3-aminopropan-1-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

3-Aminopropan-1-ol was negative in an Ames test and sister chromatid exchange assay.

3 -Aminopropan-1-ol was negative in the in vitro mammalian cell (CHO) gene mutation test HPRT.

3 -Aminopropan-1-ol was negative in the in vitro mammalian cell MNT.

In addition, 2-aminoethanol, a structural analogue, is negative in an Ames test in Salmonella typhimurium and Echerichia coli strains, and negative in an in vitro gene mutation test.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B 111 v. 22.11.16
- Test substance No.: 15/0348-2

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital i.p. and β-naphthoflavone orally induced rat liver S9-mix

Test concentrations with justification for top dose:

Based on the data and the observations from the pretest and taking into account the current guidelines, the following doses were selected in this study.
1st experiment
without metabolic activation: 50, 100, 200, 400, and 800 µg/mL
with metabolic activation: 50, 100, 200, 400, and 800 µg/mL
2nd experiment
without metabolic activation: 75, 150, 300, 600, and 800 µg/mL
with metabolic activation: 75, 150, 300, 600, and 800 µg/mL

Vehicle / solvent:

culture medium (Ham's F12); due to the good solubility of the test substance in culutre medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

whithout metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Two independent experiments (replicates) were performed.

During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.

For each test group, about 20x10^6 logarithmically growing cells per flask (300 cm²) were seeded into about 40 mL Ham's F12 medium supplemented with 10% (v/v) FCS and incubated for about 20 - 24 hours.

After the attachment period, the medium was removed from the flasks and the treatment medium (containing the test substance) with or without metabolic activation was added. The cultures were incubated for 4 h (exposure period) at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity.

The exposure period was completed by rinsing several times with HBSS. This was directly followed by the 1st passage in which 2x10^6 cells were seeded in 20 mL medium (in 175 cm2 flasks). The flasks were left to stand in the incubator for about 3 days at 37°C, relative humidity of ≥ 90% and 5% (v/v) CO2 atmosphere. After about 3 days, the cells were passaged a 2nd time in 175 cm2 flasks with 2x10^6 cells. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (3rd passage).

For selection of the mutants, two 175 cm2 flasks with 2x10^6 cells each from every treatment group, if possible, were seeded in 20 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 – 7 days. Only the cells resistant to 6-thioguanine that were assumed to be deficient of HPRT survived. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

Relative survival after treatment (Cloning efficiency 1 [CE1] adjusted by cell loss)
For the determination of the influence of the test substance after the exposure period, 200 cells per concentration were reserved from the treated cells and were seeded in petri dishes (60 mm diameter) and coated with 5 mL Ham's F12 medium incl. 10% (v/v) FCS in parallel to the 1st passage directly after test substance incubation.

Cloning efficiency 2 (CE2; viability)
For the determination of the mutation rate after the expression period, two aliquots of 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two petri dishes (60 mm diameter) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS.

In all cases, after seeding the flasks or petri dishes were incubated for 6 - 8 days to form colonies. These colonies were fixed, stained and counted.
The absolute and relative cloning efficiencies (%) were calculated for each test group.

Changes in pH were recorded by a change in the indicator color of the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured at least for the top concentrations and for the negative controls with and without S9 mix.
Osmolality was measured in at least the top concentrations and the negative controls with and without S9 mix.
Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.
The test cultures of all test substance concentrations were examined microscopically for cell morphology and cellular attachment at the end of the exposure period, which is a further indication for cytotoxicity.

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 be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range.

Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables.
However, both, biological and statistical significance are considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MUTANT FREQUENCY
No biologically relevant increase in the number of mutant colonies was observed with or without S9 mix. In both experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies (without S9 mix: MFcorr.: 0.85 – 5.48 per 10^6 cells; with S9 mix: MFcorr.: 0.28 – 6.86 per 10^6 cells) were close to or within the respective vehicle control values (MFcorr.: 1.98 – 5.87 per 10^6 cells) and clearly within the range of the 95% control limit of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 5.97 per 10^6 cells; with S9 mix: MFcorr.: 0.00 – 7.91 per 10^6 cells.
In both experiments, no statistically significant dose-related increase in the mutant frequency was found in cells after 4 hours of treatment either in the absence or presence of S9 mix.
The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 133.70 – 148.39 per 10^6 cells; with S9 mix: MFcorr.: 131.35 – 134.98 per 10^6 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 42.47 – 419.90 per 10^6 cells; with S9 mix: MFcorr.: 21.52 – 270.48 per 10^6 cells.

CYTOTOXICITY
Cytotoxic effects, as indicated by clearly reduced relative survival of about or below 20% of the respective negative control values were not observed in both experiments in the absence and presence of S9 mix, up to the highest applied concentrations.

CELL MORPHOLOGY
After 4 hours treatment in both experiments in the absence of metabolic activation and in the 1st Experiment in the presence of metabolic activation the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any test group tested for gene mutations. However, in the presence of S9 mix, after 4 hours treatment in the 2nd Experiment the morphology and attachment of the cells was adversely influenced (grade > 2) at the highest applied concentration of 800 μg/mL.

TREATMENT CONDITIONS
The pH values of the test substance preparations (stock solutions) were adjusted to a physiological value by adding small amounts of HCl.
Then, osmolality and pH values were not influenced by test substance treatment.
In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest applied test substance concentrations.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

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 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B 111 v. 22.11.16
- Test substance No.: 15/0348-2

Species / strain / cell type:

lymphocytes: human

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

first experiment (4 h exposure, w/ or w/o S9): 4.9, 8.5, 14.9, 26.1, 45.8, 80.1, 140, 245, 429, 751 µg/mL
second experiment (20 h exposure, w/o S9): 80.1, 140, 245, 429, 751 µg/mL
justification: The highest treatment concentration in this study, 751 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

4 h expo, w/o metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Demecolcin

Remarks:

20 h expo, w/o metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

4 h expo, w/ metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h stimulation with PHA
- Exposure duration: 4 h or 20 h
- Expression time (cells in growth medium): 16 h after 4 h exposure; no recovery period after 20 h exposure
- Cytochalasin B exposure: 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): The cultures were harvested by centrifugation 40 h after beginning of treatment. [...] cells were resuspended for 2 x 20 minutes in fixative and kept cold.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 (two parallel cultures)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: 1000 binucleate cells/culture = 2000

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: according to Countryman and Heddle (1976): The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI) was determined in 500 cells per culture and cytotoxicity is described as % cytostasis

Rationale for test conditions:

Positive control Demecolcin: The reference mutagen demecolcin was used as a guideline conform positive control for aneugenicity in the absence of S9 mix. The laboratory historical control data provide sufficient evidence for the responsiveness of the substance used in this system (cf. OECD 487 (2016), section 33).

Evaluation criteria:

Interpretation of Results
Many experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors. The current historical data range together with the statistical significance, confirmed by the Chi square test (α < 0.05), using a validated test script of “R” should be considered for classification of the test item.
The micronucleus assay will be considered acceptable if it meets the following criteria:
a) The rate of micronuclei in the solvent controls falls within the historical laboratory control data range.
b) The rate of micronuclei in the positive controls is statistically significant increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.
A test item can be classified as non-clastogenic and non-aneugenic if:
− the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
− no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.
A test item can be classified as clastogenic and aneugenic if:
− the number of micronucleated cells is not in the range of the historical laboratory control data and
− either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.
An increase in the number of micronucleated mononucleate cells may indicate that the test item has aneugenic potential.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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: The pH was adjusted to physiological values using HCl.
- Effects of osmolality: No relevant influence on osmolarity was observed
- Water solubility: given
- Precipitation: No precipitation of the test item in the culture medium was observed.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see attachement

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-06-15 to 1989-06-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no bacterial strain included to detect cross-linking mutagens

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 89/10

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Target gene:

histidine operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix (Aroclor 1254-induced)

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9 mix for the strains TA 100, TA 98, TA 1537 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S-9 mix for the strains TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

without S-9 mix for the strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine chloride monohydrate

Remarks:

without S-9 mix for the strain TA 1537

Details on test system and experimental conditions:

The standard plate test and the preincubation test were used

Standard plate test
The experimental procedure is based an the method of Ames et al.
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 bacteria 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. and Matsushima et al.
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.

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed only in the preincubation test using TA 1535, TA 1537 and TA 98 at doses of 2500 µg/plate and greater

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed only in the preincubation test using TA 1535, TA 1537 and TA 98 at doses of 2500 µg/plate and greater

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed only in the preincubation test using TA 1535, TA 1537 and TA 98 at doses of 2500 µg/plate and greater

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed only in the preincubation test using TA 1535, TA 1537 and TA 98 at doses of 2500 µg/plate and greater

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This test has been held according to the Notification stipulating the standard provided by the Minister of Labour based on the Industrial Safety and Health Law Article 57-2 Paragraph 1 (Notification No.77 of the Ministry of Labour, dated September 1, 1988) and related Notice (No.603, dated September 16, 1988) .

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AQ01

Target gene:

his

Species / strain / cell type:

S. typhimurium, other: TA98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA1538

Species / strain / cell type:

E. coli, other: WP2uvrA and WP2uvrA/pKM101

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 (Sodium phenobarbital and 5,6-benzofravone-induced)

Test concentrations with justification for top dose:

50 - 5000 µg/plate

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

The preincubation procedure was performed as described previously (Matsushima et al., 1980). The test compound dissolved in 0.05 or 0.1 mL of solvent was supplemented with 0.5 mL of S9 mix (metabolic activation method) or 0.1M phosphate buffer pH 7.4 (direct method) and 0.1 mL of tester strains which had been cultured in nutrient broth. The mixture was incubated for 20 min at 37°C, then rapidly mixed with 2 mL of molten top agar containing 0.05 µmol/ml of L-histidine and biotin for the Salmonella test. In the E. coli test 0.05 µmol/mL of L-tryptophan was used instead of L-histidine and biotin. Then the top agar mixture was rapidly poured onto a 30 ml of Vogel -Bonner minimal agar plate. All plates were incubated for 48 hours at 37°C and the numbers of revertant colonies were scored.

Evaluation criteria:

Two-hold rule criteria was used for data evaluation (Ames et al., 1975). The chemicals are considered to be mutagenic when a dose-related increase in revertant colony count is observed and the number of revertant colonies per plate with the test substance is more than twice that of the negative control (solvent control) and when a reproducibility of test result is observed. Mutagenic potency was calculated by following equation and maximum value of mutagenic potency was expressed as a specific acitivity on the data sheet: mutagenic potency (induced revertants / mg test substance) _(number of induced revertants on the dose X - number of revertant on the solvent control) = mg of test chemical on the dose X .

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=2000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=2000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=2000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA104

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=2000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=2000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=2000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=2000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=2000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=2000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

control values (mean ± SD):

Tester strain	Solvent control		positive control
	- S9	+ S9	- S9	+ S9
TA100	138 ± 27	139 ± 29	728 ± 196	1011 ± 210
TA1535	14 ± 5	13 ± 4	300 ± 81	255 ± 70
TA98	20 ± 8	26 ± 7	413 ± 82	404 ± 118
TA1538 (2NF)	16 ± 3	23 ± 5	376 ± 79	556 ± 216
TA1538 (4NQO)	16 ± 3	23 ± 5	343 ± 34	556 ± 216
TA1537	8 ± 2	11 ± 4	497 ± 254	196 ± 70
TA102	260 ± 49	317 ± 52	758 ± 175	1676 ± 562
TA104	269 ± 40	332 ± 48	1973 ± 755	1196 ± 252
WP2uvrA	29 ± 11	34 ± 11	273 ± 126	879 ± 177
WP2uvrA/pKM101	141 ± 43	198 ± 49	2080 ± 884	928 ± 250

 

results with 2 -aminoethanol

conc (µg/plate)	Number of revertants/plete (mean of 2 Experiments)
	Base-substitution						Frame-shift
Strain	TA100		TA1535		WP2uvrA		TA98		TA1537		TA1539
Metabolic acitvation	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
pos control	942	462	258	172	274	1071	293	301	307	255	296	227
neg control	205	197	17	11	23	22	23	31	11	9	9	16
50	203	215	26	12	29	26	19	38	9	22	11	13
100	198	208	30	10	15	28	21	32	9	13	9	15
200	199	218	17	12	23	23	16	32	10	14	9	20
500	205	220	22	12	19	21	19	30	6	20	7	15
1000	188	191	15	19	26	26	22	28	6	17	8	8
2000	72	148	12	15	15	29	14	21	4	8	6	8
5000	0	0	0	0	0	0	0	0	0	0	0	0
Judgement	-	-	-	-	-	-	-	-	-	-	-	-

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

2-aminoethanol, a structural analogue, is negative in an in vivo micronucleus test with mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

An Ames test, a HPRT test, an in vitro micronucleus test and a sister-chromatid exchange (SCE) assay are available for 3 -aminopropan-1-ol. However, in the Ames test, no bacterial strain is included to detect cross-linking mutagens. Furthermore, information of 2 -Aminoethanol, a structural analogue of 3 -Aminopropan-1 -ol, is used to determine the genotoxicity of 3 -Aminopropan-1 -ol.

In vitro

Ames test

3-aminopropan-1-ol was not mutagenic in a standard plate and pre-incubation Ames test with and without metabolic activation according to OECD guideline 471 (tested up to 5000 μg/plate in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100; metabolic activation: liver S-9 mix from Aroclor-induced male Sprague-Dawley rats) (BASF 1989). Cytotoxicity (reduction of the background lawn) was observed at the highest concentration tested in TA 1535, TA 97 and TA 98 without S-9 mix at concentrations of 2500 µg/plate and above.

In an Ames test, performed according to protocols similar to OECD guideline 471 and using test substance concentrations up to 5000 μg/plate (JETOC, 1996), mutagenicity of the structural analogue 2-Aminoethanol (MEA) was tested in Salmonella typhimurium (TA 98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA 1538) strains and Escherichia coli (WP2uvrA and WP2uvrA/pKM101) strains. In this test, MEA was found to be not mutagenic, both in the presence and the absence of metabolic activation. Concentrations ≥ 2000 µg/plate of the test substance were cytotoxic.

HPRT

In a GLP-compliant OECD 476 guideline study, 3-Aminopropanol was assessed for its potency to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix.

Following concentrations were tested:

1st Experiment with or without S9 mix: 0; 50.0; 100.0; 200.0; 400.0; 800.0 μg/mL

2nd Experiment with or without S9 mix: 0; 75.0; 150.0; 300.0; 600.0; 800.0 μg/mL

Cells were treated with the test substance for 4 hours +/- metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

No cytotoxicity was observed up to the highest concentrations tested for gene mutations.

Based on the results of the present study, the test substance did not cause any statistically significant or biologically relevant increase in the mutant frequencies either with or without S9 mix.

Thus, under the experimental conditions of this study, the test substance 3-Aminopropanol is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

In an in vitro gene mutation study in mammalian cells, performed according to OECD guideline 476 and GLP, MEA was tested in the mouse lymphoma (L5178Y TK+/-) forward mutation assay, with and without metabolic activation, using test substance concentrations up to 610 μg/mL (Linscombe et al., 1999). No cytotoxicity was observed, but the highest concentration corresponded to the limit dose of 10 mM.

MNT

In a GLP-compliant OECD 487 guideline study, 3-Aminopropanol, dissolved in deionized water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analyzed. 1000 binucleate cells per culture were evaluated for cytogenetic damage. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis. The highest treatment concentration in this study, 751 μg/mL (approx.10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487.

No precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity was observed. The pH was adjusted to physiological values.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed.

Demecolcin, MMC or CPA were used as positive controls and showed distinct increases in cells with micronuclei.

3-Aminopropanol is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Sister-chromatid exchange (SCE) assay

3-aminopropan-1-ol was also not mutagenic in Chinese hamster ovary cells when incubated at concentrations up to 8.1 mM with and without metabolic activation (liver S-9 mix from Aroclor-induced rats) (Sorsa 1988). Cytotoxicity was observed at the highest concentration level without additional metabolic activation

In vivo

In an in vivo micronucleus test, performed according to OECD guideline 474 and GLP, 375, 750 and 1500 mg/kg bw of the test substance was administered orally by gavage to groups of 5 male and female NMRI mice (BASF 1995). Signs of toxicity were observed in the mid and high dose level groups. After single application of the test substance, the animals were sacrificed 24 or 48 h post-dosing and bone marrow slides were prepared. There were no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups or between the two sacrifice intervals. Based on the results of the study, it was concluded that 2 -Aminoethanol has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available information on the test item regarding genetic toxicity are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information,the test substance is not classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.