Fatty acids, tall-oil, esters with ethylene glycol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Sep 2004 - 13 Oct 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted in 2000

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, coco, esters with 1,3-butanediol

EC Number:

277-288-1

EC Name:

Fatty acids, coco, esters with 1,3-butanediol

Cas Number:

73138-39-3

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

Fatty acids, coco, esters with 1,3-butanediol

Test material form:

liquid

Method

Target gene:

his operon for Salmonella strains
trp operson for E. coli strain

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:

Rat liver S9 mix (10% S9 fraction) provided by MOLTOX TM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 -USA) - no further information in study report given

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500, and 5000 µg/plate.
No cytotoxicity seen up to top dose of 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: paraffin oil;
- Justification for choice of solvent/vehicle: not stated

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine, ß-Propiolactone, cis-Platinum (II) Diammine Dichloride, Dimethyl- benzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); Experiment 2 was done with pre-incubation
For each of the strains, 100 µL of the bacterial suspension (1-5 x 10e9 bacteria/mL) and 100 µL of the test substance were mixed with 2.0 mL of overlay agar and poured over the surface of a minimal agar plate (90 mm in diameter) (n = 3). The overlay agar was allowed to solidify before incubation.

DURATION
- Preincubation period: Experiment 1 was done without pre-incubation, Experiment 2 was done with pre-incubation (test substance preincubated with the test strain, and 500 µL of S9-mix fraction for 1 hour at 37° C prior to mixing with the overlay agar)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates in each of the 2 experiments
NUMBER OF ASSAYS: 2 per bacteria strain

DETERMINATION OF CYTOTOXICITY
Reduction in the number of spontaneous revertants.

Bacteriostatic activity was tested in Salmonella strain TA100 in two different assays with three replicates each. 100 µL of the bacterial suspension (1-5x10e9 bacteria/mL) and the different concentrations of the test substance were added to 2 mL of overlay agar at 45°C, containing 10% (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogeneization, the content of the tube was poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration were incubated for 48 hours at 37° C, and the number of colonies counted. A negative control containing the solvent alone was run in parallel.

For test substances that were cytotoxic already below 5000 µg/plates, the highest concentration to be retained was that exhibiting a bacteriostatic activity of 75% or less. The precipitate, if present, should not interfere with the scoring.

Evaluation criteria:

The result of the test is considered positive if a concentration - related increase is obtained in one, or several of the 5 strains, with and/or without metabolic activation; a mutagenic effect is taken into account for a given concentration of the test substance if the number of revertant colonies is at least two fold that of spontaneous revertant colonies number for TA 98, TA 100 and Escherichia coli WP2(uvrA) (pKM 101), and three fold for TA 1535 and TA 1537.

Statistics:

Descriptive statistics (mean, standard error, R)

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not stated
- Precipitation: not stated

HISTORICAL CONTROL DATA
- Positive historical control data (means and standard deviation): The historical values of the laboratory (January 2003 - December 2003) were within the expected range for all five strains tested with and without S9 (with and without pre-incubation with S9).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Salmonella strain TA 100 was successfully tested for absence of any bacteriostatic activity at doses ranging from 50 to 5000 µg/plate in two assays

Any other information on results incl. tables

Table 2: Mean (triplicates) number of revertant colonies ±standard error

Experiment I	without S9
	TA1535	TA1537	TA98	TA100	WP2 uvrA pKM101
Vehicle	12±1.0	2.67±2.08	15.67±1.53	83.67±8.08	64.33±8.33
Negative control	11±2.0	3.67±1.15	14.33±1.53	80.67±3.79	62.33±7.02
5000	7.67±2.08	3.33±0.58	11.33±3.21	49±18.33	30±8
1500	11.0±1.0	3.33±0.58	16.33±3.51	46.33±10.26	55.33±14.29
500	8.67±0.58	3.33±1.15	16.33±2.52	70.33±13.2	51.67±10.02
150	11.33±1.15	3.67±1.53	18.0±2.0	113±14.73	59.33±24.83
50	14.0±4.58	2.67±0.58	14.67±1.15	105.33±5.51	61.33±21.78
beta propiolactone [50 µg in 10 µL]	76.33±12.06
9-aminoacridine [50 µg in 20 µL]		536.67±106.93
2-nitrofluorene [2 µg in 20 µL]			807.67±164.63
Sodium azide [20 µg in 20 µL]				915.33±135.66
cis-platinum diamine dichloride [1 µg in 10 µL]					636.33±61.45

 

Experiment I	with S9
	TA1535	TA1537	TA98	TA100	WP2 uvrA pKM101
Vehicle	12.67±2.08	3.0±1.73	22.0±2.0	96.33±13.8	126±10.82
Negative control	13.33±2.08	4.0±1.0	22.0±2.0	105.33±8.74	145±15.72
5000	11.0±2.0	3.0±1.0	12.33±3.06	71±11.53	46±9.54
1500	16.33±4.62	2.33±0.58	19.0±2.0	93.33±7.77	64.33±17.9
500	9.33±0.58	3.67±1.15	24.67±19.4	80.33±6.03	106±32.05
150	12.67±2.08	4.0±1.0	25.67±2.08	115.67±7.57	124±50.21
50	13.0±1.0	5.0±2.0	29.33±4.16	130.67±12.06	123.67±42.44
2-anthramine [2 µg in 20 µL]	72.0±11.14	51.33±8.62	908.33±108.64	821.67±30.73
Dimethylbenzanthracene [5 µg in 5 µL]					699.33±108.82

 

Experiment II	without S9
	TA1535	TA1537	TA98	TA100	WP2 uvrA pKM101
Vehicle (ethanol)	12.67±1.53	2.33±0.58	17±2	93±3	72.67±8.74
Negative control	14.67±2.08	3.67±0.58	15.33±2.52	85±6.56	70.33±12.5
5000	8.33±3.06	4.0±5.2	13.67±0.58	96.67±14.57	65.67±13.32
1500	8.67±2.52	4.0±5.2	14.67±2.08	83.33±4.04	68.33±18.77
500	11.33±3.21	3.0±1.0	14.67±2.0	96.67±8.33	99.67±11.93
150	11.67±1.53	2.33±0.58	15.0±2.0	104.67±13.5	72.33±16.86
50	11.0±2.65	2.0±1.0	14.33±1.53	95.67±9.45	71.0±14.11
beta propiolactone [50 µg in 10 µL]	70.67±16.2
9-aminoacridine [50 µg in 20 µL]		667.67±169.52
2-nitrofluorene [2 µg in 20 µL]			826.67±63.81
Sodium azide [20 µg in 20 µL]				768±118.19
cis-platinum diamine dichloride [1 µg in 10 µL]					523.33±148.44

 

Experiment II	with S9
	TA1535	TA1537	TA98	TA100	WP2 uvrA pKM101
Vehicle (ethanol)	11.67±3.06	2.67±1.15	23±1	118.33±3.06	128±10.82
Negative control	14.0±3.0	4.0±1.0	20±2	117±7.21	137±6.24
5000	7.33±4.04	4.0±5.2	15±2	102±9.85	95±7.21
1500	10.0±2.65	4.33±4.93	18.33±3.51	128.33±13.58	149.67±13.01
500	11.0±3.0	1.67±0.58	20.67±4.04	107.67±28.73	135±50.12
150	8.0±1.0	1.67±0.58	24.67±3.21	114.33±14.01	157.67±14.29
50	12.3±1.53	3.0±1.0	22.67±2.52	101.33±7.77	147.33±6.66
2-anthramine [2 µg in 20 µL]	63.67±5.51
2-anthramine [1 µg in 20 µL]		53.33±4.04
2-anthramine [1 µg in 10 µL]			481.33±81.68	823.33±94.71
Dimethylbenzanthracene [2.5 µg in 5 µL]					599.33±76.06

 

Applicant's summary and conclusion

Conclusions:

No mutagenic potential in any of the 5 bacterial strains tested seen in 2 independent experiments with 3 replicates each.