Isooctadecyl pivalate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvrA pKM 101
Chromosome aberration (OECD 473): negative in primary human lymphocytes with and without metabolic activation (read-across)
Gene mutation in mammalian cells (OECD 476): negative in L5178Y mouse lymphoma cells with and without metabolic activation (read-across)

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

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

Target gene:

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

Species / strain / cell type:

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

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

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.

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

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.

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)

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

weight of evidence

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: cultured human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 150 µg/mL (exposure period 24 h, fixation time 24 h, -S9 mix) and at 125 µg/mL (exposure period 48 h, fixation time 48 h, -S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Source: CAS 10233-13-3

A second chromosome aberration test with the source substance CAS 163961-32-8 was taken into account for the evaluation of the clastogenic potential. In this study, the numbers of chromosome aberrations and polyploid cells were not increased at any dose level in comparison to the control. This source substance is considered to be non-clastogenic in mammalian cells in vitro.

Conclusions:

There are two chromosome aberration tests with the appropriate source substances CAS 163961-32-8 and CAS 10233-13-3 available, respectively. Based on the results, the numbers of chromosome aberrations and polyploid cells were not increased at any dose level in comparison to the control. Both source substances were non-clastogenic in mammalian cells in cultured human peripheral lymphocytes. Applying the read-across aprroach, similar results are expected for the target substance CAS 58958-60-4.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

weight of evidence

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Positive controls validity:

valid

Remarks on result:

other: Source: CAS 10233-13-3

A second mouse lymphoma assay with the source substance CAS 163961-32-8 was taken into account for the assessment of the mutagenic potential. Based on the results of this study, no toxicologically significant increases in the mutant frequency at the TK +/- locus in L5l78Y cells were observed.

Conclusions:

No significant increases in the mutation frequency at the TK locus were observed after treatment with both source substances CAS 10233-13-3 and CAS 163961-32-8 either in the absence or in the presence of S9-mix. It is concluded that both source substance are not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions. Applying the read-across approach, the same results are expected for the target substance CAS 58958-60-4.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for read-across

There are limited data available on the genetic toxicity of Isooctadecyl pivalate (CAS 58958-60-4). The assessment was therefore partly based on studies conducted with analogue (source) substances as part of a read-across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 58958-60-4

The potential mutagenicity of Isooctadecyl pivalate was assessed in a bacterial reverse mutation assay (Ames test) performed according to OECD guideline 471 and under GLP conditions (Vivotecnia, 2015). In two independent experiments, the bacterial strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli WP2 uvrA pKM 101 were exposed to the test substance at concentrations of 0.06, 0.19, 0.56, 1.67 and 5.0 μL/plate. The plate incorporation method was applied in the first experiment with and without metabolic activation, while the pre-incubation method was applied in the second experiment with and without metabolic activation. Cytotoxicity (50%) was observed at 1.67 and 5.0 μL/plate in the E. coli strain without metabolic activation. No increase in the mean number of revertants was observed in any tester strain at any concentration tested. The negative and positive controls were shown to be valid. Based on the results of this experiment, the test substance was considered to be non-mutagenic in the presence and absence of metabolic activation.

Genetic toxicity (clastogenicity) in mammalian cells in vitro

CAS 10233-13-3

An in vitro mammalian chromosome aberration test was performed with Isopropyl laurate (CAS 10233-13-3) in primary human lymphocytes, according to OECD guideline 473 and under GLP conditions (Notox, 2010). In the first experiment cells were exposed for 3 hours to test substance concentrations of 10, 33 and 100 µg/mL in ethanol, with 24 hours expression time (with and without metabolic activation) and 24 hours expression time (with metabolic activation). In the second experiment cells were exposed for 24 hours to 66, 150 and 250 µg/mL followed by 24 hours expression time, and exposed for 48 hours to 3, 125 and 150 µg/mL followed by 48 hours expression time. The second experiment was performed without metabolic activation. 250 µg/mL was chosen as maximum dose as precipitation was observed at ≥ 100 µg/mL. The positive and negative controls were valid. 100 well-spread metaphase cells from each culture were evaluated. Some cytotoxicity was noted at the highest dose level without metabolic activation at the expression times of 24 and 48 hours (the mitotic index was 41% and 46%, respectively). No increase in the frequency of chromosome aberrations and polyploid cells was observed at any dose level. The test material was therefore non-clastogenic to human lymphocytes in vitro.

CAS 163961-32-8

An in vitro mammalian chromosome aberration test was performed with Fatty acids, C16-18 and C18-unsaturated, branched and linear, butyl esters in primary human lymphocytes according to OECD guideline 473 under GLP conditions (Safepharm, 2004). For each experiment, whole blood was drawn from the peripheral circulation of a volunteer who had previously been screened for suitability. The cell cycle length of 17 h was determined by BrdU incorporation. In the first experiment test substance concentrations of 312.5, 468.75 and 625 µg/mL were exposed for 24 hours without metabolic activation, with 24 hours expression time. In addition, cells were exposed to 625, 1250 and 2500 µg/mL for 4 hours with metabolic activation, followed by 20 hours expression time. In the second experiment cells were exposed to 625, 1250 and 2500 µg/mL for 4 hours with and without S9 followed by 20 hours expression time. 2500 µg/mL was chosen as maximum dose due to limited solubility. 100 well-spread metaphase cells from each culture were evaluated for structural chromosomal aberrations. Limited cytotoxicity (38%) was noted at 468.75 µg/mL in the first experiment. The positive and negative controls were valid. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolizing system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 10233-13-3

An in vitro mammalian cell gene mutation assay was performed according to OECD guideline 476 under GLP conditions with Isopropyl laurate (CAS 10233-13-3) in ethanol (Notox, 2010). The test substance was applied to mouse lymphoma L5178Y cells at concentrations up to and including 10 μg/mL, which was the precipitation level. The cells were treated for 3 and 24 hours without metabolic activation, for 3 hours with 8% (v/v) S9-mix, and for 3 hour with 12% (v/v) S9-mix, respectively. No cytotoxicity was observed. The positive and negative controls were valid. No significant increase in mutation frequency occurred. Therefore, Isopropyl laurate was not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions in this study.

 CAS 163961-32-8

An in vitro mammalian cell gene mutation assay was performed according to OECD guideline 476 under GLP conditions with Fatty acids, C16-18 and C18-unsaturated, branched and linear, butyl esters in mouse lymphoma L5178Y cells (Safepharm, 2007).The cells were exposed for 4 hours with and without activation in experiment l. In experiment 2, the exposure time with metabolic activation remained 4 hours, while the exposure time without activation was increased to 24 hours. A dose range of 156.25 - 5000 μg/mL was used in the first and second experiment. A confirmatory third experiment was performed because a statistically significant response was observed in the lower/ mid-dose range in the presence of metabolic activation in experiment 2 only. In experiment 3, cells were exposed to concentrations of 39.06 - 1250μg/mL. The effect observed in experiment 2 could not be reproduced and is therefore considered not to be valid. Precipitation was observed at and above 78.13 μg/mL. The positive and negative controls were valid. The test material did not induce any toxicologically significant increases in the mutant frequency at any dose level in any of the exposure groups, which included dose levels up to and including the maximum recommended dose level of 5000 μg/mL. The test substance was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Overall conclusion for genetic toxicity

There is one study available on the in vitro mutagenicity in bacterial cells of the target substance Isooctadecyl pivalate. Therefore, analogue read-across from source substances was applied from in vitro studies on genetic toxicity in mammalian cells using 2 source substances. The results of the available in vitro studies on target and source substances were consistently negative. Based on the available data and following the analogue approach, Isooctadecyl pivalate is considered to be non-mutagenic in vitro.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Isooctadecyl pivalate (CAS 58958-60-4), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the available data on the target substance and on analogue read-across approach, the results on genetic toxicity for the target and source substances do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.