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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 22 to August 28, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented guideline study according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: 2-hexyl-1-decanol
- Substance type: pure active substance
- Physical state: liquid
- Analytical purity: 98.6 %
- Lot/batch No.: 03507
- Expiration date of the lot/batch: 2012-04-01
- Storage condition of test material: room temperature, protected from moisture and light

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from S9 fraction obtained from male rats, dosed with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
First test:
-S9 mix: 16, 22 and 24 µg/ml
+S9 mix: 25, 65 and 80 µg/ml
Second test:
-S9 mix: 9, 15 and 17 µg/ml
+S9 mix: 120, 130 and 150 µg/ml
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix: mitomycin C; +S9 mix: Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
1. Experiment: -S9/+S9: 3 hours
2. Experiment: -S9: 21 hours; +S9: 3 hours
Harvesting and fixation: Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid. After 2 hours incubation , each cells suspension was centrifuged. The cell pellets were treated with hypotoic solution , incubated for 10 minutes, centrifuged and fixed by addition of cold fixative.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Other: mitotic index
Evaluation criteria:
The assay is considered accepltable if the solvent and positive control values lie within the current histrorical control range.

The test substace is considerd to cause a positive response if the following conditions are met:

- Statistically significant increases (P<0.01) in the frequency of methaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
- The increases exceed the solvent control range of this laboratory, taken at the 99% confidence limit.
- The increases are reproducible between replicate cultures.
- The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the numer of aberrant cells above concurrent control frequencies are observed, at any concentration.
Statistics:
The number aberrant metaphase cells in each test substance group was compared with the solvent control valueusing the one-tailed Fisher exact test.
A Colchran-Armitage test for trend was applied to the control and all test sbstance groups. If this is significant at the !% level, the test is reiterated excluding the highest concentration - this process continues until the trend test is no longer significant.

D20s (the minimum concentration (mg/ml) at which aberrations were found in 20% of metaphases) were estimated using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero. The following model was used

p = C + [1-C/(1+ exp(-intercept-slopeln(conc)))]


P is the proportion of cells with aberrations, conc is the concentration of the test substance. C is a parameter estimating the control proportion of aberrations.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no changes in pH of more than 1.0 unit at doses up to 2424.5 µg/ml
- Effects of osmolality: no fluctuations in osmolality of more than 50 mOsm/kg at doses up to 2424.5 µg/ml
- Precipitation: -S9: At treatment a visible oily film was present at final concentrations of 872.82 µl/ml and above. After 3 hours treatment an oily layer was still present at final concentrations of 188.53 µg/ml and above. +S9: At treatment a visible oily film was present at final concentrations of 872.82 µl/ml and above. After 3 hours treatment an oily layer was still present at final concentrations of 314.22 µg/l and above.

Any other information on results incl. tables

Test 1
Exposure period S9 mix
(v/v)
Nominal concentration of test substance (µg/ml Cells with aberrations excluding gaps Cells with aberrations including gaps Relative mitotic index (%) Polyploid mean incidence (%)
Individual values (%) Mean (%) Individual values (%) Mean (%)
3 - 0 (DMSO) 0.0 1.0 0.5 0.0 1.0 0.5 100 0.0
16 0.0 0.0 0.0 0.0 1.0 0.5 95 0.5
22 0.0 0.0 0.0 2.0 0.0 1.0 60 1.0
24 2.0 2.0 2.0 2.0 3.0 2.5 47 0.5
0.2 (Mitomycin) 14.7 14.7 14.7*** 17.6 16.2 16.9*** - 0.5
3 + 0 (DMSO) 0.0 0.0 0.0 2.0 1.0 1.5 100 0.5
(2%) 25 0.0 0.0 0.0 2.0 0.0 1.0 92 1.5
65 0.0 0.0 0.0 0.0 1.0 0.5 67 3.0
80 1.0 2.0 1.5 1.0 2.0 1.5 48 4.0
5 (Cyclophosphamide) 19.2 23.3 21.1*** 19.2 23.3 21.1*** - 0.0
One tailed Fisher's exact test
*** p<0.001
Otherwise p>=0.01
Test 2
Exposure period S9 mix
(v/v)
Nominal concentration of test substance (µg/ml Cells with aberrations excluding gaps Cells with aberrations including gaps Relative mitotic index (%) Polyploid mean incidence (%)
Individual values (%) Mean (%) Individual values (%) Mean (%)
21 - 0 (DMSO) 2.0 1.0 1.5 3.0 1.0 2.0 100 1.5
9 1.0 1.0 1.0 2.0 1.0 1.5 90 0.5
15 0.0 3.0 1.5 1.0 3.0 2.0 73 0.0
17 3.0 2.0 2.5 4.0 4.0 4.0 50 0.0
0.1 (Mitomycin) 25.0 45.5 32.3*** 30.0 45.5 35.5*** - 0.0
3 + 0 (DMSO) 1.0 0.0 0.5 1.0 1.0 1.0 100 0.0
(5%) 120 2.0 1.0 1.5 3.0 1.0 2.0 105 2.0
130 1.0 0.0 0.5 2.0 0.0 1.0 82 0.0
150 1.0 1.0 1.0 2.0 1. 1.5 48 2.0
5 (Cyclophosphamide) 17.2 17.2 17.2*** 19.0 19 19.0*** - 0.0
One tailed Fisher's exact test
*** p<0.001
Otherwise p>=0.01

Applicant's summary and conclusion

Conclusions:
2-Hexyldecan-1-ol has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.
Executive summary:

2-Hexyldecan-1-ol has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.