2-ethylhexanoyl chloride

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:

guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Reference 2

Reference Type:

secondary source

Title:

SIDS Initial Assessment Report for SIAM 27 - Acid Chlorides (AC) Category

Author:

OECD SIDS

Year:

2008

Bibliographic source:

OECD SIDS

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

strain missing to detect crosslinking/ oxidizing agents

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexanoyl chloride

EC Number:

212-081-1

EC Name:

2-ethylhexanoyl chloride

Cas Number:

760-67-8

Molecular formula:

C8H15ClO

IUPAC Name:

2-ethylhexanoyl chloride

Details on test material:

The purity of the test material was > 99%.

Method

Target gene:

his-

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

liver S-9 mix from Aroclor 1254 induced rats

Test concentrations with justification for top dose:

20 - 5000 micrograms/plate (Standard Plate test; TA 100, TA 98)
4 - 2500 micrograms/plate (Standard plate test; TA 1535, TA 1537 and preincubation test on all strains)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

S-9 was prepared from at least 5 male Sprague-Dawley rats (200 - 300 g) that received an i.p. injection of 500 mg Aroclor 1254 (as a 20% solution in peanut oil) five days before liver collection. S-9 was stored at -70 to -80 degrees C for up to 2 months. S-9 mix was prepared freshly prior to each experiment. Three volumes of S-9 were mixed with 7 volumes of a cofactor mix containing 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phsophate, 4 mM NADP and 100 mM sodium phosphate buffer (pH 7.4).
The Salmonella strains (TA98, TA100, TA1535, and TA1537) were checked periodically for deep rough character (rfa), UV sensitivity (uvrB), and ampicillin resistance (R factor plasmid). Deep-frozen bacterial cultures were thawed, and 0.1 ml of the bacterial suspension was inoculated in nutrient broth and incubated in a shaking water bath at 37 degrees C for about 16 hours (to an approximate density of > = 10E8 bacteria/ml). The cultures were then placed in ice water to prevent further growth.
For the standard plate test, test solution (0.1 ml), bacterial suspension (0.1 ml), and phosphate buffer or S-9 mix (0.5 ml) were added to tubes containing 2 ml soft agar. For the preincubation test, the test solution, bacterial suspension and S-9 mix were incubated at 37 degrees C for 20 minutes before being added to the soft agar. After mixing, the samples for either test were poured onto minimal glucose agar plates. Cells were incubated at 37 degrees C for 48 hours in the dark, and the number of colonies was counted. All tests were performed in triplicate.
The concentrations tested varied according to experiment: (standard incubation experiment 1 (with and without S-9 mix): 0, 20, 100, 500, 2500 and 5000 micrograms/plate in strains TA98 and TA100; standard incubation experiment 2 (with and without S-9 mix): 0, 4, 20, 100, 500, and 2500 micrograms/plate in strains TA1535 and 1537; preincubation experiment (with and without S-9): 0, 4, 20, 100, 500 and 2500 micrograms/plate in all strains.
A solvent control (DMSO) and the positive controls N-methyl-N'-nitro-N-nitroso-guanidine (5 micrograms/plate for strains TA100 and TA1535), 4-nitro-o-phenylenediamine (10 micrograms/plate, strain TA98), 9-aminoacridine (100 micrograms/plate for strain TA1537), and 2-aminoanthracene (10 micrograms/plate for all strains with S-9) were tested in each experiment. The positive control chemicals were dissolved in DMSO.
Precipitation of the test material was recorded (if present).
Toxicity was detected by a decrease in the number of revertants, a clearing or dimunition of the background lawn or a reduction in the titer.

Evaluation criteria:

A substance was considered mutagenic if it caused a doubling in the spontaneous (control) mutation rate, and the effect was dose-dependent and reproducible.
The experiment was considered valid if the number of colonies in the negative controls was within the normal range of the historical data for the strain, sterility controls had no evidence of contamination, the positive controls induced a significant increase in the number of revertants and the titer of viable bacteria was >/= 10E9/ml.

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:

cytotoxicity

Remarks:

5000 micrograms/plate (Standard Plate test; TA100 and TA98), >= 500 micrograms/plate (preincubation test, all strains)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Standard plate test

Dose (µg/plate)	TA 98		TA100			TA1535		TA 1537
	-S9	+S9	-S9	+S9		-S9	-S9	-S9	+S9
DMSO	21±1	34±3	108±3	113±18	DMSO	18±4	17±3	8±1	9±2
20 µg	22±2	36±6	136±7	107±2	4 µg	19±6	23±5	8±2	10±2
100 µg	20±1	30±3	141±10	122±12	20 µg	15±3	22±4	12±2	11±1
500 µg	19±3	35±10	124±10	108±4	100 µg	21±4	19±4	8±3	11±2
2500 µg	17±5	30±4	134±10	114±3	500 µg	20±2	26±1	10±2	14±8
5000 µg	9±4	B	123±11	B	2500 µg	18±6	23±4	9±2	8±2
2-AA		1160±20		1967±176			193±14		177±15
MNNG			1587±185			2077±219
NPD	922±11
9-AAC								656±58

Preincubation test

Dose (µg/plate)	TA 1535		TA100		TA1537		TA98
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	20±5	13±1	106±9	109±12	7±1	6±1	18±2	20±2
4 µg	23±5	16±4	98±4	101±7	8±2	8±1	22±3	33±1
20 µg	22±2	19±2	113±1	108±13	10±2	10±1	22±1	34±7
100 µg	23±4	14±3	133±7	115±6	10±4	8±1	25±3	32±5
500 µg	10B±5	2B±0	B	80B±18	B	3B±1	B	13B±3
2500 µg	B	B	0	B	0	B	0	B
2-AA		227±43		1313±83		112±17		545±45
MNNG	1237±45		1187±15
NPD							678±4
9-AAC					361±16

B: reduced his-background

2-AA: 2-aminoanthracene

MNNG: N-methyl-N´-nitor-N-nitrosoguanidine

NPD: 4-nitro-o-phenylendiamine

9-AAC: 9-aminoacridine chloride monohydrate

Applicant's summary and conclusion

Conclusions:

The test substance 2-ethylhexanoyl chloride is not mutagenic in the Ames test under the experimental conditions chosen here. .