Isooctadecanoic acid, mixed esters with oxybis[propanediol]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro gene mutation study in bacteria (OECD TG 471; S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A): negative

Tested with the registered substance

Chromosome aberration (OECD TG 473; V79 cells and human peripheral blood lymphocytes; with and without metabolic activation): negative

Read-across from analogue source substances 1,2,3-Propanetriol, homopolymer, diisooctadecanoate (CAS No. 63705-03-3), Hexanedioic acid, mixed esters with decanoic acid, 12-hydroxyoctadecanoic acid, isostearic acid, octanoic acid, 3,3'-oxybis[1,2-propanediol] and stearic acid (CAS No. 130905-60-1) and Oleic acid, monoester with oxybis(propanediol) (CAS No. 49553-76-6) in a Weight-of-Evidence approach

Gene mutation in mammalian cells (OECD TG 476; mouse lymphoma L5178Y and CHO cells; with and without metabolic activation): negative

Read-across from analogue source substances Oleic acid, monoester with oxybis(propanediol) (CAS No. 49553-76-6) and 1,2,3-Propanetriol, homopolymer, diisooctadecanoate (CAS No. 63705-03-3) in a Weight-of-Evidence approach

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:

14 July - 05 August 2017

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

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

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, induced with phenobarbital and beta-naphtha flavone

Test concentrations with justification for top dose:

Experiment 1 - Plate Incorporation Method (all tester strains): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
The maximum concentration was chosen as recommended in the guideline followed.

Experiment 2 – Pre-Incubation Method (all tester strains): 15, 50, 150, 500, 1500, 5000 µg/plate
Six test item dose levels per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Lot/Batch: 1679355 (experiment 1); 1719831 (experiment 2)
- Purity: 99.98% (experiment 1); > 99% (experiment 2)
- Expiry date: 11/2021 (experiment 1); 07/2022 (experiment 2)

- Justification for choice of solvent/vehicle: The test item was immiscible in dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed. Acetone was therefore selected as the vehicle. Distilled water was not evaluated as a vehicle in this test system as information provided by the sponsor suggested it was unstable.

Untreated negative controls:

yes

Remarks:

untreated plates

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 for experiment 1) and preincubation (experiment 2)

DURATION
- Preincubation period: 20 min (at 37 ± 3 °C)
- Exposure duration: approx. 48 h (at 37 ± 3 °C)

NUMBER OF REPLICATIONS: triplicate in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn and number of revertant colonies

Evaluation criteria:

Acceptance criteria:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls)
- All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per mL
- Positive controls must be included to demonstrate both the intrinsic sensitivity of the tester strains and the integrity of the S9-mix
- All of the positive controls should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation
- There should be a minimum of four non-toxic test item dose levels
- There should be no evidence of excessive contamination

Evaluation criteria:
Any, one, or all of the following are used to determine the overall result of the study.
- A dose-related increase in mutant frequency over the dose range tested
- A reproducible increase at one or more concentrations
- Fold increase greater than two times the concurrent solvent control for any tester strain

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Individual plates were counted for revertant colonies. The average and standard deviation of the number of revertant colonies were calculated. Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. No further statistical analysis was not performed.

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

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

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

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

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
- Precipitation: Precipitation was observed at 1500 and 5000 µg/plate

HISTORICAL CONTROL DATA: data are attached as separated pdf document (History Profile of Vehicle and Positive Control Values.pdf)

Table 1: Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1 (Plate Incorporation)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
67		14		21		18		13
90	(82)	20	(21)	20	(20)	24	(21)	15	(12)
90		28		20		22		9

   Experiment 2 (Pre-Incubation) 

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
80		30		18		25		9
73	(78)	19	(23)	23	(26)	25	(24)	12	(11)
82		21		27		21		13

Table 2: Test Results Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 24 July 2017					To: 27 July 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	78 83 89	(83) 5.5	28 27 31	(29) 2.1	20 22 26	(23) 3.1	23 14 21	(19) 4.7	18 9 12	(13) 4.6
	1.5 µg	79 88 97	(88) 9.0	23 26 22	(24) 2.1	21 13 21	(18) 4.6	19 25 23	(22) 3.1	9 14 13	(12) 2.6
	5 µg	82 74 77	(78) 4.0	29 22 31	(27) 4.7	19 19 29	(22) 5.8	11 11 12	(11) 0.6	10 8 8	(9) 1.2
	15 µg	101 86 62	(83) 19.7	21 24 24	(23) 1.7	25 15 30	(23) 7.6	25 24 13	(21) 6.7	16 16 22	(18) 3.5
	50 µg	75 90 81	(82) 7.5	18 23 32	(24) 7.1	12 23 20	(18) 5.7	22 23 27	(24) 2.6	7 20 19	(15) 7.2
	150 µg	69 90 87	(82) 11.4	32 14 21	(22) 9.1	25 29 27	(27) 2.0	16 16 20	(17) 2.3	20 25 11	(19) 7.1
	500 µg	86 93 88	(89) 3.6	25 22 28	(25) 3.0	12 21 23	(19) 5.9	19 20 16	(18) 2.1	14 13 14	(14) 0.6
	1500 µg	84 78 68	(77) 8.1	21 24 25	(23) 2.1	26 22 28	(25) 3.1	11 16 19	(15) 4.0	15 18 12	(15) 3.0
	5000 µg	70 P 97 P 63 P	(77) 18.0	18 P 22 P 23 P	(21) 2.6	23 P 29 P 25 P	(26) 3.1	27 P 13 P 18 P	(19) 7.1	17 P 21 P 15 P	(18) 3.1
Positive controls S9-Mix (-)	Name DoseLevel No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		598 522 475	(532) 62.1	318 290 352	(320) 31.0	804 596 630	(677) 111.6	200 254 261	(238) 33.4	127 157 305	(196) 95.3

Table 3: Test Results Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 24 July 2017					To: 27 July 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	80 79 102	(87) 13.0#	24 24 21	(23) 1.7	25 38 27	(30) 7.0	16 15 22	(18) 3.8	14 19 13	(15) 3.2
	1.5 µg	79 102 67	(83) 17.8	26 20 24	(23) 3.1	31 26 26	(28) 2.9	18 23 22	(21) 2.6	8 17 13	(13) 4.5
	5 µg	98 83 100	(94) 9.3	20 27 24	(24) 3.5	18 30 26	(25) 6.1	22 19 21	(21) 1.5	10 11 14	(12) 2.1
	15 µg	85 100 87	(91) 8.1	18 24 26	(23) 4.2	23 34 25	(27) 5.9	25 26 34	(28) 4.9	13 10 9	(11) 2.1
	50 µg	78 114 80	(91) 20.2	24 20 28	(24) 4.0	20 26 17	(21) 4.6	17 21 19	(19) 2.0	17 16 12	(15) 2.6
	150 µg	84 86 68	(79) 9.9	25 29 15	(23) 7.2	30 36 30	(32) 3.5	16 30 16	(21) 8.1	16 22 13	(17) 4.6
	500 µg	102 94 103	(100) 4.9	20 22 20	(21) 1.2	30 28 30	(29) 1.2	24 25 17	(22) 4.4	13 19 21	(18) 4.2
	1500 µg	65 89 106	(87) 20.6	26 20 29	(25) 4.6	22 23 17	(21) 3.2	14 26 30	(23) 8.3	10 21 12	(14) 5.9
	5000 µg	106 P 104 P 92 P	(101) 7.6	22 P 22 P 34 P	(26) 6.9	28 P 36 P 30 P	(31) 4.2	12 P 21 P 18 P	(17) 4.6	16 P 16 P 12 P	(15) 2.3
Positive controls S9-Mix (+)	Name DoseLevel No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		614 554 574	(581) 30.6	317 336 356	(336) 19.5	143 145 143	(144) 1.2	182 203 226	(204) 22.0	409 440 514	(454) 53.9

