2-ethyl-N,N-bis(2-ethylhexyl)hexylamine

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:

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

Test material information

Constituent 1

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.