9-Octadecenoic acid (Z)-, isooctyl ester, reaction products with glycerol trioleate and sulfur

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:

15 June 1992 to 17 September 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

9-Octadecenoic acid (Z)-, isooctyl ester, reaction products with glycerol trioleate and sulfur

EC Number:

306-111-3

EC Name:

9-Octadecenoic acid (Z)-, isooctyl ester, reaction products with glycerol trioleate and sulfur

Cas Number:

96152-40-8

IUPAC Name:

9-Octadecenoic acid (Z)-, isooctyl ester, reaction products with glycerol trioleate and sulfur

Test material form:

liquid: viscous

Details on test material:

- Physical state: liquid

Method

Target gene:

S. typhimurium: Histidine synthesis

E. coli: Tryptophan synthesis

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: The broth used to grow overnight cultures of tester strains was Vogel-Bonner salt solution supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2. Bottom agar (25 mL per 15 x 100 mm petri dish) was added to the Vogel-Bonner minimal medium E and supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose. Top agar consisting of 0.7 % w/v agar and 0.5 % w/v sodium chloride was supplemented with 10 mL of 0.5 mM histidine/ biotin solution per 100 mL agar. When S9 mix was required, 2.0 mL of the supplemented top agar was used in the overlay. However, when S9 was not required, water was added to the supplemented top agar (0.5 mL of water per 2 mL of supplemented top agar) and the resulting 2.5 mL of diluted supplemented top agar was used for the overlay.
- Periodically checked for genotype stability: Yes. The presence of the pKM101 plasmid was confirmed for tester strains TA98 and TA100 by demonstration of resistance to ampicillin (10 µg). The presence of the rfa wall mutation was confirmed by testing the sensitivity of each culture to crystal violet (10 µg).
- Periodically "cleansed" against high spontaneous background: Yes. The mean numbers of spontaneous revertants per plate in the vehicle controls that are characteristic of the respective strains were demonstrated by plating 100 µg aliquots of the culture along with the appropriate vehicle on selective media.

Additional strain / cell type characteristics:

other: rfa and uvrB mutations present in all strains together with R factor in TA98 and TA100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: The broth used to grow overnight cultures of tester strains was Vogel-Bonner salt solution supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2. Bottom agar (25 mL per 15 x 100 mm petri dish) was added to the Vogel-Bonner minimal medium E and supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose. Top agar consisting of 0.7 % w/v agar and 0.5 % w/v sodium chloride was supplemented with 10 mL of 0.11 mg/mL tryptophan solution per 100 mL agar. When S9 mix was required, 2.0 mL of the supplemented top agar was used in the overlay. However, when S9 was not required, water was added to the supplemented top agar (0.5 mL of water per 2 mL of supplemented top agar) and the resulting 2.5 mL of diluted supplemented top agar was used for the overlay.
- Periodically checked for genotype stability: Yes. The presence of the pKM101 plasmid was confirmed for tester strains TA98 and TA100 by demonstration of resistance to ampicillin (10 µg). The presence of the rfa wall mutation was confirmed by testing the sensitivity of each culture to crystal violet (10 µg).
- Periodically "cleansed" against high spontaneous background: Yes. The mean numbers of spontaneous revertants per plate in the vehicle controls that are characteristic of the respective strains were demonstrated by plating 100 µg aliquots of the culture along with the appropriate vehicle on selective media.

Additional strain / cell type characteristics:

other: contains uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

MUTAGENICITY ASSAY
Salmonella typhimurium
3330, 1000, 333, 100, 33.3, 10.0 µg/plate (with S9)
100, 33.3, 10.0, 3.33, 1.00, 0.33 µg/plate (without S9)

Escherichia coli
10000, 3330, 1000, 333, 100, 33.3 µg/plate (with and without S9)

CONFIRMATORY ASSAY
Salmonella typhimurium
3330, 1000, 333, 100, 33.3, 10.0 µg/plate (with S9)
333, 100, 33.3, 10.0, 3.33, 1.00 µg/plate (without S9)

Escherichia coli
3330, 1000, 333, 100, 33.3, 10.0 µg/plate (with S9)
10000, 3330, 1000, 333, 100, 33.3 µg/plate (without S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

plated for all tester strains both in the presence and absence of S9 with no test material

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene, ICR-191

Details on test system and experimental conditions:

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

DURATION
- Exposure duration: plates were inverted and incubated for 48 ± 2 hours at 37 ± 2 ºC.

