9-Octadecenamide, N-[(1S,2S,3R)-2,3-dihydroxy-1-(hydroxymethyl)heptadecyl]-, (9Z)-

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:

2017-02-01 to 2017-02-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 July 1997

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:

9-Octadecenamide, N-[(1S,2S,3R)-2,3-dihydroxy-1-(hydroxymethyl)heptadecyl]-, (9Z)-

EC Number:

812-963-1

Cas Number:

178436-06-1

Molecular formula:

C36H71NO4

IUPAC Name:

9-Octadecenamide, N-[(1S,2S,3R)-2,3-dihydroxy-1-(hydroxymethyl)heptadecyl]-, (9Z)-

Test material form:

solid

Specific details on test material used for the study:

- Characteristics: (Off-) white powder
- Storage conditions: at room temperature (+10 °C to +25 °C)

Method

Target gene:

histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB-, rfa-, pKM 101, resistance to Ampicillin

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB-, rfa-, pKM 101, resistance to Ampicillin

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: rfa-, pKM 101/pAQ1, resistance to Ampicillin

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: uvrB-, rfa-, non-resistance to Ampicillin

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: uvrB-, rfa-, non-resistance to Ampicillin

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, Aroclor 1254-induced, from male Sprague-Dawley rat liver (Trinova Biochem GmbH, Gießen, Germany

Test concentrations with justification for top dose:

1, 3.16, 10, 31.6, 100 and 316 µg/plate. Two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) were carried out in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Concentrations of 1000 µg/plate and higher were not evaluable due to pronounced test item precipitation (see tables X and X). Hence, 316 µg/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (Batch no. F2540; Honeywell Specialty Chemicals)
- Justification for choice of solvent/vehicle: The test item was not soluble in water or dimethylsulfoxide (DMSO).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 0.1 mL of approximately 1.00E+08 viable cells in the late exponential or early stationary phase
DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 37°C for 48 -72 h
NUMBER OF REPLICATIONS:
- Plates: 3 per concentration and experiment
- Experiments: 2 independent experiments, each with and without metabolic activation
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures. Insolubility could have been assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye.

Evaluation criteria:

Acceptance Criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by the testing facility. The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20-60
TA100: 100-200
TA102: 240-320
TA1535: 10-35
TA1537: 3-20
The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment. Cytotoxicity is defined as a reduction in the number of colonies by more than 50 % compared with the vehicle control and/or a scarce background lawn.
Historical background data of revertant colony numbers of the negative and positive controls without and with metabolic activation for the experiments of the years 2015 to 2017 (most recent background data, not audited by the QAU-department) are given in Table 1 in section “any other details on results incl. tables”.

Statistics:

Interpretation of results:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first. A test item for which the results do not meet the above-mentioned criteria is considered as non-mutagenic in the AMES test.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

not examined

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:

not examined

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolarity: not reported
- Evaporation from medium: not reported
- Water solubility: the test item is insoluble in water
- Precipitation: see section “any other information on results incl. tables”.
RANGE-FINDING/SCREENING STUDIES: see section “any other information on results incl. tables”.
HISTORICAL CONTROL DATA see tables 1 and 2 in section “any other information on results incl. tables”.

Any other information on results incl. tables

Preliminary test

The test substance was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Test item precipitation was noted starting at the concentration of 316 µg/plate. Concentrations of 1000 µg/plate and higher were not evaluable due to pronounced test item precipitation (see tables 2 and 3). Hence, 316 µg/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 1.0 to 316 µg of the test item per plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity 

Test item precipitation was noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at the top concentration of 316 µg/plate in all test strains. Reduction of the number of revertants by more than 50% was noted in both experiments at the top concentration of 316 µg/plate in the following test strains:  

Plate incorporation test:

- S9: TA1535 and TA1537;

+S9: TA1537;

Preincubation test:

- S9: TA98, TA102, TA1535 and TA1537;

+S9: TA1535 and TA1537.  

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed up to a concentration of 316 µg/plate, that led to test item precipitation, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the range of the historical data generated by the test facility (see table 1). A summary of the results is given in tables 4 to 7.

Table 1: history profile of negative and positive control values of the years 2015 to 2017 (n= 80 studies). Data obtained from plate incorporation and preincubation tests.

Negative reference item
Strain	TA 98		TA 100		TA 102		TA 1535		TA 1537
S9-mix	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
Mean	30.3	32.1	145.1	145.2	277.3	279.0	19.7	19.9	6.7	6.7
SD	5.6	5.9	18.4	19.2	16.5	17.2	4.4	4.6	1.7	1.8
Min	20	20	107	101	245	203	10	10	2	3
Max	49	49	195	198	323	324	34	36	10	10
Positive reference item
Strain	TA 98		TA 100		TA 102		TA 1535		TA 1537
S9-mix	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
	2-nitro-fluorene	Benzo[a]-pyrene	Sodium azide	2-amino-anthracene	Mitomycin C	Benzo[a]-pyrene	Sodium azide	2-amino-anthracene	9-amino-acridine	Benzo[a]-pyrene
Mean	151.2	150.4	952.9	948.8	1029.7	1024.3	135.6	135.4	76.7	77.6
SD	27.9	28.8	99.7	103.7	102.3	97.1	28.8	28.4	26.5	26.4
Min	91	96	677	703	756	781	51	49	28	31
Max	293	291	1213	1195	1637	1366	266	270	185	184

 Table 2: Preliminary cytotoxicity test without metabolic activation. Plate incorporation test with TA 100.

