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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
other: The study was conducted according to the OECD Guidelines in compliance with GLP.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1998
Reference Type:
study report
Title:
Unnamed
Year:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(2-aminoanthracene was used as the sole indicator of the efficacy of the S9-mix)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
yes
Remarks:
(2-aminoanthracene was used as the sole indicator of the efficacy of the S9-mix)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Kokosmorpholin
- Physical state: Liquid, colourless, clear
- Analytical purity: 93.5% (GC analysis)
- Lot/batch No.: V. 917 - Qualitaet II
- Stability of the test substance over the study period: Proven by reanalysis (BASF Analytical Report 97C00155, as given in the amendment)
- Storage condition of test material: Room temperature, in absence of moisture

Method

Target gene:
his operon (for S. typhimurium strains) and trp operon (for E. coli).
Species / strainopen allclose all
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:
cofactors supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
1st experiment (all S. typhimurium and E.coli strains, standard plate test with and without S-9 mix, 3 plates/dose): 0; 20; 100; 500; 2500 and 5000 μg/plate
2nd experiment (all S. typhimurium strains, standard plate test with and without S-9 mix, 3 plates/dose): 0; 3.125; 6.25; 12.3; 25 and 50 μg/plate
3rd experiment (all S. typhimurium strains, standard plate test with and without S-9 mix, 3 plates/dose): 0; 0.625; 1.25; 2.5; 5 and 10 μg/plate
4th experiment (all S. typhimurium and E.coli strains, preincubation test with and without S-9 mix, 3 plates/dose): 0; 0.625; 1.25; 2.5; 5 and 10 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Insolubility of the test substance in water.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
(sterility control)
Negative solvent / vehicle controls:
yes
Remarks:
(acetone)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA): 2.5 μg/plate dissolved in DMSO for strains TA 1535, TA 100, TA 1537 and TA 98; 60 μg/plate dissolved in DMSO for Escherichia coli WP2 uvrA
Remarks:
With S9-mix
Untreated negative controls:
yes
Remarks:
(sterility control)
Negative solvent / vehicle controls:
yes
Remarks:
(acetone)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 5 μg/plate (DMSO), TA1535,TA100; 4-nitro-ophenylendiamine (NOPD) 10 μg/plate (DMSO), TA98; 9-aminoacridine (AAC) 100 μg/plate (DMSO), TA1537; N-ethyl-N'-nitro-N-nitrosoguanidin (ENNG) 10 μg/plate (DMSO), E.coli
Remarks:
Without S9-mix
Details on test system and experimental conditions:
TEST DESIGN
Standard plate test (SPT) and preincubation test (PIT), both with and without metabolic activation, were performed.

Standard plate test (SPT, Experiment 1 - 3):
The experimental procedure was based on Ames et al. (Mut. Res. 31: 347-364, 1975) and Maron & Ames (Mut. Res. 113: 173-215, 1983).
Test tubes containing 2 mL portions of soft agar [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 histidine + 0.5 mM biotin for S. typhimurium or 0.5 mM tryptophan for E.coli)] were kept in a water bath at about 45°C. 0.1 mL test solution or vehicle (negative control), 0.1 mL fresh bacterial culture and 0.5 mL S9 mix (in case of metabolic activation) or 0.5 mL phosphate buffer (in case of no metabolic activation) were added. After mixing, the samples were poured onto minimal agar plates and incubated at 37°C for 48 to 72 h in the dark. After incubation, the bacterial colonies were counted for revertant colonies.

Preincubation Test (PIT, Experiment 4):
The experimental procedure was based on the method described by Yahagi et al. (Mut. Res. 48: 121-130, 1977) and Matsushima et al. (In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980). 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (in case of metabolic activation) or phosphate buffer (in case of no metabolic activation) were incubated at 37°C for about 20 min using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates and incubated at 37°C for 48 to 72 h in the dark. After incubation, the bacterial revertant colonies were counted.

PARAMETERS EXAMINED
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.
Titer: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest test concentrations in all experiments.
Cytotoxicity: Toxicity was detected by (1) decrease in the number of revertants, (2) clearing or diminution of the background lawn (= reduced his- or trp- background growth) and (3) reduction in the titer. Cytotoxicity was recorded for all test groups both with and without S9 mix in all experiments.
Solubility: Precipitation of the test substance was recorded and indicated. As long as precipitation does not interfere with colony scoring, 5000 μg/plate is 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.
Evaluation criteria:
The test substance is positive if 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 or with S9-mix, is observed.

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Remarks:
(from 6.5 to 12.5 μg/plate for the S. typhimurium strains, and at doses >= 2500 μg/plate for E. coli)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
cytotoxicity
Remarks:
(from 5 to 10 μg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MUTAGENICITY
When tested in the S. typhimurium strains TA1535, TA1537, TA100 and TA98 and in E. coli WP2uvrA under standard plate test (SPT) and preincubation test (PIT) conditions, with and without S9 mix, the test substance did not induce an increase in his+ or trp+ revertant colonies.

CYTOTOXICITY
A cytototoxic effect was observed in the SPT from about 6.25 - 12.5 μg/ plate onward for the Salmonella strains, and at doses >= 2500 μg/plate for E. coli WP2 uvrA. In the PIT, cytotoxicity was observed for all strain at about 5 to 10 μg/plate.

PRECIPITATION
Test substance precipitation was observed at 5000 μg/plate.

POSITIVE CONTROLS
The positive control substances induced the expected increases in revertant colonies, both with and without S9- mix.
Remarks on result:
other: other: Standard plate test (SPT, Experiment 1, 2 and 3)
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions chosen, the test substance is not mutagenic in bacteria in the absence and presence of metabolic activation.
Executive summary:

A study was conducted to determine the mutagenic potential of C12-14 alkylmorpholine according to the OECD Guideline 471 and 472 in compliance with GLP.

Tester strains TA98, TA100, TA1535, TA1537 of Salmonella typhimurium and WP2uvrA of Escherichia coli were exposed to the test substance in the standard plate test (SPT) and the preincubation test (PIT).The test concentrations were as follows: 1st Experiment (all S. typhimurium and E.coli strains, standard plate test with and without S-9 mix, 3 plates/dose): 0; 20; 100; 500; 2500 and 5000 μg/plate; 2nd Experiment (all S. typhimurium strains, standard plate test with and without S-9 mix, 3 plates/dose): 0; 3.125; 6.25; 12.3; 25 and 50 μg/plate; 3rd Experiment (all S. typhimurium strains, standard plate test with and without S-9 mix, 3 plates/dose): 0; 0.625; 1.25; 2.5; 5 and 10 μg/plate; 4th Experiment (all S. typhimurium and E.coli strains, preincubation test with and without S-9 mix, 3 plates/dose): 0; 0.625; 1.25; 2.5; 5 and 10 μg/plate.

When tested in all strains mentioned above under SPT and PIT conditions, with and without S9 mix, the test substance did not induce an increase in his+ or trp+ revertant colonies at concentrations up to 5000 μg/plate. A cytototoxic effect was observed in the SPT from about 6.25 - 12.5 μg/ plate onward for the Salmonella strains, and at doses >= 2500 μg/plate for E. coli WP2 uvrA. In the PIT, cytotoxicity was observed for all strain at about 5 to 10 μg/plate. Test substance precipitation was observed at 5000 μg/plate. The positive control substances induced the expected increases in revertant colonies, both with and without S9-mix.

Thus, under the experimental conditions chosen, the test substance was not mutagenic in bacteria in the absence and presence of metabolic activation.