Styrene

Genetic toxicity: in vivo

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

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Reference 2

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(2003)

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

Test material information

Constituent 1

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