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

Endpoint summary

Description of key information

Key in vitro studies were available for undiluted (97,1% pure) bis[2-(2-butoxyethoxy)ethyl]butanedioate for both skin and eye irritation potential. In the  EPISKIN-Standard Method,  a reconstituted three-dimensional human epidermis model, cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post incubation period  at 37 ± 1°C and compared to those of the concurrent negative controls. The relative mean tissue viability after 15 min of exposure and 42 h post incubation was 104.4%  which was > 50%.  In the BCOP (bovine corneal opacity and permeability) assay, three isolated corneas per group obtained freshy slaughtered, were evaluated for opacity and permeability after 10 minutes incubation at 32 ± 1°C of 750 µL of test/control substance and washing/2h incubation of the epithelium with RPMI medium at 32 ± 1°C. The Mean in vitro irritation score (IVIS) for the tested group was -1.05, which was <3.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

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 439 and GLP, and is considered to be relevant, adequate and reliable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: in vitro

Strain:

other: human skin model EPISKIN-SM (TM)

Details on test animals or test system and environmental conditions:

EPISKIN-SMTM(SkinEthic). This skin model consists of normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell cultured inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum

Amount / concentration applied:

The test item was applied undiluted. 10µL (26.3µL/cm2) of the test item were dispensed directly atop the EPISKIN-SMTM tissue.

Irritation / corrosion parameter:

other: other: mean relative tissue viability (% versus negative control)

Value:

104.4

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 15 min treatment and 42 h postincubation. Max. score: 50.0. Remarks: Test group . (migrated information)

Irritation / corrosion parameter:

other: other: relative tissue viability (% versus negative control)

Value:

8.4

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 15 min treatment and 42 h postincubation. Max. score: 50.0. Remarks: Positive control group. (migrated information)

Pre-Experiments:

The mixture of 10 µL test item per 2 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.

The mixture of 10 µL of the test item per 90 µL aqua dest. showed no colouring detectable by unaided eye-assessment.

Since the test item was not solvable in aqueous solutions, emulsification was observed for both pre-experiments.

 

Experiment:

Results

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
absolute OD550	0.937 0.911	0.923 0.960	0.934 0.937	0.116 0.119	0.129 0.125	0.115 0.110	1.098 1.069	0.966 0.912	0.906 0.886
OD550(blank-corrected)	0.893 0.86	0.880 0.916	0.890 0.893	0.072 0.076	0.085 0.081	0.071 0.066	1.055 1.025	0.922 0.868	0.862 0.843
total mean OD550of 3 replicate tissues (blank-corrected)	0.890*			0.075			0.929
SD OD550	0.009			0.007			0.098
relative tissue viabilities [%]	98.9	100.9	100.2	8.3	9.3	7.7	116.9	100.6	95.8
mean relative tissue viability[%]	100.0			8.4**			104.4
SD tissue viability [%]***	1.0			0.8			11.0
CV [% viability]	1.0			9.9			10.6

 

*    Corrected mean OD₅₅₀ of the negative control corresponds to 100% absolute tissue viability.

**  Mean relative tissue viability of the three positive control tissues is ≤ 40%.

*** The standard deviation (SD) obtained from the three concurrently tested tissues is < 18%.

 

Quality Criteria

	Value	Cut off	pass/fail
Mean OD550nm Blank	0.044	< 0.1	pass
Mean Absolute OD550nmNC	0.934	0.6 ≤ NC ≤ 1.5	pass
Mean Relative Viability [%] PC	8.4	≤ 40%	pass
SD of % Viability [%]	0.8 – 11.0	< 18%	pass

 

Historical data

	OD550Blank	Mean Absolute OD550 NC	Relative Viability [%] PC	SD of Viability [%]
Mean	0.043	0.869	12.50	8.01
SD	0.002	0.122	8.67	8.63
n	44	44	44	176

 

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM^TM (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum.

In the present study bis[2-(2-butoxyethoxy)ethyl]butanedioate was applied topically to the EPISKIN-SM^TM tissue for 15 min. followed by a 42 h postincubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with PBS.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (104.4%) after 15 min. treatment and 42 h post incubation. Furthermore, no effects of any kind were observed for the test item treated tissues.

 

The controls confirmed the validity of the study. The mean OD₅₅₀ of the six blank values was < 0.1. The mean absolute OD₅₅₀ of the three negative control tissues was ≥ 0.6 and ≤ 1.5. The mean relative tissue viability (% negative control) of the positive control was ≤ 40% ( 8.4%) . The maximum standard deviation of viability of replicate tissues of all dose groups was < 18% (0.8% - 11.0%).

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM^TM (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum.

In the present study bis[2-(2-butoxyethoxy)ethyl]butanedioate was applied topically to the EPISKIN-SM^TM tissue for 15 min. followed by a 42 h postincubation period and immediate determination of cytotoxic effects via MTT reduction assay.
Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with PBS.

 

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (104.4%) after 15 min. treatment and 42 h post incubation. Furthermore, no effects of any kind were observed for the test item treated tissues. The controls confirmed the validity of the study. The mean OD₅₅₀ of the six blank values was < 0.1. The mean absolute OD₅₅₀ of the three negative control tissues was ≥ 0.6 and ≤ 1.5. The mean relative tissue viability (% negative control) of the positive control was ≤ 40% ( 8.4%) . The maximum standard deviation of viability of replicate tissues of all dose groups was < 18% (0.8% - 11.0%). 

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In the present study the skin irritant potential of bis[2-(2-butoxyethoxy)ethyl]butanedioate was analysed. The EPISKIN-Standard Model^TM(EPISKIN-SM^TM), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, undiluted (97.1% pure) test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post incubation period at 37 + 1°C and compared to those of the concurrent negative controls.
In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 15 min of exposure and 42 h post incubation was 104.4% which was > 50%. The test item is therefore not classified by the UN GHS or EU CLP criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

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 437 and GLP, and is considered to be relevant, adequate and reliable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: ex vivo

Strain:

other: Bovine cornea

Details on test animals or tissues and environmental conditions:

The assay uses isolated corneas obtained as a by-product from animals freshy slaughtered at the abattoir A. Moksel AG, Buchloe, Germany. On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.

Amount / concentration applied:

The test item was used as provided by the sponsor.

Irritation parameter:

other: IVIS

Basis:

mean

Score:

-1.05

Max. score:

3

Remarks on result:

other: Test group

Irritation parameter:

other: IVIS

Basis:

mean

Score:

2.28

Max. score:

3

Remarks on result:

other: Negative control group

Irritation parameter:

other: IVIS

Basis:

mean

Score:

72.12

Max. score:

55

Remarks on result:

other: Positive control group

The eye irritancy potential of bis[2-(2-butoxyethoxy)ethyl]butanedioate was investigated in the bovine corneal opacity and permeability assay.
The test item was tested as provided by the sponsor.

The following mean in vitro irritation score was calculated: -1.05.

Therefore the test item was classified into UN GHS No Category.
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

 

Table 1. Opacity

Cornea No.	Test item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	4	7	3
2		4	4	0
3		4	5	1
MV		4.00	5.33	1.33
4	Positive Control	5	58	53	51.67
5		5	61	56	54.67
6		5	55	50	48.67
MV		5.00	58.00	53.00	51.67
7	Test item	3	4	1	-0.33
8		5	5	0	-1.33
9		5	5	0	-1.33
MV		4.33	4.67	0.33	-1.00

MV = mean value

 

Table 2. Permeability

Cornea No.	Test item	OD490	Corrected OD490 Value
1	Negative Control	0.043
2		0.082
3		0.065
MV		0.063
4	Positive Control	1.778	1.715
5		2.010	1.947
6		0.493	0.430
MV		1.427	1.364
7	Test item	0.083	0.020
8		0.072	0.009
9		0.025	-0.038
MV		0.060	-0.003

MV = mean value

 

Table 3. In Vitro Irritation Score

Cornea No.	Test item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	3.00	0.043	2.28
2		0.00	0.082
3		1.00	0.065
MV		1.33	0.063
4	Positive Control	51.67	1.715	72.12
5		54.67	1.947
6		48.67	0.430
MV		51.67	1.364
7	Test item	-0.33	0.020	-1.05
8		-1.33	0.009
9		-1.33	-0.038
MV		-1.00	-0.003

MV = mean value

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the evaluation criteria the test item bis[2-(2-butoxyethoxy)ethyl]butanedioate is classified into UN GHS No Category.

Executive summary:

The eye irritancy potential of bis[2-(2-butoxyethoxy)ethyl]butanedioate was investigated in the bovine corneal opacity and permeability assay.

Preparation of the test item was tested as provided by the sponsor (undiluted, 97.1% purity).The assay used three isolated corneas per group obtained as a by-product from animals freshy slaughtered, and both opacity and permeability were measured after 10 minutes incubation at 32+1°C of 750 µL of test/control substance and washing/2h incubation of the epithelium with RPMI medium at 32+1°C. The Mean in vitro irritation score (IVIS) for the tested group was -1.05, which was <3.

The in vitro irritation score obtained with the positive control fell within the expected ranges (<3 and >55, respectively).

According to the evaluation criteria the test item bis[2-(2-butoxyethoxy)ethyl]butanedioate is not classified by the UN GHS or EU CLP criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

A key study for skin irritant potential of bis[2-(2-butoxyethoxy)ethyl]butanedioate was conducted by means of the EPISKIN-Standard Model^TM(EPISKIN-SM^TM). Hereby, the undiliuted (97.1% pure) test item was applied to a reconstituted three-dimensional human epidermis model (BioService, 2015a). Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post incubation period at 37 ± 1°C and compared to those of the concurrent negative controls. In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 15 min of exposure and 42 h post incubation was 104.4% which was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS or EC CLP criteria.

 

The eye irritancy potential of bis[2-(2-butoxyethoxy)ethyl]butanedioate was investigated in the bovine corneal opacity and permeability assay (BioSerivice, 2015b). Preparation of the test item was tested as provided by the sponsor (undiluted, 97.1% purity).The assay used three isolated corneas per group obtained as a by-product from animals freshy slaughtered, and both opacity and permeability were measured after 10 minutes incubation at 32 ± 1°C of 750 µL of test/control substance and washing/2h incubation of the epithelium with RPMI medium at 32 ± 1°C. The Mean in vitro irritation score (IVIS) for the tested group was -1.05, which was <3. The in vitro irritation score obtained with the positive control fell within the expected ranges (<3 and >55, respectively). According to the evaluation criteria the test item bis[2-(2-butoxyethoxy)ethyl]butanedioate is not classified by the UN GHS or EU CLP criteria.

Justification for selection of skin irritation / corrosion endpoint:
Key study

Justification for selection of eye irritation 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 skin irritation and eye irritation.