Table 4: Test Results Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 02 August 2017					To: 05 August 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	72 86 81	(80) 7.1#	15 18 23	(19) 4.0	31 24 32	(29) 4.4	27 23 25	(25) 2.0	16 9 11	(12) 3.6
	15 µg	84 80 75	(80) 4.5	17 27 18	(21) 5.5	31 27 29	(29) 2.0	18 23 21	(21) 2.5	9 11 15	(12) 3.1
	50 µg	81 74 70	(75) 5.6	12 17 21	(17) 4.5	37 20 26	(28) 8.6	29 20 19	(23) 5.5	12 8 11	(10) 2.1
	150 µg	73 81 90	(81) 8.5	23 23 15	(20) 4.6	30 30 28	(29) 1.2	21 24 23	(23) 1.5	19 11 8	(13) 5.7
	500 µg	86 74 81	(80) 6.0	17 14 17	(16) 1.7	26 34 31	(30) 4.0	26 28 19	(24) 4.7	18 11 9	(13) 4.7
	1500 µg	73 70 85	(76) 7.9	14 14 18	(15) 2.3	28 26 31	(28) 2.5	19 21 27	(22) 4.2	14 11 9	(11) 2.5
	5000 µg	83 P 86 P 87 P	(85) 2.1	23 P 16 P 19 P	(19) 3.5	29 P 29 P 29 P	(29) 0.0	24 P 19 P 24 P	(22) 2.9	7 P 15P 8 P	(10) 4.4
Positive controls S9-Mix (-)	Name DoseLevel No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		1290 1191 1001	(1161) 146.9	1617 1787 1834	(1746) 114.2	1241 1249 1147	(1212) 56.7	342 298 379	(340) 40.6	386 327 427	(380) 50.3

Table 5: Test Results Experiment 2 – With Metabolic Activation (Pre-Incubation) 

Test Period		From: 02 August 2017					To: 05 August 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	76 82 88	(82) 6.0#	28 26 20	(25) 4.2	30 35 38	(34) 4.0	23 25 34	(27) 5.9	15 17 18	(17) 1.5
	15 µg	74 77 83	(78) 4.6	22 27 21	(23) 3.2	35 35 30	(33) 2.9	24 33 28	(28) 4.5	16 13 11	(13) 2.5
	50 µg	72 64 78	(71) 7.0	20 21 20	(20) 0.6	27 34 32	(31) 3.6	30 32 32	(31) 1.2	18 11 17	(15) 3.8
	150 µg	87 81 87	(85) 3.5	27 14 27	(23) 7.5	28 22 36	(29) 7.0	24 31 27	(27) 3.5	16 14 16	(15) 1.2
	500 µg	85 83 73	(80) 6.4	17 27 24	(23) 5.1	39 32 25	(32) 7.0	34 31 32	(32) 1.5	19 21 17	(19) 2.0
	1500 µg	80 70 78	(76) 5.3	25 20 21	(22) 2.6	26 25 29	(27) 2.1	26 23 28	(26) 2.5	17 14 14	(15) 1.7
	5000 µg	69 P 78 P 86 P	(78) 8.5	13 P 19 P 20 P	(17) 3.8	31 P 35 P 29 P	(32) 3.1	32 P 28 P 35 P	(32) 3.5	11 P 19 P 18 P	(16) 4.4
Positive controls S9-Mix (+)	Name DoseLevel No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		1095 979 1053	(1042) 58.7	291 266 256	(271) 18.0	120 138 147	(135) 13.7	196 180 174	(183) 11.4	455 444 437	(445) 9.1

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

P: Test itemprecipita

Conclusions:

Interpretation of results: negative

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

Justification for type of information:

Please refer to the Analogue Approach Justification provided in Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the 2nd Experiment at 800 μg/mL and in the 3rd Experiment from 200 μg/mL onward at 18 hours sampling time and from 400 μg/mL onward at 28 hours sampling time

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Source, CAS 63705-03-3, BASF, 2013b

Additional studies and results considered in the Weight-of-Evidence approach:

CAS 130905-60-1, Sasol, 1996a: not clastogenic

CAS 49553-76-6, DuPont, 2012a: not clastogenic

Conclusions:

Interpretation of results: negative

Executive summary:

The potential of the target substance to induce chromosome aberrations in mammalian cells is estimated based on adequate and reliable in vitro studies performed with structural analogue source substances. Experiments have been performed both in the presence as well as in the absence of metabolic activation in chinese hamster lung fibroblasts and human peripheral blood lymphocytes. All results obtained are negative, i.e. no chromosome aberrations in the cells investigated were observed. Therefore, no hazard with regard to clastogenicity is identified for the target substance. As explained in the analogue justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in the potential to form chromosome aberrations.

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

Justification for type of information:

Please refer to the Analogue Approach Justification provided in Section 13.

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:

cytotoxicity

Remarks:

-S9 mix: ≥ 30 µg/mL; +S9: ≥ 50 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Source, CAS 49553-76-6, DuPont, 2012b

Additional study considered in the Weight-of-Evidence approach:

CAS 63705-03-3, BASF, 2013c: not mutagenic (HPRT locus in Chinese hamster Ovary (CHO) cells)

Conclusions:

Interpretation of results: negative

Executive summary:

The potential of gene mutation in mammalian cells of the target substance is estimated based on adequate and reliable in vitro studies of a structural analogue source substances. Experiments have been performed both in the presence as well as in the absence of metabolic activation in chinese hamster ovary cells (HPRT locus) and in mouse lymphoma L5178Y cells (TK locus). All results obtained are negative, i.e. no gene mutations in the cells investigated were observed. Therefore, no hazard with regard to gene mutation in mammalian cells is identified for the target substance. As explained in the analogue justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in the potential of gene mutations in mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mammalian erythrocyte micronucleus formation in vivo (OECD TG 474; oral application): negative

Read-across from analogue source substance Hexanedioic acid, mixed esters with decanoic acid, 12-hydroxyoctadecanoic acid, isostearic acid, octanoic acid, 3,3'-oxybis[1,2-propanediol] and stearic acid (CAS No. 130905-60-1)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Please refer to the Analogue Approach Justification provided in Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Dose tested was near MTD (max. tolerable dose), causing reduced activity and piloerection

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Source: CAS 130905-60-1, Sasol, 1990g

Conclusions:

Interpretation of results: negative

Executive summary:

The potential to induce the formation of erythrocyte micronuclei in vivo of the target substance is estimated based on an adequate and reliable in vivo study of a structural analogue source substance. Oral exposure via gavage of a single doses of 15000 mg/kg bw did not induce erythrocyte micronuclei in male and female mice. Therefore, no hazard with regard to in vivo erythrocyte micronuclei formation is identified for the target substance. As explained in the analogue justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in the genotoxic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Justification for read-across

The read-across from structural analogue source substances approach comprises aliphatic esters of the poly-functional alcohols di- and triglycerol. The fatty acid rests of the esters exhibit carbon chain lengths in the range C5 - C20. They are either linear and saturated in nature but also unsaturated C16 and C18 and branched C6 and C18 moieties are present. Since the alcohols employed provide 4 - 5 reactive hydroxyl functions, the esterification degree found in source and target substances ranges from mono- to penta-esters.

The available data allows for an accurate hazard and risk assessment of the target substance and the read-across concept is applied for the assessment of environmental fate and environmental and human health hazards. Thus, where applicable, environmental and human health effects are predicted from adequate and reliable data for source substances by interpolation to the target substance applying the read-across concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No. 1907/2006 (REACH). In particular, for each specific endpoint the source substances structurally closest to the target substance 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 substances are the basis of read-across. A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID section 13).

In vitro gene mutation in bacteria

The potential to induce mutations in bacteria has been tested with the target substance Isooctadecanoic acid, mixed esters with oxybis[propanediol] in a study according to OECD TG 471 under GLP conditions (Envigo, 2017). Salmonella typhimurium strains A 1535, TA 1537, TA 98 and TA 100 and Eschericia coli strain WP2 uvr A were exposed to 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate and 15, 50, 150, 500, 1500, 5000 µg/plate in two independent experiments following the plate incorporation method and the pre-incubation procedure, respectively. Acetone was used as vehicle and the experiments were performed both in the absence and the presence of a metabolic activation system (cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, induced with phenobarbital and beta-naphtha flavone). No cytotoxicity was observed at any concentration of the test item in any of the experiments but precipitation occurred at 1500 and 5000 µg/plate. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation. Vehicle and positive controls showed the expected results, demonstrating the validity of the experiments and the activity of the metabolising system. Under the experimental conditions applied, the test substance was found not to induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

In vitro chromosome aberration in mammalian cells

No data are available for the target substance and hence read-across from several analogue source substances has been applied to assess the endpoint of clastogenicity in mammalian cells in a Weight-of-Evidence approach.

