3-methyl-5-phenylpentanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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:

1999-08-20 to 1999-11-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A GLP compliant study performed to standardised guidelines (OCED 473 and EU Method B.10) with a sufficient level of detail to assess the quality of the presented data. The study was performed on a similar substance to 3-methyl-5-phenylpentanol. The test substance in this study is the aldehyde of 3-methyl-5-phenylpentanol and as such is considered to be sufficient for read-across to address the endpoint.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UK Department of Health Committee on Mutagenicity Guidelines for the Mutagenicity Testing of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

55066-49-4

EC Number:

611-227-5

Cas Number:

55066-49-4

IUPAC Name:

55066-49-4

Constituent 2

Reference substance name:

-

EC Number:

433-900-0

EC Name:

-

IUPAC Name:

433-900-0

Constituent 3

Reference substance name:

3-methyl-5-phenylpentan-1-al

IUPAC Name:

3-methyl-5-phenylpentan-1-al

Test material form:

other: liquid (not specified)

Details on test material:

- Physical state: Liquid, colourless
- Storage condition of test material: room temperature in the dark until 20 August 1999, thereafter room temperature, in the dark, under nitrogen.

Method

Species / strain

Species / strain / cell type:

lymphocytes: human, peripheral

Details on mammalian cell type (if applicable):

- Source: Whole blood was drawn from the peripheral circulation of a volunteer (screened for suitability). The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time study was determined using BrdU (bromodeoxyuridine) incorporation. The number of first, second and third division metaphase cells was determined in order to calculate the average generation time (AGT). The average AGT for donors used by the laboratory was determined as approximately 16.7 hours under experimental growth conditions.
- Culture: Eagle’s minimal essential medium, (supplemented with sodium bicarbonate, HEPES buffer, L-glutamine, penicillin/streptomycin, amphotericin B and 15 % foetal calf serum. Cultures were incubated at 37 °C with 5 % CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide with phytohaemagglutinin (PHA) at 90 µg/mL final concentration.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Microsomal enzyme fraction (S9)

Test concentrations with justification for top dose:

Experiment 1 (4 hour exposure, 20 hour recovery)
- S9: 0, 55, 110, 220, 440, 880 and 1760 µg/mL of test substance with 0.25 µg/mL mitomycin C as the positive control
+ S9: 0, 55, 110, 220, 440, 880 and 1760 µg/mL of test substance with 25 µg/mL cyclophosphamide as the positive control

Experiment 2:
- S9, 24 hour exposure: 0, 6.88, 13.76, 27.3, 55 and 82.5 µg/mL of test substance with 0.4 µg/mL mitomycin C as the positive control
+ S9, 4 hour exposure: 0, 110, 220, 440, 880 and 1760 µg/mL of test substance with 25 µg/mL cyclophosphamide as the positive contr

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours both with and without S9 (with a 20 hour recovery period) or 24 hours without S9
- Expression time (cells in growth medium): 20 hours in the 4 hour exposure period
- Fixation time (start of exposure up to fixation or harvest of cells):

SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (2000 nuclei counted)

Evaluation criteria:

- Mitotic index:
2000 lymphocyte nuclei were counted. The number of metaphase cells was recorded and expressed as the mitotic index and also as a percentage of the vehicle control value.

- Chromosome damage:
The first 100 (where possible) consecutive well-spread metaphases from each culture were counted, and if the cell had 44-48 chromosomes, any gaps, breaks or rearrangements, were noted according to the simplified system recommended in the 1983 UKEMS guidelines for mutagenicity (Savage (1976))

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the vehicle control, where necessary using Fisher’s Exact test.

Results and discussion

Test results

Species / strain:

lymphocytes: human, peripheral

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no observable change in pH with the addition of the test material into the media
- Effects of osmolality: The osmolality did not increase by more than 50 mOSM with the addition of the test material into the media
- Precipitation: An oily precipitate was observed in the range finding study above 880 µg/mL, but not in the main tests (experiments 1 and 2).

RANGE-FINDING/SCREENING STUDIES:
In all except the pulse exposure without S9, there was evidence of toxicity, especially in the 24-hour exposure without S9. Microscopic assessment of the slides prepared from the treatment cultures demonstrated that metaphase cells were present at dose levels up to the 10 mM concentration 1760 µg/mL in the 4(20) hour treatment in the absence and presence of metabolic activation. The maximum dose with metaphases present in the 24 hour continuous exposure was 55 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The controls were found to be in agreement with the historical control data.

Any other information on results incl. tables

Experiment 1

Unlike the preliminary test, no precipitation was observed, however, haemolysis of the blood cultures was observed at and above 440 µg/mL. These differences in observations were not considered to be significant. From the mitotic index data, it was observed that the test substance was more toxic than observed in the preliminary test. There was an increase in mitotic inhibition at 1760 µg/mL in both treatment groups, such that approximately 50 % mitotic inhibition was achieved in both cases with evidence of a toxic dose response relationship.

When assessing the chromosome aberration data, both of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was considered to be satisfactory and the method was operating as expected.

The test substance induced statistically significant increases in the frequency of cells with aberrations in the absence of metabolic activation at the 10 mM dose level only. The aberrations were predominantly chromatid gaps and breaks and there was no excess of chromatid exchange aberrations.

The test substance did not induce a significant increase in the number of polyploidy cells at any dose level in either of the treatment cases.

 

Experiment 2

As in experiment 1, no precipitation was observed. The qualitative assessment of the slides determined (as in experiment 1) that there were scorable metaphases at up to the maximum dose levels tested.

The mitotic index data confirmed the qualitative observations. A dose related inhibition of mitotic index was observed and an approximate 50 % mitotic inhibition was achieved in one or both cultures at 55 µg/mL in the absence of S9 and at 1760 µg/mL in the presence of S9.

The chromosome aberration data indicated that the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method was demonstrated to be working.

The test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the presence or absence of metabolic activation. Small increases in the frequency of cells with aberrations were noted in the absence of metabolic activation. However these were predominantly due to chromatid gaps and breaks and associated with high levels of toxicity and were therefore considered to have no biological significance.

The test substance did not induce a significant increase in the number of polyploidy cells at any dose level in either of the treatment cases.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance did not induce a biologically significant increase in the frequency of cell with chromosome aberrations in either the presence or absence of a liver enzyme metabolising system in either of two separate experiments. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

In a GLP compliant clastogenicity study conducted in accordance with standardised guidelines OECD 473 and EU Method B.10, the clastogenic potential of the test substance was assessed in a chromosome aberration study in human peripheral blood lymphocytes. The test substance in this study was the aldehyde of 3-methyl-5-phenylpentanol and as such is considered representative of the clastogenic effects expected with 3-methyl-5-phenylpentanol. Under the conditions of the test, the test substance was found to be non-clastogenic in human peripheral blood lymphocytes.