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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study complies to OECD Test Guidance 429 and GLP, and is considered to be relevant, adequate and reliable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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; DBEESU
- Substance type: Organic mono constituent substance
- Physical state: 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

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories GmbH, 5800 AN Venray, The Netherlands
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 18-21 g
- Fasting period before study: no data
- Housing: The animals were kept in groups of 5 animals in IVC cages, type 11 L, polysulphone
cages on Altram in saw fibre bedding (prescreen test and main study: lot no. 290114)
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice (prescreen test and main test study: lot no. 1526)
- Water (e.g. ad libitum): Free access to tap water, Sulphur acidified to a pH of approximately 2.8
- Acclimation period: Adequate acclimatisation period (at least five days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): At least 10 x I hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Study Initiation date: 23 June, 2014
Experimental Completion Date: 29 July, 2014

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25%,50% (each diluted with AOO 4:1, v/v) and 100% (undiluted test item)

No. of animals per dose:

5 mice/dose group (main test); 5 mice/prescreen test

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Due to the solubility properties of the test item the vehicle AOO (4:1 (v/v) acetone I olive oil) was used.
- Irritation: No specific findings
Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals.
The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in AOO (Acetone, Sigma, lot no. SZBD1425V, expiry date: 12/2014; olive oil highly refined, Sigma, lot no. BCBM3643V, expiry date: 06/08/2014).
In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination. Both ears were observed for erythema and scored according to Table 1. Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours
after the first dose) and day 6. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%.

Four animals were treated by topical application with the test item on three consecutive days at the following concentrations to the entire dorsal surface of each ear:
Animals no. 1 and no. 2 were treated with a test item concentration of 100% (undiluted)
Animals no. 3 and no. 4 were treated with a test item concentration of 50% (diluted with AOO)
One further animal was treated with 100% AOO and served as negative control.
Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of bath ears of all animals was measured (see Table 2).

During this period also all clinical signs were recorded.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
Neither signs of systemic toxicity nor signs of irritation at any application site could be detected in any animal. For details see Table 3.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the prescreen test (see Table 4).

- Lymph node proliferation response: Not assessed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+((3-d)/(b-d)]x(a-c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. lf all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine – incorporation into lymph node cells of the test group animals, relative to that recorded for the lymphnodes of control group animals (Stimulation Index equal to or greater than 3.0).
On the basis of !he test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/2011 as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, fifth revised edition, 2013 :
Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1 A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤ 2%
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2%
WARNING, exclamation mark. May cause an allergic skin reaction.

TREATMENT PREPARATION AND ADMINISTRATION:
Based on the results observed in the prescreen test the following test item concentrations were selected for the main study: 25%,50% (each diluted with AOO 4:1, v/v) and 100% (undiluted test item). The preparations were made immediately prior to each dosing.
The animals were randomly selected.
Identification was ensured by cage number and individual marking (tail).
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity. including dermal irritation at site of application.
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days.

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/mL.

Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4 °C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of lncorporated 3H -Methyl Thymidine
The 3H-methyl thymidine - incorporation was measured in a 15-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

None of the three tested concentrations of the test item reached the stimulation index of 3. The stimulation index at a concentration of 25% was 0.9 The stimulation index at a concentration of 50% was 0.9 The stimulation index at a concentration of 100% was 1.0 All animals survived throughout the test period without showing any clinical signs.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table 1. Individual Data : Radioactive Determination of the Test Substance Groups under Any other information on results incl. tables.

Any other information on results incl. tables

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Table 1. Individual Data : Radioactive Determination of the Test Substance Groups

POS	CPM	Test item	Conc. [%]	Animal number	DPM	DPM mean background	DPM/Node	Stimulation- index
21	738.0	Negative Control	100	16	1974.0	1943.6	971.8
22	1163.0			17	3134.0	3103.6	1551.8
23	812.0			18	2213.0	2182.6	1091.3
24	1470.0			19	3919.0	3888.6	1944.3
25	964.0			20	2577.0	2546.6	1273.3
MV	1029.4			MV	2763.4	2733.0	1366.5	1.0
SD	264.0			SD	697.7	697.7	348.8
31	431.0	bis[2-(2-butoxyethoxy) ethyl] butanedionate in AOO	25	1	1123.0	1092.6	546.3	0.4
32	1350.0			2	3605.0	3574.6	1787.3	1.3
33	676.0			3	1801.0	1770.6	885.3	0.6
34	1250.0			4	3350.0	3319.6	1659.8	1.2
35	854.0			5	2361.0	2330.6	1165.3	0.9
MV	912.2			MV	2448.0	2417.6	1208.8	0.9
SD	345.4			SD	931.0	931.0	465.5	0.3
36	841.0	bis[2-(2-butoxyethoxy) ethyl] butanedionate in AOO	50	6	2296.0	2267.6	1133.8	0.8
37	1024.0			7	2744.0	2713.6	1356.8	1.0
38	664.0			8	1773.0	1742.6	871.3	0.6
39	1223.0			9	3273.0	3242.6	1621.3	1.2
40	1130.0			10	3006.0	2975.6	1487.8	1.1
MV	976.4			MV	2618.8	2588.4	1294.2	0.9
SD	201.3			SD	531.4	531.4	265.7	0.2
41	1093.0	bis[2-(2-butoxyethoxy) ethyl] butanedionate	100	11	2948.0	2917.6	1458.8	1.1
42	1909.0*			12	5095.0*	n.d.	n.d.	n.d.
43	870.0			13	2343.0	2312.6	1156.3	0.8
44	755.0			14	2027.0	1996.6	998.3	0.7
45	1318.0			15	3527.0	3496.6	1748.3	1.3
MV	1009.0			MV	2711.3	2680.9	1340.4	1.0
SD	215.9			SD	575.6	575.6	287.8	0.2
71	13.0	Background Szinti and TCA			34.0
72	13.0				33.0
73	10.0				26.0
74	11.0				29.0
75	12.0				30.0
MV	11.8			MV	30.4	0.0	0.0	0.0
SD	1.2			SD	2.9

* = outlier, failed Nalimov, n.d. = not determined

If not noted individually, the results include both lymph nodes of an animal.

POS = position in counter; CPM = counts per minute; DPM = disintegrations per minute; SD = standard deviation; MV = mean value; Szinti = scintillation fluid; TCA = trichloroacetic acid

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.
Consequently, according to OECD 429 the test item bis[2-(2-butoxyethoxy)ethyl]butanedioate as described in this report is expected to have no sensitising properties and therefore should not be regarded as a dermal sensitiser.
According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item bis[2-(2-butoxyethoxy)ethyl]butanedioate has no obligatory labelling requirement for skin sensitisation and is unclassified.

Executive summary:

Based on the results of a prescreen test, the test item was assessed for sensitising properties in an LLNA assay in CBA/CaOlaHsd mice (5 females/group) at concentrations of 25% (v/v), 50% (v/v) , each diluted with AOO 4:1, (v/v) and 100% (undiluted test item). The vehicle used was AOO (4: 1 (v/v) acetone/olive oil). At the daily clinical observation the animals did not show any visible clinical symptoms and no case of mortality was observed. None of the three tested concentrations of the test item reached the stimulation index of 3: the stimulation index at a concentration of 25, 50 and 100% was 0.9, 0.9 and 1.0, respectivey. The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3. Consequently, according to OECD 429 the test item bis[2-(2butoxyethoxy)ethyl]butanedioate as described in this report is expected to have no sensitising properties and therefore should not be regarded as a dermal sensitiser.

According to Commission Regulation (EU) No 286/2011 as well as UN GHS or EC CLP, the test item bis[2-(2-butoxyethoxy)ethyl]butanedioate has no obligatory labelling requirement for skin sensitisation and is unclassified.