Alcohols, C13-15-branched and linear

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material did not induce mutagenic effects in two Ames tests performed according to OECD 471.

 

The test substance is assumed to be not genotoxic, as deduced from an in vitro mammalian cell gene mutation test in CHO cells performed according to OECD 476 with structural analogue CAS 10042-59-8.

 

The test substance is assumed not to induce structural chromosomal aberrations, as deduced from an in vitro mammalian chromosome aberration test in human lymphocytes performed according to OECD 473 with structural analogue CAS 2425-77-6.

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

Study period:

April 2021 - July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Aug 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 Jun 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution.
The test substance was dissolved in dimethyl sulfoxide (DMSO).
To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
The further concentrations were diluted according to the planned doses.
All test substance formulations were prepared immediately before use.

The stability of the test substance in the vehicle DMSO was not determined analytically, because the test substance was administered immediately after preparation and is usually stable.

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction
The S9 fraction was prepared according to Ames et al. at BASF SE in an AAALAC-approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive.
At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and beta-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days. During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am to 6 pm and darkness from 6 pm to 6 am.
Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed, and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

S9 mix
The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on ice until used. The concentrations of the cofactors in the S9 mix were:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.
To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.

Test concentrations with justification for top dose:

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.

Vehicle / solvent:

Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Remarks:

Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strain.

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains. Vehicle controls were used for several BASF projects done in parallel.

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other:

Remarks:

The stability of the selected positive controls was well-defined under the selected culture conditions, since they were well-established reference mutagens. Positive controls were used for several BASF projects done in parallel.

Details on test system and experimental conditions:

Test strain
For testing, a deep-frozen (-70°C to -80°C) bacterial culture (E. coli WP2 uvrA) is thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The optical density of the fresh bacteria culture was determined-Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL). This culture grown overnight was kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strain mentioned was in accordance with the current scientific recommendations for the conduct of this assay.
E. coli WP2 uvrA was checked for UV sensitivity.
Tryptophan auxotrophy was checked in each experiment via the spontaneous rate.

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al..
• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager.
Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al..
0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Rationale for test conditions:

Scope of tests and test conditions
1st Experiment
Strain: E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control
2nd Experiment
Strain: E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

Evaluation criteria:

Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.

Toxicity
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Solubility
If precipitation of the test material was observed, it would be recorded and indicated in the tables. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

SOLUBILITY AND STERILITY CONTROL
Test substance precipitation was observed at and above 2500 μg/plate with and without S9 mix.
The additional treated plates for sterility control showed no contamination in all performed experiments.

For detailed result tables and historical control data see "Attached background material"

Conclusions:

Under the experimental conditions chosen here, it is concluded that C13-C15-Alcohol is not a
mutagenic test substance in the bacterial reverse mutation test in the absence and the
presence of metabolic activation.

Reference 2

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:

yes

Remarks:

(only four strains tested, 2-aminoanthracene was the sole indicator of the efficacy of the S9-mix)

GLP compliance:

no

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): C13 - C15 - Alkohol
- Physical state: colorless liquid
- Analytical purity: 99.3% alcohols (0.5% low boiling substances, 0.2% high boiling substances)
- Composition of test material, percentage of components: 29.5% i-C13-alcohol, 33% n-C13-alcohol, 19% i-C15-alcohol, 17.8% n-C15-alcohol
- Lot/batch No.: from continuous production
- Sustance number: 88/577
- Storage: room temperature

Target gene:

his-gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9-mix of Aroclor 1254 induced rat livers (Sprague-Dawley)

Test concentrations with justification for top dose:

20 - 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Complete solubility of the test substance in 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 for details

Details on test system and experimental conditions:

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

Standard plate test:
The experimental procedure is based on the method of Ames et al ., 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.

Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate

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

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

NUMBER OF REPLICATIONS: triplicate

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;

Key result

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Standard plate test (20 - 5000 µg/plate)
Strain	Metabolic activation system	mean his^+/trp^+revertant colonies (negative control)	maximum revertant factor (conc. (µg/plate))	dose dependency	Assessment	maximum revertant factor (positive control)
TA 98	no	23	1.0 (20/500/2500/5000)	no	negative	29 (NPD)
	yes	34	1.1 (20/100)	no	negative	34.7 (2-AA)
TA 100	no	114	0.9 (100)	no	negative	14.8 (MNNG)
	yes	103	1.3 (100)	no	negative	17.2 (2-AA)
TA 1537	no	9	1.3 (20)	no	negative	47 (AAC)
	yes	12	0.9 (20/100/500)	no	negative	11.7 (2-AA)
TA 1535	no	15	1.0 (20)	no	negative	128.4 (MNNG)
	yes	15	1.2 (2500)	no	negative	10.5 (2-AA)
Preincubation test (20 - 5000 µg/plate)
Strain	Metabolic activation system	mean his^+/trp^+revertant colonies (negative control)	maximum revertant factor (conc. (µg/plate))	dose dependency	Assessment	maximum revertant factor (positive control)
TA 98	no	24	1.1 (500)	no	negative	39.9 (NPD)
	yes	35	1.2 (500)	no	negative	26.5 (2-AA)
TA 100	no	110	1.1 (500)	no	negative	10.6 (MNNG)
	yes	125	1.1 (20)	no	negative	9.5 (2-AA)
TA 1537	no	8	1.2 (100)	no	negative	43.9 (AAC)
	yes	12	0.8 (20/100/2500)	no	negative	9.3 (2-AA)
TA 1535	no	19	1.2 (5000)	no	negative	59 (MNNG)
	yes	18	1.0 (20/5000)	no	negative	7.1 (2-AA)
2-AA = 2-aminoanthracene
NPD = N-nitro-o-phenylendiamine
MNNG = N-methyl-N-nitro-N-nitrosoguanidine
AAC = 9-aminoacridine chloride monohydrate

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See 'Attached justification'.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks on result:

other: Result from read-across CAS 10042-59-8

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The genetic toxicity for alcohols C13 - 15 branched and linear (CAS 85566-16-1) was determined in two Ames tests (BASF SE 1989 and 2021). Additionally, the genetic toxicity was determined in an HPRT-assay and an in vitro chromosome aberration assay for the structural analogues CAS 10042-59-8 (BASF SE, 2011) and CAS 2425-77-6 (Sasol/Cognis, 2009), respectively. Furthermore, toxicity data on "alcohols C9 -11-iso-, C10-rich" with CAS number 68526-85-2 was obtained from its disseminated dossier.

 

In two Ames-tests performed according to OECD guideline study 471, Salmonella strains S. typhirium TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA were exposed to the test material in doses up to 5000 µg/plate via a standard plate test and a preincubation test in triplicate with and without the presence of S-9 mix. DMSO was used as vehicle. No cytotoxicity was observed and the test material did not induce mutagenic effects.

 

Furthermore, a GLP-compliant in vitro mammalian cell gene mutation test was performed according to OECD guideline 476 with structural analogue CAS 10042-59-8. In this study, chinese hamster ovary (CHO) cells were exposed in medium to concentrations of 3.1, 6.3, 12.5, 25, 50, 100 µg/mL for 4 hours in the presence and absence of a metabolic activation system (S9-mix). In a second experiment, cells were exposed to the previously mentioned concentrations for 24 hours where in addition cells were exposed to concentrations of 10, 20, 40, 60, 80, 100 µg/mL for 4 hours in the presence of S9-mix. In a third experiment in the presence of S9 mix, cells were exposed to concentrations of 2.5, 5, 10, 20, 40, 60, 80 µg/mL for 4 hours. For all experiments duplicate cultures were used. The expression time was 7 - 9 days and the selection time was 6 - 7 days. Cytotoxicity was determined based on the cloning efficiency. No genotoxicity was observed in any experiment performed and all controls were considered valid. Cytotoxic effects were observed in all experiments in the presence and absence of S9-mix at least in the highest applied concentration.

 

Additionally, a GLP- compliant in vitro mammalian chromosome aberration test performed according to OECD 473 was conducted with structural analogue CAS 2425-77-6. In the first test of this study, human lymphocytes were exposed to test substance concentrations of 16, 22 and 24 µg/mL (no metabolic activation system) and 25, 65 and 80 µg/mL (including S9-mix as metabolic activation system) for 3 hours. In the second test, human lymphocytes were exposed to concentrations of 9, 15 and 17 µg/mL (no metabolic activation system; exposure duration 21 hours) and 120, 130 and 150 µg/mL (including S9-mix as metabolic activation system; exposure duration 3 hours). DMSO was used as vehicle. The mitotic index was determined as basis for cytotoxicity in addition to determination of polyploidy. No genotoxic effects were observed at any test concentration in the presence or absence of a metabolic activation system. The test substance 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.

 

Supporting information is dissiminated on the ECHA Homepage for a structurally similar substance, CAS 68526 -85 -2. In a GLP-compliant study performed according to OECD guideline 473, chinese hamster ovary cells were exposed to test concentrations of 5, 10, 20, 30, 40, 80, and 160 µg/mL (test 1) and 5, 10, 20, 30, 40, 50, 60, 70, 80 and 100 µg/mL (test 2) in the presence and absence of S9-mix (functioning as metabolic activation system). DMSO was used as vehicle. The mitotic index was determined as the basis for cytotoxicity. At the end of the study period, cytotoxicity was observed and all controls were considered valid. No genotoxicity was observed in any of the previously described test conditions. In addition an in vitro gene mutation was conducted for this substance, performed according to OECD guideline 476 and GLP-compliant. In this test, mouse lymphoma L5178Y cells were exposed to test concentrations of 0, 0.45, 0.9, 1.8, 3.6, 7.2, 14, 21, 29, 42 µg/mL (test 1) and 0, 2.5, 4.9, 9.8, 14, 20, 29, 32, 35, 39, 44 µg/mL (test 2) in the presence and absence of S9 -mix, that functioned as metabolic activation system. Cells were exposed for 4 or 24 hours and the expression time was 2 days, followed by selection for 10-14 days. The relative total growth was determined as the basis for cytotoxictiy. Overall, all controls were considered valid and cytotoxicity was observed in doses above 21 µg/mL. No genotoxicity was observed. It was concluded that the test material is not a mutagenic agent.

Justification for classification or non-classification

Based on the available information the substance does not need to be classified for genetic toxicity, as in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.