Fatty acids C18-C22 (even numbered), tetraesters with pentaerythritol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 100, TA 98, E. coli WP2 uvrA

Chromosome aberration: negative in Chinese hamster ovary cells with and without metabolic activation.

Read-across from structural source substance fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS 189200-42-8)

Gene mutation in mammalian cells: negative in mouse lymphoma L5178Y cells with and without metabolic activation

Read-across from structural source substance 2,2-bis[[(1-oxoisopentyl)oxy]methyl]propane-1,3-diyl divalerate (PE C5 tetraester) (CAS 15834-04-5)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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 189200-42-8, 1995, RL2

Conclusions:

Based on the results of the in vitro chromosome aberration test with the source substance fatty acids, C8-10 mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS 189200-42-8) no increase of chromosome aberrations were observed in CHO cells with and without metabolic activation. Applying the read-across approach, similar results are expected for the target substance fatty acids C18-C22 (even numbered), tetraesters with pentaerythritol.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

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

Remarks:

and above (precipitating concentration: 100 µg/mL, tested up to 250 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Source: CAS 15834-04-5, 2010, RL1

Conclusions:

Based on the results of the mouse lymphomas assay, the source substance 2,2-bis[[(1-oxoisopentyl)oxy]methyl]propane-1,3-diyl divalerate (PE C5 tetraester) (CAS 15834-04-5) did not reveal mutagenic properties in mouse lymphoma cells with and without metabolic activation. Applying the read-across approach, similar results are expected for the target substance fatty acids C18-C22 (even numbered), tetraesters with pentaerythritol.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Nov - 17 Dec 1999

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

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon, trp operon

Species / strain / cell type:

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

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 and β-naphthoflavone

Test concentrations with justification for top dose:

First experiment (served as range finding study):
5, 15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation (TA 1535, TA 1537, TA 98 and TA 100)
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation (WP2uvrA)
50, 150, 500, 1500 and 5000 µg/plate with metabolic activation (all strains)

Second experiment:
50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation (all strains)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

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: +S9: 1 ,2 or 10 µg/plate for TA 1535, TA 1537, TA 100 and WP2uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: growth of bacterial background lawn

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression(S)) significant increase in the revertant count in at least one strain of bacteria.

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:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was observed at 500 µg/plate and higher for all tester strains with and without metabolic activation

RANGE-FINDING/SCREENING STUDIES: In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The dose range of the test material was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 pg/plate. The test material exhibited toxicity at and above 500 µg/plate to tester strain TA 100 (without S9-mix only). This response however, was not observed in either the range-finding or main studies and was therefore considered artefactual and of no toxicological significance.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control values were within the range of the historical control data (please refer to Table 1 under "any other information on material and methods incl. tables") except of all three values for TA 98 and a single value for TA100 and TA 1535, respectively, in Experiment I without metabolic activation, a single value for TA 98 in Experiment I with metabolic activation, and all three values for TA 98 in Experiment II with metabolic activation. These deviations were not considered to invalid the test since a distinct increase of the number of revertants was seen when compared with the control values.
- Negative historical control data: The negative control values were within the range of the historical control data (please refer to Table 1 under "any other information on material and methods incl. tables") except of a single value for TA 1535 in Experiment II with metabolic activation, which was not considered to influence the validity of the test.

Table 2: Summar of Results of Experiment I

Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)					Number of revertants (mean number of colonies per plate)
	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Without metabolic activation						With metabolic activation
0	117	24	24	27	13	90	20	15	24	14
	73	15	18	18	8	82	14	11	24	20
	95	12	14	19	6	72	18	15	22	14
5	76	28	nt	28	9	nt
	73	11		15	11
	82	22		24	8
15	86	18	nt	20	12
	63	25		21	11
	79	9		26	10
50	89	16	14	19	10	72	15	15	20	24
	101	21	9	17	9	80	22	20	27	7
	85	15	13	18	18	90	20	23	18	22
150	80	15	10	17	11	82	18	17	23	20
	95	22	11	22	8	101	24	13	21	18
	91	22	13	17	15	102	25	17	16	15
500 P	99	16	14	28	16	95	18	16	21	22
	90	22	10	15	13	81	17	20	23	12
	94	13	17	18	12	88	25	21	24	10
1500 P	104	19	9	30	13	77	35	10	30	33
	115	25	15	21	16	66	13	12	35	12
	96	21	14	26	14	91	21	21	16	15
5000 P	106	17	19	36	7	114	14	16	33	20
	105	15	23	23	14	93	21	13	33	23
	115	14	28	30	11	89	12	13	26	12
Positive controls	ENNG	ENNG	ENNG	4NQO	9AA	2AA	2AA	2AA	BP	2AA
Concentration (µg/plate)	3	5	2	0.2	80	1	2	10	5	2
No. Colonies	307	158	411	98	584	507	401	788	242	333
	297	190	362	101	586	518	292	680	263	373
	274	182	618	93	769	824	308	716	167	284

ENNG: N-ethyl-N`-nitro-N-nitrosoguanidine

9AA: 9-Aminoacridine

4NQO: 4-Nitroquinoline-1-oxide

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

P: Precipitation

Table 3: Summary of results of Experiment II

Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)					Number of revertants (mean number of colonies per plate)
	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Without metabolic activation						With metabolic activation
0	136	14	23	28	11	108	14	24	28	12
	130	22	27	23	11	96	9	31	33	8
	133	18	27	33	8	109	12	30	39	7
50	142	14	27	26	5	112	18	19	21	13
	140	16	19	23	9	99	16	23	26	9
	130	18	18	20	10	123	12	26	27	23
150	131	13	22	25	6	79	18	26	14	9
	156	21	23	23	14	91	5	24	21	4
	118	20	28	23	6	81	6	24	15	13
500 P	157	20	22	17	9	93	14	16	13	13
	100	17	19	17	15	92	7	42	17	11
	103	19	16	25	7	89	20	28	10	10
1500 P	106	13	23	29	8	100	20	24	17	11
	91	15	27	16	11	78	21	29	15	16
	121	21	22	22	22	114	11	26	15	6
5000 P	150	17	35	25	15	104	17	40	27	11
	116	15	37	20	6	119	20	39	28	11
	107	14	37	20	6	90	9	35	18	15
Positive controls	ENNG	ENNG	ENNG	4NQO	9AA	2AA	2AA	2AA	BP	2AA
Concentration (µg/plate)	3	5	2	0.2	80	1	2	10	5	2
No. Colonies	734	215	878	140	980	832	188	260	156	321
	589	276	727	159	983	917	186	318	161	310
	676	248	891	163	926	909	188	336	158	269

ENNG: N-ethyl-N`-nitro-N-nitrosoguanidine

9AA: 9-Aminoacridine

4NQO: 4-Nitroquinoline-1-oxide

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

P: Precipitation

Conclusions:

Based on the results of the present study with the Fatty Acids C18-C22 (even numbered), tetraesters with pentaerythitol, no mutagenic properties in bacterial cells were observed with and without metabolic activation.

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

Data on mutagenicity in bacterial cells with fatty acids C18-C22 (even numbered), tetraesters with pentaerythritol are available, whereas in vitro studies in mammalian cells of fatty acids C18-C22 (even numbered), tetraesters with pentaerythritol are missing.

The assessment was therefore based on studies conducted with analogue 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

The mutagenic potential of fatty acids C18-C22 (even numbered), tetraesters with pentaerythritol was tested in a reverse mutation assay according to OECD 471 under GLP conditions (Safepharm Laboratories, 2000). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA were used. Tester strains were incubated with test material at concentrations of 0, 50, 150, 500, 1500, and 5000 µg/plate with and without the addition of a metabolic activation system (phenobarbitone and β-naphthoflavone induced rat liver S9 mix). Precipitation occurred at doses ≥ 500 µg/plate. Vehicle and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. Thus, Fatty acids C18-C22 (even numbered), tetraesters with Pentaerythritol (CAS 61682-73-3) did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested, and thus, was not mutagenic in bacteria.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 189200-42-8

A study investigating the in vitro mammalian chromosome aberration was performed with fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS 189200-42-8) in Chinese hamster ovary cells (CHO cells) comparable to OECD 473 and under GLP conditions (ExxonMobil, 1995). Duplicate cultures of CHO cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In the first experiment, cells were exposed to the test substance for 3 h and for 16 h followed by 16 h expression time with and without metabolic activation, respectively. The test substance was dissolved in acetone and used at concentrations of 40, 80 and 160 µg/mL. In the second experiment cells were again exposed for 3 h and for 16 h followed by 16 h expression time with and without metabolic activation, respectively. Additionally, cells were exposed for 3 and 16 h followed by 40 h expression time with and without metabolic activation, respectively. The same substance concentrations as in first experiment were used. The test substance did not induce cytotoxicity but a precipitate was visible in the second experiment at 160 µg/mL after 16 h incubation without metabolic activation. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. N-Methyl-N-Nitro-N-Nitrosoguanidine and 7,12-Dimethylbenz[a]anthracene were used as positive control materials inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level tested in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to CHO cells in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 15834-04-5

An in vitro mammalian cell gene mutation assay according to OECD 476 and under GLP conditions was performed with 2,2-bis[[(1-oxoisopentyl)oxy]methyl]propane-1,3-diyl divalerate (PE C5 tetraester) (CAS 15834-04-5) in mouse lymphoma L5178Y cells (Notox, 2010). In the first experiment, the cells were treated for 3 h with 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL in the presence or absence of S9-mix (8% (v/v)). In the second experiment, concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL were applied with metabolic activation (12%, v/v) for 3 h and 0.1, 1, 3, 10, 33, 100, 200, 250 µg/mL without metabolic activation for 24 h. The test substance was tested up to precipitating concentration (100 µg/mL and above). Cyclophosphamide and methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. Positive and negative controls were valid and in range of historical control data. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. It was concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

Overall conclusion for genetic toxicity

There are no available studies on the genetic toxicity of the target substance fatty acids C18-C22 (even numbered), tetraesters with pentaerythritol. Therefore analogue read-across from source substances was applied for in vitro studies on cytogenicity and in vitro studies on gene mutation in mammalian cells. The results of the available studies were consistently negative. Based on the available data and following the analogue approach, no hazard regarding genotoxicity is identified for the target substance fatty acids C18-C22 (even numbered), tetraesters with pentaerythritol.

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 fatty acids C18-C22 (even numbered), tetraesters with pentaerythritol, 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 analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) No 1272/2008 and are therefore conclusive but not sufficient for classification.