2-propylheptan-1-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria:
OECD 471 (Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA (pKM101); with and without metabolic activation): negativ (Evonik Oxeno GmbH, 2009; reliable)

Gene mutation in mammalian cells:

OECD 476 with Chinese hamster ovary cells (with and without metabolic activation): negative (BASF, 2011; reliable)

Cytogenicity in mammalian cells:

QSAR (Times): no genotoxic potential (substance was in domain of the system)

OECD 473 in Chinese hamster fibroblasts (with and without metabolic activation) with the structural analogue Isodecanole (CAS 25339 -17 -7) : negative (Fraunhofer Institute, 1993; reliable)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Propylheptan-1-ol
- Physical state: Clear liquid
- Analytical purity: 96.20 area %
- Isomers composition: isomers of C10 alcohols 2.94 area %
- Lot/batch No.: 16.06.2008
- Test substance No.: 0649/82526
- Expiration date of the lot/batch: July 2009
- Storage condition of test material: Room temperature

Target gene:

his- and trp-gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

5 - 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: 2-propylheptan-1-ol was insoluble in water. Its solubility was assessed at 50 mg/mL in dimethyl sulphoxide (DMSO) in which it was dissolved. DMSO was, therefore, used as the vehicle for this study.

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;

First test:

Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 μg/plate), positive control or negative control were placed in glass vessels. The negative control was the chosen vehicle, DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter. Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four nonprecipitating concentrations should be obtained, unless otherwise justified by the Study Director.

Second test:

As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 μg/plate, and seven concentrations were
used in order to ensure that at least four non-toxic concentrations were tested.

Positive controls:

In the absence of S9 mix:
- Sodium azide (in DMSO): 2 μg/plate for strains TA100 and TA1535
- 9-Aminoacridine (in DMSO): 50 μg/plate for strain TA1537
- 2-Nitrofluorene ( in DMSO): 2 μg/plate for strain TA98
- 4-Nitroquinoline-1-oxide (in DMSO): 2 μg/plate for strain WP2 uvrA (pKM101)

In the presence of S9 mix:
- 2-Aminoanthracene (in DMSO): 5 μg/plate for strains TA100 and TA1535, 10 μg/plate for strain WP2 uvrA (pKM101)
- Benzo[a]pyrene: (in DMSO): 5 μg/plate for strains TA98 and TA1537

Evaluation criteria:

Acceptance criteria:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL.

Criteria for assessing mutagenic potential
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important.

Statistics:

The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

Species / strain:

other: Salmonella typhimurium: strains TA1535, TA1537, TA98 and TA100; Escherichia coli: strain WP2 uvrA (pKM101)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in all strains at one or more concentrations in both tests)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: yes

1st experiment:Standard plate test (5 - 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)	comments
TA 98	no	36.7	1.1 (50 / 500)	no	negative	9 (2NF)	Thinning of background lawn at 5000 µg/plate
	yes	9.3	1.0 (5 / 500 / 1500)	no	negative	3.3 (B[a]P)	Slight thinning of background lawn at 5000 µg/plate
TA 100	no	150	1.0 (15 / 50)	no	negative	4.3 (NaN3)	Slight thinning of background lawn at 5000 µg/plate
	yes	178	1.0 (50)	no	negative	15.7 (AAN)	Slight thinning of background lawn at 5000 µg/plate
TA 1537	no	8.7	1.4 (150)	no	negative	40.3 (AAC)	Slight thinning of background lawn at 1500 µg/plate; Thinning of background lawn at 5000 µg/plate;
	yes	32	0.9 (15 / 50 / 150 / 500)	no	negative	4.6 (B[a]P)	Slight thinning of background lawn at 1500 µg/plate; Severe thinning of background lawn at 5000 µg/plate;
TA 1535	no	26.7	1.2 (50)	no	negative	43.6 (NaN3)	Slight thinning of background lawn at 1500 µg/plate; Thinning of background lawn at 5000 µg/plate;
	yes	28	0.9 (15)	no	negative	14.8 (AAN)	Thinning of background lawn at 5000 µg/plate
WP2 uvrA	no	117.3	1.3 (500)	no	negative	26.6 (NQO)	Thinning of background lawn at 5000 µg/plate;
	yes	171	1.2 (500)	no	negative	2.5 (AAN)	Slight thinning of background lawn at 5000 µg/plate
2nd experiment:Preincubation test (5 - 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)	comments
TA 98	no	36.7	1.0 (5 / 15 / 50 / 150)	no	negative	7.5 (2NF)	Slight thinning of background lawn at 500 µg/plate; Severe thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;
	yes	48.7	1.1 (50)	no	negative	8.2 (B[a]P)	Slight thinning of background lawn at 500 µg/plate; Severe thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;
TA 100	no	174.7	1.1 (5)	no	negative	5.1 (NaN3)	Slight thinning of background lawn at 500 µg/plate; Severe thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;
	yes	197	0.9 (5 / 15)	no	negative	14.6 (AAN)	Slight thinning of background lawn at 500 µg/plate; Severe thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;
TA 1537	no	12	0.9 (5)	no	negative	87.2 (AAC)	Thinning of background lawn at 500 µg/plate; Lawn absent at 1500 and 5000 µg/plate;
	yes	30.7	1.0 (5)	no	negative	7.6 B[a]P)	Thinning of background lawn at 500 µg/plate; Lawn absent at 1500 and 5000 µg/plate;
TA 1535	no	22	1.2 (15)	no	negative	54.5 (NaN3)	Thinning of background lawn at 500 µg/plate; Lawn absent at 1500 and 5000 µg/plate;
	yes	26	1.1 (15)	no	negative	13.1 (AAN)	Thinning of background lawn at 500 µg/plate; Lawn absent at 1500 and 5000 µg/plate;
WP2 uvrA	no	178.3	1.0 (5)	no	negative	18 (NQO)	Thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;
	yes	206.3	1.0 (5 / 50)	no	negative	2.9 (AAN)	Thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;

Conclusions:

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

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce back mutations in selected loci in several strains of Salmonella typhimurium and one Escherichia coli strain in the Ames test.

 

Strains:                       TA 1535, TA 100, TA 1537, TA 98, Escherichia coli WP2 uvrA

Dose range:                15 µg - 5,000 µg/plate (SPT); 15 µg -  250 µg/plate (PIT)

Test conditions:           Standard plate test (SPT) and pre­ incubation test (PIT) both with and without metabolic activation (Aroclor induced rat liver S-9 mix).

Solubility:                    No precipitation of the test sub­stance was found.

Toxicity:                     A bacteriotoxic effect was observed depending on the strain and test conditions from about 120 µg - 250 µg/plate onward.

 

 

Mutagenicity:

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.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mutagenicity:

An  increase in the number of his• revertants was not observed both in the standard plate test

andin the preincubation test either without S-9 mix or after the addition of a metabolizing system.

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Propylheptanol
- Physical state: liquid (colorless, clear)
- Analytical purity: 99.8 area % 2-Propylheptanol and isomeres
- Lot/batch No.: Tk2012_20110113
- Expiration date of the lot/batch: 2012-01-13
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle DMSO for 4 hours was determined analytically. The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE.

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.

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

 

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: chromosomal aberration

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OASIS TIMES v2.27.19.13

2. MODEL (incl. version number)
Chromosomal Aberrations S9 activated; v. 12.12

3. SMILES IDENTIFIERS USED AS INPUT FOR THE MODEL
CCCCCC(CCC)CO

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: In Vitro Mammalian Chromosome Aberration Test
- Unambiguous algorithm: refer to QMRF
- Defined domain of applicability:
1. General parametric requirements - includes ranges of variation of log KOW and MW. It specifies in the domain only those chemicals that fall in the range of variation of the MW and log Kow defined on the bases of the correctly predicted training set chemicals. This layer of the domain is applied only on parent chemicals.
2. Structural domain - it is represented by list of atom - centered fragments extracted from the chemicals in the training set. The training chemicals were split into two subsets: chemicals correctly predicted by the model and incorrectly predicted
chemicals. These two subsets of chemicals were used to extract characteristics determining the "good" and "bad" space of the domain. Extracted characteristics were split into three categories: unique characteristics of correct and incorrect chemicals (presented only in one of the subsets) and fuzzy characteristics presented in both subsets of chemicals. Structural domain is applied on parent chemicals, only.
3. Mechanistic domain - in SS model it includes: Interpolation space: this stage of the applicability domain of the model holds only for chemicals for which an additional COREPA model is required. It estimates the position of the target chemicals in the population density plot built in the parametric space defined by the explanatory variables of the model by making use the training set chemicals. Currently, the accepted threshold of population density is 10%.
The mechanistic domain is applied on the parent structures and on their metabolites.

- Appropriate measures of goodness-of-fit and robustness and predictivity:
Statistics of the model:
• Sensitivity = (predicted positive/observed positive) = 71%
• Specificity = (predicted negative/observed negative) = 73%
• Concordance = (correct predicted positive and negative chemicals in respect to all training set chemicals) = 73%

5. APPLICABILITY DOMAIN
- Descriptor domain:
Log(Kow): range = [ -13.2 .. 15.4 ]
calculated: 3.71 (In domain)
MOL._WEIGHT: range = [ 30 .. 738 ]Da
calculated: 158Da (In domain)
--> Conclusion: The chemical fulfils the general properties requirements.

- Structural fragment domain: The following ACF are identified: Fragments in correctly predicted training chemicals – 100.00%, Fragments in non-correctly predicted training chemicals – 0.00%, Fragments not present in the training chemicals – 0.00%
--> Conclusion: The chemical is in the interpolation structural space

- Mechanistic domain: Interpolation space
- Similarity with analogues in the training set: not reported

6. ADEQUACY OF THE RESULT
The substance falls in the applicability domain of the model. The model was found to give reliable predictions for industrial chemicals. It is therefore considered to be acceptable for REACH.

The substance is predicted to be in vitro CA negative.

Qualifier:

according to guideline

Guideline:

other: REACH guidance on QSARs R.6, May/July 2008

Principles of method if other than guideline:

TIMES-SS v.2.27.19.13 - In Vitro Mammalian Chromosome Aberration Test v.12.12 (structure-toxicity and structure-metabolism relationships)

GLP compliance:

no

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

The registrant considers the prediction as valid because this model was validated with more than 1000 substances. The model was found to have 71% sensitivity, 73% specificity and 73% concordance for training set chemicals..

The QSAR program calculated no genotoxic potential of the test substance. The substance is in domain of the system.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

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.

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

Conclusions:

Under the test conditions, Isodecanol (CAS 25339-17-7) induced no chromosomal aberrations in cultured mammalian V79 cells with and without metabolic activation.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to the attached justification in chapter 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Gene mutation in bacteria:

2-Propylheptan-1-ol (96.2%) was not mutagenic in a reliable preincubation and standard plate Ames test (according to OECD 471, under GLP) with and without metabolic activation (tested up to 5000 µg/plate in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA (pKM101); metabolic activation: liver S-9 mix from Phenobarbital- and 5,6-benzoflavone-induced rats; Evonik Oxeno GmbH, 2009). Cytotoxicity (reduction of the background lawn) was observed in all strains at one or more concentrations in both tests.

 

Gene mutation in mammalian cells:

2 -Propylheptanol (99.8%) was tested in a reliable HPRT-test (according to OECD TG 476, under GLP) with Chinese hamster ovary cells (CHO) with and without metabolism (metabolic activation: S-9 mix from Phenobarbital / β-naphthoflavone induced rat liver). The test concentrations were 2.5 - 100 µg/mL (treatment 4 and 24 h, respectively). No increase of the mutant frequency was observed after treatment with the test substance. 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 (BASF, 2011).

Cytogenicity in mammalian cells:

There were no experimental data for the cytogenicity of 2-Propylheptan-1-ol in mammalian cells available.

Therefore, a QSAR was conducted using the model "Chromosomal Aberrations S9 activated" of the software OASIS TIMES. The QSAR program calculated no genotoxic potential of the test substance. This QSAR prediction is considered to be valid, because the substance was in domain of the system.

Furthermore, the prediction is confirmed by a reliable chromosomal aberration test in Chinese hamster fibroblasts with and without metabolic activation (metabolic activation: Aroclor 1254-induced rat liver S-9 mix) with the structural analogue Isodecanol (CAS 25339-17-7). The test concentrations were 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) and the test was negative (Fraunhofer Institute, 1993).

Justification for classification or non-classification

The available experimental test data and QSAR data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.