Reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and 2-[2-(2-butoxyethoxy)ethoxy]ethyl (3,6,9,12-tetraoxahexadecyl)adipate

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, TA 100 and E. coli WP2 uvr A pkM 101

Read-across from the source substance bis(2-(2-butoxyethoxy)ethyl)adipate (CAS No. 141-17-3)

Chromosome aberration (OECD 473): negative in CHO cells with and without metabolic activation

Gene mutation in mammalian cells (OECD 490): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jan - 08 Feb 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: JHSF (Japan Health Science Foundation)

MEDIA USED
- Type and identity of media: RPMI 1640 medium supplemented with horse serum, sodium pyruvate and penicillin/streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

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 for 5 days

Test concentrations with justification for top dose:

Cytotoxicity test, short-term treatment (4 h), with and without metabolic activation: 5000, 2500, 1250, 625, 312, 156, 78 and 39 µg/mL
Cytotoxicity test, long-term treatment (24 h), without metabolic activation: 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.8 µg/mL

In each assay 4 doses were selected for the gene mutation test:
Short-term treatment (4 h), without metabolic activation: 625, 312, 156 and 78 µg/m:
Short-term treatment (4 h), with metabolic activation: 2500, 1250, 625 and 312 µg/mL
Long-term treatment (24 h), without metabolic activation: 500, 250, 125 and 62.5 µg/mL

Considering that the test item is a multi-constituent substance, the maximum concentration tested was 5000 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION:
Short-term treatment (4 h), with and without metabolic activation: 5000, 2500, 1250, 625, 312, 156, 78 and 39 µg/mL
Long-term treatment (24 h), without metabolic activation: 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.8 µg/mL

EXPERIMENTAL PROCEDURE AND DETAILS:
Short-term treatment (4 h), -/+S9:
- A series of tubes containing 6x10E+6 cells in suspension/tube in medium was used. The different concentrations of the test item were added directly into the tubes (2 cultures per concentration).
- At the end of the incubation period of 4 h, the tubes were centrifuged and the medium was aspirated. 2 washes were performed and 10 ml of medium were deposited in each culture. A count of the cells was performed on each tube, then treated cells were transferred into flasks and incubated 24 h (± 1 h) at 37 °C (± 0.5 °C), 5% CO2.
- After the incubation period, cell counting was performed and cell cultures were adjusted if necessary to 2x10E+5 cells/mL and incubated for 24 h (± 1 h) at 37 °C (± 0.5 °C), 5% CO2.
- After the incubation period, cell counting was performed again, i.e. 48 h after the end of the treatment. These counts allowed assessing the cytotoxicity expressed as a percentage of relative cell growth (RSG). The actual gene mutation test was performed using 4 selected concentrations of the test item as determined by cytotoxicity.
- The cells were seeded in 96-well plates at 2 cells/200µL in non-selective culture medium (2 plates/concentration) to determine viability. Plates were incubated at 37 °C (± 0.5 °C), 5% CO2 for 10 to 12 days. Empty wells were recorded to calculate the survival rate (cloning efficiency, CE) to determine the viability.
- In parallel, 96-well plates were seeded at 2x10E+3 cells/200µL in selective culture medium to determine the mutation frequency (4 plates/concentration). Plates were incubated at 37 °C (± 0.5 °C), 5% CO2 for 10 to 12 days. Empty wells were recorded to calculate the mutation frequency.

Long-term treatment (24 h), -S9:
- A series of flasks containing 1.5x10E+6 cells/flask in culture medium was used. The different concentrations of the test item were added directly to the flasks (2 cultures per concentration) and then incubated for 24 h (± 1 h) at 37 °C (± 0.5 °C), 5% CO2.
- After the incubation period, the cultures were transferred into sterile tubes, centrifuged and the medium was aspirated. 2 washes were performed and 10 ml of medium were deposited in each culture. A count of the cells was performed on each tube. The treated cells were placed in new culture flasks at 2x10E+5 cells/mL and incubated for 24 h (± 1 h) at 37 °C (± 0.5 °C), 5% CO2.
- All further steps are identical to the procedure for the short-term treatment (4 h).

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cytotoxicity corresponds to relative survival compared to the respective negative control values)

OTHER EXAMINATIONS:
Cloning efficiency was determined by seeding exposed cells in one microtiter plate with a density of 2 cells/well in medium without TFT.
Small and large colonies were differentiated as small colonies are capable to indicate chromosomal mutations.

Evaluation criteria:

A test item is considered potentially mutagenic if all of the following conditions are met:

- The mutation rate induced is significantly higher than that of the negative control,
- The observed effect is dose-dependent,
- The observed effect is reproducible,
- The rate of mutation is greater than the value of 126 plus the mutation frequency of the negative control (Moore et al., 2006).

The evaluation results are based on statistical methods, but the statistical significance should not be the only deciding factor.

If the criteria given above are not met, the test item is considered not mutagenic in this test.

Moore et al. (2006). Env. Mol. Mut. 2006, 47: 1-5

Statistics:

The calculations and summary of the results were performed using the Excel software. The mutant frequency for each series studied was subjected to a Chi-2 test.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9: starting at 1250 µg/mL (4 h) and 1000 µg/mL (24 h); +S9: starting at 5000 µg/mL (4 h)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

VALIDATION AND COMPARISON WITH HISTORICAL CONTROL DATA:
The negative control shows a cloning efficiency (CE) between 65 and 120%, a mutation frequency (MF) between 5x10E-5 and 17x10E-5) cells and a suspension growth (SG) between 8 and 32 (4 h-treatment) and between 32 and 180 (24 h-treatment).

The positive control shows a mutation frequency (MF) higher than 3x10E-4 with at least 40% in small colonies.

Except for the positive control for the long-term treatment (24 h), without S9, these values are slightly outside the IC (inhibition concentration) 95% of the historical control data. However, they are all consistent with the validity criteria and no deviation to the study plan or incidents have been observed during the study. The control values will, therefore, be added to the historical control data of the laboratory. The results validate the test.

Tables and further details on results are included in a separeate pdf document attached to this robust study summary.

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Aug - 5 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GROUPE lNTERMlNISTERIEL DES PRODUITS CHIMIQUES, 67, rue Barbes, 94201 Ivry-sur-Seine CEDEX, France

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC CCL 61, ECACC 85051005
- Suitability of cells: Chinese Hamster Ovary (CHO) cultures are recommended by the OECD TG 473
- Number of passages if applicable: 29 - 31
- Methods for maintenance in cell culture if applicable: Mc Coy’s + 10 % Fetal Calf Serum (FCS)

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

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:

Assay 1 (short-term treatment): 2000, 1400, 800, 560, 320, 128 µg/mL (-/+ S9)
Assay 2 (long-term treatment): 1400, 800, 560, 320, 224, 128, 51.2 µg/mL (-S9)
If no precipitate or limiting cytotoxicity is observed, the highest test concentration should correspond to 10 mM, 2000 µg/mL or 2 μL/mL, whichever is the lowest.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: Ethanol was selected as most suitable vehicle, which has been demonstrated to be suitable in in vitro genotoxicity studies and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

yes

Remarks:

McCoy

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 40 h
- Exposure duration: 4 h (assay 1, -S9), 3 h (assay 1, +S9), 20 h (assay 2, -S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 20 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid® (NA) (0.15 μg/mL)

STAIN (for cytogenetic assays): Giemsa at 0.4% (w/v) in phosphate buffer (0.01 M, pH 6.8)

NUMBER OF REPLICATIONS: duplicate cultures in two independent experiments

NUMBER OF CELLS EVALUATED:
- 300 metaphases minimum are analysed for the highest non-cytotoxic concentration of each solution (if necessary)
- 300 metaphases minimum are analysed for the absolute negative control and solvent control
- 25 metaphases minimum are analysed for the positive controls

DETERMINATION OF CYTOTOXICITY
- Method: Preliminary study using Balb/c 3T3

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The test item will be considered as clastogen in vitro with regards to CHO cells according to the following criteria:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control,
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.

The test item for which the results do not meet all above criteria is considered non-clastogenic in this system.

Positive results indicate that the test item induces structural chromosome aberrations in CHO cell cultures.

The positive control shall largely fulfil to all three criteria.

The number of cells with chromosomal aberrations in the negative control shall be less than 5%.

Equivocal or disputable results may not allow a clear positive response. Results shall be clarified by further testing using modification of experimental conditions (concentration spacing and metabolic activation conditions).

Statistics:

The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test item solution with a concentration of 2000 μg/mL show a complete cell death and those with a concentration of 800 μg/mL show a statistically significant inhibition of cell growth of 52%

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH was measured, at least for the two top doses and for the vehicle control with and without S9 mix. The pH values were not influenced by test substance treatment.
- Effects of osmolality: Osmolarity was measured, at least for the top dose and for the vehicle control with and without S9 mix. Osmolarity was not influenced by test substance treatment.

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test (using Balb/c 3T3 mouse embryo fibroblast) was performed to obtain a first estimate of the maximum concentration which should be tested in the chromosomal aberration test.

COMPARISON WITH HISTORICAL CONTROL DATA:
The structural chromosome aberration rates of the vehicle and positive control groups were within the historical control data range and, thus, fulfilled the acceptance criteria of this study.

Table 1: Type and number of chromosome aberrations (without metabolic activation)

Serie		Assay	Concentration	Cellsobserved	Aberrations (type and number)
					Chromosomal						Chromatidic						Other
					G***	C	M	D	CR	R	g***	c	ic	tr	qr	d	CP	cp
Without metabolic activation
Absolute negative control		1*	-	300	2	1	0	0	0	0	1	1	0	0	0	0	0	0
		2**	-	300	2	1	0	0	0	0	0	1	0	0	0	0	0	0
Positive control(mitomycin C)		1*	0.25 µg/mL	26	0	0	0	0	0	0	0	5	0	2	0	1	0	0
		2**	0.125 µg/mL	52	3	5	0	0	0	0	1	4	0	2	1	0	0	0
Solvent control (Ethanol)		1*	-	300	0	0	0	0	0	0	2	4	0	0	0	0	0	0
		2**	-	300	2	0	0	0	0	0	2	0	0	0	0	0	0	0
Solution obtained from Reaction mass of bis[2-[2-(2- butoxyethoxy)ethoxy]ethyl]adipate and 2-[2-(2-butoxyethoxy)ethoxy]ethyl (3,6,9,12-tetraoxahexadecyl)adipate	LEMI code:   GNY310817-S1	1*	128 µg/mL	300	0	2	0	0	0	0	2	1	0	0	0	0	0	0
			320 µg/mL	300	2	0	0	0	0	0	3	2	0	0	0	0	0	0
			560 µg/mL	302	3	1	0	0	0	0	6	2	0	0	0	0	0	0
	LEMI code:   GNY070917-S1	2**	224 µg/mL	300	1	0	0	0	0	0	3	2	0	0	0	0	0	0
			320 µg/mL	300	4	0	0	0	0	0	2	1	0	0	0	0	0	0
			560 µg/mL	300	3	1	0	0	0	0	1	2	0	0	0	0	0	0

G, C, M, D, CR, R, g, c, ic, tr, qr, d, CP and cp: type of aberration

*1: Assay 1

** 2: Assay 2

***not taken into account.

Table 2: Number and percentage of cells with aberrations (without metabolic activation)

Serie		Assay	Concentration	Cells observed	Total aberrations	Aberrations /cell	Cells with aberration	Cells with aberration (% )	c²	P***
Without metabolic activation
Absolute negative control		1*	-	300	2	0.007	2	0.7%	-	-
		2**	-	300	2	0.007	2	0.7%	-	-
Positive control (mitomycin C)		1	0.25 µg/mL	26	8	0.308	8	30.8%	55.34	< 0.001
		2	0.125 µg/mL	52	12	0.231	12	23.1%	46.96	< 0.001
Solvent control (Ethanol)		1*	-	300	4	0.013	4	1.3%	-	-
		2**	-	300	0	0.000	0	0.0%	-	-
Solution obtained from Reaction massofbis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and2-[2-(2- butoxyethoxy)ethoxy]ethyl (3,6,9,12- tetraoxahexadecyl)adipate	LEMI code:    GNY310817-S1	1*	128 µg/mL	300	3	0.010	3	1.0%	0.14	NS****
			320 µg/mL	300	2	0.007	2	0.7%	0.66	NS****
			560 µg/mL	302	3	0.010	3	1.0%	0.15	NS****
	LEMI code:    GNY070917-S1	2**	224 µg/mL	300	2	0.007	2	0.7%	1.99	NS****
			320 µg/mL	300	1	0.003	1	0.3%	1.00	NS****
			560 µg/mL	300	3	0.010	3	1.0%	2.99	NS****

*1: Assay 1

**2: Assay 2

***P: Statistical significance

****NS: Not statistically Significant, P ≥ 0.005

Table 3: Type and number of chromosome aberrations (with metabolic activation)

Serie		Assay	Concentration	Cells observed	Aberrations (type and number)
					Chromosomal						Chromatidic						Other
					G**	C	M	D	CR	R	g**	c	ic	tr	qr	d	CP	cp
With metabolic activation (S9 mix)
Absolute negative control		1*	-	300	1	0	0	0	0	0	5	1	0	0	0	1	0	0
Positive control(cyclophosphamide)		1*	10 µg/mL	25	0	0	0	0	0	0	0	5	0	4	0	0	0	0
Solvent control (Ethanol)		1*	-	300	2	0	0	0	0	0	1	2	0	0	0	0	0	0
Solution obtained from Reaction massofbis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipateand2-[2-(2-butoxyethoxy)ethoxy]ethyl(3,6,9,12-tetraoxahexadecyl)adipate	LEMI code:   GNY310817-S1	1*	800 µg/mL	300	2	3	0	0	0	0	3	1	0	0	0	0	0	0
			1400 µg/mL	300	2	1	0	0	0	0	3	3	0	0	0	0	0	0
			2000 µg/mL	300	1	1	0	0	0	0	3	3	0	0	0	0	0	0

G, C, M, D, CR, R, g, c, ic, tr, qr, d, CP and cp: type of  aberration

*1: Assay 1

** 2: Assay 2

***not taken into account.

Table 4: Number and percentage of cells with aberrations (with metabolic activation)

	Serie			Assay	Concentration	Cells observed	Total aberrations	Aberrations /cell	Cells with aberration	Cells with aberration (% )	c²	P**
	With metabolic activation (S9 mix)
	Absolute negative control			1*	-	300	2	0.007	2	0.7%	-	-
	Positive control (cyclophosphamide)			1*	10 µg/mL	25	9	0.360	8	32.0%	57.12	< 0.001
	Solvent control (Ethanol)			1*	-	300	2	0.007	2	0.7%	-	-
	Solution obtained from Reaction massofbis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and2-[2-(2- butoxyethoxy)ethoxy]ethyl (3,6,9,12- tetraoxahexadecyl)adipate	LEMI code:    GNY310817-S1		1*	800 µg/mL	300	4	0.013	4	1.3%	0.66	NS***
					1400 µg/mL	300	4	0.013	4	1.3%	0.66	NS***
					2000 µg/mL	300	4	0.013	4	1.3%	0.66	NS***
*1: Assay 1 **2: Assay 2 ***P: Statistical significance
****NS:  Not statistically Significant, P ≥ 0.005

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July 2010 to 17 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (Salmonella typhimurium strains); trp operon (E.coli strains)

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:

Preliminary Toxicity Test
0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 1 and 2
50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide

The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.

Untreated negative controls:

no

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

benzo(a)pyrene

other: 2-aminoanthtracene (2AA)

Remarks:

+S9: 2AA (all strains); -S9: N-ethyl-N-nitro-N-nitrosoguanidine (ENNG), benzo(a)pyrene (BP), 9-aminoacridine (9AA), 4-nitroquinoline-N-oxide (4NQO)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Standard plate incorporation method (experiment 1) and preincubation method (experiment 2)

DURATION
- Preincubation period: 20 min (only exp 2)
- Exposure duration: 48 h at 37°C (exp 1 and 2)

NUMBER OF REPLICATIONS:
3 replicates/strain

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.

Evaluation criteria:

Acceptance Criteria:

The following criteria must be met for acceptance:
- All tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate in vehicle and untreated controls.
- The appropriate characteristics of each tester strain must be confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor.
- All tester strain cultures should be in the range of 0.9 to 9.9x10E9 bacteria per mL.
- Each mean positive control value should be at least 2x the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains and the integrity of the S9 mix
- The test should include a minimum of four non-toxic dose levels.

Evaluation Criteria:

There are several criteria for determining a positive result. Any, one, or all of the following may be 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.
- The biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by the UKEMS (Mahon et al., 1989).
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

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

Statistics:

Mean values with standard deviations were calculated.

Species / strain:

S. typhimurium, other: 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

Species / strain:

E. coli WP2 uvr A pKM 101

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

Additional information on results:

RANGE-FINDING STUDIES
In order to select appropriate dose levels for use in the main test, a preliminary test were conducted in the presence or absence of metabolic activation. Ten dose levels and controls were tested up to and including 5000 µg/plate at approximately half-log intervals. The assay was conducted by mixing 0.1 mL the bacterial culture (TA 100 or WP2uvrA-), and 0.1 mL of the vehicle or test chemical mixture, 0.5 mL of S9 mix or phosphate buffer and 2.0 mL of molten agar supplemented with trace histidine or tryptophan and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37°C, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Table 1: Numbers of revertant colonies in the preliminary toxicity test

With (+) or without (-) S9 mix	Strain	Dose (µ/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA 100	90	88	99	108	113	114	107	86	114	118	113*
+	TA 100	120	112	108	121	100	98	92	96	90	98	105*
-	WP2 uvrA-	43	40	40	43	48	42	38	38	45	41	42*
+	WP2 uvrA-	47	43	48	43	38	37	42	41	37	38	42*

* Precipitate present

Table 2: Range-finding test without metabolic activation

	Revertant colony counts (mean of 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	105	19	28	24	13
50	111	16	24	25	10
150	120	14	28	29	12
500	105	15	31	25	11
1500	109P	17P	26P	28P	12P
5000	115P	14P	30P	24P	13P
ENNG (3)	497
ENNG (5)		162
ENNG (2)			283
4NQO (0.2)				116
9AA (80)					2530

P - precipitate

Abbreviations: ENNG, N-ethyl-N’-nitro-N-nitrosoguanidine; 9AA, 2-aminoacridine; BP, benzo(a)pyrene; 2AA, 2-aminoanthracene; 4NQO, 4- nitroquinoline-1-oxide

Table 3: Range-finding test with metabolic activation

	Revertant colony counts (mean 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	115	14	33	29	12
50	98	13	32	27	12
150	109	16	32	30	11
500	99	10	29	29	12
1500	115P	14P	31P	29P	12P
5000	116P	14P	32P	29P	14P
2AA (1)	801
2AA (2)		292			95
2AA (10)			130
BP (5)				189

P - preicipitate

Table 4: Main Test without metabolic activation

	Revertant colony counts (mean 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	117	21	26	34	9
50	115	20	35	27	11
150	117	20	30	27	9
500	115	21	31	30	10
1500	117P	20P	31P	31P	11P
5000	124P	18P	30P	32P	7P
ENNG (3)	439
ENNG (5)		174
ENNG (2)			182
4NQO (0.2)				122
9AA (80)					730

P - precipitate.

Table 5: Main Test with metabolic activation

	Revertant colony counts (mean 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	124	24	41	29	13
50	117	18	38	24	10
150	127	18	36	30	9
500	119	11	34	33	12
1500	114P	13P	29P	26P	10P
5000	130P	18P	35P	22P	9P
2AA (1)	952
2AA (2)		261			217
2AA (10)			202
BP (5)				309

P - precipitate

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for grouping of substances and read-across

The read-across approach uses 'bis(2-(2-butoxyethoxy)ethyl)adipate' (CAS No. 141-17-3) as structurally similar source substance to transfer (read-across) data to the target substance 'reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and [2-[2-(2-butoxyethoxy)ethoxy]ethyl](3,6,9,12-tetraoxahexadecyl)adipate' (EC No. 943-330-9). The common feature of the source substance and the target substance is that they are diester derivatives of adipic acid, containing only even numbered and linear ethoxylated side chains. Both carboxylic functions of adipic acid are used to form esters with ethylene glycol monobutyl ethers of varying length. While the side chains of the target substance contain tri- and/or tetraethylene glycol monobutyl ether moieties, the side chains in the source substance are made up solely of diethylene glycol monobutyl ether. Thus, the ethylene glycol monobutyl ether substituents in the target substance contain 1 or 2 additional ethylene glycol monobutyl ether units. Although the constituents of the target substance are hence larger in size and have a higher molecular weight, it can be assumed that no significant steric hindrance is introduced when compared to the smaller side chains of the source substance. All parts of the ethylene glycol monobutyl ether side chains are freely rotatable due to the fact that neither a ring system nor π-bonds exert any constraints on rotatability. Therefore, it is feasible to assume an identical environmental and metabolic fate of both substances. In order to avoid the need to test every substance for every endpoint, the read-across from an analogue substance 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 using adequate and reliable data from the source substance in accordance with Annex XI, Item 1.5, of Regulation (EC) No. 1907/2006 (REACH). Structural similarity and similarities in properties and/or activities of the source and target substance are the basis of read-across.

In vitro gene mutation in bacteria

No study performed with the target substance is available. Hence, data obtained with the source substance bis(2-(2-butoxyethoxy)ethyl)adipate (CAS No. 141-17-3) is used to read-across information regarding gene mutation in bacteria. A bacterial gene mutation assay (Ames test) with the source substance was performed according to OECD TG 471 and in compliance with GLP (Harlan, 2011). Two independent experiments were performed (plate incorporation and pre-incubation), both in the presence or absence of metabolic activation (S9 mix), in the S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A pKM 101 with test substance concentrations ranging from 0.15 to 5000 µg/plate for an exposure period of 48 h. No cytotoxicity was observed in any of the tester strains up to the limit concentration of 5000 µg/plate. No increase in mean revertant number was observed in any bacterial strain after exposure to the test substance at any test concentration in both experiments. The solvent and positive controls showed the expected results and thus confirmed the efficiency of the assay. Under the conditions of this assay, the test substance did not induce mutagenicity in the selected strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A pKM 101 in the absence and presence of metabolic activation, respectively.