An in vitro mammalian chromosome aberration test was performed with Oleic acid, monoester with oxybis(propanediol) (CAS 49553-76-6) in cultured peripheral human lymphocytes according to OECD TG 473 and under GLP conditions (DuPont, 2012a). Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). Cells were exposed for 4 hours to the test substance dissolved in DMSO at concentrations of 25, 50, 100, 150, 200 µg/mL without metabolic activation and for 4 hours at concentration of 50, 100, 200, 300 and 400 µg/mL with metabolic activation and for 22 hours without metabolic activation at concentration of 10, 25, 50, 75 and 100 µg/mL. The test substance induced cytotoxicity at 150 μg/mL in the 4-hour non activated test condition and at 100 μg/mL in the 22-hour non-activated test and in the 4-hour S9-activated test at 200 µg/mL and above. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. Mitomycin C and Cyclophosphamide were used as positive control substances inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolising 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 human lymphocytes in vitro.

A chromosome aberration test was conducted according to OECD TG 473 and under GLP conditions with 1,2,3-Propanetriol, homopolymer, diisooctadecanoate (CAS 63705-03-3) in chinese hamster lung fibroblasts V79 cells (BASF, 2013b). Cultures of V79 cells, treated with the test item, were evaluated for chromosome aberrations in three experiments at dose levels of 1, 3.13, 6.25, 12.5, 25, 50, 100, 200 µg/mL in the first experiment and of 12.5, 25, 50, 100, 200, 400, 800 µg/mL in the second and third experiments. Experiments were performed in the presence and the absence of a cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes and all the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and the activity of the metabolising system. Cytotoxicity was observed in the 2nd experiment at 800 µg/mL and in the 3rd experiment from 200 µg/mL onward at a sampling time of 18 hours and from 400 µg/mL onward at 28 hours sampling time. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either the absence or presence of S9. In conclusions the test item was considered to be non-clastogenic in V79 cells in vitro.

In another in vitro mammalian chromosome aberration test, the clastogenic potential of Hexanedioic acid, mixed esters with decanoic acid, 12-hydroxyoctadecanoic acid, isostearic acid, octanoic acid, 3,3'-oxybis[1,2-propanediol] and stearic acid (CAS 130905-60-1) was investigated in chinese hamster lung fibroblasts (V79). The study was performed according to OECD TG 473 and GLP conditions were observed (Sasol, 1996a). Duplicate cultures of V79 cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In both experiments, cells were exposed for 3 h to the test substance dissolved in ethanol at concentrations of 40, 200, 400 µg/mL with metabolic activation and for 16 and 26 h without metabolic activation. The substance was tested up to the limit concentration and did not induce cytotoxicity. Mitomycin C and Cyclophosphamide were used as positive control substances inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolising 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 V79 cells in vitro.

In vitro gene mutation in mammalian cells

Again no data obtained with the target substance are available regarding the genetic toxicity endpoint of gene mutation in mammalian cells. Therefore, adequate studies with analogue source substances are used to assess this endpoint by means of Weight-of-Evidence.

The in vitro mammalian cell gene mutation of with Oleic acid, monoester with oxybis(propanediol), (CAS 49553-76-6) was investigated according to OECD TG 476 under GLP conditions (DuPont, 2012b). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Aroclor 1254 rat liver S9). In a preliminary toxicity assay, the maximum concentration of test substance in medium was 5000 µg/mL. In the first experiment, cells were exposed for 4 h to test substance at concentrations of 10 - 150 µg/mL (in DMSO) with and without metabolic activation. The concentrations chosen for cloning were 10, 20, 30, 40 and 50 µg/mL in the presence and absence of metabolic activation. No cloned cultures exhibited induced mutant frequencies ≥ 90 mutants per 10E6 clonable cells. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 5 - 75 µg/mL. The concentrations chosen for cloning were 5, 10, 20, 30 and 40 µg/mL. No cloned cultures exhibited induced mutant frequencies ≥ 90 mutants per 10E6 clonable cells. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data. Cytotoxicity was observed at concentrations ≥ 30 µg/mL without metabolic activation and ≥ 50 µg/mL with metabolic activation. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study, the test substance did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.

An in vitro mammalian cell gene mutation assay according to OECD TG 476 and GLP was performed with 1,2,3-Propanetriol, homopolymer, diisooctadecanoate (CAS 63705-03-3) in Chinese hamster ovary (CHO) cells (BASF, 2013c). The test substance was dissolved in DMSO as it proved to be insoluble in water. Experiments were performed with and without a metabolic activation system (co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254). Concentrations used were 3.13, 6.25, 12.50, 25, 50, 100 µg/mL (experiment 1, with and without S9), 9.38, 18.75, 37.50, 75, 150, 200 µg/mL (experiment 2, without S9) and 4.69, 9.38, 18.75, 37.50, 75, 150 µg/mL (experiment 2, with S9). Ethyl methanesulfonate (EMS) and Dimethylbenz[a]anthracene (DMBA) were used as positive control substances. Cytotoxicity of the test substance was observed in experiment 1 in the absence of S9 mix at 100 µg/mL and in the presence of S9 mix at 50 µg/mL onward as well as in the second experiment in the absence of S9 mix at 150 µg/mL onward and in the precense of S9 mix at 75 µg/mL onward. Positive and solvent controls were valid and in range of historical control data. No significant increase in the mutation frequency at the HPRT 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 CHO HPRT test system under the experimental conditions described.

Genetic toxicity in vivo

In addition to the in vitro tests on genetic toxicity, an in vivo study investigating micronucleus formation in erythrocytes is available for an adequate source substance. The study is used to provide key information for assessment of this endpoint.

Hexanedioic acid, mixed esters with decanoic acid, 12-hydroxyoctadecanoic acid, isostearic acid, octanoic acid, 3,3'-oxybis[1,2-propanediol] and stearic acid (CAS 130905-60-1) was found to be not genotoxic in the micronucleus assay in vivo (Sasol, 1990g). A single dose of 15000 mg/kg bw/day administered by oral gavage to groups of 5 male and 5 female mice did not induce micro-nuclei formation. The dose level was based on a dose-range finding experiment. A dose of 15,000 mg/kg body weight was considered to be near the MTD (maximal tolerated dose) and was therefore chosen for the main study. Bone marrow samples were taken 24, 48 and 72 h after dosing. In none of the parameters investigated, a significant difference was found between female animals treated with the test article and negative control animals. The number of polychromatic erythrocytes without micronuclei in group males (48 h p.a.) as well as the ratio of polychromatic to normochromatic erythrocytes was significantly increased. The same parameters were decreased in group males (72 h p.a.). These effects might be explained by a stimulation of the formation of polychromatic erythrocytes in male mice after 48 h by the test article resulting in a subsequent increase of normochromatic erythrocytes, which arise from polychromatic erythrocytes, after 72 h. The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system. In conclusion, the test substance was considered to be non-mutagenic under the experimental conditions of the study.

Conclusion on genetic toxicity

Several reliable studies performed with either the target substance or analogue source substances are available investigating their genotoxic potential. Genotoxic effects considered include gene mutation in bacteria and mammalian cells as well as clastogenicity both in vitro and in vivo. The available data demonstrate the lack of genotoxic effects since all tests performed were negative. Thus, no hazard regarding genotoxicity is identified for the target substance Isooctadecanoic acid, mixed esters with oxybis[propanediol].

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 (REACH) information on intrinsic properties of substances may be generated by means other than tests, e.g. using information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI 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 read-across concept is applied to the target substance Isooctadecanoic acid, mixed esters with oxybis[propanediol], data gaps can be filled by interpolation from representative structural analogue source substances to avoid unnecessary animal testing.

The read-across concept is also used to derive the classification of the target substance taking the properties of the source substances into account. Based on the read-across concept, all available data on genetic toxicity both in vitro and in vivo do not meet the classification criteria according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.