engelhardt, G. et al.
Year:
2003
Bibliographic source:
Arch Toxicol 77: 56-61
Reference Type:
secondary source
Title:
European risk assessment report, Styrene CAS No. 100-42-5, EINECS No. 202-851-5, Draft for submission to SCHER, November 2007.
Author:
European Union
Year:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Inhalation exposure for up to 14 days according to OECD Guideline 412.
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesanstalt für Pflanzenbau und Pflanzenschutz Rheinland-Pfalz
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Styrene
EC Number:
202-851-5
EC Name:
Styrene
Cas Number:
100-42-5
Molecular formula:
C8H8
IUPAC Name:
ethenylbenzene
Details on test material:
- Name of test material (as cited in study report): styrene
- Physical state: clear colourless liquid
- Analytical purity: > 99.9%
- Purity test date: 11 Jul 2001, 30 Oct 2001
- Lot/batch No.: Tank 21/22
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: mean weight of 32.6 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: singly in wire cages
- Diet (e.g. ad libitum): rodent laboratory diet, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Switzerland) free from contaminants
- Water (e.g. ad libitum): tap water free from contaminants
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 18 Sep 2001 To: 17 Oct 2001

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
activated-charcoal-filtered air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass-steel inhalation chamber (1.4 m3)
- Method of holding animals in test chamber: cage
- Source and rate of air: ca. 28 m3/h
- System of generating particulates/aerosols: Piston metering pumps (Sarstedt DESAGA), Glass vaporizers with thermostat (BASF AG)
- Temperature, humidity, pressure in air chamber: 22 ± 2 °C, 50 ± 20%, slight negative pressure


TEST ATMOSPHERE
- Brief description of analytical method used: calibrated gas chromatographic analyses
- Samples taken from breathing zone: yes, 2 measured samples per concentration and exposure


OTHER:
During exposure, food and water were withdrawn.
Duration of treatment / exposure:
6 h/day
Frequency of treatment:
one, three, seven, 14 or 21 exposures
Post exposure period:
Animals were killed immediately after the end of the daily exposure by cervical dislocation.
Doses / concentrations
Remarks:
Doses / Concentrations:
0.757 ± 0.0318 and 1.508 ± 0.0593 mg/L
Basis:
analytical conc.
No. of animals per sex per dose:
5 per exposure group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneally, once
- Doses / concentrations: 20 mg/kg bw
Animals were sacrifieced 24 hours after treatment.

Examinations

Tissues and cell types examined:
polychromatic and normochromatic erythrocytes from the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Reproduction of an experiment reported by Koskinen et al. (2000) and Vodicka et al. (2001).


DETAILS OF SLIDE PREPARATION:
The two femora from the animal were prepared. After cutting of the epiphyses, the bone marrow was flushed out into a centrifigation tube using prewarmed fetal calf serum (FCS). The suspension was mixed, centrifugated, the supernatant removed and the pellet resuspended in fresh FCS. One drop of the resulting suspension was dropped on clean microscopic slides and smears prepared. The air-dried slides were stained with modified May Grünwald and Giemsa solution.


METHOD OF ANALYSIS:
In general, 2000 polychromatic erythrocytes (PCEs) from each animal from each test group were microscopically evaluated and investigated for micronuclei (MN). Thenormochromatic erythrocytes (NCEs) with and without MN were also scored.
Ratio of PCEs to NCEs.
Number of small and large MN.
Evaluation criteria:
Positive:
- Dose-related and significant increase in the number of PCEs with MN at any of the intervals.
- The proportion of cells containing MN exceeded both the values of the current negative control and the negative historical control range.
Statistics:
one-sided Wilcoxon test

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Test group [mg/L]

Sampling time [days]

No. of animals

No. of PCEs

No. of NCEs

Micronuleated PCEs/2000 PCEs [Group mean ± SD]

Air control

1

5

10000

5202

1.3 ± 1.52

0.75

5

10000

4391

2.5 ± 3.24

1.5

5

10000

5109

2.4 ± 1.10

Cyclophosphamide

5

10000

4451

20.7 ± 14.74

Air control

3

5

10000

4543

2.6 ± 2.28

0.75

5

10000

5763

2.9 ± 2.86

1.5

5

10000

6580

2.7 ± 1.14

Cyclophosphamide

5

10000

4829

17.2 ± 6.73

Air control

7

5

10000

4914

2.9 ± 0.45

0.75

5

10000

4531

2.9 ± 2.17

1.5

5

10000

4555

3.2 ± 2.79

Cyclophosphamide

5

10000

3991

19.2 ± 11.78

Air control

14

5

10000

5237

3.0 ± 1.87

0.75

5

10000

4458

2.5 ± 1.00

1.5

5

10000

4987

1.7 ± 1.34

Cyclophosphamide

5

10000

3797

17.3 ± 5.55

Air control

21

4

8000

4423

3.0 ± 3.37

0.75

5

10000

5108

1.7 ± 1.95

1.5

5

10000

5850

2.1 ± 3.42

Cyclophosphamide

5

10000

4964

17.5 ± 15.8

After exposure to 1.5 mg/L lethality was observed in7 of 35 animals between study days 2 and 6. Nonspecific clinical symptoms indicative of some irritation of the respiratory tract and systemic toxicity were also observed in this treatment group. The body weight gain of the animals was depressed in relation to the concentration, most prominently during the first days (3 - 6) of exposure.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative