Isotridecan-1-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: neg (BASF 1989) - same results for all three RA substances

HPRT: neg: (BASF 2011) - data obtained for propylheptanol

CA: neg (Fraunhofer 1993) - data obtained for CAS 25339 -17 -7

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only four strains tested; 2-aminoanthracene was the sole indicator of S9 efficancy)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his-gene

Species / strain / cell type:

other: Salmonella typhimurium TA1535, TA100, TA1537, TA98

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix (rats pretreated with Aroclor 1254)

Test concentrations with justification for top dose:

20 µg - 5000 µg/plate (SPT), 20 µg - 5000 µg/plate (PIT)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see: Details on test system and conditions

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

Standard plate test
-------------------
The experimental procedure is based on the method of Ames et al. (1973, 1975).
Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6% agar + 0.6 % NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:

0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test
-------------------
The experimental procedure is based on the method described by Yahagi et al. (1977) and Matsushima et al. (1980)

0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.

After incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

Positive controls
-----------------
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:

with S-9 mix:
10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535

without S-9 mix:
5 µg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537

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:

other: Salmonella typhimurium TA1535, TA100, TA1537, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance was completely soluble in DMSO.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

1st/2nd experiment, Standard plate test (20 - 5000 µg/plate)
Strain	Metabolic activation system	mean his^+revertant colonies (negative control)	maximum revertant factor (conc. (µg/plate))	dose dependency	Assessment	maximum revertant factor (positive control)
TA 98	no	23	1.2 (2500)	no	negative	29 (NPD)
	yes	34	1.1 (100/500)	no	negative	34.7 (2-AA)
TA 100	no	114	1.0 (20/100)	no	negative	14.8 (MNNG)
	yes	103	1.2 (100)	no	negative	17.2 (2-AA)
TA 1537	no	9	1.1 (20)	no	negative	186.7 (AAC)
	yes	10	1.0 (500)	no	negative	17.6 (2-AA)
TA 1535	no	16	1.4 (500)	no	negative	87.3 (MNNG)
	yes	18	1.1 (5000)	no	negative	8.4 (2-AA)
3rd experiment, Preincubation test (20 - 5000 µg/plate)
Strain	Metabolic activation system	mean his^+revertant colonies (negative control)	maximum revertant factor (conc. (µg/plate))	dose dependency	Assessment	maximum revertant factor (positive control)
TA 98	no	24	1.1 (20/100)	no	negative	39.2 (NPD)
	yes	35	1.1 (100/500)	no	negative	40.2 (2-AA)
TA 100	no	125	0.9 (100/500)	no	negative	9.8 (MNNG)
	yes	111	1.1 (20)	no	negative	16.4 (2-AA)
TA 1537	no	9	1.2 (100)	no	negative	48.6 (AAC)
	yes	12	1.1 (20)	no	negative	14.5 (2-AA)
TA 1535	no	18	1.2 (2500)	no	negative	86.4 (MNNG)
	yes	21	1.0 (2500/5000)	no	negative	7.8 (2-AA)
2-AA: 2-aminoanthracene
MNNG: N-methyl-N'-nitro-N-nitroso-guanidine
NPD: 4-nitro-o-phenylendiamine
AAC: 9-aminoacridine chloride monohydrate

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham´s F12 medium supplemented with penicillin/streptomycin and amphotericine B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and beta-naphthoflavone.

Test concentrations with justification for top dose:

Experiment 1:
Without S9 mix: 3.1, 6.3, 12.5, 25, 50, 100 µg/mL (4 h)
With S9 mix: 3.1, 6.3, 12.5, 25, 50, 100 µg/mL (4 h)

Experiment 2:
Without S9 mix: 3.1, 6.3, 12.5, 25, 50, 100 µg/mL (24 h)
With S9 mix: 10, 20, 40, 60, 80, 100 µg/mL (4 h)

Experiment 3:
With S9 mix: 2.5, 5, 10, 20, 40, 60, 80 µg/mL (4 h)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was selected as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available. The final concentration of the vehicle DMSO in the culture medium was 1% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: 300 μg/mL ethyl methanesulfonate (EMS) With metabolic activation: 20 μg/mL methylcholanthrene (MCA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 7-9 days
- Selection time: 6-7 days

SELECTION AGENT (mutation assays): 6-Thioguanine (6-TG)

NUMBER OF REPLICATIONS: Duplicate cultures


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (CE)

Evaluation criteria:

Acceptance criteria:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0 – 15.95 mutants per 10E06 clonable cells.
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies.
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment criteria:
A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10E06 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control were observed in all experiments in the absence and the presence of S9 mix at least in the highest applied concentrations.

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: pH value was not influenced by test substance treatment.
- Effects of osmolality: Osmolality was not influenced by test substance treatment.
- Precipitation: In the absence and the presence of S9 mix no precipitation in culture medium was observed up to the highest applied test substance concentration.

RANGE-FINDING/SCREENING STUDIES:
Without S9:
4 h treatment: reduced CE at 25 µg/mL (67%); CE of 0% at 50 µg/mL
24 h treatment: reduced CE at 50 µg/mL (14%); CE of 0% at 100 µg/mL

With S9:
4 h treatment: reduced CE at 50 µg/mL (18%); CE of 0% at 100 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA:
The solvent control and positive control data were within the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In detail, without S9 mix, there was a strong decrease in the number of colonies from 50 μg/mL (CE1 relative: 0.0%) onward after an exposure period of 4 hours in the 1st experiment. The cell densities were distinctly reduced. In the 2nd experiment after an exposure period of 24 hours cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% relative survival was observed at 50 μg/mL (CE1 relative: 27.8%) and above. In addition, with S9 mix, there was a strong decrease in the number of colonies at 100 μg/mL (CE1 relative: 0.0%) in the 1st experiment and from 60 μg/mL (CE1 relative: 1.9% and 0.5%) onward in the 2nd and 3rd experiment, respectively. The cell densities were distinctly reduced. Although using dilution steps of 2 or less in all experiments in the absence and presence of S9 mix due to steep test substance cytotoxicity cloning efficiency values in the range of 10 to 20% were not obtained.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Results of experiment I (4 h exposure - without metabolic activation).

Concentration [µg/mL]	Cloning efficiency after treatment [%]	Cloning efficiency after expression time [%]	Mutants per 1E+06 surviving cells
0 (DMSO)	100	100	6.84
3.1	99	103	4.70
6.3	98	104	2.21
12.5	106	100	6.01
25	100	94	3.82
50	0	n.c.	n.c.
100	0	n.c.	n.c.
EMS, 300 µg/mL	69	82	65.51

 

Table 2: Results of experiment I (4 h exposure - with metabolic activation).

Concentration [µg/mL]	Cloning efficiency after treatment [%]	Cloning efficiency after expression time [%]	Mutants per 1E+06 surviving cells
0 (DMSO)	100	100	6.83
3.1	106	94	3.13
6.3	115	105	6.67
12.5	110	102	3.64
25	89	98	8.61
50	97	96	4.56
100	0	n.c.	n.c.
MCA, 20 µg/mL	112	96	80.97

 

Table 3: Results of experiment II (24 h exposure - without metabolic activation).

 

Concentration [µg/mL]	Cloning efficiency after treatment [%]	Cloning efficiency after expression time [%]	Mutants per 1E+06 surviving cells
0 (DMSO)	100	100	1.33
3.1	93	84	6.34
6.3	83	83	1.22
12.5	88	89	3.36
25	87	84	2.79
50	28	96	4.36
100	0	n.c.	n.c.
EMS, 300 µg/mL	55	55	674.02

 

Table 4: Results of experiment III (4 h exposure - with metabolic activation).

Concentration [µg/mL]	Cloning efficiency after treatment [%]	Cloning efficiency after expression time [%]	Mutants per 1E+06 surviving cells
0 (DMSO)	100	100	0.41
2.5	104	112	1.08
5	95	105	2.79
10	102	102	4.85
20	93	103	0.80
40	92	104	1.15
60	0.5	n.c.	n.c.
80	0	n.c.	n.c.
MCA, 20 µg/mL	98	106	96.08

 n.c.: Culture was not continued due to strong cytotoxicity

 

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-10 medium (+ FCS, + streptomycin, + penicillin)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat iver S-9 mix

Test concentrations with justification for top dose:

without S9- mix, fixation time 21 h after treatment start: 5, 10 and 20 µg Isodecanol/ml;
with S9-mix, exposure 3 h, fixation time 18 h after treatment start: 5, 10 and 20 µg Isodecanol/ml;
with S9-mix, exposure 3 h, fixation time 21 h after treatment start: 5, 10 and 20 µg Isodecanol/ml;

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
Ethylmethanesulfonate: DMSO;
Cyclophosphamide: medium (Ham's F12);
test substance: ethanol;

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethanesulonate (500 µg/ml), and Cyclophosphamide (5 µg/ml)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

Isodecanol was soluble in ethanol, but precipitated at a dose of more than 100 µg/ml in culture medium. A stock dilution of Isodecanol was prepared by dissolving it in ethanol. From such a stock dilution all required concentrations were made (Stock dilution: 20 µl and 380 µl ethanol = 4.5 µg Isodecanol/µl.

Approximately 18-20 hours before treatment an appropriate number of flasks for the experiment were prepared from a single pool of cells. Each 25 sq. cm flask was seeded with 300000 cells in supplemented Ham's F10 medium. These cells should be in exponential growth phase at the time of treatment.
Treatment without metabolic activation:
The culture medium was removed, fresh culture medium and Isodecanol added to the cultures for 21 hours. The cells were thus treated for a period of 1-2 cell cycles, which should ensure a yield of first post-treatment mitoses.

Treatment with metabolic activation:
In order to avoid toxicity on the cells, treatment with S9-mix and Isodecanol was performed for a period of three hours. During this incubation time the culture medium was serum free.Then the medium was removed and the cells washed twice. As treatment did not cover the length of one whole cell cycle, two fixation times were chosen to sample cells, which were in different stages on cell cycle during treatment; the following times were chosen: 18 and 21 hours after treatment commenced.

Harvesting and Slide preparation:

2.5 hours before the end of the incubation period, colcemid (0.25 µg/ml medium) was added to each culture, and, after gentle mixing, the cultures were returned to the incubator. At the end of the incubation period, the medium was removed from the flasks, and the cells were brought into suspension with trypsin/EDTA. These cell suspensions were centrifuged at ca. 300 g for 8 min. The supernatant was discarded and the pellet of cells resuspended in hypotonic solution freshly prepared. The tubes were placed in a water bath at 37°C for 15 min. Then the centrifugation process was repeated and the supernatant discarded. The cells volume suspended by dropwise addition of fixative up to a total of approximately 10 ml; fixative = ethanol and acetic acid, 3: 1. The centrifugation procedure was repeated two times, each time discarding the old fixative and resuspending the cells in fresh fixative . After the final addition of fixative, the tubes were left standing in an upright position for at least 30 min. The tubes were then centrifuged as above, the fixative discarded and the cells resuspended in sufficient fixative, e.g. 0.2 - 0.5 ml, to give a slightly cloudy suspension. 2 - 3 drops of the suspension were dropped onto a microscope slide previously soaked and cooled to 4°C, from about 30 - 50 cm above the slide. Two - four slides were made from each culture, and left to air dry. The slides were stained the day after preparation according to conventional methods with Giemsa stain (5- 10 % Giemsa solution, freshly prepared and filtered).


SPINDLE INHIBITOR (cytogenetic assays): Colcemid (10 µg colcemid/ml; this concentration was added 2.5 hours before the end of the incubation time;)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: two experiments were performed;

NUMBER OF CELLS EVALUATED: 100 well spread metaphases per treatment group and experiment were examined for structural chromosome aberrations;

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Generally, the interpretation of a result as positive or negative is supported on statistical evaluation, but up to now no unequivocal statistical methods have been developed for evaluating chromosomal aberrations. This situation is due to the differences in ranking the types of aberrations. Gaps are ranked lowest and exchanges are ranked highest. Pulverisation or shattering of chromosomes is assumed to indicate a toxic effect rather than a clastogenic one.
It is generally accepted that a substance has chromosome damaging activity when parallel cultures at one dose level repeatedly produce aberrations in more than 10 % of analyzed cells (gaps excluded). Dose dependency may provide further evidence for clastogenicity. If only gaps are increased, and this in single test group without any dose relation, the result can be considered to be negative; in this case gaps express cytotoxicity rather than genotoxic effects because there is no unequivocal mechanistic explanation for the origin of gaps. It is difficult to estimate the background level of exchanges and endoreplication. But in most of these cases there is also a similar increase in gaps and breaks.

Statistics:

A statistical analysis was not done.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(without S-9: at 20 µg/ml; with S-9: at 10 µg/ml))

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:

The cytotoxicity was estimated by measuring the Mitotic Index of 5000 cells at each dose with and without S9-mix.

Stock dilution: 20 µI and 980 µl ethanol = 16.8 µg Isodecanol/µl. Cultures with and without S9-mix were carried out with the concentrations: 100, 80, 60, 40, 20 and 10 µg Isodecanol/ml.

In cultures without S9-mix the cells were exposed to the test substance for 21 hours. Then the cells were harvested for slide preparation.
The cells in S9-mix activated cultures were exposed to the test substance in medium without FCS for 3 hours. Then the medium was removed, cells washed twice with prewarmed medium (Ham's F10) before replenishment with fresh culture medium; cell harvesting for slide preparation was 21 hours after treatment start.

Non-activated assay:
Treated cultures with 20 µg Isodecanol/ml there was an approximately 56% reduction of mitoses; treated cultures with 80 µg/ml or more no metaphases could be observed. Therefore the main study was limited with the following concentrations: 5, 10 and 20 µg Isodecanol/ml.
Activated assay:
Treated cultures with 20 µg Isodecanol/ml, there was an approximately 55% inhibition of mitoses; treated cultures with 60 µg/ml or more no metaphases could be observed. Therefore the main study was limited with the following concentrations: 5, 10 and 20 µg lsodecanol/ml.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

without S9 mix, fixation time: 21 h after treatment start
	Replicate No.	Chromatid			Chromosome			Others	Total numbers of Aberrations		Cells with Aberrations
		Chromatid Gap	Chromatide Break or Fragment	Chromatide Type exchange	Chromosome Gap	Chromosome Break or Isochromatide Fragment	Chromosome Type exchange		+ Gaps	- Gaps	+ Gaps	- Gaps
negative control (ethanol)	A	4	---	1	---	---	---	---	5	1	4	1
	B	8	1	---	---	---	---	---	9	---	8	---
	mean (%)	6	0.5	0.5	0	0	0	0	7	0.5	6	0.5
500 µg/ml EMS	A	17	2	2	5	14	---	---	40	21	30	19
	B	21	---	2	3	4	---	1 DI	31	10	24	9
	mean (%)	19	1	2	4	9	0	0.5	35.5	15.5	27	12.5
5 µg/ml	A	5	1	---	---	---	---	---	6	---	6	---
	B	2	---	---	1	---	---	---	3	1	3	1
	mean (%)	3.5	0.5	0	0.5	0	0	0	4.5	0.5	4.5	0.5
10 µg/ml	A	6	---	---	---	---	---	---	6	---	6	---
	B	8	1	---	---	---	---	---	9	---	8	---
	mean (%)	7	0.5	0	0	0	0	0	7.5	0	7	0
20 µg/ml	A	4	---	---	---	---	---	---	4	---	4	---
	B	3	---	---	---	---	---	1 DI	4	1	3	1
	mean (%)	3.5	0	0	0	0	0	0.5	4	0.5	3.5	0.5
DI = Dicentric (+ fragment)
with S9 mix (3 h), fixation time: 18 h after treatment start
	Replicate No.	Chromatid			Chromosome			Others	Total numbers of Aberrations		Cells with Aberrations
		Chromatid Gap	Chromatide Break or Fragment	Chromatide Type exchange	Chromosome Gap	Chromosome Break or Isochromatide Fragment	Chromosome Type exchange		+ Gaps	- Gaps	+ Gaps	- Gaps
negative control (ethanol)	A	11	---	---	---	---	---	1 E	12	1	10	1
	B	10	1	---	---	1	---	1 E	13	2	13	2
	mean (%)	10.5	0.5	0	0	0.5	0	1	12.5	1.5	11.5	1.5
5 µg/ml CP	A	15	2	3	4	10	---	1 DI	35	18	24	14
	B	15	---	1	11	12	---	---	39	24	24	17
	mean (%)	15	1	2	7.5	11	0	0.5	37	21	24	15.5
5 µg/ml	A	7	1	1	---	---	---	---	9	1	9	1
	B	7	---	---	1	---	---	---	8	1	8	1
	mean (%)	7	0.5	0.5	0.5	0	0	0	8.5	1	8.5	1
10 µg/ml	A	15	---	---	---	2	---	---	17	2	133	2
	B	10	---	---	4	2	---	1 DI	17	7	10	3
	mean (%)	12.5	0	0	2	2	0	0.5	17	4.5	11.5	2.5
20 µg/ml	A	9	3	---	3	2	---	---	17	5	13	3
	B	11	1	---	1	---	---	---	13	1	13	1
	mean (%)	10	2	0	2	1	0	0	15	3	13	2
E = Endoreduplication
DI = Dicentric (+ fragment)
with S9 mix (3 h), fixation time: 21 h after treatment start
	Replicate No.	Chromatid			Chromosome			Others	Total numbers of Aberrations		Cells with Aberrations
		Chromatid Gap	Chromatide Break or Fragment	Chromatide Type exchange	Chromosome Gap	Chromosome Break or Isochromatide Fragment	Chromosome Type exchange		+ Gaps	- Gaps	+ Gaps	- Gaps
negative control (ethanol)	A	10	---	---	---	---	---	---	10	0	10	0
	B	7	---	---	1	1	---	---	9	2	7	2
	mean (%)	8.5	0	0	0.5	0	0	0	9.5	1	8.5	1
5 µg/ml CP	A	8	---	3	1	13	1	---	26	18	19	12
	B	13	---	---	4	31	1	1 DI	50	37	35	26
	mean (%)	10.5	0	1.5	2.5	22	1	0.5	36.5	27.5	27	19
5 µg/ml	A	10	---	---	---	1	---	---	11	1	8	1
	B	7	---	1	4	---	---	1 DI	13	6	11	5
	mean (%)	8.5	0	0.5	2	0.5	0	0.5	12	3.5	9.5	3
10 µg/ml	A	1	---	---	---	1	---	---	2	1	2	1
	B	10	2	---	1	2	---	3 E	18	6	13	6
	mean (%)	5.5	1	0	0.5	1.5	0	1.5	10	3.5	7.5	3.5
20 µg/ml	A	11	---	---	2	1	---	---	14	3	12	3
	B	9	2	2	2	1	---	---	16	5	13	4
	mean (%)	10	1	1	2	1	0	0	15	4	12.5	3.5

	Mitotic indices
	without S-9	with S-9
negative control	83	134.4
positive control	81.4	136.4
10 µg/ml	88.6	75.6
20 µg/ml	36	74

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Mutagenicity was only tested with iso-Tridecan-1-ol in bacteria, but no data are available for cytogenicity and gene mutation in mammalian cells. For these endpoints data were adopted by read-across from related branched long chain primary alcohols, i.e., propylheptanol (CAS 10042-59-8) and iso-Decanol (CAS 25339-17-7). A detailed justification for this approach has been attached to IUCLID chapter 13.

Gene mutation in bacteria:

Iso-Tridecan-1-ol was not mutagenic in a reliable preincubation and standard plate Ames test (according to OECD 471 and GLP) with and without metabolic activation (tested up to 5000 µg/plate in Salmonella typhimurium TA 98, TA 100, TA 1535, and TA 1537; metabolic activation: rat liver S-9 mix (rats pretreated with Aroclor 1254); BASF, 1989). No cytotoxicity (reduction of the background lawn) was observed.

To support the read across approach, mutagenicity data have also been included for the RA substances:

Propylheptanol also gave a negative result in the Ames assay when tested with or without metabolic activation in TA 1535, 1537, 98, 100, and e.coli up to 5000µg/plate (Evonik 2009).

Isodecanol did not cause an increase in revertants of S. typhimurium strains TA 1535, 1537, 98, and 100 with or without rat liver S9 (BASF 1989). Plates in the standard plate test were treated with up to 5000µg/plate. In the pre-incubation assay, concentrations were reduced 2500 or 250µg/plate depending on the strain due to cytotoxicity.

Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were treated with Isononanol following the Ames test plate incorporation method as well as the pre-incubation method (Infracor 1986). Dose levels of 10 to 5000 µg/plate with or without the addition of S9 mix gave no indication of a mutagenic activity.

Cytogenicity in mammalian cells:

A reliable chromosomal aberration test in Chinese hamster fibroblasts with and without metabolic activation (metabolic activation: Aroclor 1254-induced rat liver S-9 mix) was conducted with iso-Decanol (CAS: 25339-17-7). Test concentrations of 5, 10 and 20 µg/ml (without S9-mix: exposure time: 21 h, fixation time 21 h after treatment start; with S9-mix: exposure time: 3 h, fixation time 18 h or 21 h after treatment start) did not lead to an increase in mutant colonies (Fraunhofer Institute, 1993).

Gene mutation in mammalian cells:

Propylheptanol (99.8%) was tested in a reliable HPRT-test (according to OECD TG 476 and GLP) with Chinese hamster ovary cells (CHO) with and without metabolic activation (BASF 2011). Test concentrations ranged from 2.5 to 100 µg/mL for 4 and 24 h treatments, respectively. No increase of the mutant frequency was observed. Solvent control and positive control results were valid and were within the historical control data. Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control were observed in all experiments in the absence and the presence of S9 mix at least in the highest applied concentrations.

Justification for classification or non-classification

No classification according to 1272/2008/EC (CLP) criteria is required, since all assays for gene mutations in bacteria or mammalian cells or for clastogenic activity gave negative results.