Fatty acids, C16-18, isononyl esters

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:

14 - 25 Jul 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997; corrected in 2020

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:

Fatty acids, C16-18, isononyl esters

EC Number:

292-960-4

EC Name:

Fatty acids, C16-18, isononyl esters

Cas Number:

91031-57-1

Molecular formula:

not available - multi constituent substance

IUPAC Name:

Fatty acids, C16-18, isononyl esters

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for the E.coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: rfa (deep rough (defective lipopolysaccharide cellcoat)), gal (mutation in galactose metabolism); chl (mutation in nitrate reductase); bio (defective biotin synthesis); uvrB (loss of excision repair system (deletion of ultraviolet-repair B gene))

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH (Giessen, Germany); prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw)
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA, USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components, 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix in the final culture medium: 0.5 mL S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Test concentrations with justification for top dose:

Experiment 1 (Direct plate assay):
- all strains: 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate with and without metabolic activation,

Experiment 2 (Pre-incubation assay):
- all strains: 17, 52, 164, 512, 1600, 5000 µg/plate with and without metabolic activation

5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances according to OECD 471.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol (Merck, Darmstadt, Germany)
- Justification for choice of solvent/vehicle: Ethanol was selected for use in the assay as it has been routinely used and validated for use in the bacterial reverse mutation assay. The test material formed a clear colourless solution in ethanol.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: ICR-191; in DMSO; -S9 / TA1537 / 2.5 µg/plate (Exp 1); Methylmethanesulfonate - MMS; in DMSO; -S9 / TA 100 / 650 µg/plate (Exp 1 and 2);

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: three replicate plates / dose
- Number of independent experiments: 2 (direct plate and pre-incubation assay)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^9 cells/mL
- Test substance added in agar (plate incorporation; Experiment 1); pre-incubation (Experiment 2)

TREATMENT AND HARVEST SCHEDULE:
Direct plate assay
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0 °C
Pre-incubation assay
- Preincubation period: 30 ± 2 min at 37.0 ± 1.0 °C
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies per plate.

METHODS FOR MEASUREMENTS OF GENOTOXICITY
- The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

OTHER
- The Salmonella typhimurium strains were checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
- The Escherichia coli WP2uvrA strain was checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year.

Rationale for test conditions:

Test conditions according to OECD 471

Evaluation criteria:

A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537 or TA 98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA 1535, TA 1537 or TA 98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow-up experiment.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Test results 1

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:

Precipitation at concentrations of 1600 μg/plate and upwards in Experiment 1 and 2 with and without S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation at concentrations of 512 μg/plate and upwards in Experiment 1 and 1600 μg/plate and upwards in Experiment 2 with and without S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

Precipitation at concentrations of 512 μg/plate and upwards in Experiment 1 (with S9-mix) and 1600 μg/plate and upwards in Experiment 1 (without S9-mix) and in Experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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:

Precipitation at concentrations of 1600 μg/plate and upwards in Experiment 1 and 2 with and without S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

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:

Precipitation at concentrations of 1600 μg/plate and upwards in Experiment 1 and 2 with and without S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- The test material formed a clear colourless solution in ethanol.
- Precipitation: In Experiment 1, precipitation of the test material on the plates was observed at concentrations of 512 μg/plate and upwards in tester strains TA100 (absence and presence of S9-mix) and TA98 (presence of S9-mix). Precipitation of the test material on the plates was observed at concentrations of 1600 μg/plate and upwards in the other tester strains. In Experiment 2, precipitation of the test material on the plates was observed at concentrations of 1600 μg/plate and upwards in all tester strains in the absence and presence of S9-mix.

STUDY RESULTS:
In the Direct Plate Assay (Experiment 1) and Pre-Incubation Assay (Experiment 2), there was no increase in the number of revertants observed upon treatment with the test material under all conditions tested (refer to 'Any other information on results incl. tables', table 1 and 2).
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants observed upon treatment with the test material under all conditions tested.

HISTORICAL CONTROL DATA
See 'Any other information on results incl. tables', table 3 and 4.

