Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

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:

24 Apr - 18 Jun 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

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:

Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

EC Number:

260-257-1

EC Name:

Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

Cas Number:

56554-53-1

Molecular formula:

C30H56O6

IUPAC Name:

1,3-bis[(3,5,5-trimethylhexanoyl)oxy]propan-2-yl 3,5,5-trimethylhexanoate

Method

Target gene:

his operon for S. typhimurium strains and trp operon for E. coli strain

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Experiment 1 (plate incorporation): 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation
Experiment 2 (pre-incubation): 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/ml in solubility checks performed in-house. Acetone was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

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

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min (Experiment 2 only)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn

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.
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).
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 judgement about test item activity. Results of this type will be reported as equivocal.

Statistics:

Mean values and standard deviation were calculated.

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

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

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

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was immiscible in sterile distilled water at 50 mg/ml.
- Precipitation: A test item precipitate (oily in appearance) was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
- Stability in vehicle: There was no visible change in test item formulations throughout each experiment, therefore, it was assumed to be stable under the test conditions.

RANGE-FINDING/SCREENING STUDIES:
The preliminary toxicity test and Experiment 1 can be considered as range-finding studies. No cytotoxicity and no effects on the number of revertant colonies were observed at any test concentration in both tests. Therefore, in Experiment 2 the test item concentration range was the same as the range-finding tests, i.e. up to the limit concentration of 5000 µg/plate.
COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertant colonies in the vehicle, untreated and positive controls was within the ranges of the reported history profile of vehicle (combined historical negative and solvent control ranges) and positive control values of the testing facility for the two last calendar years.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the preliminary toxicity test, the test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.
No cytotoxic effects (reduction in the growth of the bacterial background lawn) were observed at any concentration in Experiments 1 and 2.

Any other information on results incl. tables

Table 1. Preliminary toxicity test (numbers of revertant colonies).

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	65	63	69	68	65	66	62	63	63	66P	69P
+	TA100	73	71	62	63	73	64	79	67	69	65P	69P
-	WP2uvrA	29	27	27	25	24	29	31	35	36	33P	36P
+	WP2uvrA	43	44	44	41	48	43	27	34	38	46P	49P

P: Precipitate

 

Table 2. Spontaneous mutation rates (concurrent negative controls)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
EXPERIMENT 1 (plate incorporation)
TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
106 99 136 (114)	23 31 24 (26)	44 41 28 (38)	35 28 31 (31)	11 12 13 (12)
EXPERIMENT 2 (pre-incubation)
134 128 110 (124)	27 20 21 (23)	57 31 44 (44)	40 33 25 (33)	11 15 7 (11)

 

Table 3. Test results Experiment 1

EXPERIMENT 1 (plate incorporation) Number of revertants (mean ± standard deviation)
S9-Mix	Without
Test item (µg/plate)	TA 100	TA 1535	WP2uvrA	TA 98	TA1537
Solvent control (acetone)	112 120 138 (123 ± 13.3)	19 24 17 (20 ± 3.6)	51 52 40 (48 ± 6.7)	35 24 36 (32 ± 6.7)	16 15 16 (16 ± 0.6)
50	114 124 112 (117 ± 6.4)	17 19 21 (19 ± 2.0)	37 49 43 (43 ± 6.0)	32 31 32 (32 ± 0.6)	16 15 15 (15 ± 0.6)
150	111 114 115 (113 ± 2.1)	17 17 19 (18 ± 1.2)	47 43 35 (42 ± 6.1)	29 27 36 (31 ± 4.7)	15 16 17 (16 ± 1.0)
500	120 122 130 (124 ± 5.3)	17 16 23 (19 ± 3.8)	43 41 44 (43 ± 1.5)	29 27 21 (26 ± 4.2)	19 19 17 (18 ± 1.2)
1500	134 P 120 P 110 P (121 ± 12.1)	25 P 17 P 24 P (22 ± 4.4)	40 P 52 P 41 P (44 ± 6.7)	31 P 31 P 20 P (27 ± 6.4)	11 P 8 P 12 P (10 ± 2.1)
5000	123 P 114 P 111 P (116 ± 6.2)	24 P 13 P 13 P (17 ± 6.4)	39 P 33 P 33 P (35 ± 3.5)	29 P 24 P 29 P (27 ± 2.9)	16 P 9 P 8 P (11 ± 4.4)
ENNG	649 549 575 (591 ± 51.9)	640 678 1024 (781 ± 211.6)	942 1016 956 (971 ± 39.3)	-	-
4NQO	-	-	-	167 174 158 (166 ± 8.0)	-
9AA	-	-	-	-	474 289 516 (426 ± 120.8)
S9-Mix	With
Test item (µg/plate)	TA 100	TA 1535	WP2uvrA	TA 98	TA1537
Solvent control (acetone)	95 119 86 (100 ± 17.1)	11 5 13 (10 ± 4.2)	55 49 55 (53 ± 3.5)	27 27 33 (29 ± 3.5)	15 13 17 (15 ± 2.0)
50	102 69 116 (96 ± 24.1)	19 8 13 (13 ± 5.5)	55 41 49 (48 ± 7.0)	27 24 29 (27 ± 2.5)	12 19 16 (16 ± 3.5)
150	90 131 103 (108 ± 21.0)	13 17 8 (13 ± 4.5)	48 43 48 (46 ± 2.9)	27 35 37 (33 ± 5.3)	12 8 11 (10 ± 2.1)
500	123 79 91 (98 ± 22.7)	15 16 13 (15 ± 1.5)	48 52 53 (51 ± 2.6)	27 25 27 (26 ± 1.2)	15 16 17 (16 ± 1.0)
1500	90 P 112 P 99 P (100 ± 11.1)	15 P 9 P 15 P (13 ± 3.5)	44 P 47 P 48 P (46 ± 2.1)	39 P 25 P 25 P (30 ± 8.1)	15 P 12 P 19 P (15 ± 3.5)
5000	106 P 103 P 103 P (104 ± 1.7)	9 P 15 P 9 P (11 ± 3.5)	48 P 43 P 43 P (45 ± 2.9)	31 P 31 P 29 P (30 ± 1.2)	13 P 15 P 15 P (14 ± 1.2)
2AA	1407 1922 1370 (1566 ± 308.6)	238 253 243 (245 ± 7.6)	513 537 581 (544 ± 34.5)	-	249 385 295 (310 ± 69.2)
BP	-	-	-	200 262 210 (224 ± 33.3)	-

