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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test and a HPRT test, both performed according to the current guideline requirements, propan-1-ol was shown to be not mutagenic. In an OECD guideline conform in vitro chromosome abberration test, no clastogenic or aneugenic properties have been observed.

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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)
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): N-Propanol
- Physical state: Liquid, colorless, clear
- Analytical purity: 99.95 area-%
- Lot/batch No.: TK504_20081022
- Storage condition of test material: Room temperature
Target gene:
S. typhimurium: his-
E. coli: trp-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital i.p. and β-naphthoflavone orally induced rats
Test concentrations with justification for top dose:
0; 20; 100; 500; 2500 and 5000 μg/plate (SPT)
0; 312.5; 625; 1250; 2500 and 5000 μg/plate (PIT)
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see "Details on test system"
Details on test system and experimental conditions:
Experiment 1: Standard plate test (SPT)
Test tubes containing 2 mL soft agar kept in a water bath at 45°C, and remaining components added in the following order:
0 .1 mL test solution or vehicle
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.

Experiment 2: Preincubation assay (PIT)
0.1 mL test solution or vehicle, 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.

Experiment1&2
In each experiment 3 test plates per dose or per control used; after incubation at 37°C for 48 hours in the dark, the bacterial colonies ( his+/trp+ revertants) are counted.
Positive control:
with metabolic activation: 2-aminoanthracene: 2.5 μg/plate for each S. typhimurium strain; 60 μg/plate for E. coli WP2 uvrA
without metabolic activation: 5 μg/plate N-methyl-N'-nitro-N-nitrosoguanidine for TA 100 and TA 1535, 10 μg/plate 4-nitro-o-phenylendiamine
for TA 98, 100 μg/plate 9-aminoacridine chloride for TA 1537 and 5 µg/plate N-Ethyl-N-nitro-N-nitrosoguanidine for E. coli WP2 uvrA; all substances were dissolved in DMSO.
The titer was determined and in regularly measurements the strain characteristics were checked. Sterility control was performed.
Evaluation criteria:
Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• The titer of viable bacteria was ≥ 10e8/mL.

Assessment criteria
The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Species / strain:
other: S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test substance was found with and without S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mutagenicity

Standard plate test (20 - 5000 µg/plate)
Strain Metabolic activation system mean revertants in Controls maximum revertant factor dose dependency Assessment
TA 98 no 28 1.0 no negative
  yes 33 1.2 no negative
TA 100 no 100 1.1 no negative
  yes 99 1.3 no negative
TA 1535 no 17 1.0 no negative
  yes 15 1.0 no negative
TA 1537 no 11 1.3 no negative
  yes 9 1.4 no negative
WP2 uvr A no 34 1.1 no negative
  yes 39 1.1 no negative
Preincubation test (312.5 - 5000 µg/plate)
Strain Metabolic activation system mean revertants in Controls maximum revertant factor dose dependency Assessment
TA 98 no 26 1.1 no negative
  yes 32 1.0 no negative
TA 100 no 102 1.0 no negative
  yes 103 1.1 no negative
TA 1535 no 14 1.0 no negative
  yes 14 1.0 no negative
TA 1537 no 8 1.0 no negative
  yes 8 1.0 no negative
WP2 uvr A no 36 1.1 no negative
  yes 51 1.0 no negative
Endpoint:
in vitro gene mutation study in mammalian cells
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 476 (In Vitro Mammalian Cell Gene Mutation Test)
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): N-Propanol
- Physical state: Liquid, colorless, clear
- Analytical purity: 99.95 area-%
- Lot/batch No.: TK504_20081022
- Storage condition of test material: Room temperature
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from phenobarbital i.p. and β-naphthoflavone orally induced rat livers
Test concentrations with justification for top dose:
1st Experiment
without S9 mix (4-hour exposure period)
0; 75; 150; 300; 600 μg/mL
with S9 mix (4-hour exposure period)
0; 75; 150; 300; 600 μg/mL
2nd Experiment
without S9 mix (24-hour exposure period)
0; 75; 150; 300; 600 μg/mL
with S9 mix (4-hour exposure period)
0; 100; 200; 400; 600 μg/mL
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see "details on test system"
Details on test system and experimental conditions:
Preparation of test cultures
The stocks of cells (1.0-mL portions) were thawed at 37°C in a water bath. 0.5 mL were pipetted into 25 cm2 plastic flasks containing 5 mL Ham's F12 medium (incl. 10% [v/v] FCS). The medium was replaced after 24 hours to remove any dead cells. After at least 2 passages, cells were taken for the experiment, and for these there was another passage to prepare test cultures.

Pretreatment of cells with "HAT" medium
During the week prior to treatment, spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.
3 – 5x10e5 cells were seeded per flask (75 cm²) and incubated with "HAT" medium for 3 - 4 days. After that, a passage in Ham's F12 medium incl. 10% (v/v) FCS was followed with a subsequent incubation for a further 3 - 4 days.

Attachment period
For each test group, about 1x10e6 logarithmically growing cells per flask (175 cm²; after the 2nd passage) were seeded into about 20 mL Ham's F12 medium supplemented with 10% (v/v) FCS and incubated for about 20 - 24 hours. Two flasks were used for each test group.

Exposure period
After the attachment period, the medium was removed from the flasks and the treatment medium was added (without S9 mix: 18 mL medium + 2 mL vehicle/test substance/ pos. control; with S9 mix: 14 mL medium + 2 mL vehicle/test substance/ pos. control + 4 mL S9 mix) For exposure periods of more than 4 hours medium with 10% (v/v) FCS was used.
The cells were exposed for 4 hours in the 1st Experiment without S9 mix and in both experiments with S9 mix. In the 2nd Experiment without S9 mix the exposure period was 24 hours.

Expression period
The exposure period was completed by rinsing several times with HBSS. Then the flasks were topped up with at least 20 mL Ham's F12 medium incl. 10% (v/v) FCS and left to stand in the incubator for about 3 days (4-hour treatment) or 2 days (24-hour treatment). This was followed by the 1st passage. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (2nd passage).

Selection period
For selection of the mutants, six 75 cm2 flasks with 3x10e5 cells each from every treatment group, if possible, were seeded in 10 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 - 7 days. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.
Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• 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 mutants per 10e6 clonable cells
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data)
• 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
The test item is considered 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 doseresponse relationship. Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 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 item generally is considered as “negative” if the following criteria are met:
• 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.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary of results

Exp. Exposure Test S9 Precipitation Genotoxicity Cytotoxicity
  period  groups mix MFcorr. CE1 CE2
  (hrs) (µg/mL) [ per 10e6 cells] [%] [%]
1 4 Negative control - - 2.16 100 100
  75 - - 3.24 92.1 100.9
  150 - - 2.72 100.4 96.9
  300 - - 0.91 95.5 93.8
  600 - - 2.27 89 97.9
  Positive control 1 - - 176.37 95.4 85.9
2 24 Negative control - - 1.92 100 100
  75 - - 4.73 84.5 109.2
  150 - - 2 97.4 118.9
  300 - - 3.19 99.2 105.6
  600 - - 4.62 101 99.8
  Positive control 1 - - 439.36 65.6 63.3
1 4 Negative control + - 3.74 100 100
  75 + - 0.89 90.7 103
  150 + - 5.22 87.6 99.2
  300 + - 3.21 91 92.7
  600 + - 1.89 85.9 108.6
  Positive control 2 + - 103.94 77.7 90.9
2 4 Negative control + - 2.51 100 100
  100 + - 4.1 103.1 93.2
  200 + - 2.52 90.1 90.9
  400 + - 2.33 94.1 94.7
  600 + - 6.49 103 98.7
    Positive control 2 + - 47.29 96.9 109.7
Positive control 1 = EMS 300 mg/mL
Positive control 2 = MCA 20 mg/mL
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
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)
Version / remarks:
(July 21, 1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Source: no data
- Analytical purity: 99.82
- Batch No.: 6 v. 17 .09 .2002 -14:00 Uhr
- Physical state: Colourless, Liquid
- Storage: RT
- Stability: The stability of the test substance under storage conditions throughout the study period was guaranteed
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
high proliferation rate (doubling time of about 12 - 16 hours), high plating efficiency (>= 90% ), stable karyotype (modal number of 22 chromosomes). The cells used were checked for mycoplasma cotamination, karyotype stability, plating efficiency
Metabolic activation:
with and without
Metabolic activation system:
S9 mix derived from the livers of Aroclor 1254 treated Sprague-Dawley rats
Test concentrations with justification for top dose:
EXPERIMENT 1 (4h exposure, 18 hour harvest time)
- With S9 mix : 0; 75; 150; 300; 600 µg/ml
- Without S9 mix : 0; 75; 150; 300; 600 µg/ml