Test item (µg/plate)	Background lawn	Revertants plate 1	Revertants plate 2
0.316	normal	121	127
1	normal	144	142
3.16	normal	116	149
10	normal	144	134
31.6	normal	142	149
100	normal	146	146
316	test item precipitation	110	100
1000	test item precipitation	0	0
3160	test item precipitation	0	0
5000	test item precipitation	0	0
Solvent control 100 µL/plate	normal	147	149

 Table 3: Preliminary cytotoxicity test with metabolic activation. Plate incorporation test with TA 100.

Test item (µg/plate)	Background lawn	Revertants plate 1	Revertants plate 2
0.316	normal	130	120
1	normal	163	127
3.16	normal	120	153
10	normal	131	131
31.6	normal	158	161
100	normal	144	143
316	test item precipitation	114	97
1000	test item precipitation	0	0
3160	test item precipitation	0	0
5000	test item precipitation	0	0
Solvent control 100 µL/plate	normal	153	157

Table 4: Plate incorporation test without metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
	TA98	TA100	TA102	TA1535	TA1537
1.0	22.7	112.3	281.7	24.3	6.3
3.16	24.0	119.3	292.0	15.0	6.0
10.0	28.3	118.0	287.7	18.7	6.0
31.6	26.0	120.3	284.0	17.3	5.3
100	25.7	128.0	251.0	16.3	6.7
316	22.7 test item precipitation	109.0 test item precipitation	260.7 test item precipitation	11.0 test item precipitation	2.0 test item precipitation
Vehicle control 100 µL/plate	32.7	128.0	271.3	26.7	6.7
Positive reference item	2-nitro-fluorene	Sodium azide	Mitomycin C	Sodium azide	9-amino-acridine
Concentration µg/plate	10	10	10	10	100
	193.7	993.0	1093.3	136.0	73.3

 Table 5: Plate incorporation test with metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
	TA98	TA100	TA102	TA1535	TA1537
1.0	29.7	119.3	255.3	23.0	4.3
3.16	29.3	132.0	293.0	19.0	6.0
10.0	24.7	119.0	264.3	22.7	4.0
31.6	24.3	132.0	277.7	16.7	5.7
100	30.7	118.7	258.3	19.0	5.0
316	22.7 test item precipitation	108.3 test item precipitation	245.0 test item precipitation	10.0 test item precipitation	2.0 test item precipitation
Vehicle control 100 µL/plate	30.7	120.0	274.0	17.0	5.3
Positive reference item	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene
Concentration µg/plate	10	2	10	2	10
	184.0	992.7	1088.3	137.7	77.3

 Table 6: Preincubation test without metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
1.0	31.7	117.3	281.7	16.7	8.0
3.16	29.3	113.0	276.0	17.0	8.3
10.0	28.7	118.7	258.7	18.0	6.3
31.6	28.3	129.0	267.0	16.0	6.7
100	29.7	120.3	288.7	20.7	5.7
316	12.0 test item precipitation	79.7 test item precipitation	126.7 test item precipitation	9.0 test item precipitation	2.3 test item precipitation
Vehicle control 100 µL/plate	30.7	125.0	286.7	18.3	6.0
Positive reference item	2-nitro-fluorene	Sodium azide	Mitomycin C	Sodium azide	9-amino-acridine
Concentration µg/plate	10	10	10	10	100
	165.7	998.3	1036.7	139.7	32.3

 Table 7: Preincubation test with metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
	TA98	TA100	TA102	TA1535	TA1537
1.0	32.0	116.3	267.3	22.7	9.7
3.16	32.0	118.3	273.7	23.3	9.3
10.0	33.3	109.3	276.3	26.3	6.7
31.6	32.3	118.7	271.0	20.7	4.3
100	29.0	111.3	252.3	18.3	7.0
316	15.3 test item precipitation	75.3 test item precipitation	202.7 test item precipitation	9.0 test item precipitation	2.0 test item precipitation
Vehicle control 100 µL/plate	22.7	131.0	302.0	25.0	7.3
Positive reference item	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene
Concentration µg/plate	10	2	10	2	10
	163.3	1029.7	997.3	137.7	65.0

 

Applicant's summary and conclusion

Conclusions:

Under the described conditions and tested up to a concentration of 316 µg/plate, that led to test item precipitation, the test substance caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation. Therefore, the test substance is considered non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

The potential of Ceramide IIIB to induce gene mutations was examined in 5 Salmonella typhimurium strains in two independent experiments without and with metabolic activation (plate incorporation test and preincubation test).

The test item was completely dissolved in ethanol for concentrations lower than 1000 µg/plate. The vehicle ethanol was employed as the negative control.

In two preliminary cytotoxicity tests, test item precipitation was noted starting at the concentration of 316 µg/plate. Concentrations of 1000 µg/plate and higher were not evaluable due to pronounced test item precipitation. Thus, six concentrations ranging from 1.0 to 316 µg/plate were employed in the main experiment.

Cytotoxicity as determined by the reduction of the number of revertants by more than 50% was noted at the top concentration of 316 µg/plate in some of the test strains with and without metabolic activation.

No increase in revertant colony numbers as compared with control up to a concentration of 316 µg/plate was observed in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

In conclusion, the test substance was tested up to a concentration of 316 µg/plate, which led to precipitation but caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.