9-Octadecenedioic acid

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:

09 Mar - 3 Apr 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

802-122-7

EC Number:

802-122-7

IUPAC Name:

802-122-7

Constituent 2

Reference substance name:

Octadec-9-enedioic acid

IUPAC Name:

Octadec-9-enedioic acid

Details on test material:

- Name of test material (as cited in study report): NEWDIOIC 12358
- Physical state: white powder
- Analytical purity: 100%
- Lot/batch No.: B1
- Expiration date of the lot/batch: 16 Jan 2017
- Storage condition of test material: between 15 and 25 °C, under nitrogen

Method

Target gene:

his operon

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation method), 2 (preincubation method) and 3 (plate incorporation method): 50, 160, 500, 1600 and 5000 μg/plate, with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solubility in the vehicle was 50 g/L

Controls

Untreated negative controls:

yes

Remarks:

solvent control without treatment

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene

Remarks:

-S9: 2-nitrofluorene (5 µg/plate) for TA98; sodium azide (10 µg/plate) for TA100 and TA1535; 9-aminoacridine (30 µg/plate) for TA1537; mitomycin (0.5 µg/plate) for TA 102; +S9: 2-aminoanthracene (5 or 25 µg/plate) all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment 1 and 3: in agar (plate incorporation); experiment 2: preincubation

DURATION
- Preincubation period: 20-30 min
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: three replications each in 3 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies, or a clearing or diminuition of the background lawn

OTHER EXAMINATIONS:
- Other: the test substance, solvent S9 mix and top agar sterility was tested by adding 0.1 mL/plate of the above-mentioned to a minimal agar plate and incubating at 37 ºC for 48-72 h. No baterial growth must be observed.

OTHER: 2-aminoanthracene was used as the only positive control with metabolic activation. However, the ability of the S9-mix to activate B[a]P and 2-aminoanthracene was confirmed in an assay prior to the main study.

Evaluation criteria:

The test substance is considered to be mutagenic if a reproducible dose-effect relation is observed in one or more of the 5 strains with and/or without metabolic activation. The mutagenicity is taken into account for a given concentration only when the number of revertants is equal at least to the double of the spontaneous rate of reversion for TA 98, TA 100 and TA 102 strains (R ≥ 2) and the triple of the spontaneous rate of reversion for TA 1535 and TA 1537 strains (R ≥ 3).
The test substance is considered non-mutagenic if, in the outcome of all tests the rate of revertants always remained lower than the double of the rate of spontaneous reversion for all the concentrations of tested substance, for TA 98, TA 100 and TA 102 strains (R < 2) and the triple of the sponstaneous rate of reversion for TA 1535 and TA 1537 strain (R < 3), with and without metabolic activation, and on the condition of having made sure that the abscence of mutagen effect is not bound to the toxicity of the test conditions.

Statistics:

The average plate count was presented with the mean and standard deviation for each set of triplicates per test item concentration.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity was observed in strain TA 100 from 1600 μg/plate and in TA 1537 at 5000 μg/plate in the presence of metabolic activation in experiment 2; and in strain TA 100 and TA 1535 at 5000 μg/plate in the presence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at the highest concentration of 5000 µg/plate, with and without metabolic activation

RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity assay (plate incorporation test), concentrations of 50 - 5000 μg/plate were tested with S. typhimurium TA100. No cytotoxicity was observed at any concentration level. Therefore, 5000 μg/plate was selected as the highest concentration in the main study. The results for TA 100 were included in the main study.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, results were within the historical positive and vehicle control data range

ADDITIONAL INFORMATION ON CYTOTOXICITY: In experiment 2, cytotoxicity was observed in strain TA 100 starting at 1600 μg/plate and in TA 1537 at 5000 μg/plate in the presence of metabolic activation, however this effect was not observed in experiment 3. In experiment 3, cytotoxicity was observed in strain TA 100 (visible as dose-related decrease in revertants, number of revertants at highest concentration > 50%) and TA 1535 at 5000 μg/plate in the presence of metabolic activation.