Any other information on results incl. tables

Table 1: Experiment 1 – Direct plate assay

Dose (µg/plate)	Mean number of revertant colonies / 3 replicate plates (± SD)
	TA1535		TA1537		TA98		TA100		WP2 uvrA
	Mean	± SD	Mean	± SD	Mean	± SD	Mean	± SD	Mean	± SD
Without metabolic activation
Solvent control	9	4	5	4	20	2	94	20	12	2
1.7	8	4	3	2	10	2	94	12	15	4
5.4	8	3	3	2	10	2	86	20	17	4
17	7	2	2	1	15	5	90	5	15	3
52	5	1	4	2	19	7	89	2	14	4
164	6	2	3	1	13	2	77	6 NP	15	1
512	10	2 NP	2	2 NP	11	6 NP	84	14 SP	18	4 NP
1600	6	2 SP	3	1 SP	9	3 SP	93	13 SP	15	8 SP
5000	10	3 n SP	4	2 n SP	11	3 n SP	86	9 n SP	22	3 n SP
Positive control	903	83	1147	129	1044	242	662	106	984	250
With metabolic activation
Solvent control	9	3	7	4	22	7	59	13	20	2
1.7	9	5	2	2	15	7	72	8	17	2
5.4	5	3	4	1	17	3	58	7	13	3
17	8	5	7	6	21	6	74	13	21	3
52	5	3	4	1	18	5	77	11	24	6
164	8	3	2	2	19	1 NP	72	4 NP	15	1
512	5	3 NP	2	1 NP	23	4 SP	62	6 SP	17	5 NP
1600	9	2 SP	6	5 SP	20	3 SP	78	12 SP	19	4 SP
5000	5	3 n SP	4	1 n SP	22	11 n SP	80	13 n SP	20	5 n SP
Positive control		26	196	24	1176	196	812	173	339	23
NP = No precipitate SP = Slight precipitate n = Normal bacterial background lawn   Positive controls (without metabolic activation): TA1535 – sodium azide (SA) TA1537 – ICR-191 TA98 – 2-nitrofluorene (NF) TA100 – methylmethanesulfonate (MMS) WP2 uvrA - 4-nitroquinoline N-oxide (4-NQO)   Positive controls (with metabolic activation) All strains – 2-aminoanthracene (2AA)

 

Table 2: Experiment 2 – Pre-incubation assay

Dose (µg/plate)	Mean number of revertant colonies / 3 replicate plates (± SD)
	TA1535		TA1537		TA98		TA100		WP2 uvrA
	Mean	± SD	Mean	± SD	Mean	± SD	Mean	± SD	Mean	± SD
Without metabolic activation
Solvent control	6	5	4	1	13	1	89	10	19	10
17	7	3	4	1	6	3	95	7	19	1
52	9	6	3	2	9	1	95	6	19	3
164	10	1	2	1	12	2	89	10	23	2
512	6	2 NP	3	2 NP	8	3 NP	101	15 NP	22	11 NP
1600	9	6 SP	6	2 SP	8	4 e MC m SP^1	89	8 SP	15	8 SP
5000	10	3 n SP	4	1 n SP	15	8 n SP	116	5 n SP	20	9 n SP
Positive control	723	65	1038	231	1362	155^2	537	30	187	73
With metabolic activation
Solvent control	9	2	3	2	21	8	94	4	24	6
17	10	3	8	3	16	4	83	13	19	1
52	10	3	4	1	23	3	84	8	27	1
164	8	3	4	4	17	2	72	10	23	4
512	5	2 NP	5	4 NP	22	2 NP	84	10 NP	26	8 NP
1600	11	7 SP	6	2 SP	12	3 SP	91	4 SP	31	7 SP
5000	10	5 n SP	4	1 n SP	24	10 n SP	93	10 n SP	27	6 n SP
Positive control	125	50	259	110	614	26	470	400	446	25
^1 = Considered a outlier due to effects only seen at one dose in the middle of the tested dose range ^2 = Outlier excluded: Mean of two plates MC = Microcolonies NP = No precipitate SP = Slight precipitate e = Bacterial background lawn extremely reduced m = Bacterial background lawn moderately reduced n = Normal bacterial background lawn   Positive controls (without metabolic activation): TA1535 – sodium azide (SA) TA1537 and TA98 – 2-nitrofluorene (NF) TA100 – methylmethanesulfonate (MMS) WP2 uvrA - 4-nitroquinoline N-oxide (4-NQO)   Positive controls (with metabolic activation) All strains – 2-aminoanthracene (2AA)

 

Table 3: Historical control data of the solvent control (Jun 2019 – Jun 2022)

	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	2 – 22	3 – 28	1 – 24	1 – 20	3 – 32	5 – 62	56 – 188	34 – 169	9 – 54	9 – 73
Mean	9	10	4	5	12	17	99	86	19	22
SD	2.6	2.8	2.1	2.3	3.6	5.1	16	20	5.0	6.4
Total number of plates	2242	2282	2274	2323	2337	2413	2347	2390	2137	2158
95% upper limit	14	15	8.1	9.5	19	27	130	126	29	35
95% lower limit	3.9	4.5	-0.12	0.49	4.8	7.0	68	46	9.2	9.5
SD = Standard deviation

 

Table 4: Historical control data of the positive control (Jun 2019 – Jun 2022)

	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	95 – 1373	78 – 1145	71 – 2175	46 – 1989	379 – 2141	251 – 3369	167 – 1870	166 – 2185	89 – 2027	115 – 1642
Mean	863	261	863	253	1393	978	783	1259	1281	437
SD	176	109	377	164	319	416	189	426	361	209
Total number of plates	2054	2107	1609	2133	2180	2186	2133	2212	2002	1983
95% upper limit	1207	474	1603	574	2017	1794	1152	2093	1988	846
95% lower limit	519	48	123	-68	769	162	414	425	574	28
SD = Standard deviation

 

 

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, Fatty acids, C16-18, isononyl esters showed negative mutagenic responses in all bacterial strains over the entire dose-range.