Octadecyl docosanoate

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:

12 Oct - 11 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Octadecyl docosanoate

EC Number:

246-115-1

EC Name:

Octadecyl docosanoate

Cas Number:

24271-12-3

Molecular formula:

C40H80O2

IUPAC Name:

octadecyl docosanoate

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 male rats, treated with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

Prior to the main experiments, a range-finding study was performed using the TA 98 and TA 100 strains with following test concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 μg/plate with and without metabolic activation.

Experiment 1: 10.0, 31.6, 100, 316, 1000, 2500, 5000 μg/plate with and without metabolic activation
Experiment 2: 1.58, 5.00, 15.8, 50, 158, 500, 1580, 5000 μg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity. (A correction factor of 1.09 was applied to consider the purity of the test item)

Controls

Untreated negative controls:

yes

Remarks:

destilled water

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (NPD); 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn (toxicity is determined by a clearing or diminution of the background lawn)

Evaluation criteria:

Acceptance criteria
The study was considered valid if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative/solvent control plates with and without S9 mix are within the historical control data ranges (Control limit ranges (Poisson-based) of mean values)
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Evaluation criteria
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A reduced background lawn was observed in exp.1 starting at a concentration of 2500 µg/plate and in exp. 2 at 5000 µg/plate, with and without S9-Mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A reduced background lawn was observed in exp.1 starting at a concentration of 2500 µg/plate and in exp. 2 at 5000 µg/plate, with and without S9-Mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A reduced background lawn was observed in exp.1 starting at a concentration of 2500 µg/plate and in exp. 2 at 5000 µg/plate, with and without S9-Mix.

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

Remarks:

A reduced background lawn was observed in exp.1 starting at a concentration of 2500 µg/plate and in exp. 2 at 5000 µg/plate, with and without S9-Mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A reduced background lawn was observed in exp.1 starting at a concentration of 2500 µg/plate and in exp. 2 at 5000 µg/plate, with and without S9-Mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Reduced background lawn was observed in both tested strains (TA 98 and TA 100) starting at a test substance concentration of 2500 µg/plate with and without metabolic activation. There was no relevant increase in the number of revertants in cells treated with the test substance. The positive controls induced a clear increase in the number of revertants (at least 4.2-fold).

HISTORICAL CONTROL DATA
please refer to Table 3 in "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1:Summary of test results (Experiment 1; Plate Incorporation Method)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	TA102
-	Negative control (distilled water)	21 ± 3.8	26 ± 2.3	106 ± 6.4	23 ± 1.5	217 ± 24.8
	Solvent control (tetrahydrofuran)	19 ± 3.5	18 ± 2.9	77 ± 2.6	26 ± 1.0	191 ± 7.6
	10.0	18 ± 1.5	27 ± 5.7	73 ± 6.1	23 ± 1.0	203 ± 25.2
	31.6	16 ± 0.6	26 ± 6.5	65 ± 13.4	24 ± 2.1	207 ± 41.4
	100	20 ± 2.1	25 ± 5.8	71 ± 9.1	27 ± 1.0	246 ± 25.2
	316	17 ± 0.6	25 ± 5.5	95 ± 23.0	26 ± 5.3	225 ± 23.5
	1000	19 ± 2.5	24 ± 1.0	74 ± 11.8	26 ± 1.0	227 ± 35.2
	2500	1 ± 1.2 B, P	9 ± 1.2 B, P	26 ± 5.1 B, P	10 ± 1.5 B, P	218 ± 9.6 P
	5000	0 ± 0.0 B, P	1 ± 1.0 B, P	7 ± 4.6 B, P	1 ± 0.6 B, P	12 ± 1.5 B, P
	Positive controls (unit/plate)	NPD (40 µg)	NPD (10 µg)	SA (10 µg)	SA (10 µg)	MMS (1 µL)
	Mean No. of colonies/plate (average of 3 plates)	173 ± 16.6	297 ± 65.3	591 ± 41.8	380 ± 39.5	817 ± 17.6
+	Negative control (distilled water)	15 ± 2.5	25 ± 3.5	84 ± 4.7	29 ± 63.1	209 ± 10.0
	Solvent control (tetrahydrofuran)	15 ± 0.6	21 ± 1.2	77 ± 10.4	31 ± 2.5	207 ± 6.0
	10.0	13 ± 1.0	26 ± 3.2	76 ± 4.9	24 ± 4.0	238 ± 9.5
	31.6	14 ± 2.5	23 ± 3.5	78 ± 7.8	29 ± 3.8	238 ± 9.2
	100	16 ± 2.6	25 ± 2.3	80 ± 10.1	27 ± 4.5	259 ± 30.3
	316	12 ± 1.0	23 ± 4.5	83 ± 10.0	27 ± 1.2	210 ± 38.7
	1000	14 ± 1.5	27 ± 1.0	89 ± 9.0	28 ± 7.0	255 ± 15.8
	2500	0 ± 0.0 B, P	12 ± 1.0 B, P	24 ± 2.6 B, P	7 ± 0.6 B, P	260 ± 11.0 P
	5000	0 ± 0.0 B, P	2 ± 1.0 B, P	11 ± 1.0 B, P	0 ± 0.6 B, P	15 ± 0.0 B, P
	Positive controls  (µg/plate)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (10)
	Mean No. of colonies/plate (average of 3 plates)	106 ± 6.2	128 ± 38.7	320 ± 16.5	217 ± 35.2	715 ± 21.1