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535

9AA: 9-Aminoacridine: 80 µg/plate for TA1537

4NQO: 4-Nitroquinoline-1-oxide: 0.2 µg/plate for TA98

2AA: 2-Aminoanthracene: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA

BP: Benzo(a)pyrene: 5 µg/plate for TA98

P: Precipitate

 

Table 4. Test results Experiment 2

EXPERIMENT 2 (pre-incubation) Number of revertants (mean ± standard deviation)
S9-Mix	Without
Test item (µg/plate)	TA 100	TA 1535	WP2uvrA	TA 98	TA1537
Solvent control (acetone)	118 132 114 (121 ± 9.5)	25 23 25 (24 ± 1.2)	57 27 21 (35 ± 19.3)	35 41 33 (36 ± 4.2)	17 8 13 (13 ± 4.5)
50	112 119 120 (117 ± 4.4)	19 27 13 (20 ± 7.0)	40 35 49 (41 ± 7.1)	25 41 27 (31 ± 8.7)	13 12 7 (11 ± 3.2)
150	114 111 122 (116 ± 5.7)	21 24 17 (21 ± 3.5)	41 31 41 (38 ± 5.8)	23 32 15 (23 ± 8.5)	11 8 12 (10 ± 2.1)
500	124 127 136 (129 ± 6.2)	28 29 29 (29 ± 0.6)	41 37 43 (40 ± 3.1)	31 28 31 (30 ± 1.7)	9 13 4 (9 ± 4.5)
1500	115 P 126 P 126 P (122 ± 6.4)	23 P 25 P 20 P (23 ± 2.5)	39 P 39 P 32 P (37 ± 4.0)	31 P 29 P 33 P (31 ± 2.0)	9 P 16 P 12 P (12 ± 3.5)
5000	107 P 104 P 116 P (109 ± 6.2)	16 P 31 P 19 P (22 ± 7.9)	31 P 49 P 49 P (43 ± 10.4)	33 P 35 P 23 P (30 ± 6.4)	17 P 8 P 13 P (13 ± 4.5)
ENNG	448 453 502 (468 ± 29.8)	233 192 231 (219 ± 23.1)	541 492 501 (511 ± 26.1)	-	-
4NQO	-	-	-	158 140 106 (135 ± 26.4)	-
9AA	-	-	-	-	604 442 535 (527 ± 81.3)
S9-Mix	With
Test item (µg/plate)	TA 100	TA 1535	WP2uvrA	TA 98	TA1537
Solvent control (acetone)	130 132 128 (130 ± 2.0)	9 9 23 (14 ± 8.1)	53 59 41 (51 ± 9.2)	32 29 33 (31 ± 2.1)	12 8 12 (11 ± 2.3)
50	120 114 115 (116 ± 3.2)	13 13 21 (16 ± 4.6)	39 49 41 (43 ± 5.3)	27 41 31 (33 ± 7.2)	8 19 19 (15 ± 6.4)
150	130 134 116 (127 ± 9.5)	17 17 15 (16 ± 1.2)	44 48 56 (49 ± 6.1)	45 33 40 (39 ± 6.0)	16 20 9 (15 ± 5.6)
500	114 124 135 (124 ± 10.5)	16 9 8 (11 ± 4.4)	53 48 53 (51 ± 2.9)	25 32 39 (32 ± 7.0)	17 19 16 (17 ± 1.5)
1500	123 P 118 P 108 P (116 ± 7.6)	9 P 9 P 8 P (9 ± 0.6)	45 P 43 P 43 P (44 ± 1.2)	37 P 39 P 33 P (36 ± 3.1)	15 P 23 P 13 P (17 ± 5.3)
5000	122 P 126 P 115 P (121 ± 5.6)	12 P 11 P 8 P (10 ± 2.1)	51 P 49 P 49 P (50 ± 1.2)	35 P 32 P 31 P (33 ± 2.1)	11 P 15 P 19 P (15 ± 4.0)
2AA	1270 1262 1407 (1313 ± 81.5)	184 212 234 (210 ± 25.1)	275 255 350 (293 ± 50.1)	-	279 277 289 (282 ± 6.4)
BP	-	-	-	176 204 203 (194 ± 15.9)	-

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535

9AA: 9-Aminoacridine: 80 µg/plate for TA1537

4NQO: 4-Nitroquinoline-1-oxide: 0.2 µg/plate for TA98

2AA: 2-Aminoanthracene: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA

BP: Benzo(a)pyrene: 5 µg/plate for TA98

P: Precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative