reaction mass of (9Z,11E)-octadeca-9,11-dienoic acid and (10E,12Z)-octadeca-10,12-dienoic acid and octadec-9-enoic acid

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:

2003-10-13 to 2004-01-08

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:

1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000-05-19

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (9Z,11E)-octadeca-9,11-dienoic acid and (10E,12Z)-octadeca-10,12-dienoic acid and octadec-9-enoic acid

Molecular formula:

C18H32O2, C18H32O2, C18H34O2

IUPAC Name:

Reaction mass of (9Z,11E)-octadeca-9,11-dienoic acid and (10E,12Z)-octadeca-10,12-dienoic acid and octadec-9-enoic acid

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: GR31143161
- Expiration date of the batch: 24.04.2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

OTHER SPECIFICS: colourless-yellowish liquid

Method

Target gene:

his

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and ß-Naphthoflavone induced male rat liver.

Test concentrations with justification for top dose:

Experiment I and II ( except for strains TA 1537 and TA 102 without 89 mix): 33, 1 00, 333, 1000, 2500, and 5000 µg/plate
Experiment II (strains TA 1537 and TA 102 without 89 mix): 0.3, 1, 3, 10, 33, 100, 250, and 500 µg/plate
ln the pre-experiment the concentration range of the test item was 3- 5000 µg/plate. The pre-experiment is reported as part of experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration for experiment I.

Vehicle / solvent:

- Solvent used: ethanol
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix, TA1535, TA100, 10 µg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without S9 mix, TA1537, 50 µg/plate; TA98, 10 µg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix, TA102, 4 µL/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix, TA1535, TA1537, TA98, TA100, 2.5 µg/plate; TA102, 10 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the number of spontaneaus revertants or a clearing of the bacterial background lawn.

Rationale for test conditions:

To establish a dose response effect six dose Ievels with adequately spaced concentrations were tested. The maximum dose Ievel was 5000 µg/plate.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice ( strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent secend experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent contros such an increase is not considered biologically relevant.

Statistics:

A statistical analysis of the data is not required.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium 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

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Cytotoxic effects

Strain	Experiment I		Experiment II
	- S9 mix	+ S9 mix	- S9 mix	+ S9 mix
TA1535	-	-	5000	-
TA1537	2500	5000	100	2500, 5000
TA98	-	-	-	-
TA100	-	-	-	-
TA102	-	-	33 - 500	-

 Table 2: Summary of results, without S9 mix

Concentration [µg/plate]	Revertants/plate [Mean]
	TA1535		TA1537		TA98		TA100		TA102
	I	II	I	II	I	II	I	II	I	II
Neg. Control	11	19	6	5	25	28	185	17	200	234
Solvent Control	16	22	6	5	25	29	188	183	177	169
Pos. Control #	602	1075	54	73	165	199	557	502	1339	917
0.3	-	-	-	7	-	-	-	-	-	150
1	-	-	-	4	-	-	-	-	-	166
3	-	-	-	6	-	-	-	-	-	172
10	-	-	-	6	-	-	-	-	-	175
33	13	18	6	6	23	20	222	158	152	52
100	11	18	5	8	20	21	225	174	161	32
250	-	-	-	8	-	-	-	-	-	18
333	9	14	4	-	21	17	148	152	159	-
500	-	-	-	4	-	-	-	-	-	0
1000	7	14	3	-	19	14	206	124	152	-
2500	11	16	3	-	19	20	232	154	155	-
5000	9	9	5	-	20	21	208	178	153	-

- Not performed

# Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate), Methylmethanesulfonate (4 1 µL/plate) strain TA 102

Table 3: Summary of results, with S9 mix 

Concentration [µg/plate]	Revertants/plate [Mean]
	TA1535		TA1537		TA98		TA100		TA102
	I	II	I	II	I	II	I	II	I	II
Neg. Control	11	11	10	14	21	18	179	188	184	216
Solvent Control	19	27	9	13	28	31	202	190	176	134
Pos. Control ##	312	274	265	224	498	472	801	709	1355	1246
33	20	29	10	8	30	29	257	156	156	122
100	18	19	10	12	33	25	211	196	172	108
333	16	17	9	11	32	25	210	167	153	116
1000	12	14	6	6	29	19	198	114	152	98
2500	13	19	6	4	31	26	179	137	162	102
5000	13	18	4	3	30	27	174	142	109	156

## 2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98, and TA 100; 2-aminoanthracene (10.0 µg/plate) strain TA 102