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Genetic toxicity in vitro

Description of key information

It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Isoprenol is considered to be non-mutagenic in this HPRT assay.

According to the results of the present study, the test substance 3-Methylbuten-3-ol-1 is not mutagenic in the Ames test under the experimental conditions chosen here.

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.03.2010 - 17.05.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver microsome preparations (S9 mix)
Test concentrations with justification for top dose:
concentrations in μg/mL
Experiment I
without S9 mix: 55.0 110 220 440 880
with S9 mix: 55.0 110 220 440 880

Experiment II
without S9 mix: 55.0 110 220 330 440
with S9 mix: 55.0 110 220 440 880
Vehicle / solvent:
Culture medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
As solvent/vehicle is culture medium, untreated negative controls and negative solvent/vehicle controls are the same.
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent
increase of mutant frequencies using SYSTAT􀂣11 (SYSTAT Software, Inc., 501, Canal
Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Isoprenol is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of Isoprenol to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a

treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The highest concentration of the test item (880 μg/mL) was equal to a molar concentration of about 10 mM.

The tested concentrations are described in Table 2 (page 19). The evaluated experimental points and the results are summarised in Table 1 (page 13).

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-03-03 and 1989-03-09 (testing days)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without GLP, four strains only.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9
Test concentrations with justification for top dose:
20, 100, 500, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine chloride monohydrate
Remarks:
positive control substance depending on tester strain and activation condition
Details on test system and experimental conditions:
Two independent experiments were carried out.

1st Experiment:
METHOD OF APPLICATION: in agar (plate incorporation)
Standard plate incorporation test with and without metabolic activation with S-9 mix prepared from liver homogenate of Aroclor 1254-pretreated Sprague-Dawley rats.
TEST CONCENTRATION: 0, 20, 100, 500, 2500 and 5000 µg/plate
SOLVENT: DMSO
NUMBER OF REPLICATIONS: 3 test plates per dose or per control
DETERMINATION OF CYTOTOXICITY
- Method: reduced his- background growth, decrease in the number of his+ revertants

2nd Experiment
METHOD OF APPLICATION: preincubation;
Preincubation test with and without metabolic activation with S-9 mix prepared from liver homogenate of Aroclor 1254-pretreated Sprague-Dawley rats.
TEST CONCENTRATION: 0, 20, 100, 500, 2500 and 5000 µg/plate
SOLVENT: DMSO
NUMBER OF REPLICATIONS: 3 test plates per dose or per control
DETERMINATION OF CYTOTOXICITY
- Method: reduced his- background growth, decrease in the number of his+ revertants
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 2500 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system. Positive control experiments gave the expected increase in the number of revertants.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Complete solubility of the test substance in DMSO.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect was observed only in the preincubation test without S-9 mix using TA 1537 and TA 98 at doses >= 2500 µg/plate .
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

According to the results of the present study, the test substance 3-Methylbuten-3-ol-1 is not mutagenic in the Ames test under the experimental conditions chosen here.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

It can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow

cells of the mouse. Therefore, Isoprenol is considered to be non-mutagenic in this micronucleus assay.

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Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26.04.2010 - 17.05.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Since no gender specific differences concerning signs of toxicity in the pre-experiment were observed, the main study was performed using males only.
Sex:
male
Details on test animals or test system and environmental conditions:
Age at start of experiment: 8-9 weeks
Mean inital body weight: 39.3 g (SD +/- 2.3 g)

The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type 111111, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
Bedding: granulated soft wood bedding (Rettenmaier & Sohne GmbH + Co. KG, 73494 Rosenberg, Germany)
Feed: pelleted standard diet, ad libitum (Hartan Laboratories B.V. Postbus 6174,5960 AD Horst,The Netherlands)
Water: tap water, ad libitum, (Gemeindewerke, 64380 Rossdorf, Germany)
Environment: temperature 22 +/- 2 GC
relative humidity 45 - 65 %
artificial light 6.00 a.m. - 6.00 p.m.
Route of administration:
oral: unspecified
Vehicle:
Corn oil
Details on exposure:
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test item, the vehicle or the positive control substance once orally.
Frequency of treatment:
Once
Post exposure period:
24 h
Additional sample for the highest dose level at 48 h after treatment.
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
7 males per group (5 for contol groups)
Control animals:
yes, concurrent vehicle
Positive control(s):
5 males, 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control.
Tissues and cell types examined:
Polychromatic erythrocytes
Details of tissue and slide preparation:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald.
At least one slide was made from each bone marrow sample.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
Statistical significance at the five per ceAt level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Therefore, Isoprenol is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

This study was performed to investigate the potential of Isoprenol to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 mLlkg b.w.. 24 hand 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Seven males per test group (except the vehicle and positive control groups with 5 males each) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that Isoprenol

did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Cyclophosphamide as positive control showed a substantial increase of induced micronucleus frequency.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The available information is conclusive but not sufficient for classification.