Fatty acids, C16-18 and C18-hydroxy, oligomeric reaction products with adipic acid, decanoic acid, isooctadecanoic acid, octanoic acid, pentaerythritol and stearic acid

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:

08 Mar - 04 Apr 2017

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 and C18-hydroxy, oligomeric reaction products with adipic acid, decanoic acid, isooctadecanoic acid, octanoic acid, pentaerythritol and stearic acid

EC Number:

500-320-5

EC Name:

Fatty acids, C16-18 and C18-hydroxy, oligomeric reaction products with adipic acid, decanoic acid, isooctadecanoic acid, octanoic acid, pentaerythritol and stearic acid

Cas Number:

130353-58-1

Molecular formula:

not applicable, the substance is UVCB

IUPAC Name:

1,6-bis({2-[(hexadecanoyloxy)methyl]-3-hydroxy-2-(hydroxymethyl)propyl}) hexanedioate; 1-{2-[(decanoyloxy)methyl]-3-hydroxy-2-[(octadecanoyloxy)methyl]propyl} 6-[3-hydroxy-2-(hydroxymethyl)-2-{[(3-methylheptadecanoyl)oxy]methyl}propyl] hexanedioate; 3-hydroxy-2-{[(6-{[3-hydroxy-2-(hydroxymethyl)-2-[(octadecanoyloxy)methyl]propyl]peroxy}-6-oxohexanoyl)oxy]methyl}-2-(hydroxymethyl)propyl 12-hydroxyoctadecanoate

Method

Target gene:

his operon (S. typhimurium)
trp operon (E. coli)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphthoflavone.

Test concentrations with justification for top dose:

First experiment: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Second experiment: 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: In solubility checks performed in–house, the test item was noted as insoluble in sterile distilled water and dimethyl sulphoxide. The test item was noted as initially soluble in dimethyl formamide (50 mg/mL) and acetone (100 mg/mL), however these formulations came out of solution shortly after removal from sonication/heating and could not be considered. The test item was fully soluble in tetrahydrofuran at 200 mg/mL (this formulation stayed in solution), therefore, this solvent was selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene (1, 2 and 10 µg/plate for TA100, TA1535 and TA1537, and WP2uvrA, respectively, + S9)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min (preincubation)
- Exposure duration: Approximately 48 h (plate incorporation and preincubation)

NUMBER OF REPLICATIONS: Triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the growth of the bacterial background lawn
The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 μg/plate because of test item precipitation. A number of further manual counts were also performed, predominantly due to colonies spreading and bubble interference, thus distorting the actual plate count. Occasional plates were also manually assessed for accuracy (verification) against the automated counts.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Experiment I: starting at 1500 µg/mL; Experiment II: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Experiment I: starting at 1500 µg/mL; Experiment II: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Experiment I: starting at 1500 µg/mL; Experiment II: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Experiment I: starting at 1500 µg/mL; Experiment II: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Experiment I: starting at 1500 µg/mL; Experiment II: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at concentrations ≥ 1500 µg/plate in Experiment I (plate incorporation) and at 5000 µg/plate in Experiment II (pre-incubation).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control data were within the range of the historical control data.
- Negative (solvent/vehicle) historical control data: The solvent and untreated control data were within the range of the historical control data.

Any other information on results incl. tables

Table 1: Results of Experiment I (plate incorporation)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate
		(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	Untreated	112	14	28	20	17
–	Solvent	126 ± 13	15 ± 2.5	39 ± 12	22 ± 6.1	12 ± 3.5
–	1.5	122 ± 19.1	16 ± 2.0	43 ± 8.4	37 ± 6.6 **	15 ± 7.8
–	5	120 ± 10.6	16 ± 5.7	37 ± 18.5	32 ± 2.5 *	17 ± 5.0
–	15	127 ± 6.7	16 ± 4.5	42 ± 3.5	34 ± 3.8 **	16 ± 1.5
–	50	117 ± 2.8	17 ± 5.0	40 ± 7.4	36 ± 4.2 **	19 ± 6.0
–	150	117 ± 12.3	14 ± 4.7	31 ± 18.6	42 ± 2.0 ***	22 ± 3.5 *
–	500	117 ± 14.6	15 ± 3.1	39 ± 10.7	33 ± 5.2 *	24 ± 4.6 *
–	1500 P	103 ± 15.6	13 ± 4.9	45 ± 3.6	39 ± 1.0 ***	20 ± 3.1
–	5000 P	123 ± 13.1	10 ± 4.0	41 ± 5.5	31 ± 5.5 *	16 ± 6.9
Positive controls, –S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0,2	80
	Mean No. of colonies/plate (average of 3 ± SD)	440 ± 4.4	480 ± 53.5	844 ± 20.6	196 ± 15.5	405 ± 65.6
+	Solvent	98 ± 6.0	11 ± 1.5	37 ± 6.4	34 ± 6.0	17 ± 2.1
+	1.5	111 ± 5.3	15 ± 1.5	37 ± 12.2	25 ± 2.3	17 ± 2.3
+	5	110 ± 4.7	15 ± 2.6	39 ± 7.5	26 ± 14.2	13 ± 3.1
+	15	107 ± 15.5	13 ± 3.8	40 ± 3.1	29 ± 4.6	12 ± 2.5
+	50	105 ± 11.7	18 ± 1.7 **	38 ± 2.1	29 ± 6.5	17 ± 1.0
+	150	97 ± 9.3	13 ± 4.0	34 ± 3.8	26 ± 3.5	11 ± 4.4
+	500	104 ± 2.6	11 ± 0.6	40 ± 2.5	26 ± 5.1	13 ± 2.6
+	1500 P	100 ± 6.81	10 ± 0.0	32 ± 6.1	26 ± 4.0	22 ± 3.5
+	5000 P	104 ± 2.5	10 ± 2.1	33 ± 9.8	23 ± 4.4	13 ± 1.0
Positive controls, +S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	700 ± 54.6	253 ± 16.5	337 ± 16.5	273 ± 25.1	278 ± 11.5