OTHER RESULTS: During the first step of experiment 1 without metabolic activation, a ratio R higher than the double of the spontaneous rate of reversion was shown for strain TA 98 at 50 μg/plate and a ratio higher to the triple of the spontaneous rate of reversion was observed for strain TA 1535 at 50 μg/plate. As these results were not reproducible in experiment 2, experiment 3 was performed according to the same protocol as experiment 1. The results were not reproducible in experiment 3 either, and therefore these results of experiment 1 are not taken into consideration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1

EXPERIMENT 1 (plate incorporation test)
S9-mix	Without
Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Untreated control	31.0 ± 3.6	74.0 ± 13.0	15.7 ± 2.3	21.7 ± 0.6	381.0 ± 11.5
NC	25.3 ± 3.8	65.3 ± 6.8	15.7 ± 1.2	20.3 ± 4.7	352.3 ± 10.1
50	66.3 ± 11.5	67.7 ± 12.1	69.0 ± 9.2	46.0 ± 12.3	369.7 ± 21.2
160	47.7 ± 8.6	53.0 ± 13.2	46.0 ± 5.3	57.0 ± 14.9	379.0 ± 7.0
500	38.7 ± 6.1	63.0 ± 10.5	28.7 ± 3.8	40.0 ± 13.1	416.3 ± 20.4
1600	24.3 ± 9.0	59.0 ± 14.9	19.7 ± 7.8	39.3 ± 12.7	395.3 ± 10.5
5000	22.7 ± 1.2P	56.3 ± 16.6P	14.0 ± 3.0P	26.0 ± 5.6P	356.7 ± 55.1P
2-NF	1391.7 ± 86.4	-	-	-	-
SA	-	2380.7 ± 49.0	2401.7 ± 179.7	-	-
MM	-	-	-	-	2366.7 ± 176.3
9-AA	-	-	-	142.7 ± 83.7	-
S9-mix	With
Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Untreated control	39.3 ± 6.0	101.3 ± 14.8	16.3 ± 2.9	28.0 ± 8.9	435.3 ± 10.0
NC	34.0 ± 4.6	122.3 ± 10.5	15.3 ± 3.2	30.3 ± 5.8	437.7 ± 17.0
50	30.7 ± 2.5	81.7 ± 4.0	14.3 ± 5.5	31.7 ± 3.2	441.7 ± 47.1
160	28.7 ± 2.3	85.3 ± 6.5	17.3 ± 2.5	30.0 ± 2.0	444.7 ± 43.1
500	41.7 ± 6.8	101.3 ± 12.5	18.3 ± 1.5	34.0 ± 7.0	442.3 ± 33.5
1600	44.3 ± 4.0	83.7 ± 11.0	11.3 ± 3.5	30.3 ± 4.0	441.0 ± 41.6
5000	32.3 ± 3.1	80.0 ± 5.2P	11.7 ± 1.5P	31.0 ± 7.8P	457.3 ± 60.2P
2AA	2688.0 ± 74.5	2933.0 ± 113.7	297.7 ± 12.7	261.0 ± 20.0	3828.0 ± 208.5
NC = vehicle control, DMSO P = precipitate 2-NF: 2 -nitrofluorene (5 µg/plate) SA: sodium azide (10 µg/plate) MM: mitomycin C (0.5 µg/plate) 9AA:9-aminocridine (30 µg/plate) 2AA: 2-aminoanthracene (5 or 25 µg/plate)

