Fatty acids, C18-unsatd., dimers, di-Me esters, hydrogenated

Genetic toxicity: in vitro

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:

14 Jan – 05 Mar 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP – 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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

C36 fatty diol

EC Number:

604-608-2

Cas Number:

147853-32-5

Molecular formula:

not available UVCB

IUPAC Name:

C36 fatty diol

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Physical state: colourless viscous liquid
- Substance type: UVCB
- Analytical purity: no data
- Lot/batch No.: 091728
- Storage condition of test material: room temperature, in the dark

Method

Target gene:

his operon (for S. typhimurium strains) and trp operon (for the E. coli strain).

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with pheobarbitone/ß-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Experiments 1 and 2: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Other: Prior to use, the solvent was dried using molecular sieves.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: N-ethyl-N’-nitro-N-nitrosoguanidine (3 µg/plate) for TA100, (5 µg/plate) for TA1535 and (2 µg/plate) for WP2uvrA-; 9-Aminoacridine (80 µg/plate) for TA1537; 4-Nitroquinoline-1-oxide (0.2 µg/plate) for TA98

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: +S9: 2-Aminoanthracene (1 µg/plate) for TA100, (2 µg/plate) for TA1535 and TA1537, (10 µg/plate) for WP2uvrA-; Benzo(a)pyrene (5 µg/plate) for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration:
Preliminary toxicity test: 48 h
Main experiment: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn.

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett´s method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A clear, oily precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA: See Table 4 under `Any other information on results incl. tables`.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Preliminary toxicity test: The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-) see Table 1 under `Any other information on results incl. tables`.
Main experiment: The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Preliminary toxicity test.

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.05	1.5	5	15	50	150	500	1500	5000
-	TA100	117	134	111	133	124	109	109	121	117	103P	94P
+	TA100	112	100	96	108	115	88	114	108	104	106P	113P
-	WP2uvrA-	30	36	27	32	17	26	22	26	22	17P	38P
+	WP2uvrA-	18	21	17	24	31	5	27	27	31	25P	37P

P: Precipitate

 

Table 2. Test results of experiment 1 (plate incorporation).

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA-	TA98	TA1537
–	0	116 ± 8.1	14 ± 4.6	25 ± 3.1	32 ± 4.0	21 ± 2.1
–	50	112 ± 6.1	21 ± 3.0	24 ± 4.0	24 ± 4.0	24 ± 4.0
–	150	109 ± 17.0	15 ± 6.4	23 ± 5.5	21 ± 6.0	19 ± 6.9
–	500	113 ± 7.4	19 ± 4.0	18 ± 5.0	30 ± 2.5	20 ± 6.7
–	1500	104 ± 10.5P	19 ± 3.2P	18 ± 3.2P	29 ± 10.1P	23 ± 6.7P
–	5000	102 ± 3.5P	21 ± 8.5P	23 ± 2.1P	27 ± 1.5P	20 ± 6.6P
Positive controls, –S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	511 ± 27.5	480 ± 228.2	906 ± 75.5	238 ± 13.5	795 ± 42.5
+	0	123 ± 12.1	8 ± 0.6	38 ± 3.1	40 ± 6.7	24 ± 1.0
+	50	129 ± 5.8	13 ± 5.0	36 ± 6.5	53 ± 4.9	21 ± 5.7
+	150	131 ± 4.7	11 ± 3.8	43 ± 1.7	36 ± 13.6	20 ± 2.6
+	500	117 ± 7.5	9 ± 3.1	33 ± 4.0	27 ± 6.5	18 ± 9.2
+	1500	135 ± 5.6P	9 ± 0.6P	33 ± 6.5P	32 ± 6.5P	22 ± 4.9P
+	5000	121 ± 7.6P	11 ± 7.6P	40 ± 9.3P	27 ± 6.7P	22 ± 5.8P
Positive controls, +S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1186 ± 229.6	284 ± 20.7	417 ± 33.5	222 ± 22.9	314 ± 74.3

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate

Table 3. Test results of experiment 2 (plate incorporation).

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA-	TA98	TA1537
–	0	81 ± 2.8	23 ± 6.6	21 ± 3.1	29 ± 1.5	17 ± 5.8
–	50	71 ± 13.0	13 ± 4.4	26 ± 6.6	33 ± 11.3	13 ± 3.1
–	150	75 ± 12.7	13 ± 2.6	23 ± 2.6	26 ± 7.8	12 ± 5.5
–	500	66 ± 2.1	19 ± 2.1	18 ± 2.6	35 ± 9.1	8 ± 2.5
–	1500	82 ± 17.4P	12 ± 4.0P	22 ± 4.0P	44 ± 9.0P	13 ± 3.8P
–	5000	92 ± 13.0P	18 ± 2.5P	21 ± 2.5P	40 ± 1.2P	14 ± 5.6P
Positive controls, –S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	602 ± 92.8	175 ± 29.3	652 ± 89.8	168 ± 35.2	586 ± 156.0
+	0	88 ± 15.4	12 ± 4.0	25 ± 5.3	48 ± 3.2	18 ± 2.1
+	50	86 ± 3.5	9 ± 3.8	31 ± 2.3	49 ± 1.0	19 ± 2.9
+	150	83 ± 3.5	16 ± 4.6	27 ± 4.7	43 ± 3.1	12 ± 5.1
+	500	79 ± 8.5	9 ± 4.4	31 ± 1.7	44 ± 3.1	18 ± 7.4
+	1500	76 ± 10.4P	9 ± 2.3P	31 ± 3.5P	50 ± 5.6P	18 ± 2.1P
+	5000	91 ± 7.0P	12 ± 1.2P	30 ± 0.7P	44 ± 7.2P	16 ± 3.6P
Positive controls, +S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1129 ± 60.1	232 ± 19.2	592 ± 118.1	161 ± 10.1	202 ± 15.4

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

BP  = Benzo(a)pyrene

P = Precipitate

Table 4. History profile of vehicle and positive control values.

Strain S9-Mix	TA100		TA1535		WP2uvrA-
	-S9	+S9	-S9	+S9	-S9	+S9
Combined vehicle and untreated control values 2002 Mean ± SD	91 ± 16.6	100 ± 18.0	20 ± 5.5	17 ± 4.3	23 ± 4.8	27 ± 5.3
Positive control values 2002 Mean ± SD	460 ± 118.2	1880 ± 594.3	347 ± 204.0	310 ± 119.7	621 ± 235.5	866 ± 350.5
Combined vehicle and untreated control values 2003 Mean ± SD	98 ± 20.7	106 ± 23.4	20 ± 8.5	15 ± 3.9	23 ± 5.6	28 ± 7
Positive control values 2003 Mean ± SD	429 ± 108.1	1529 ± 493.1	297 ± 156.8	305 ± 82.6	649 ± 175.7	656 ± 264.4

 

Strain S9-Mix	TA98		TA1537
	-S9	+S9	-S9	+S9
Combined vehicle and untreated control values 2002 Mean ± SD	22 ± 5.2	35 ± 6.8	12 ± 4	17 ± 4.7
Positive control values 2002 Mean ± SD	136 ± 38.2	239 ± 74.7	1953 ± 803.1	453 ± 145.9
Combined vehicle and untreated control values 2003 Mean ± SD	20 ± 5.1	30 ± 7.1	10 ± 3.9	14 ± 5
Positive control values 2003 Mean ± SD	174 ± 64.6	249 ± 72.2	1273 ± 623.1	370 ± 109.9

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative