Octadecanamide, N-[(1S,2S,3R)-2,3-dihydroxy-1-(hydroxymethyl)heptadecyl]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1. Key study, read-across, source substance Ceramide IIIB: guideline study, GLP, Reliability 1: non-mutagenic in the bacterial reverse mutation assay (5 strains tested)

2. Supporting study, target substance Ceramide III: guideline study, Reliability 2: non-mutagenic in the bacterial reverse mutation assay (4 strains tested)

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

30 May 2008

Deviations:

no

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

Specific details on test material used for the study:

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

Target gene:

histidine operon

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

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

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

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

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

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

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

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

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. 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).

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.

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

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

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

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

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”.

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

 

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993-12-21 to 1994-01-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May, 1983

Deviations:

yes

Remarks:

only 4 strains tested, no historical data were reported

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted December, 1992

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix (Aroclor 1254-induced rat liver S-9 plus cofactors)

Test concentrations with justification for top dose:

Range finding test with strains S. typhimurium TA 100: 1, 3.3, 10, 33.3, 100, 333, 1000, 3330, 5000 µg/plate with and without mammalian metabolic activation
Main study (1st and 2nd mutation assay): S. typhimurium strains: 100, 333, 1000 3330, 5000 µg/plate with and without mammalian metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide of spectroscopic quality (Merck)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycine

Remarks:

without metabolic activiation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activiation

Details on test system and experimental conditions:

METHOD OF APPLICATION: 1st mutation assay: plate incorporation; 2nd mutation assay: plate incorporation (to confirm the first assay)
DURATION (plate incorporation)
- Preincubation period: 48 h
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: reduction of the background lawn, viability or reduction of the spontaneous reversion rate
RANGE FINDING TEST
- In order to determine the toxicity and to select the appropriate test article concentrations for the Mutation Assay,
a Range Finding Test was performed using strains TA100.
- Seven doses of test article, ranging from 1.0 to 5000 µg/plate, were evaluated with and without Aroclor-induced rat liver S-9, using two plate per dose.

Evaluation criteria:

Criteria for a Negative Response.
A response is considered to have caused a negative response if all of the strains treated with the test article have mean reversion frequencies that are no greater than twice that of the mean reversion frequencies of the corresponding solvent control plates in any tester strain

Criteria for a Positive Response.
A response is considered to have caused a positive response if any strain has a dose that produces a mean reversion frequency that is two times or more greater than the mean reversion frequency of the corresponding solvent control plates. In addition, the response must be dose dependent or increasing concentrations of the extract must show increasing mean reversion frequencies. In evaluating the results, consideration will be given to the degree of toxicity exhibited by the dose causing the two-fold/three-fold or greater increase in reversion frequency and the magnitude of the increase in reversion frequency.

Criteria for an Equivocal Response.
A response is considered to have caused an equivocal response if it does not fulfill the criteria of either a negative or a positive response and/or the Study Director does not consider the response to be either positive or negative.

Statistics:

No formal hypothesis testing was done.

Key result

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

TOXICITY OF THE TEST SUBSTANCE

The survival of the TA100 culture was determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plates.

Both in the absence and presence of S9-mix the survival of strain TA100 was not reduced up to and including test substance concentrations of 5000 µg/plate (Table 1). Based on these data, the test substance was tested up to and including a concentration of 5000 µg/plate in the absence and presence of S9-mix.

THE AMES SALMONELLA/MICROSOME PLATE TEST

Tables 2 and 3 show the results of the Salmonella/microsome plate test. All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory background historical ranges indicating that the test conditions were optimal, and that the metabolic activation system functioned properly.

Table 1: PRELIMINARY TOXICITY DETERMINATION OF THE TEST SUBSTANCE IN TA100. At doses of 1000 to 5000 µg/plate, the test substance precipitated on the plates.

	Viable counts/plate (duplicate plates)
Concentration (µg/plate)	Without S9-mix		With S9-mix
Control (0.1 mL DMSO)	255	270	291	282
1.0	283	286	251	278
3.3	296	290	265	275
10.0	291	269	287	293
33.3	277	281	294	274
100	244	254	262	221
333	266	278	240	288
1000	283	274	288	304
3330	273	279	277	267
5000	280	276	279	302

Table 2: Experiment 1. MUTAGENIC RESPONSE IN THE AMES SALMONELLA/MICROSOME PLATE TEST.¹= Test substance precipitated slightly on the plates.²= Test substance precipitated moderately on the plates.

	Mean number of revertant (His+) colonies / (3 replicate plates with different strains of S. typhimurium
	TA1535	TA1537	TA98	TA100
Without S9-mix
100	4	5	27	112
333	6	5	31	111
1000^1	8	8	26	129
3330^1	7	9	23	141
5000^2	6	6	27	124
Solvent control (0.1 mL DMSO)	6	6	29	109
Positive control	382	357	581	720
With S9-mix
100	7	8	36	146
333	6	7	34	133
1000^1	8	6	42	150
3330^1	9	6	30	139
5000^2	7	7	29	156
Solvent control (0.1 mL DMSO)	9	9	41	128
Positive control	382	206	352	545

Table 3: Experiment 2. MUTAGENIC RESPONSE IN THE AMES SALMONELLA/MICROSOME PLATE TEST.¹= Test substance precipitated slightly on the plates.²= Test substance precipitated moderately on the plates.

	Mean number of revertant (His+) colonies / (3 replicate plates with different strains of S. typhimurium
	TA1535	TA1537	TA98	TA100
Without S9-mix
100	7	4	23	126
333	8	5	25	117
1000^1	8	2	24	112
3330^1	5	3	24	125
5000^2	6	4	26	118
Solvent control (0.1 mL DMSO)	7	6	32	117
Positive control	280	546	478	1268
With S9-mix
100	6	5	38	120
333	7	5	33	122
1000^1	9	4	35	127
3330^1	7	5	29	134
5000^2	6	3	30	132
Solvent control (0.1 mL DMSO)	6	4	37	124
Positive control	327	214	584	680

PRECIPITATION CODE

Slight Precipitate: distinguished by noticeable precipitate on the plate. The precipitate does not influence automated counting of the plate.

Moderate Precipitate: Distinguished by marked amount of precipitate on the plate, requiring the plate to be hand counted.

Heavy Precipitate: Distinguished by a large amount of precipitate on the plate, making the required hand count difficult.

Conclusions:

Based on the results of this study it is concluded that the test substance is not mutagenic in the Ames Salmonella/microsome assay.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted May 26, 1983) and EEC Directive 67/548/EEC B.14 (adopted December 1992), four strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) were exposed to Ceramide III at concentrations of 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. Two tests were performed using the plate incorporation method.

In the first mutation assay, Ceramide III did not induce a significant dose-related increase in the number of revertant colonies in all four tested strains, both in the absence and presence of mammalian metabolic activation. These results were confirmed in the second mutation assay.

Slight (at doses of 1000 and 3330 µg/plate) to moderate precipitation (at a dose of 5000 µg/plate) was observed. No cytotoxic effects of Ceramide III were described.

The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed.

There was no evidence of induced mutant colonies over background related to Ceramide III.

Based on the results of this study it is concluded that Ceramide III is not mutagenic in the Salmonella typhimurium reverse mutation assay.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The source substance Ceramide IIIB did not reveal mutagenic potential in a guideline study under GLP.

The target substance Ceramide III was not mutagenic in a study according to the principle described by Ames.

Therefore, based on the information of both source and target substance, Ceramide III ist considered to be non-mutagenic in the bacterial reverse mutation assay.