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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
only four strains used in study, only one positive control used for the assay with metabolic activation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no strain used for the detection of oxidising mutagens and cross-linking agents; 2-aminoanthracene used as single positive control for the assays with metabolic activation
Principles of method if other than guideline:
The study was conducted according to the testing protocol published by Ames (1973, 1975) and in accordance with OECD 471 from 26 May 1983, which was replaced.
Ames BN et al. (1973). Proc. Nat. Acad. Sci. USA 70: 2281 -2285
Ames BN et al. (1975). Mut. Res. 31: 347 - 364
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethyl-N,N-bis(2-ethylhexyl)hexylamine
EC Number:
217-461-0
EC Name:
2-ethyl-N,N-bis(2-ethylhexyl)hexylamine
Cas Number:
1860-26-0
Molecular formula:
C24H51N
IUPAC Name:
tris(2-ethylhexyl)amine
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Tri-2-Aethylhexylamin
- Physical state: liquid
- Analytical purity: approx. 98%
Specific details on test material used for the study:
- Analytical purity: approx. 98 %
- Impurities (identity and concentrations): approx. 1.5 % Di-2-ethylhexylamine

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
MEDIA USED
- Type and composition of media: nutrient broth solution (8 g Difco bacto nutrient broth + 5 g NaCl/liter)
- temperature: 37°C
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : 5 male Sprague-Dawley rats, injected with Aroclor 1254, 5 days before sacrifice
- method of preparation of S9 mix: livers washed, homogenized in three volumes of KCl solution (150 mM), centrifugation at 9,000 g for 10 min, use of supernatent
Test concentrations with justification for top dose:
20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
- Ssolvent used: acetone
Controls
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
water control
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
other: with S-9 mix: all strains 2-aminoanthracene; without S-9 mix: strains TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine; TA98: 4-nitro-o-phenylendiamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate test

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 test plates per test concentration or control; two experiments were carried out independently.
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
- 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 S-9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test results
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Complete solubility of the test substance in acetone up to the highest dose of 5000 µg/plate.

Any other information on results incl. tables

Table 1: Standard plate test 1 (Mean ± SD)

Dose (µg/plate) TA 1535 TA 100 TA 1537 TA 98
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 17±4 14±5 130±6 113±6 6±1 6±1 27±3 37±5
20 14±3 11±1 135±26 142±9 10±2 9±2 21±4 34±7
100 16±5 14±1 127 136±9 10±2 12±5 18±6 34±4
500 17±5 13±1 152±17 139±10 9±2 12±5 22±3 33±6
2500 16±2 16±3 118±20 132±5 11±2 10±1 25±1 28±6
5000 17±4 13±6 124±6 152±8 5±3 10±1 23±3 19±1
2-AA - 453±76 - 2500±278 - 141±42 - 1423±45
MNNG 2367±29 - 2467±333 - - - - -
AAC - - - - 60±16 - - -
NPD - - - - - - 563±140 -

Table 2: Standard plate test 2 (Mean ± SD)

Dose (µg/plate) TA 1535 TA 100 TA 1537 TA 98
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 15±3 14±4 101±6 108±4 7±2 7±1 27±4 41±6
20 14±4 12±2 94±12 128±10 7±1 6±2 28±6 31±3
100 14±4 13±1 107±7 113±10 5±1 8±2 21±9 31±3
500 10±1 16±5 96±11 107±3 4±1 8±2 25±5 31±3
2500 11±1 16±2 87±8 110±15 5±1 4±1 27±5 30±4
5000 11±1 13±5 83±4 115±3 4±2 3±1 29±5 37±9
2-AA - 319±20 - 2017±206 - 211±15 - 1533±166
MNNG 1640±284 - 1910±62 - - - - -
AAC - - - - 632±35 - - -
NPD - - - - - - 873±44 -

2-AA: 2-aminoanthracene;

MNNG; N-methyl-N-nitro-N-nitrosoguanidine

NPD: 4-nitro-o-phenylendiamine

AAC: 9-aminoacridine chloride monohydrate

Applicant's summary and conclusion

Conclusions:
Under the conditions tested the test substance is non mutagenic in the bacterial AMES-test.
The positive control gave the expected values.