Isooctadecyl pivalate

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:

27 Apr - 18 Jun 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Regulation and Inspection of Community of Madrid, Spain

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

his operon (S. typhimurium strains)
trp operon (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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation method) and 2 (preincubation method): 0.06, 0.19, 0.56, 1.67 and 5.0 μL/plate, with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was soluble in DMSO at a 1/3 dilution

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Remarks:

-S9: 2-nitrofluorene (5 µg/plate) for TA98; sodium azide (2.5 or 3.5 µg/plate) for TA100 and TA1535; 9-aminoacridine (45 µg/plate) for TA1537; 4-nitroquinoline-N-oxide (0.4 µg/plate) for E coli; +S9: 2-aminoanthracene (1.5, 2.5 or 30 µg/plate) all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment 1: in agar (plate incorporation); experiment 2: preincubation

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

NUMBER OF REPLICATIONS: three replications each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies, or a clearing or diminuition of the background lawn

OTHER EXAMINATIONS:
- Other: the test item sterility was tested by adding 5 μL/plate to a minimal agar plate and incubating at 37 ºC for 48 h.

OTHER: 2-aminoanthracene was used as the only positive control with metabolic activation. However, the ability of the S9-mix to activate B[a]P and 2-aminoanthracene was confirmed in an assay prior to the main study and 2-aminoanthracene is considered to be an appropriate positive control.

Evaluation criteria:

The criteria used for determining a positive result take into account a dose-response effect in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate, in at least one strain, with or without metabolic activation.
A result is considered positive when the number of revertants of the test item-treated plates is increased 2-fold (S. typhimurium TA98, TA100 and E .coli EP2(pKM101) or 3-fold (S. typhimurium TA1535, TA1537) when compared to the solvent-treated plates.

Statistics:

The average plate count was presented with the mean and standard deviation for each set of triplicates per test item concentration.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic from 1.67 μL/plate without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed in the solubility test in 1/3 dilution in DMSO

RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity assay, concentrations of 0.06 - 5 μL/plate were tested with S. typhimurium TA100. No cytotoxicity was observed at any concentration level. Therefore, 5.0 μL/plate was selected as the highest concentration in the main study.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, results were within the historical control data range

ADDITIONAL INFORMATION ON CYTOTOXICITY: in the main study, clear cytotoxicity (50%) was observed at 1.67 and 5.0 μL/plate in the E. coli strain. This is not considered to have affected the results of the study.

Any other information on results incl. tables

Table 1: Experiment 1

EXPERIMENT 1 (direct incorporation test)
S9-Mix	Without
Test item (µL/plate)	TA 98	TA 100	TA 1535	TA 1537	E. coli WP2(pKM101)
NC	16 ± 1.5	82 ± 10.7	14 ± 3.0	7 ± 2.0	217 ± 28.9
0.06	16 ± 3.6	95 ± 19.5	16 ± 4.0	7 ± 3.2	209 ± 47.5
0.19	18 ± 2.0	102 ± 15.3	14 ± 2.9	7 ± 1.5	218 ± 29.2
0.56	18 ± 4.0	104 ± 15.6	13 ± 4.4	4 ± 3.0	237 ± 34.5
1.67	16 ± 3.8	95 ± 15.7	13 ± 1.0	6 ± 1.5	248 ± 8.7
5.0	18 ± 3.1	107 ± 21.9	17 ± 5.1	5 ± 1.5	179 ± 11.1
2-NF	462 ± 57.2	-	-	-	-
SA	-	675 ± 34.7	762 ± 58.5	-	-
4NQO	-	-	-	-	1999 ± 65.5
9-AA	-	-	-	172 ± 34.9	-
S9-Mix	With
Test item (µL/plate)	TA 98	TA 100	TA 1535	TA 1537	E. coli WP2(pKM101)
NC	19 ± 5.0	92 ± 4.0	16 ± 5.3	6 ± 2.5	215 ± 13.9
0.06	23 ± 8.0	101 ± 12.9	12 ± 5.8	5 ± 0.6	190 ± 41.3
0.19	19 ± 8.5	100 ± 15.0	16 ± 6.7	5 ± 4.2	212 ± 5.3
0.56	23 ± 3.2	104 ± 11.5	17 ± 6.1	6 ± 1.5	204 ± 24.3
1.67	20 ± 2.5	90 ± 20.1	15 ± 4.4	8 ± 1.2	213 ± 34.6
5.0	26 ± 3.6	91 ± 2.1	15 ± 6.1	7 ± 2.1	218 ± 24.4
2AA	614 ± 101.5	1434 ± 161.2	380 ± 81.1	185 ± 15.4	1901 ± 138.8
NC = Vehicle Control, DMSO 2-NF: 2-nitrofluorene SA: sodium azide 4NQO: 4-nitroquinoline-N-oxide 9AA:9-aminoacridine 2AA: 2-aminoanthracene  (for details see method description)

 

Table 2: Experiment 2

EXPERIMENT 2 (preincubation test)
S9-Mix	Without
Test item (µL/plate)	TA 98	TA 100	TA 1535	TA 1537	E. coli WP2(pKM101)
NC	19 ± 4.7	74 ± 10.1	16 ± 3.5	5 ± 1.2	234 ± 22.2
0.06	17 ± 2.5	88 ± 5.6	17 ± 5.0	8 ± 2.1	218 ± 3.5
0.19	17 ± 2.3	74 ± 10.4	16 ± 2.1	5 ± 2.9	227 ± 33.7
0.56	20 ± 4.6	102 ± 4.0	12 ± 3.2	5 ± 2.1	93 ± 32.0
1.67	17 ± 1.2	87 ± 5.3	13 ± 4.4	8 ± 1.2	17 ± 10.0
5.0	17 ± 3.6	77 ± 11.4	15 ± 4.4	6 ± 2.5	0 ± 0.0
2-NF	372 ± 45.0	-	-	-	-
SA	-	727 ± 12.0	837 ± 26.9	-	-
4NQO	-	-	-	-	2186 ± 244.5
9-AA	-	-	-	168 ± 15.0	-
S9-Mix	With
Test item (µL/plate)	TA 98	TA 100	TA 1535	TA 1537	E. coli WP2(pKM101)
NC	23 ± 2.6	91 ± 17.0	13 ± 1.2	5 ± 2.5	224 ± 37.0
0.06	23 ± 0.6	90 ± 5.0	15 ± 1.5	5 ± 0.6	187 ± 33.1
0.19	24 ± 0.6	84 ± 8.6	13 ± 5.2	6 ± 1.2	211 ± 49.5
0.56	20 ± 3.1	83 ± 16.0	15 ± 3.5	7 ± 2.5	219 ± 20.0
1.67	25 ± 3.8	81 ± 4.0	16 ± 2.1	9 ± 0.6	223 ± 20.3
5.0	22 ± 7.2	85 ± 6.4	12 ± 1.2	6 ± 2.5	230 ± 41.4
2AA	490 ± 41.2	1311 ± 67.5	327 ± 51.7	169 ± 25.7	1849 ± 58.1
NC = Vehicle Control, DMSO 2-NF: 2-nitrofluorene SA: sodium azide 4NQO: 4-nitroquinoline-N-oxide 9AA:9-aminoacridine 2AA: 2-aminoanthracene  (for details see method description)