In vitro chromosome aberration in mammalian cells

The potential of reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and [2-[2-(2-butoxyethoxy)ethoxy]ethyl](3,6,9,12-tetraoxahexadecyl)adipate (EC No. 943-330-9) to induce chromosome aberrations has been assessed in a study performed according to OECD TG 473 and observing GLP provisions (LEMI, 2017). Solutions of the test substance were tested in cultured CHO (Chinese Hamster Ovary) cells in the absence and presence of metabolic activation (S9 mix). 2 independent experiments were performed, a short-term and a long-term treatment. For experiment 1 (short-term treatment), CHO cells were exposed to concentrations of 128, 320, 560, 800, 1400, and 2000 µg/mL of the test item in ethanol for 4 h in the absence and for 3 h in the presence of metabolic activation, respectively. In experiment 2 (long-term treatment), CHO cells were exposed for 20 h to concentrations of 51.2, 128, 224, 320, 560, 800, and 1400 µg/mL in the absence of metabolic activation. For both experiments, positive and negative controls were carried out in parallel. Positive controls induced a statistically significant increase in the number of chromosomal aberrations in comparison with corresponding negative controls. The values of negative and positive controls do not show a significant difference with the historical control data of the laboratory. Negative controls and positive controls, thus, validate the two experiments. Cytotoxicity was observed. The experiments with a concentration of 2000 μg/mL (with and without S9 mix) showed a complete cell death and the experiments with concentrations of 800 μg/mL showed a statistically significant inhibition of cell growth of 52%. No genotoxic effect after exposure to the test substance was observed with any test concentration, neither in the absence nor in the presence of metabolic activation. Reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and [2-[2-(2-butoxyethoxy)ethoxy]ethyl](3,6,9,12-tetraoxahexadecyl)adipate (EC No. 943-330-9) is therefore not considered to be clastogenic in CHO cells under the conditions of the experiment.

In vitro gene mutation in mammalian cells

The ability of reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and [2-[2-(2-butoxyethoxy)ethoxy]ethyl](3,6,9,12-tetraoxahexadecyl)adipate (EC No. 943-330-9) to induce gene mutations in the thymidine kinase locus of mouse lymphoma L5178Y cells was investigated in a study according to OECD TG 490 under GLP conditions (IDEA, 2017). Two independent gene mutation experiments were performed. The first experiment was performed as short-term treatment (4 h) with and without metabolic activation (S9 mix). As a negative response was obtained after 4 h of treatment, a second experiment was carried out without metabolic activation in a long-term treatment (24 hours). Cytotoxicity tests were included in the gene mutation experiments using concentrations of 39, 78, 156, 312, 625, 1250, 2500, and 5000 µg/mL (+/-S9 mix) in the short-term (4 h) treatment and of 7.8, 15.63, 31.25, 62.5, 125, 250, 500, and 1000 µg/mL (-S9 mix) in the long-term (24 h) treatment. Based on cytotoxicity observed, 4 concentrations in each experiment were selected to be evaluated for gene mutations: 78, 156, 312, and 625 µg/mL for the short-term (4 h) treatment (-S9 mix), 312, 625, 1250, and 2500 µg/mL for the short-term (4 h) treatment (+S9 mix) and 62.5, 125, 250, and 500 µg/mL in the long-term (24 h) treatment (-S9 mix). The mutant frequency and hence the genotoxic potential of the test substance was determined by a comparison of the number of mutant colonies in selective medium with the number of colonies on non-selective medium. Positive controls resulted in a statistically significant increase in mutation frequency, validating the test. The mutation frequencies observed for the test item were not statistically different from those observed for the negative controls. It is therefore concluded that the test substance did not show any mutagenic nor pro-mutagenic activity in the in vitro gene mutation assay on the thymidine kinase locus of mouse lymphoma L5178Y cells under the experimental conditions applied and the concentrations tested.

Conclusion on genetic toxicity

In vitro studies with the source substance and the target substance investigating the induction of gene mutation in bacteria and mammalian cells as well as chromosome aberration in mammalian cells did not result in any positive, i.e. genotoxic, response. All available data on in vitro genetic toxicity indicate a lack of genotoxic potential of the source and the target substance. No hazard for genetic toxicity is therefore identified for the target substance.

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 physico-chemical, 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". Thus, data gaps can be filled by a read-across approach from a structural analogue source substance to avoid unnecessary animal testing.

The analogue concept is also used to derive the C&L of the target substance taking the properties of the source substance into account. Based on the analogue concept and on data obtained with the target substance, all available data regarding genetic toxicity do not meet the classification criteria according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.