Fatty acids, C16-18 (even numbered, C18 unsaturated), 2-ethylhexyl esters, epoxidized

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:

Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 Jun 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Kaiser-Friedrich-Straße 7, 55116 Mainz

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered, C18 unsaturated), 2-ethylhexyl esters, epoxidized

Molecular formula:

Unspecified

IUPAC Name:

Fatty acids, C16-18 (even numbered, C18 unsaturated), 2-ethylhexyl esters, epoxidized

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0022865630
- Test substance No.: 12/0076-2
- Date of production: 27 May 2020
- Expiry date: 27 May 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity: The homogeneity of the test substance was guaranteed on
account of the high purity and was ensured by mixing before preparation of the test substance solutions.
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions was guaranteed until 27 May 2021 as indicated by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.
- Stability of the test substance in the solvent/vehicle: The stability of the test substance in the vehicle acetone was not determined analytically, because the test substance was administered immediately after preparation and is usually
stable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in acetone. To achieve a clear solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before use.

FORM AS APPLIED IN THE TEST (if different from that of starting material):
- dissolved in the vehicle acetone

OTHER
- Physical state, appearance: liquid, yellowish, clear

Method

Target gene:

his, trp

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:

Type and composition of metabolic activation system:
- source of S9: BASF SE; in an AAALAC approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive
- method of preparation of S9 mix: At least 5 male Wistar rats [Crl:WI(Han)] received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. 24 hours after the last administration, the rats were sacrificed, and the livers were weighed, washed and homogenized. After centrifugation of the homogenate at 9000 x g, portions of the supernatant (S9 fraction) were stored at -70°C to -80°C. The S9 mix was prepared freshly prior to each experiment (1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors))

Test concentrations with justification for top dose:

1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

3rd Experiment
Strain: TA 1537
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: Due to technical reason an evaluation of the revertant colonies of the
tester strain TA1537 was not possible; therefore, the experimental
part was repeated.

As long as precipitation did not interfere with the colony scoring, 5 mg/plate was
generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, acetone was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: with S9 mix: 2-aminoanthracene (2-AA); without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD)

Remarks:

Sterility control: Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at testing: approx. 10^9 cells per mL

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours

DETERMINATION OF CYTOTOXICITY
- Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Evaluation criteria:

Acceptance criteria
The experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and
TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TOXICITY
- A weak bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions at 5000 μg/plate.
In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions at and above 2500 μg/plate. (see table 1)

HISTORICAL CONTROL DATA
- Positive historical control data: see table 2
- Negative (solvent/vehicle) historical control data: see table 3

Any other information on results incl. tables

Table 1: Toxicity: Decreased revertant numbers were observed at following concentrations (μg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	Without	-
	With	5000	-	-	5000	-
2nd-PIT	Without	5000	-	-	-	-
	With	-
3rd-PIT	Without	not tested	not tested	5000	not tested	not tested
	With	not tested	not tested	2500 - 5000	not tested	not tested

Table 2: Historical Positive Controls

Strain	S9 Mix	Positive control	No. of plates	No. of values	Min	Max	Mean	SD
TA 1535                    Without		MNNG	200	73	406	8303	4032	1720.0
With		2-AA	207	73	99	433	235	100.3
TA 100                   Without		MNNG	201	73	611	6430	2853	1237.9
With		2-AA	204	73	534	4037	2080	965.0
TA 1537                 Without		AAC	204	73	342	2381	1008	364.6
With		2-AA	210	73	46	301	161	66.7
TA 98                    Without		NOPD	198	73	391	1711	693	204.0
With		2-AA	201	73	337	3025	1676	754.9
E. coli                   Without		4-NQO	195	73	344	2265	987	423.9
With		2-AA	198	72	98	276	206	36.2

Table 3: Historical Negative Controls (based on Sorcerer Counter / Ames Study Mananger System (Dec 2019 - Dec 2020))

Strain	S9 Mix	Vehicle	No. of plates		No. of values		Min	Max	Mean	SD
TA 1535                    Without		(Alle)	288		107		6	20	13	2.8
With		(Alle)	297		107		7	21	13	2.7
TA 100                      Without		(Alle)	288		107		73	157	112	13.5
With		(Alle)	300		107		78	145	113	14.0
TA 1537                    Without		(Alle)	288		107		4	16	10	2.1
With		(Alle)	294		107		5	14	10	2.0
TA 98                        Without		(Alle)	282		107		13	31	22	3.2
With		(Alle)	294		107		15	53	26	5.4
E. coli                        Without		(Alle)	285		107		18	38	27	4.3
With		(Alle)	288		107		19	39	27	3.9

 

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5000 μg/plate (SPT); 33 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: Precipitation of the test substance was observed at and above 1000 μg/plate with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 2500 μg/plate.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.