bis[2-(2-butoxyethoxy)ethyl] succinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A key mouse lymphoma thymidine kinase assay using the cell line L5178Y was conducted up to 10 mM in Experiment I without and with metabolic activation and in Experiment II with metabolic activation; in Experiment II without metabolic activation testing was done up to 7.5 mM. Growth inhibition was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation. In experiment II with metabolic activation no growth inhibition was observed. No precipitation of the test item was noted in the experiments. In experiment I without metabolic activation the relative total growth (RTG) was 60.6% for the highest concentration (10 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 64.3%. In experiment II without metabolic activation the relative total growth (RTG) was 11.3% for the highest concentration (7.5 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 110%. In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study complies to OECD Test Guidance 476 and GLP, and is considered to be relevant, adequate and reliable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase TK+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

-Type and identity of media:
Complete Culture Medium
RPMI 1640 medium supplemented with 10% horse serum, 100U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 1.5 µg/mL amphotericin B

Treatment Medium
RPMI 1640 medium supplemented with 5% horse serum (in case of short term exposure) or 7.5 % horse serum (in case of long term exposure), 100U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B

Selective Medium
RPMI 1640 medium supplemented with 20% horse serum, 100U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 5 µg/mL TFT

-Properly maintained: yes
-Periodically checked for Mycoplasma contamination: yes
-Periodically checked for karyotype stability: yes
-Periodically”cleansed” against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment I without and with metabolic activation: 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, 9.0 and 10 mM
Experiment II without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0 and 7.5 mM
Experiment II with metabolic activation: 0.1, 0.3, 0.7, 2.0, 4.0, 6.0, 8.0 and 10.0 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (1% DMSO v/v)
- Justification for choice of solvent/vehicle: As for this test system the use of AOO (4:1 (v/v) acetone/olive oil;) was technically not possible, another vehicle was used according to OECD Guideline. A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 10 mM. Based on the results of the solubility test DMSO was used as solvent (1% DMSO v/v). Different test item stock solutions were prepared and added to the cells. The solution was treated with ultrasound for 12 minutes at 37°C and diluted prior to treatment. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

1% DMSO v/v)

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9, 200 µg/mL and 300 µg/mL

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9, 8 µg/mL and 10 µg/mL

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9, 2.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Experiment I without and with metabolic activation and Experiment II with metabolic activation (4h short-term exposure)
- Preincubation period: 4 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Cloning efficieny: 6 days
- Selection time (if incubation with a selection agent): 14 days

Experiment II without activation (24h long-term exposure)
- Preincubation period: 24 h
- Exposure duration: 24 h
- Expression time (cells in growth medium): 2 days
- Cloning efficieny: 6 days
- Selection time (if incubation with a selection agent): 14 days



SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS:
Cloning efficiency: duplicate
Mutagenicity : ?

NUMBER OF CELLS EVALUATED: ?

DETERMINATION OF CYTOTOXICITY
- Method: other: measuring the colony-forming ability and the growth rate of cultures

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT:
-At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable
-The cloning efficiency of the negative and/or solvent controls is in the range 65% - 120%
-The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50 – 170 mutants per 106 cells
-The number of the negaztive/solvent controls shouls undergo 8-32 fold increase during a 2 day growth period (short-term treatment) or 32-180 fold increase during a 3 day growth period (long-term treatment)
-The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 106 cells with at least 40% of the colonies being small colonies or with an induiced small colony mutant frequency of at least 150 mutants per 106 cells. The RTG must be greater than 10%.

EVALUATION OF RESULTS
The test item is considered mutagenic if the following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation Factor (GEF) of 126 mutants per 106 cells
-A dose-dependent increase in mitant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥ 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: growth inhibition was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation; no growth inhibition in experiment II with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was noted in the experiments.
- The pH-value detected with the test item was within the physiological range.

RANGE-FINDING/SCREENING STUDIES:
Pre-Experiment for Toxicology:
Experiment I: 0.2, 0.5, 2.5, 5.0, 7.5 and 10 mM were tested without and with metabolic activation.
Experiment II : 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM were testes without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
BioService historical data for mutatnt frequancies were available (negative, solvent and positive controls).
All frequencies were within the historical control range, except the mutant frequencies for the solvednt of expderiment I with metabolic actiation, which were below the range of historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Growth inhibition was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Summary: Experiment I and II, without metabolic activation

	Test Group	Conc. [mM]	RCE^a [%]	RTG^b [%]	MF^c[mutants/10^6cells]	IMF^d[mutants/10^6cells]	GEF^eexceeded	Statistical Significant Increase^f	Precipitate
Exp I	C1	0	105.3	112.1	67.2	/	/	/	-
	C2		81.8	89.2		/	/	/	-
	S1	0	100.0	100.0	53.5	/	/	/	-
	S2					/	/	/	-
	3	0.2	87.2	82.0	77.9	24.4	-	+	-
	4	0.5	90.1	92.6		0.0	-	-	-
	5	1.0	99.7	93.2	53.5	-1.1	-	-	-
	6	2.5	80.6	77.2	52.4	32.3	-	+	-
	7	5.0	78.2	63.4	85.8	33.2	-	+	-
	8	7.5	74.7	63.6	86.7	9.6	-	-	-
	9	9.0	93.1	70.1	63.1	-1.6	-	-	-
	10	10	80.6	60.6	51.9	42.2	-	+	-
	EMS	300 µg/mL	84.4	70.2	95.7	574.4	+	+	-
	MMS	10 µg/mL	57.6	49.2	627.9	631.9	+	+	-
Exp II	C1	0	116.9	135.9	66.0	/	/	/	-
	C2		116.9	128.7		/	/	/	-
	S1	0	100.0	100.0	93.1	/	/	/	-
	S2					/	/	/	-
	1	0.05	97.0	100.3	90.6	-2.5	-	-	-
	2	0.1	106.3	102.6	86.1	-7.1	-	-	-
	3	0.2	101.5	91.8	90.3	-2.8	-	-	-
	4	0.5	115.0	92.9	89.0	-4.2	-	-	-
	5	1.0	103.0	79.1	108.0	14.9	-	-	-
	6	2.5	111.4	67.1	90.0	-3.1	-	-	-
	7	5.0	107.9	31.9	84.8	-8.3	-	-	-
	8	7.5	80.3	11.3	150.0	56.9	-	+	-
	EMS	200 µg/mL	51.5	27.2	2490.8	2397.7	+	+	-
	MMS	8 µg/mL	63.1	39.8	1179.4	1086.3	+	+	-

 

C: Negative Controls

S: Solvent Controls (DMSO)

a: Relative Cloning Efficiency, RCE = [(CE_{dose group}/ CE_{of corresponding controls}) x 100]

    Cloning Efficiency, CE = ((-LN(((96-(mean P1,P2)) / 96)) / 1.6) x 100)

b: Relative Total Growth, RTG = (RSG x RCE)/100

c: Mutant Frequency,

   MF = {-In [negative cultures/total wells (selective medium)] / -In [negative cultures/total wells (non selective medium)]} x 800

d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f: Statistical significant increase in mutant frequency compared to solvent controls (Mann-Whitney test, p < 0.05). +: significant; - not significant

EMS: Ethylmethanesulfonate [200 and 300 µg/ml]

MMS: Methyl methanesulfonate [8 and 10 µg/ml]

 

Table 2: Summary: Experiment I and II, witth metabolic activation

	Test Group	Conc. [mM]	RCE^a [%]	RTG^b [%]	MF^c[mutants/10^6cells]	IMF^d[mutants/10^6cells]	GEF^eexceeded	Statistical Significant Increase^f	Precipitate
Exp I	C1	0	86.4	81.9	58.3	/	/	/	-
	C2		95.0	93.6		/	/	/	-
	S1	0	100.0	100.0	55.1	/	/	/	-
	S2					/	/	/	-
	3	0.5	83.8	73.5	53.7	-1.4	-	-	-
	4	0.5	89.1	92.1	56.5	1.3	-	-	-
	5	1.0	90.5	79.3	33.5	-21.7	-	-	-
	6	2.5	95.0	89.4	56.0	0.9	-	-	-
	7	5.0	106.9	97.7	32.1	-23.0	-	-	-
	8	7.5	93.4	85.4	54.1	-1.1	-	-	-
	9	9.0	77.7	60.5	52.6	-2.5	-	-	-
	10	10	74.4	64.3	57.1	1.9	-	-	-
	B[a]P	2.5 µg/mL	72.2	35.7	494.9	439.7	+	+	-
Exp II	C1	0	105.9	92.6	84.1	/	/	/	-
	C2		102.7	98.7		/	/	/	-
	S1	0	100.0	100.0	89.9	/	/	/	-
	S2					/	/	/	-
	3	0.1	93.7	95.3	80.1	-9.8	-	-	-
	4	0.3	109.4	117.7	79.6	-10.3	-	-	-
	5	0.7	95.1	93.8	98.6	8.7	-	-	-
	6	2.0	81.0	83.6	131.4	41.5	-	-	-
	7	4.0	104.3	104.5	87.9	-2.0	-	-	-
	8	6.0	113.0	118.9	78.4	-11.5	-	-	-
	9	8.0	93.7	91.8	94.9	5.0	-	-	-
	10	10	105.9	110.0	77.5	-12.4	-	-	-
	B[a]P	2.5 µg/mL	92.3	62.6	575.9	+	+	+	-

 

C: Negative Controls

S: Solvent Controls (DMSO)

a: Relative Cloning Efficiency, RCE = [(CE_{dose group}/ CE_{of corresponding controls}) x 100]

    Cloning Efficiency, CE = ((-LN(((96-(mean P1,P2)) / 96)) / 1.6) x 100)

b: Relative Total Growth, RTG = (RSG x RCE)/100

c: Mutant Frequency,

   MF = {-In [negative cultures/total wells (selective medium)] / -In [negative cultures/total wells (non selective medium)]} x 800

d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f: Statistical significant increase in mutant frequency compared to solvent controls (Mann-Whitney test, p < 0.05). +: significant; - not significant

B[a]P: Benzo[a]pyrene [2.5 µg/mL]

 

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item bis[2-(2-butoxyethoxy)ethyl]butanedioate is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L1578Y cells.

Executive summary:

The test item bis[2-(2-butoxyethoxy)ethyl]butanedioate was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

 

The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I 10 mM (without and with metabolic activation) was selected as the highest concentrations. In experiment II 7.5 mM (without metabolic activation) and 10 mM (with metabolic activation) were selected as the highest concentrations. Experiment I without and with metabolic activation and Experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

 

The test item was investigated at the following concentrations:

Experiment I without and with metabolic activation: 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, 9.0 and 10 mM

Experiment II without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0 and 7.5 mM; with metabolic activation:  0.1, 0.3, 0.7, 2.0, 4.0, 6.0, 8.0 and 10.0 mM

 

No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation. In experiment II with metabolic activation no growth inhibition was observed.

 

In experiment I without metabolic activation the relative total growth (RTG) was 60.6% for the highest concentration (10 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 64.3%. In experiment II without metabolic activation the relative total growth (RTG) was 11.3% for the highest concentration (7.5 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 110%.

 

In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A key study for mammalian gene mutation with bis[2-(2-butoxyethoxy)ethyl]butanedioate was conducted in the mouse lymphoma thymidine kinase assay using the cell line L5178Y (BioService, 2015d).The selection of the concentrations used in the main experiments was based on data from pre-experiments. In experiment I 10 mM (without and with metabolic activation) was selected as the highest concentrations. In experiment II 7.5 mM (without metabolic activation) and 10 mM (with metabolic activation) were selected as the highest concentrations. Experiment I without and with metabolic activation and Experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

 

The test item was investigated at the following concentrations:

- Experiment I without and with metabolic activation: 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, 9.0 and 10 mM

- Experiment II

without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0 and 7.5 mM;

with metabolic activation: 0.1, 0.3, 0.7, 2.0, 4.0, 6.0, 8.0 and 10.0 mM.

 

No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation. In experiment II with metabolic activation no growth inhibition was observed.

 

In experiment I without metabolic activation the relative total growth (RTG) was 60.6% for the highest concentration (10 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 64.3%. In experiment II without metabolic activation the relative total growth (RTG) was 11.3% for the highest concentration (7.5 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 110%.

 

In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed.

Justification for selection of genetic toxicity endpoint
Key study

Justification for classification or non-classification

Based on the results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008), bis[2-(2-butoxyethoxy)ethyl]butanedioate does not have to be classified and has no obligatory labelling requirement for mammalian muagenicity.