NUMBER OF REPLICATIONS: test concentrations were performed in triplicate

EVALUATION PROCEDURE
The condition of the bacterial background lawn was evaluated for evidence of cytotoxicity and test material precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate. The numbers of revertant colonies per plate for the vehicle and negative controls and all plates containing test material were counted manually. The numbers of revertant colonies per plate for positive controls were counted by automated colony counter.

Evaluation criteria:

Test data from individual experiments was considered valid if:
a) tester strain cultures exhibit sensitivity to crystal violet
b) cultures of tester strains TA98 and TA100 exhibit resistance to ampicillin
c) tester strain cultures exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions
d) positive controls must exhibit a 3-fold increase over the value of the vehicle control for that strain
e) a minimum of three non-toxic doses are required to evaluate assay data.

Once the criteria for a valid assay have been met, responses observed in the assay are evaluated as follows:

Tester strains TA98, TA100 and WPuvrA-
For a test material to be considered positive, it must produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test material.

Tester strains TA1535 and TA1537
For a test material to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test material.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Test material handling
In acetone the test material formed a cloudy yellow homogenous suspension at 200 mg/mL which was the most concentrated stock dilution of the test material prepared. The test material formed a solution at 20 mg/mL and remained a solution at all subsequent dilutions prepared for the mutagenicity assay.

- Dose Range Finding Study
Doses to be tested in the mutagenicity assay were selected based on the results of the dose rangefinding study conducted on the test material using tester strains TA100 and WP2uvrA- in both the presence and absence of a 10% S9 mix (one plate per dose). Ten doses of test material, from 10,000 to 10.0 µg/plate, were tested. Under the conditions of the study, cytotoxicity was observed with tester strain TA100 at 667 µg/plate and above in the presence of S9 and at 33.3 µg/plate and above in the absence of S9 as evidenced by a thinning and/or disappearance of the bacterial background lawn and a reduction in the number of revertants per plate. With tester strain WP2uvrA-, cytotoxicity was observed at 10,000 µg/plate in the presence of S9 and at 667 µg/plate in the presence of S9 and at 667 µg/plate in the absence of S9 as evidenced by a slight thinning of the bacterial background lawn and/or a reduction in the number of revertants per plate.

- S9 Optimisation Study
An S9 optimisation study was performed using tester strains TA98 and TA100 with the highest non-cytotoxic dose of test material, 333 µg/plate, and four concentrations of S9 mix (5%, 10%, 20% and 80% S9 homogenate per mL of S9 mix). Under the conditions of the study, no effect was observed due to varying the S9 homogenate concentration in this experiment. In the absence of an effect, a 10% S9 mix was used in the mutagenicity assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenicity Assay
In the initial mutagenicity assay, all data were acceptable and no positive increases in the number of revertants per plate were observed with any of the tester strains either in the presence or absence of S9. After a review of the data generated in the initial mutagenicity assay, it was decided to increase the doses treated with the Salmonella tester strains in the absence of S9 and to decrease the doses tested with the E. coli tester strain in the presence of S9 due to the observed levels of cytotoxicity.

 

Confirmatory Mutagenicity Assay
In the confirmatory mutagenicity assay, all data were acceptable and no positive increases in the number of revertants per plate were observed with any of the tester strains either in the presence or absence of S9. In the absence of S9, greater cytotoxicity was observed than demonstrated in the initial mutagenicity assay. For this reason, it was decided to retest all strains in the absence of S9 in a further confirmatory assay.

 

Further Confirmatory Assay (in the absence of S9)
All data with the Salmonella tester strains were acceptable and no positive increases in the number of revertants per plate were observed. The cytotoxicity observed in this experiment was similar to that observed in the initial mutagenicity assay. No data were generated with tester strain WP2uvrA- due to an unacceptable mean vehicle control value for this strain. Tester strain WP2uvrA- was retested in a further experiment in which all data were acceptable and no positive increase in the number of revertants per plate was observed in the absence of S9.

