Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA 98, 100, 1535, 1537 and E. coli tester strain WP2uvrA with and without metabolic activation

Chromosome aberration (Read-across, OECD 473): negative in Chinese hamster lung fibroblasts (V79) cells with and without metabolic activation.

Gene mutation in mammalian cells (Read-across, OECD 476): negative in mouse lymphoma L5178Y cells with and without metabolic activation.

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

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:

09 Aug 2016 - 20 Jan 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:

adopted in 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Medicines & Healthcare products Regulatory Agency, United Kingdom

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The test item dilutions were prepared in acetone on the day of each experiment. No correction for purity was required. Acetone is toxic to the bacterial cells at 0.1 mL (100 μL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 μL) aliquots (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined.

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of California, Berkeley, on culture discs, on 04 August 1995, British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987

MEDIA USED
- Type and identity of media: Top agar was prepared using 0.6% Bacto agar (lot number 5223985 06/20) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 43372 02/17 and 43406 02/17).
- Properly maintained: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

microsomal preparations (S9 mix), prepared from rat livers treated with a mixture known to induce an elevated level of these enzymes.

Test concentrations with justification for top dose:

1st Experiment: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
2nd Experiment:
Salmonella strain TA100 (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
Salmonella strains TA1535, TA98 and TA1537 (absence of S9): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Salmonella strains TA1535 and TA100 (presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
E.coli strain WP2uvrA (absence and presence of S9) and Salmonella strains TA98 and TA1537 (presence of S9): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
The top dose was selected based on cytotoxicity in each strain with or without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide and dimethyl formamide at 50 mg/mL but was fully soluble in acetone at 100 mg/mL in solubility checks performed in-house

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

positive controls in the absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Remarks:

positive controls in the presence of S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation (in buffer without metabolic activation or instead in S9-mix for metabolic activation)

All of the plates were incubated at 37 ± 3°C for approximately 48 h and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were required at 5000 µg/plate because of test item precipitation.

DURATION
- Exposure duration: 48 h after 20 min preincubation

NUMBER OF REPLICATIONS: 3 (triplicates)
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method with and without metabolic activation.

Without metabolic activation:
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 min (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method.

With metabolic activation:
The procedure was the same as without metabolic activation, except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media.

Evaluation criteria:

A test item was considered non-mutagenic (negative) in the test system if the following criteria were not met:
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 as determined by UKEMS (Mahon et al., 1989).
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 (Cariello and Piegorsch, 1996)).

Statistics:

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.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (white and particulate in appearance) was noted under a low power microscope at 1500 μg/plate and by eye at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
The results of the range finding study were included as experiment 1.

CYTOTOXICITY
First experiment: maximum dose of 5000 µg/plate. The test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains initially from 150 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA in either the absence or presence S9-mix. Consequently, the toxic limit or the maximum recommended dose concentration of the test item was employed as the maximum dose in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. The test item induced a much stronger toxic response in the second mutation test (employing the pre-incubation method), with weakened bacterial background lawns noted in the absence of S9-mix from 15 μg/plate (TA100), 50 μg/plate (TA1535, TA98 and TA1537) and 150 μg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were noted to the Salmonella strains only from 500 μg/plate (TA100, TA1535 and TA98) and 1500 μg/plate (TA1537). No toxicity was noted to Escherichia coli strain WP2uvrA dosed in the presence S9-mix in the second mutation test. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.


HISTORICAL CONTROL DATA
- Positive historical control data: provided for 2015 and 2016
- Negative (solvent/vehicle) historical control data: provided for 2015 and 2016
see attached document

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA98 revertant colony frequency was observed in the presence of S9-mix at 50 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Table 1: Mean number of revertants in experiment 1

Experiment I	without S9
	TA100	TA1535	WP2 uvrA	TA98	TA1537
Vehicle (acetone)	87	12	23	13	10
1.5 µg	89	14	13	15	12
5 µg	76	12	13	18	12
15 µg	101	14	17	18	13
50 µg	93	12	19	17	11
150 µg	94 S	16	17	15	7
500 µg	78 S	8 S	18	14 S	7 S
1500 µg	23 S	8 S	12	13 S	0 V
5000 µg	0 VP	0 VP	11 P	8 SP	0 VP
N-ethyl-N'-nitro-N-nitrosoguanidine	524	128	553
4-Nitroquinoline-1-oxide				173
9-Aminoacridine					610
Experiment I	with S9
	TA100	TA1535	WP2 uvrA	TA98	TA1537
Vehicle (acetone)	94	13	21	22	19
1.5 µg	97	11	20	21	15
5 µg	88	13	21	20	15
15 µg	94	11	19	25	12
50 µg	97	17	18	22	15
150 µg	98	14	19	20	12
500 µg	77	11	23	22	11
1500 µg	74 S	7 S	19	18	8
5000 µg	32 SP	7 SP	22 P	12 SP	3 SP
2-aminoanthracene	747	214	224		448
Benzo(a)pyrene				111

P = precipitation; S = sparse bacterial background law, V = very weak bacterial background lawn

 

