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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
Study period:
17 Jun 2015 - 25 Jun 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP / OECD guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
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: 2-diethylaminoethanol
- Test substance No.: 02/0430-2
- Batch Identification: 000STD77L0
- Physical state: liquid, colorless to slightly yellow, clear
- Analytical purity: 98.4 g/100g
- Stability under test conditions: guaranteed until 10 Feb 2017 as indicated by the sponsor
- Storage condition of test material: Room temperature

Method

Target gene:
Salmonella typhimurium: histidine
Escherichia coli WP2 uvrA: tryptophan
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/b-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Standard plate test and Preincubation test: 0; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was used as vehicle
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: With S9 mix: 2-aminoanthracene (2-AA). Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine (NOPD); 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO).
Details on test system and experimental conditions:
TEST SYSTEM
For testing, deep-frozen (-70°C to -80°C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) werethawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth
+ 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The optical density of the fresh bacteria cultures was
determined. Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 109 cells per mL).
These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

1. Standard plate test +/- S9 mix:
METHOD OF APPLICATION: in agar (plate incorporation) according to Ames et al. 1975, Maron et al. 1983
NUMBER OF REPLICATIONS: 3 test plates /dose or control
2. Preincubation Test +/- S9 mix (because no mutagenicity was observed in the Standard plate test)
METHOD OF APPLICATION: in medium, according to Yahagi et al. 1977 und Matsushima et al. 1980
NUMBER OF REPLICATIONS: 3 test plates /dose or control
Evaluation criteria:
Acceptance criteria
Generally, the experiment was considered valid if the following criteria were 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.
• Fresh bacterial culture containing approximately 10exp9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous
mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of
the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
depending on the strain and test conditions for about 2500 µg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No test substance precipitation was found with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2500µg/plate onward.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

For detailed results see attached document.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions chosen here, it is concluded that 2-diethylaminoethanol is not a mutagenic test substance in the bacterial
reverse mutation test in the absence and the presence of metabolic activation