2AA = 2-aminoanthracene

B = reduced background lawn

MMS = methylmethanesulfonate

NPD = 4-nitro-1,2-phenylene-diamine

P = precipitation

SA = sodium azide

SD = standard deviation

Table 2: Summary of test results (Experiment 2; Plate Incorporation Method)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	TA102
-	Negative control (distilled water)	25 ± 5.9	32 ± 3.1	98 ± 18.0	20 ± 1.5	363 ± 9.7
	Solvent control (tetrahydrofuran)	32 ± 1.5	31 ± 8.5	114 ± 2.3	18 ± 2.3	244 ± 35.5
	1.58	25 ± 1.0	24 ± 2.6	82 ± 19.3	21 ± 2.6	325 ± 62.2
	5.0	24 ± 2.1	25 ± 5.9	68 ± 2.6	19 ± 2.1	329 ± 25.6
	15.8	27 ± 1.0	36 ± 4.4	86 ± 18.0	23 ± 1.5	236 ± 21.5
	50	28 ± 5.0	26 ± 4.7	88 ± 15.6	21 ± 2.1	257 ± 20.1
	158	24 ± 8.4	29 ± 4.7	74 ± 9.0	20 ± 2.5	290 ± 89.1
	500	23 ± 1.0	28 ± 3.2	79 ± 19.2	21 ± 2.0	321 ± 66.3
	1580	20 ± 3.8 P	23 ± 3.1 P	77 ± 19.3 P	19 ± 1.2 P	308 ± 18.2 P
	5000	4 ± 1.0 B, P	13 ± 3.1 B, P	10 ± 2.0 B, P	5 ± 0.6 B, P	23 ± 4.4 B, P
	Positive controls (unit/plate)	NPD (40 µg)	NPD (10 µg)	SA (10 µg)	SA (10 µg)	MMS (1 µL)
	Mean No. of colonies/plate (average of 3 plates)	112 ± 7.0	320 ± 35.8	1376 ± 82.9	1167 ± 64.7	1404 ± 48.8
+	Negative control (distilled water)	18 ± 0.6	30 ± 3.1	72 ± 3.5	14 ± 1.5	384 ± 17.4
	Solvent control (tetrahydrofuran)	20 ± 3.8	28 ± 4.6	95 ± 9.3	15 ± 1.0	364 ± 58.6
	1.58	21 ± 1.7	29 ± 4.5	89 ± 32.7	14 ± 1.5	337 ± 10.6
	5.0	19 ± 1.5	28 ± 7.4	94 ± 23.8	14 ± 1.2	393 ± 45.6
	15.8	22 ± 1.5	24 ± 1.5	85 ± 12.5	15 ± 2.5	330 ± 16.5
	50	23 ± 1.5	26 ± 6.2	79 ± 8.1	14 ± 1.5	394 ± 37.1
	158	22 ± 2.6	21 ± 4.0	94 ± 27.0	15 ± 2.5	289 ± 27.9
	500	26 ± 2.0	20 ± 0.0	65 ± 8.4	18 ± 1.5	355 ± 8.1
	1580	17 ± 2.1 P	22 ± 4.0 P	71 ± 5.5 P	6 ± 1.7 P	358 ± 33.3 P
	5000	2 ± 1.0 B, P	4 ± 1.0 B, P	10 ± 2.0 B, P	3 ± 2.1 B, P	32 ± 6.1 B, P
	Positive controls  (µg/plate)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (10)
	Mean No. of colonies/plate (average of 3 plates)	201 ± 15.4	1731 ± 186.7	2406 ± 15.0	229 ± 25.0	1014 ± 83.5

2AA = 2-aminoanthracene

B = reduced background lawn

MMS = methylmethanesulfonate

NPD = 4-nitro-1,2-phenylene-diamine

P = precipitation

SA = sodium azide

SD = standard deviation

Table 3: Historical control data (2014 -2016)

	S9-Mix		Revertants/plate
			TA1537	TA98	TA100	TA1535	TA102
Negative control	-	Mean ± SD	8.2 ± 2.9	24.2 ± 6.7	90.7 ± 15.6	13.8 ± 6.7	270.4 ± 55.0
		Min - Max	3 - 35	11 - 58	49 - 155	4 - 41	141 - 472
		LCL99 - UCL99	0.0 - 16.9	4.1 - 44.3	43.9 - 137.5	0.0 - 33.9	105.5 - 435.2
	+	Mean ± SD	8.3 ± 3.1	29 ± 6.8	96.4 ± 14.1	10.5 ± 4.5	339.7 ± 71.3
		Min - Max	3 - 36	15 - 59	62 - 160	3 - 38	157 - 586
		LCL99 - UCL99	0.0 - 17.6	8.7 - 49.4	54.1 - 138.8	0.0 - 23.9	125.9 - 553.6
Positive control *	-	Mean ± SD	94.5 ± 22.7	430.7 ± 155.5	612.1 ± 220.0	792.0 ± 299.5	1729.2 ± 518.8
		Min - Max	35 - 273	141 - 1830	132 - 1423	38 - 1854	272 - 3321
		LCL99 - UCL99	26.5 - 162.5	0.0 - 897.2	0.0 - 1272.0	0.0 - 1690.5	172.9 - 3285.5
	+	Mean ± SD	234.1 ± 101.4	1880.5 ± 708.5	1727.7 ± 522.0	133.9 ± 134.9	801.2 ± 223.7
		Min - Max	26 - 682	70 - 3606	169 - 3132	22 - 1954	137 - 3588
		LCL99 - UCL99	0.0 - 538.3	0.0 - 4006.1	161.8 - 3293.6	0.0 - 538.7	130.3 - 1472.2

* = - S9 Mix: 4-nitro-1,2-phenylene-diamine for TA 98 and TA 1537, sodium azide for TA 100 and TA 1537, methylmethanesulfonate for TA 102; + S9 Mix: 2-aminoanthracene for all 5 strains

LCL99 = Lower Control Limit (99% Poisson-based)

Max = maximum of revertants/plate

Min = minimum of revertants/plate

UCL99 = Upper Control Limit (99% Poisson-based)

SD = standard deviation

Applicant's summary and conclusion

Conclusions:

Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells with and without metabolic activation.