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

Skin irritation / corrosion

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

807-645-4

Cas Number:

701920-77-6

Molecular formula:

C20H38O8

IUPAC Name:

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

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): bis[2-(2-butoxyethoxy)ethyl]butanedioate; Di-butoxy-ethoxy-ethyl succinate
- Substance type: Organic mono constituent substance
- Physical state: Colourless liquid
- Analytical purity: 97.1%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: 97.1%
- Purity test date: 08/07/2014
- Lot/batch No.: 20142205JC
- Expiration date of the lot/batch: Not applicable
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

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

Test system

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.

Results and discussion

In vitro

Results

Results 1

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)

Results 2

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)

Any other information on results incl. tables

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%). 

Applicant's summary and conclusion

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.