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate

*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Table 2: Comfirmatory Experiment: Without metabolic activation (plate incorporation)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate
		(average of 3 plates ± Standard deviation)
		Frameshift type
		TA98	TA1537
–	Untreated	18	16
–	Solvent	32 ± 11.0	10 ± 3.0
–	1.5	28 ± 4.0	7 ± 0.6
–	5	33 ± 2.3	10 ± 3.8
–	15	33 ± 4.7	12 ± 6.7
–	50	34 ± 1.2	9 ± 2.1
–	150	32 ± 2.0	13 ± 1.5
–	500	33 ± 3.5	12 ± 6.6
–	1500 P	35 ± 3.8	12 ± 1.7
–	5000 P	35 ± 5.3	10 ± 1.0
Positive controls, –S9	Name	4NQO	9AA
	Concentrations (μg/plate)	0,2	80
	Mean No. of colonies/plate (average of 3 ± SD)	209 ± 17.6	280 ± 30.4

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

P = Precipitate

Table 3: Results of Experiment II (pre-incubation)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate
		(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	Untreated	116	14	32	24	12
–	Solvent	85 ± 11.0	19 ± 8.1	34 ± 2.9	20 ± 3.1	10 ± 5.6
–	15	93 ± 4.0	21 ± 1.5	36 ± 4.6	20 ± 10.4	8 ± 1.2
–	50	90 ± 0.6	19 ± 6.2	24 ± 2.6	21 ± 0.6	9 ± 2.6
–	150	86 ± 7.9	17 ± 4.4	34 ± 6.0	17 ± 1.5	10 ± 5.2
–	500	94 ± 3.2	17 ± 2.6	35 ± 6.6	17 ± 8.3	10 ± 1.5
–	1500	94 ± 4.7	12 ± 0.6	29 ± 3.6	19 ± 4.2	8 ± 1.5
–	5000 P	100 ± 3.0 *	13 ± 1.5	36 ± 1.2	22 ± 3.8	12 ± 0.6
Positive controls, –S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0,2	80
	Mean No. of colonies/plate (average of 3 ± SD)	773 ± 103.9	572 ± 26.7	520 ± 72.1	240 ± 43.8	160 ± 2.9
+	Solvent	99 ± 11.7	10 ± 1.0	41 ± 4.0	25 ± 4.0	10 ± 4.0
+	15	107 ± 13.1	10 ± 2.5	48 ± 1.7	17 ± 4.0	8 ± 2.6
+	50	93 ± 1.2	13 ± 4.0	41 ± 5.1	24 ± 6.0	13 ± 5.5
+	150	95 ± 10.2	9 ± 0.6	35 ± 6.0	25 ± 7.5	10 ± 2.5
+	500	105 ± 6.5	8 ± 1.7	37 ± 15.6	17 ± 3.6	11 ± 5.0
+	1500	103 ± 11.1	15 ± 2.1 *	43 ± 3.2	26 ± 1.0	14 ± 5.1
+	5000 P	94 ± 11.5	8 ± 1.0	41 ± 7.5	24 ± 3.2	10 ± 2.5
Positive controls, +S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	980 ± 129	203 ± 15.6	234 ± 29.1	93 ± 22.5	180 ± 18.6

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate

*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Table 4: Historical control data

Combined vehicle and untreated control values: 2015
Strain S9-Mix	TA100		TA1535		WP2uvrA		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Values †	274	278	504	285	461	229	526	299	506	282
Min	60	61	7	7	10	12	11	10	4	6
Max	166	175	31	29	58	43	45	46	27	27
Mean	91	95	16	14	24	27	21	24	12	13
SD	19.3	19.1	4.5	4.0	5.6	5.9	6.2	6.1	3.8	3.4
Positive control values 2015
Strain S9-Mix	TA100		TA1535		WP2uvrA		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Values	276	280	252	264	231	227	262	276	253	261
Min	222	250	79	118	116	103	100	78	164	97
Max	2266	2402	2779	457	2769	550	502	705	2318	823
Mean	614	927	472	246	792	266	222	218	911	336
SD	260.6	452.5	434.8	55.7	342.1	97.7	70.2	107.6	412.4	135.7
Combined vehicle and untreated control values: 2016
Strain S9-Mix	TA100		TA1535		WP2uvrA		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Values	399	401	758	393	690	345	788	415	762	398
Min	63	66	8	8	10	13	8	12	3	4
Max	154	156	34	39	53	53	49	51	24	23
Mean	90	93	15	15	22	27	21	25	12	13
SD	14.5	14.3	4.5	5.2	5.8	6.3	4.8	5.7	3.5	3.5
Positive control values 2016
Strain S9-Mix	TA100		TA1535		WP2uvrA		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Values	409	406	381	386	341	335	388	385	379	381
Min	221	284	84	92	107	102	100	96	95	101
Max	2222	2863	2994	879	1611	637	449	4357	1413	639
Mean	724	1264	854	240	718	240	186	188	406	290
SD	320.4	562.9	664.9	62.1	338.6	98.2	49.8	230.8	227.0	92.7

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative