1-aminopropan-2-ol

Genetic toxicity: in vitro

Administrative data

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:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

- initial OECD TG 471 + 472, adopted 26 May 1983.
- OECD 471, adopted: 21 July 1997, corrected: 26 June 2020.

Deviations:

no

Principles of method if other than guideline:

based on Ames et al. (1975) with modifications originally described by Yahagi et al. (1975) and Maron and Ames (1983):
- Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Research 2:347-364.
- Maron, D.M., and Ames B. (1983). Revised Methods for the Salmonella Mutagenicity Test. Mutation Research 113: 173-2 15.
- Yahagi, T., Degawa, M., Seino Y., Matsushima, T., Nagao, M., Sugimura, T., and Hashimoto, Y. (1975). Mutagenicity of carcinogenic azo dyes and their derivatives. Cancer Letters 1:91-96.

This methodology (preincubation modification) has been shown to detect mutagenicity with certain classes of chemicals, such as nitrosamines or volatile test articles, which may not be detected in the standard plate incorporation method.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-aminopropan-2-ol

EC Number:

201-162-7

EC Name:

1-aminopropan-2-ol

Cas Number:

78-96-6

Molecular formula:

C3H9NO

IUPAC Name:

1-aminopropan-2-ol

Specific details on test material used for the study:

Chemical Name: Monoisopropanolamine (MIPA)

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: The Dow Chemical Co., Midland, MI,
lot MM930105
- Purity, including information on contaminants, isomers, etc.: 99.63%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Actual concentrations of the stock test articles dosing solutions ranged from 87% to 116% of target. Clear, colorless solution at 100 mg/ml in sterile deionized water.

Method

Species / strain

Species / strain / cell type:

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

Remarks:

WP2uvrA

Details on mammalian cell type (if applicable):

deficiencies: Histidine (Salmonella) and tryptophan (E. coli)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 -induced rat liver

Type and composition of metabolic activation system:
- source of S9: Liver microsomal enzymes (S9 homogenate) were purchased from Molecular Toxicology, Inc., Annapolis, MD 2 140 1, Batch 05 15 (40.0 mg of protein per ml). The homogenate was prepared from male Sprague-Dawley rats that had been injected (i.p.) with AroclorTM 1254 (200 mg per ml in corn oil) at 500 mg/kg as described by Ames et al, 1975.
- method of preparation of S9 mix: The S9 mix was prepared immediately prior to its use in any experimental
procedure.
0.70 ml H2O
0.10 ml 1M NaH2P04/Na2HP04, pH7.4
0.02 ml 0.25M Glucose-6-phosphate
0.04 ml 0.10 M NADP
0.04 ml 0.825M KCl/0.2M MgCl2
0.10 ml S9 Homogenate
--> 1.00 ml
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µl vehicle or test article dilution, 100 µl tester strain, 500 µl aliquot of S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): check for sterility by plating 0.5 ml on selective agar

Test concentrations with justification for top dose:

100, 250, 500, 1000, 2500 and 5000 ug/plate

Doses to be tested in the mutagenicity assay were selected based on the results of the dose range finding study conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix (one plate per dose). Ten doses of test article, from 5,000 to 6.67 µg per plate, were tested (Experiment 16247-A1). Cytotoxicity was observed with both tester strains TA100 and WP2uvrA at the 3,330 and 5,000 pg per plate doses in both the presence and absence of S9 mix as evidenced by a reduction in the number of revertants per plate and/or a thinning of the bacterial background lawn. No test article precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 mix.

Vehicle / solvent:

Sterile deionized water (CHV Batch 296,297,298 and 299)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

- no MA: TA98: 1.0 µg 2-nitrofluorene, TA100, TA 1535: 2.0 µg sodium azide, TA1537: 2.0 µg ICR-191, WP2uvrA: 1.0 µg 4-nitroquinoline-N-oxide. - with MA: TA98, TA100, TA1535, TA1537: 2.5 µg 2-aminoanthracene, WP2uvrA: 25.0 µg 2-aminoanthracene.

Details on test system and experimental conditions:

- Number of replicates: 3 plates/dose
- Pre-incubation time: 20 min. 37 C

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: preincubation

TREATMENT SCHEDULE:
- Preincubation period, if applicable: The test article, the tester strain and the S9 mix (or phosphate buffer, where appropriate) were preincubated for 20 +/- 2 minutes at 37 +/- 2°C prior to the addition of molten, selective overlay agar.
- Exposure duration/duration of treatment: The agar and the preincubation reaction mixture were mixed and then overlaid onto a minimal agar plate. Following incubation at 37 +/- 2°C for 48 +/- 8 hr, revertant colonies were counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Cytotoxicity was detected as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn.

CRITERIA FOR A VALID ASSAY:
1.) Tester Strain Integrity: S. typhimurium (rfa wall mutation, pKM101 Plasmid, characteristic number of spontaneous revertants).
2.) Tester Strain Integrity: E. coli (characteristic number of spontaneous revertants).
3.) Tester Strain Culture Density: density of tester strain cultures were greater than or equal to 0.5 x 10exp9 bacteria per ml and/or had reached a target level of turbidity demonstrated to produce cultures with a density greater than or equal to 0.5 x 10exp9 bacteria per ml.
4.) Positive Control Values: without or with MA at least a 3-fold increase over the mean value of the vehicle control for that strain;
5.) Cytotoxicity: A minimum of three non-toxic doses were required to evaluate assay data.

Rationale for test conditions:

- highest test concentration: 5000 µg/plate, which is the recommended maximum test concentration for soluble non-cytotoxic substances in the OECD TG471.
- dose range findings study with 10 concentrations: 6.67, 10, 33.3, 66.7, 100, 333, 667, 1000, 3330, 5000 µg/plate TA100 and WP2uvrA
- preincubation study with 6 concentrations: 100, 250, 500, 1000, 2500, 5000 µg/plate.

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean
revertants per plate of at least one tester strain over the mean revertants per plate of the
appropriate vehicle control. This 3-fold or greater increase in the mean number of revertants per
plate had to be observed at more than one dose and had to be accompanied by a dose response to
increasing concentrations of the test article. In addition, the observed dose-responsive increase
had to be shown to be reproducible. An observed response which did not meet all three of the
above criteria (magnitude, dose-responsiveness, reproducibility) was not evaluated as positive.

Statistics:

mean revertants per plate +/- standard deviation

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with MA: >= 2500 µg/plate; without MA: 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with MA: >= 2500 µg/plate; without MA: 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with MA: >= 2500 µg/plate; without MA: 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with MA: >= 2500 µg/plate; without MA: 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with MA: >= 2500 µg/plate; without MA: 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- with MA: >= 2500 µg/plate normal-slightly reduced background lawn;
- without MA: 5000 µg/plate moderately-extremely reduced background lawn

Any other information on results incl. tables

Result tables: see attached pdf in section "Overall remarks, attachments"

Applicant's summary and conclusion

Conclusions:

Not mutagenic

Executive summary:

The results of the Salmonella - Escherichia colilMammalian-Microsome Reverse Mutation Assay, Preincubation Method with a Confirmatory Assay, indicate that under the conditions of this study, in both an initial and a confirmatory assay, the test article, Monoisopropanolamine, did not cause a positive increase in the number of revertants per plate of tester strains TA98, TA100, TA1535, TA1537, or WP2uvrA either in the presence or absence of microsomal enzymes prepared from AroclorTM-induced rat liver up to the highest dose of 5000 µg/plate tested.