3-morpholinopropylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 June 1992 to 29 June 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Organisation for Economic Co-operation and Development Guidelines for Testing Chemicals ISBN 92-64-12221-4

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no strain tested for detecting certain mutagens, cross-linking agents and hydrazines (i.e. strains TA102 or WP2uvrA)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-morpholinopropylamine

EC Number:

204-590-2

EC Name:

3-morpholinopropylamine

Cas Number:

123-00-2

Molecular formula:

C7H16N2O

IUPAC Name:

3-morpholin-4-ylpropan-1-amine

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: not specified
- Expiration date of the lot/batch: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the clear glass container received from the sponsor;
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: not specified

Method

Target gene:

Histidine locus (Salmonella strains)

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 - induced rat liver S9

Test concentrations with justification for top dose:

Toxicity Pre-screen (TA1538 and TA100): 0, 50.0, 167, 500, 1670 and 5000 µg per plate with and without S9;
Mutation Assay (all strains): 0, 167, 500, 1670, 5000, 7500 and 10000 µg per plate with and without S9;
Retest (all strains): 16.7, 50.0, 167, 500, 1670 and 5000 µg per plate with S9;

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9; 10.0 µg/plate for TA1535 and TA100

Controls 2

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9; 150 µg/plate for TA1537

Controls 3

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9; 5.00 µg/plate for TA1538 and TA98

Controls 4

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

With S9; 2.50 µg/plate for TA1535, TA1537, TA1538, TA98 and TA100

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Toxicity prescreen:
Treatment was performed by combining 0.1 mL tester strain, 0.1 mL of the appropriate concentration of the test article or solvent and 2 mL of molten top agar (supplemented with 0.5 mM histidine / 0.5 mM biotin). The tubes were vorteced and the mixture was poured onto minimal glucose plates, evenly distributed, and allowed to solidify. Within an hour the plates were inverted and incubated in the dark at 37°C for 48 hours. All cultures/plates were identified using computer-generated adhesive labels that included information regarding study number, date, strain, test/control article and +-S9.
Mutation Assay (and Retest):
Treatment for the mutation assay was performed exactly as described in the toxicity prescreen, except that the test and control articles were evaluated in triplicate cultures in all five tester strains in the presence and absence of an exogenous metabolic activation system (S9). Cultures treated in the presence of S9 containced 0.5 mL of the S9 mixture. The S9 mixture contained 8 mM MgCl2, 33 mM KCl, 4 mM NADP, 5 mM glucose-6-phosphate, 100 mM Na2HPO4 (pH 7.4) and 6% (v/v) Aroclor 1254-induced male Sprague-Dawley rat liver homogenate.

DURATION
- Exposure duration: 48 hours
- Selection time (if incubation with a selection agent): 48 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS:triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Inhibited growth: characterized by the reduced or absent bacterial lawn and/or the presence of pindot colonies

Evaluation criteria:

A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. Statistical analyses were performed using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. If the test article does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine-independent revertants.

Statistics:

Statistical analyses are performed only when a 50% increase in revertant frequency, relative to the concurrent negative controls, is observed. This 50% "trigger" was selected based upon the normal, spontaneous variation observed among replicate negative control cultures, as well as spontaneous fluctuation observed in this laboratory among groups of cultures treated with a variety of test articles judged to be negative in this assay.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate and upwards

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not specified
- Precipitation: no precipitation has been observed
- Other: sterility results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

RANGE-FINDING/SCREENING STUDIES:
Toxicity of the test article was determined in a preliminary toxicity prescreen by evaluating the growth of the background lawn and/or frequency of spontaneous revertants. The test article was evaluated at doses of 50, 167, 500, 1670 and 5000 µg/plate in the absence of S9. Each test article dose, as well as the appropriate solvent control, was evaluated in duplicate cultures in strains TA1538 and TA100.
Based upon the results of the toxicity prescreen, the dose levels selected for the mutation assay were 167, 500, 1670, 5000, 7500 and 10000 µg per plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive/Negative historical control data: All positive and negative control values were within acceptable limits.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Results of the toxicity prescreen indicated the test item was not toxic to either strain at doses of 50, 167, 500, 1670 and 5000 µg/plate.
In the Mutation Assay, inhibited growth (characterized by a reduced background lawn and/or the presence of pindot colonies) was observed in all strains at doses of 500, 1670, 5000, 7500 and/or 10000 µg/plate with S9. Therefore, the test item was reevaluated in all five strains at doses of 16.7, 50, 167, 500, 1670 and 5000 µg/plate with S9 (fewer than four acceptable doses with S9 were available for most strains in the original assay). Inhibited growth again was observed in all strains ad doses of 500, 1670 and/or 5000 µg/plate with S9.

Applicant's summary and conclusion

Conclusions:

Interpretation of the results: negative with and without metabolic activation

The results for test article were negative in the Ames/Salmonella Plate Incorporation Assay under the conditions, and according to the criteria, of the test protocol