Table 2: Mean number of revertants in experiment II

Experiment II	without S9
	TA100	TA1535	WP2 uvrA	TA98	TA1537
Vehicle (acetone)	82	18	20	15	8
0.05 µg	82	NT	NT	NT	NT
0.15 µg	80	8	NT	17	9
0.5 µg	75	11	NT	21	7
1.5 µg	82	13	17	19	8
5 µg	85	13	20	14	6
15 µg	81 S	12	20	14	5
50 µg	0 T	0 T	17	18 S	0 T
150 µg	0 T	0 T	14	0 T	0 T
500 µg	NT	0 T	18 S	0 T	0 T
1500 µg	NT	NT	0 V	NT	NT
5000 µg	NT	NT	0 VP	NT	NT
N-ethyl-N'-nitro-N-nitrosoguanidine	721	3264	96
4-Nitroquinoline-1-oxide				151
9-Aminoacridine					465
Experiment II	with S9
	TA100	TA1535	WP2 uvrA	TA98	TA1537
Vehicle (acetone)	89	13	25	16	8
0.5 µg	76	14	NT	NT	NT
1.5 µg	89	12	22	20	11
5 µg	77	10	25	23	9
15 µg	78	12	23	21	10
50 µg	86	12	27	26**	11
150 µg	81	10	26	18	8
500 µg	0 V	0 V	26	14 S	11
1500 µg	0 V	0 V	24	0 V	0 V
5000 µg	NT	NT	22 P	0 VP	0 TP
2-aminoanthracene	1314	197	260		210
Benzo(a)pyrene				64

P = precipitation; S = sparse bacterial background law, V = very weak bacterial background lawn, T = toxic, no bacterial background lawn, NT = not tested

** p<0.01

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)

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 lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

systematic influence of the test compound which led to a reduction in the mitotic index from 10 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Source: CAS 853947-59-8

Conclusions:

The read across approach is justified in the analogue justification. The target and source substance are considered unlikely to differ in their genotoxic potential. In an in vitro mammalian chromosome aberration test (OECD guideline 473) with Chinese hamster ovary cells with the source substance C8-C10-1,3-Butandiolester (CAS 853947-59-8) no clastogenic effects were seen. Therefore, no hazard with regard to chromosome aberration in mammalian cells is expected for target substance Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate (EC 908-917-6).

Reference 3

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Source: CAS 91031-31-1

Conclusions:

The read across approach is justified in the analogue justification. The target and source substances are considered unlikely to differ in their genotoxic potential. In an in vitro mammalian cell gene mutation test using the thymidine kinase gene performed according to OECD guideline 476 with the source substance Fatty acids, C16-18, esters with ethylene glycol (CAS 91031-31-1) no genotoxicity was found in mouse lymphoma L5178Y cells. Therefore, no mutagenic potential in mammalian cells is expected for target substance Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate (EC 908-917-6).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable

Additional information

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

Genetic toxicity (mutagenicity) in bacteria in vitro

The in-vitro genetic toxicity of Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate was assessed in a bacterial reverse mutation assay (Ames test) according to GLP criteria and OECD guideline 471 (key study, 2017). The test substance was dissolved in acetone. Two independent experiments (plate incorporation and pre-incubation method) were performed. The mutagenic potential of the test substance was assessed in S. typhimurium tester strains TA 98, 100, 1535, 1537 and E. coli tester strain WP2uvrA at concentrations up to 5000 µg/plate in the 1st experiment and up to cytotoxicity levels in the 2nd experiment. Cytotoxicity was observed in different bacterial strains: The test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains initially from 150 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA. Test item precipitation was seen at 5000 μg/plate.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. The vehicle and positive control results were valid and fell within the range of historical control data. Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate was considered to be non-mutagenic under the conditions of this Ames test.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 91031-31-1

Mutagenic properties of Fatty acids, C16-18, esters with ethylene glycol (CAS 91031-31-1) were characterized in an in vitro mammalian cell gene mutation study according to OECD guideline 476 under GLP conditions (key study, 2010). Gene mutations in the thymidine kinase locus were assessed in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Phenobarbital/β-naphtoflavone-induced rat liver S9). In the first experiment, cells were exposed for 3 h to test substance at concentrations of 0.1-333 µg/mL (in DMSO) with and without metabolic activation. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 3-175 µg/mL and with metabolic activation (3 h; 12% S9-mix) from 0.1-333 µg/mL. The concentrations were limited by precipitation observed at the highest concentration. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data. No cytotoxicity was observed up to the precipitating concentration of 100 µg/mL and up to 333 µg/mL, respectively. 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, Fatty acids, C16-18, esters with ethylene glycol did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 853947-59-8

An in vitro mammalian chromosome aberration test was conducted with C8-C10-1,3-Butandiolester (CAS 853947-59-8) in accordance with OECD guideline 473 under GLP conditions (key study, 1997). The induction of structural chromosome aberrations was evaluated in vitro in Chinese hamster lung fibroblasts (V79) cells, incubated for 18 and 28 h with and without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). Concentrations of 10-100 µg/mL (18 h incubation) and 80 and 100 µg/mL (28 h incubation) of the test substance in the vehicle ethanol were applied. The solubility limit of the test substance in the vehicle ethanol in the culture medium was determined to be 100 µg/mL. In the first experiment without metabolic activation, the negative controls exhibited a mitotic index of 2.0% only and the experiment was therefore repeated. In the 2nd experiment, the negative as well as the positive controls showed the expected results and were within the range of historical control data. The frequency of polyploidy cells with and without metabolic activation was within the expected range (< 10%). In the experiments both with and without metabolic activation, a systematic influence of the test substance was observed, which led to a reduction in the mitotic index. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed.

Therefore, under the conditions of the study, C8-C10-1,3-Butandiolester did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in Chinese hamster lung fibroblasts in vitro.

Overall conclusion for genetic toxicity

The available data on the target and source substances do not indicate that the target or source substances exhibit mutagenic or clastogenic properties in bacteria or mammalian cells. Therefore, no properties for genetic toxicity are expected for Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate.

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 Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate 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, 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.