Table 2: Experiment 2

EXPERIMENT 2 (preincubation test)
S9-mix	Without
Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Untreated control	28.0 ± 6.1	78.3 ± 10.3	10.7 ± 2.5	24.3 ± 4.7	372.0 ± 20.1
NC	27.3 ± 2.3	59.3 ± 11.8	13.0 ± 2.0	17.0 ± 5.0	315.3 ± 24.7
50	24.0 ± 4.4	58.3 ± 6.7	14.3 ± 2.5	19.3 ± 5.1	376.0 ± 31.9
160	22.0 ± 6.2	59.7 ± 8.5	14.7 ± 2.9	30.0 ± 4.6	358.3 ± 11.4
500	28.3 ± 5.9	59.0 ± 18.1	13.7 ± 6.7	21.7 ± 5.5	350.7 ± 14.8
1600	26.7 ± 7.6	34.7 ± 2.5	13.3 ± 1.5	14.3 ± 3.2	353.3 ± 13.7
5000	25.3 ± 8.8P	12.7 ± 12.7P	12.3 ± 5.0P	6.0 ± 2.6P	266.7 ± 11.5P
2-NF	1231.7 ± 225.0	-	-	-	-
SA	-	2560.3 ± 277.5	2211.7 ± 127.8	-	-
MM	-	-	-	-	2698.3 ± 461.9
9-AA	-	-	-	142.7 ± 24.7	-
S9-mix	With
Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Untreated control	42.7 ± 4.7	86.0 ± 1.7	18.3 ± 2.3	29.0 ± 5.3	444.0 ± 16.0
NC	30.3 ± 0.6	58.3 ± 1.2	14.7 ± 2.9	20.3 ± 1.5	388.3 ± 26.8
50	35.3 ± 11.5	48.0 ± 16.5	15.3 ± 3.8	26.7 ± 1.2	328.7 ± 28.7
160	34.0 ± 1.0	53.3 ± 0.6	12.0 ± 1.0	18.3 ± 5.1	346.3 ± 12.5
500	33.3 ± 1.5	62.0 ± 17.5	19.3 ± 2.5	15.3 ± 2.3	356.7 ± 19.2
1600	37.0 ± 9.2	68.0 ± 9.8	12.0 ± 3.5	17.0 ± 3.5	365.0 ± 14.2
5000	26.0 ± 1.7P	49.7 ± 8.1P	13.3 ± 2.3P	13.7 ± 2.5P	333.3 ± 61.1P
2AA	1447.7 ± 11.5	1173.0 ± 25.2	169.3 ± 2.9	307.7 ± 23.7	3863.7 ± 212.0
NC = vehicle control, DMSO P = precipitate 2-NF: 2 -nitrofluorene (5 µg/plate) SA: sodium azide (10 µg/plate) MM: mitomycin C (0.5 µg/plate) 9AA:9-aminocridine (30 µg/plate) 2AA: 2-aminoanthracene (5 or 25 µg/plate)

 

Table 3: Experiment 3 (repeat of experiment 1)

EXPERIMENT 3 (plate incorporation test)
S9-mix	Without
Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Untreated control	25.3 ± 8.0	62.3 ± 14.7	30.0 ± 9.6	14.3 ± 1.2	333.0 ± 4.6
NC	22.3 ± 5.5	58.7 ± 14.0	30.7 ± 6.4	15.7 ± 3.2	386.0 ± 26.9
50	23.0 ± 1.7	64.7 ± 6.7	33.3 ± 8.0	21.3 ± 6.4	401.3 ± 29.0
160	26.3 ± 11.0	73.0 ± 14.7	33.3 ± 6.0	23.0 ± 3.6	397.7 ± 14.4
500	27.0 ± 8.9	60.0 ± 14.7	36.7 ± 13.6	26.0 ± 7.5	412.3 ± 10.3
1600	20.7 ± 6.1	58.0 ± 8.0	42.7 ± 13.6	22.7 ± 2.1	438.0 ± 18.5
5000	21.7 ± 2.1P	43.7 ± 11.1P	31.3 ± 2.1P	18.3 ± 2.3P	340.0 ± 52.9P
2-NF	1151.7 ± 58.3	-	-	-	-
SA	-	2368.3 ± 182.3	2424.0 ± 88.2	-	-
MM	-	-	-	-	2349.7 ± 101.9
9-AA	-	-	-	294.7 ± 153.0	-
S9-mix	With
Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Untreated control	31.7 ± 4.9	89.0 ± 2.6	34.3 ± 7.2	14.7 ± 5.5	385.3 ± 35.0
NC	28.3 ± 8.5	90.3 ± 3.8	29.0 ± 7.9	19.0 ± 0.0	467.0 ± 31.3
50	31.3 ± 4.6	92.3 ± 13.3	37.7 ± 6.5	18.7 ± 0.6	503.7 ± 44.0
160	30.3 ± 2.1	79.0 ± 4.6	36.3 ± 4.6	26.0 ± 1.0	456.7 ± 21.0
500	37.7 ± 4.9	74.0 ± 8.7	32.0 ± 7.0	24.0 ± 1.7	455.3 ± 29.3
1600	27.0 ± 2.6	67.0 ± 4.6	34.3 ± 5.5	16.3 ± 1.2	506.7 ± 21.7
5000	24.0 ± 9.5P	58.3 ± 10.1P	15.0 ± 6.2P	14.3 ± 3.1P	390.7 ± 105.6P
2AA	2141.7 ± 45.4	2197.3 ± 91.9	292.0 ± 21.2	290.3 ± 20.4	3713.3 ± 64.3
NC = vehicle control, DMSO P = precipitate 2-NF: 2 -nitrofluorene (5 µg/plate) SA: sodium azide (10 µg/plate) MM: mitomycin C (0.5 µg/plate) 9AA:9-aminocridine (30 µg/plate) 2AA: 2-aminoanthracene (5 or 25 µg/plate)

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative