[No public or meaningful name is available]

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:

18 Nov - 07 Dec 2020

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 2020

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate

Cas Number:

18641-58-2

Molecular formula:

C69H132O8

IUPAC Name:

2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate

Method

Target gene:

His operon (S. typhimurium strains) and trp operon (E. coli strain)

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: Cofactor supplemented post-mitochondrial fraction (S9 mix).
- source of S9 : Trinova Biochem GmbH, Giessen, Germany
- method of preparation of S9 mix: S9 mix was prepared from the livers of male Sprague-Dawley rats that were induced with 500 mg/kg bw Aroclor 1254.
- concentration or volume of S9 mix and S9 in the final culture medium: 5% (Experiment 1) and 10% (Experiment 2)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each S9 batch was characterised with the mutagens benzo-(a)-pyrene and 2-aminoanthracene in tester strain TA100.

Test concentrations with justification for top dose:

Pre-experiment / Experiment 1: 0.55*, 1.7*, 5.4, 17, 52, 164, 512 and 1500 µg/plate
Experiment 2: 27, 48, 86, 154, 500 and 1000 µg/plate

*tested in strains TA100 and WP2uvrA only as part of the preliminary experiment

Justification for top dose: Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. The highest concentration of the test item used in the subsequent mutation assays was the level at which the test item exhibited limited solubility.

Vehicle / solvent:

- Vehicle/solvent used: dried tetrahydrofuran (THF)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, -S9 mix, 2.5 µg/plate in DMSO for TA1537; 2-aminoanthracene (2AA), +S9 mix, in DMSO, 1 µg/plate for TA98 and TA100, 2 µg/plate for TA100, 2.5 µg/plate for TA1535 and TA1537, 5 µg/plate for TA1537 and 15 µg/plate for WP2uvrA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
- In the first experiment, 5% S9 mix was added, whereas in the second experiment 10% S9 mix were used.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY :
- Method: reduction of the bacterial bacground lawn, increase in the size of microcolonies and reduction of the revertant colonies

Evaluation criteria:

The test item was considered negative for gene mutation in bacteria if the following criteria were met:
- The total number of revertants in the tester strain TA100 or WP2uvrA was not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 was not greater than three times the concurrent vehicle control.
- The negative response was reproducible in at least one follow-up experiment.

The test item was considered positive for gene mutation in bacteria if the following criteria were met:
- The total number of revertants in the tester strain TA100 or WP2uvrA was greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 was greater than three times the concurrent vehicle control.
- In case a follow up experiment was performed when a positive response was observed in one of the tester strains, the positive response was reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

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

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

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 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 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

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:

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: In the first experiment, precipitation of the test item on the plates was observed at concentrations of 512 µg/plate and upwards. Since the test item precipitated heavily on the plates at the test item concentration of 1500 μg/plate, the number of revertants at this dose level could not be determined. In the second experiment, precipitation was observed at 500 and/or 1000 μg/plate (except for tester strains TA100 and WP2uvrA in the presence of S9-mix for which only moderate precipitation was observed), the number of revertants at these dose levels could not be determined.

RANGE-FINDING/SCREENING STUDIES:
The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 0.55, 1.7, 5.4, 17, 52, 164, 512 and 1500 µg/plate in the absence and presence of S9 mix. Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment. The results obtained in the preliminary experiment with strains TA100 and Wp2uvrA were designated part of Experiment 1.

STUDY RESULTS
There was no increase in the number of revertant colonies observed for any strain at any concentration in any eperiment, neither in the presence nor absence of metabolic activation. The solvent controls remained within the range of historical control data, the positive controls induced a marked increase in the number of revertant colonies, thus demonstrating the functionality of the metabolic activation system and the sensitivity of the test.
Please refer to Tables No. 1 and 2 under "Any other information on results incl. tables".

CYTOTOXICITY:
In the first experiment, there was no cytotoxicity observed for any strain at any concentration with or without S9 mix. In the second experiment, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed for strain TA1537. However, since no dose relationship was observed, the reduction was not considered to be caused by toxicity of the test item. It was more likely the reduction was caused by an incidental fluctuation in the number of revertant colonies.

HISTORICAL CONTROL DATA (HCD): Please refer to Tables 1 and 2 under "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Results preliminary experiment and Experiment 1

Preliminary experiment (strains TA100 and WP2uvrA) and Experiment 1 (strains TA1535, TA1537 and TA98): Plate incorporation assay, 5% S9 mix
Strain	TA 100		Wp2uvrA		TA 1535		TA 1537		TA 98
Metabolic activation	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Vehicle control
THF mean	86	69	16	11	12	9	5	2	12	19
± SD	± 7	± 10	± 2	± 1	± 5	± 2	± 1	± 1	± 2	± 4
HCD^#mean	109	101	24	29	9	10	5	5	14	18
± SD	± 19	± 21	± 9	± 10	± 3	± 3	± 2	± 2	± 5	± 6
[range]	58 - 188	50 - 176	9 - 61	11 - 68	3 - 26	4 - 25	2 - 24	2 - 17	4 - 61	6 - 60
Test item [µg/plate]
0.55 mean	92	74	14	16
± SD	± 10	± 5	± 2	± 1
1.7 mean	84	79	9	16
± SD	± 13	± 8	± 6	± 0
5.4 mean	88	73	18	12	8	10	6	4	12	19
± SD	± 8	± 7	± 2	± 2	± 1	± 2	± 2	± 1	± 6	± 4
17 mean	89	65	18	20	13	6	3	2	12	17
± SD	± 11	± 9	± 8	± 7	± 3	± 2	± 3	± 2	± 3	± 3
52 mean	92	65	14	21	7	12	5	5	13	16
± SD	± 11	± 8	± 2	± 5	± 3	± 4	± 3	± 2	± 2	± 4
164 mean	90 NP	76 NP	13 NP	21 NP	9 NP	16 NP	4 NP	5 NP	13 NP	17 NP
± SD	± 16	± 4	± 3	± 2	± 4	± 3	± 1	± 1	± 5	± 5
512 mean	113 SP	100 SP	11 MP	13 MP	7 MP	8 MP	5 MP	4 MP	9 MP	11 MP
± SD	± 7	± 20	± 7	± 4	± 2	± 1	± 1	± 3	± 2	± 5
1500 mean	nHP	nHP	nHP	nHP	nHP	nHP	nHP	nHP	nHP	nHP
± SD
Positive control
^§mean	692	1144	1390	273	998	307	1283	249	1654	940
± SD	± 78	± 34	± 33	± 27	± 2	± 17	± 46	± 103	± 132	± 49
HCD^#mean	865	1432	1196	421	972	289	818	293	1252	945
± SD	± 181	± 398	± 526	± 186	± 170	± 108	± 370	± 142	± 251	± 378
[range]	452 - 1993	397 - 2666	93 - 1999	109 - 1968	107 - 1530	78 - 1481	64 - 1475	52 - 1843	379 - 2118	265 - 2077
^§= information on respective positive control is reported in Material and Method section
^#= Historical control data generated from Nov 2017 - Nov 2020
^HP= heavy precipitate, number of revertants could not be determined;^MP: moderate precipitate;^NP: no precipitate;^SP: slight precipitate;^n: normal backterial background lawn

Table 2: Results Experiment 2

Experiment 2 (all strains): Plate incorporation assay, 10% S9 mix
Strain	TA 100		Wp2uvrA		TA 1535		TA 1537		TA 98
Metabolic activation	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Vehicle control
THF mean	100	75	16	24	10	8	6	8	6	12
± SD	± 6	± 5	± 7	± 5	± 5	± 4	± 3	± 4	± 4	± 4
HCD^#mean	109	101	24	29	9	10	5	5	14	18
± SD	± 19	± 21	± 9	± 10	± 3	± 3	± 2	± 2	± 5	± 6
[range]	58 - 188	50 - 176	9 - 61	11 - 68	3 - 26	4 - 25	2 - 24	2 - 17	4 - 61	6 - 60
Test item [µg/plate]
27 mean	113	80	23	23	11 SP	10	7	7	9	8
± SD	± 8	± 8	± 1	± 5	± 3	± 2	± 4	± 5	± 6	± 4
48 mean	85	73	21	29	11 SP	8	1	2	10	13
± SD	± 5	± 5	± 2	± 4	± 6	± 2	± 2	± 2	± 3	± 3
86 mean	85 NP	57	17 NP	18	10 SP	12 NP	6 NP	5 NP	7 NP	9 NP
± SD	± 18	± 7	± 8	± 7	± 2	± 4	± 4	± 3	± 4	± 5
154 mean	85 SP	79 NP	13 MPSP	18NP	6 MP	9 MP NP	8 SP	7 SP	8 SP	13 SP
± SD	± 18	± 9	± 3	± 6	± 3	± 2	± 6	± 2	± 2	± 2
500 mean	85	78 SP	22 MP	24 MP	9 MP	HP	MP	8 SP	MP	19 MP
± SD	± 9	± 22	± 1	± 1	± 2			± 5		± 5
1000 mean	nHP	63 nMP	nHP	22 nMP	nHP	10 nMP HP	nHP	nHP	nHP	nHP
± SD		± 7		± 7		± 1
Positive control
^§mean	799	1347	1399	247	716	194	1125	268	1477	536
± SD	± 32	± 61	± 75	± 28	± 38	± 21	± 127	± 82	± 97	± 42
HCD^#mean	865	1432	1196	421	972	289	818	293	1252	945
± SD	± 181	± 398	± 526	± 186	± 170	± 108	± 370	± 142	± 251	± 378
[range]	452 - 1993	397 - 2666	93 - 1999	109 - 1968	107 - 1530	78 - 1481	64 - 1475	52 - 1843	379 - 2118	265 - 2077
^§= information on respective positive control is reported in Material and Method section
^#= Historical control data generated from Nov 2017 - Nov 2020; THF: tetrahydrofuran, solvent control
^HP= heavy precipitate, number of revertants could not be determined;^MP: moderate precipitate;^NP: no precipitate;^SP: slight precipitate;^n: normal backterial background lawn

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, the test item was not mutagenic in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and in E. coli strain WP2uvrA with and without metabolic activation.