EXPERIMENT 2 (18h exposure, 18 hour harvest time)
- Without S9 mix : 0; 100; 200; 400; 600 µg/ml

EXPERIMENT 3 (4h* & 18h** exposure, 28h harvest time)
- With S9 mix* : 0; 75; 150; 300; 600 µg/ml
- Without S9 mix**: 400; 600 µg/ml
Vehicle / solvent:
Due to the good solubility of the test substance in water, the aqueous culture medium (MEM) was selected as the vehicle
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 350 µg/ml Ethyl methanesulphonate (without S9 mix); 0.5 µg/ml Cyclophosphamide (monohydrate) (with S9 mix)
Details on test system and experimental conditions:
INCUBATION
- Duration: 4h (serum free), 18h (with serum)
- Time from treatment till harvest: 18h and 28h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.2 µg/ml cell culture medium, added 2-3 h prior to harvesting
STAIN (for cytogenetic assays): Giemsa and Titrisol

DETERMINATION OF CYTOTOXICITY
Growth inhibition was estimated by counting the number of cells in the dose groups in comparison with the concurrent vehicle control at the end of the culture period using a counting chamber.

CELL CYCLE TIME
The cell cycle of the untreated V79 cells lasted for about 13 - 14 hours measured by the method of Speit G et al 2002. With the choice of an 18h sampling time, a 1-1.5 folds of the normal cell cycle time was covered as recommended by the OECD Guideline No. 473 "In Vitro Mammalian Chromosome Aberration Test". The later sampling time of 28 hours was chosen to cover a possible cell cycle delay.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Determination of morphology: Yes
- Determination of mitotic index (No. of cells in mitosis divided by total No. of cells): Yes, total of 1000 cells were tested

In general, it was planned to evaluate three dose levels. The additionally selected lower doses (75 mg/µl) would be evaluated only if the results obtained on cytotoxicity deviated from those obtained in the pretest. Slides were coded before microscopic analysis. If only a few cells were found or if the
metaphases were of low quality, a chromosome analysis was not carried out

PRETEST FOR DOSE SELECTION
Cultures were exposed for the duration of 4 hours with an 18 h harvest time to a wide dose range of the test article, (1 µg/ml - 600 µg/ml culture medium) both without S-9 mix and after adding a metabolizing system. In the pretest, various parameters (pH, osmolarity, solubilty, cell attachment, cell count and quality of metaphases) were checked for all or at least some selected doses.
Evaluation criteria:
- The test chemical is considered positive in this assay if the following criteria are met: a dose-related and reproducible significant increase in the number of structural chromosomal aberrations, the proportion of aberrations exceeded both the concurrent negative control range and the negative historical control range.
- A test substance is generally considered nonclastogenic in this test system if: there was no significant increase in the number of chromosomally damaged cells at any dose above concurrent control frequencies. The aberration frequencies were within the historical control range.
Statistics:
As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for all test groups (total of 200/dose for test substance and 100/dose for the positive control), and if cells had 20 - 22 chromosomes, they were analyzed for structural chromosome aberrations. Numerical chromosome aberrations were also recorded. Structural chromosomes aberrations included chromatid and chromosome gaps, breaks, fragments and deletions as well as multiple aberrations (metaphases with 5 or more aberrations excl. gaps), pulverisation (chromosomes are present as irregular particles and a chromosomal structure can no longer be detected), exchanges and finally interchanges at the level of chromatids and chromosomes. Numerical aberrations included aneuploidy, polyploidy and endopolyploidy.

A comparison of each dose group with the vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are significant, labels (* p< 0 .05, ** p< 0.01) are printed in the tables.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not influenced up to the highest concentration
- Effects of osmolality: Not influenced up to the highest concentration
- Water solubility: Solubility was verified in MEM medium up to the highest concentration

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test substance did not exhibit any toxicity up to the highest guideline recommended concentration, i.e. 10 mM (= 600 µg/ml) with percentages of the cell counts between 90.5%-95.4% of controls without S9 mix and 95.4-108% of controls with metabolic activation in Experiment I. In experiment II % cell count ranged between 97.9 - 117.9% of controls without metabolic activation and 105.5-116.4% of controls with metabolic activation

MITOTIC INDEX
- There was only a slight suppression of the mitotic activity in the 1st experiment without S-9 mix after pulse treatment of 4 hours and a harvest time of 18 hours from about 300 µg/ml onward [88.1, 69.6 and 63.8% of controls (150, 300 and 600 µg/ml)]
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

NON HUMAN DATA

IN VITRO

In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA 1535, TA 100, TA 1537, TA 98) and E.coli (E. coli WP2 uvrA) were exposed to propan-1 -ol using the standard plate test (SPT) and the preincubation test (PIT) in the presence and the absence of mammalian metabolic activation. The tests were performed using five different concentrations up to 5000µg/plate. There was no evidence of induced mutant colonies over background. The study was performed under GLP conditions and satisfies the requirements of the OECD guideline 471 (BASF SE, 2009).

In an OECD guideline conform in vitro mammalian cell gene mutation asseay (HPRT test, OECD 476) Chinese hamster ovary (CHO) cells were exposed to propan-1 -ol at 4 different concentrations of up to 600 µg/ml (0.01M) in the presence and absence of mammalian metabolic activation. Under the experimental conditions of this study, the test item propan-1.ol was not mutagenic in the HPRT locus assay (BASF SE, 2009).

In a mammalian cell cytogenetics assay (chromosome aberration), V79 cultures were exposed to propan-1-ol (99.8% pure, vehicle: Minimum essential medium) at concentrations between 75 and 600 µg/ml (with 2 fold increment in the concentration) with and without S9 mix. The cells were exposed for 4h and 18 hours. Harvesting was performed for each time point 18 and 28 hours after the start of incubation. Tested up to the limit concentration of 10 mM (600 µg/ml), no cytotoxicity was observed in V79 cells. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background (BASF AG, 2003). The study was performed according GLP standards and also satisfies the requirement for Test Guideline (in vitro mammalian cytogenetics (chromosome aberration) OECD 473 (chinese hamster lungs fibroblast, V79).

IN VIVO

There is no reliable information on genetic toxicity in vivo available. An in vivo chromosomal aberration test with rats (bone marrow assay) led to an inconclusive finding due to limitations in data presentation (Bariljak and Kozachuk 1988). In the exposed group (8 animals, 500 cells) 2.2 % of bone marrow cells carried chromosomal aberrations (exclusive gaps), in the negative control group (10 animals) no chromosomal aberrations were found in 600 cells. Furthermore, it is also reported on polyploidy frequencies 2.4 % in the exposed group and 0.5 % in the negative control. The dose of propan-1-ol is given as 1/5 of the LD-50, the route of administration is not specified. Assessment of the study is however limited by the following factors: no historical controls, administered dose is not clear, low number of samples tested. Therefore this study can not be used for assessing the genetic toxicity of the test substance in vivo.

HUMAN DATA

No human information is available.



Justification for classification or non-classification

Based on the reliable and OECD guideline conform in vitro genetic toxicity tests there is no requirement for classification according to Directive 67/548(EEC) and Regulation (EC) No. 1272/2008.