Table 1: Mutagenicity Assay Results (means)

With S9	Dose (µg)/plate	TA98	TA100	TA1535	TA1537	Background lawn	With S9	Dose (µg)/plate	WP2uvrA-	Background lawn
Vehicle control		25	138	22	4	1	Vehicle control		24	1
Negative control		32	120	20	7	1	Negative control		23	1
Test material	10.0	31	129	21	12	1	Test material	33.3	22	1
	33.3	38	130	21	6	1		100	14	1
	100	26	135	16	7	1		333	21	1
	333	27	114	16	6	1		1000	9	1
	1000	20	41	9	4	1		3330	6	1sp
	3330	10	8	2	1	2sp		10000	0	6mp
Positive control		1118	1152	303	208	1	Positive control		302	1
Without S9							Without S9
Vehicle control		17	95	17	5	1	Vehicle control		16	1
Negative control		18	99	16	8	1	Negative control		21	1
Test material	0.33	21	93	17	7	1	Test material	33.3	17	1
	1.00	15	102	13	4	1		100	11	1
	3.33	16	94	9	8	1		333	19	1
	10.0	15	108	13	6	1		1000	21	1
	33.3	12	81	12	6	2		3330	18	2
	100	9	70	16	5	2		10000	17	2
Positive control		178	608	516	362	1	Positive control		484	1

Table 2: Confirmatory Assay Results (means)

With S9	Dose (µg)/plate	TA98	TA100	TA1535	TA1537	Background lawn	With S9	Dose (µg)/plate	WP2uvrA-	Background lawn
Vehicle control		36	132	14	8	1	Vehicle control		23	1
Negative control		35	137	11	10	1	Negative control		25	1
Test material	10.0	31	138	11	6	1	Test material	10.0	18	1
	33.3	32	149	13	11	1		33.3	13	1
	100	39	124	10	12	1		100	14	1
	333	27	133	9	9	1		333	13	1
	1000	23	47	7	5	2sp		1000	13	1
	3330	11	2	1	1	2sp		3330	4	1sp
Positive control		1434	1594	419	213	1	Positive control		516	1
Without S9							Without S9
Vehicle control		18	97	11	6	1	Vehicle control		11	1
Negative control		18	107	11	5	1	Negative control		18	1
Test material	1.00	16	107	12	5	1	Test material	33.3	15	1
	3.33	13	111	15	4	1		100	18	1
	10.0	11	76	15	3	3		333	15	2
	33.3	16	100	9	4	2		1000	8	2
	100	10	66	9	0	3		3330	1	2sp
	333	4	28	5	2	3		10000	0	3sp
Positive control		174	570	542	362	1	Positive control		1041	1

Table 3: Further Confirmatory Assay Results (means)

Without S9	Dose (µg)/plate	TA98	TA100	TA1535	TA1537	Background lawn	Without S9 (1)	Dose (µg)/plate	WP2uvrA- (1)	Background lawn (1)	WP2uvrA- (2)	Background lawn (2)
Vehicle control		12	111	12	6	1	Vehicle control		176	1	18	1
Negative control		12	109	11	5	1	Negative control		NC	1	19	1
Test material	1.00	19	112	11	8	1	Test material	33.3	NC	1	20	1
	3.33	16	111	11	3	1		100	NC	1	20	1
	10.0	11	96	11	6	1		333	NC	1	15	1
	33.3	16	98	12	5	1		1000	NC	1	16	2
	100	10	69	13	3	2		3330	NC	2	1	2sp
	333	0	35	0	0	3		10000	NC	3	0	3sp
Positive control		211	861	692	353	1	Positive control		NC	1	721	1

NC = not counted due to unacceptable mean vehicle control value

Background Lawn Evaluation Codes

1 = normal

2 = slightly reduced

3 = moderately reduced

sp = slight precipitate

mp = moderate precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of the study, in both an initial and confirmatory assay, the test material did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).

Executive summary:

The potential of the test material to cause gene mutation in bacterial strains was determined following a methodology similar to that outlined in the standardised guideline OECD 471. During the study, four strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and one strain of Escherichia coli (WP2uvrA-) were treated in the presence and absence of a rat liver derived metabolic activation system (S9 mix). In two separate assays, the test material did not induce any significant increases in the number of revertant colonies, in any of the tester strains, either in the presence or absence of S9 mix.