1,4-Butanediyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 28th to December 22nd, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted April 24th, 2002

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-{[3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoyl]oxy}butyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

EC Number:

700-652-2

Cas Number:

1259300-69-0

Molecular formula:

C20H34O8

IUPAC Name:

4-{[3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoyl]oxy}butyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories (Bar Harbor, ME).
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: animals were born on 13 Sep 2011 and 11 Oct 2011 ± 3 days
- Weight at study initiation: 19.0-23.4 g (screen), 18.5-22.7 g (main).
- Housing: housed 1 per cage. Paper bedding placed beneath cages and changed at least 3 times per week.
- Diet: fresh PMI rodent chow (diet #5001) ad libitum.
- Water: ad libtium.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: continuously monitored using automatic recording devices.
- Humidity: continuously monitored using automatic recording devices.
- Photoperiod: 12 hrs dark /12 hrs light.

IN-LIFE DATES: From: 28 Nov 2011 To: 22 Dec 2011

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

5 % (v/v), 50 % (v/v) and 100 % (v/v)

No. of animals per dose:

5

Details on study design:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Screen test: 200 µl was added to 200 µl of Dimethylformamide (DMF) to yield a 50 % (v/v) solution. An additional dilution was made in DMF. The test article was also used as received. - Main test: 300 µl was added to 300 µl of Dimethylformamide (DMF) to yield a 50 % (v/v) solution. An additional dilution was made in DMF. The test article was also used as received.

PRELIMINARY DERMAL IRRITATION SCREEN: nine groups (2 animals per group) were treated with the test substance, for three consecutive days. Treatment was made by topical application of the test substance concentrations to the dorsum of each ear once daily for three consecutive days. The test substance was spread over the entire dorsal surface of the ear using a micropipette at 25 µl/ear.

MAIN STUDY: the test substance concentrations used in the main LLNA study were chosen such that the maximum concentration tested was the highest achievable solution of the test substance in the vehicle, while avoiding both overt systemic toxicity and excessive local dermal irritation. Concentrations of 25 % (v/v), 50 % (v/v) and 100 % (v/v) were chosen. 5 animals per group were treated by topical application of the test substance concentrations, vehicle (N,N-Dimethylformamide) or positive control (25 % v/v) in the same manner as in the screen. A Naive group of 5 animals was sham-treated. DMF was chosen as the vehicle, based on solubility.

TYPE AND FREQUENCY OF OBSERVATIONS: aIl animals in the study were observed once daily throughout the study for clinical signs, either of local irritation at the application site or systemic toxicity, and for mortality.
 
MEASUREMENTS
- Body weight measuremetns: recorded on Day 1, immediately prior to dosing, and on Day 6 (prior to sacrifice in the screen, and prior to BrdU injection in the main test).
- Ear thickness measurements: on Day 1 prior to dosing, on Day 3 before the third test substance application (approximately 48 h after the first test substance application), and on Day 6 before sacrifice (approximately 120 h after the first dose and 72 h after the third dose).
- Changes in ear thickness on Day 3 and Day 6 relative to Day 1 were expressed as a percent of the Day 1 pre-dose values. Ear thickness increases of 25 % or more were considered biologically significant (based on the scientific literature and historical laboratory data) and deemed indicative of a greater than moderate local dermal irritation response.
 
NODE ISOLATION AND PROCESSING
On Day 6 of the main test, approximately 120 h following the initial dose, and 5 h prior to sacrifice, the mice were injected with S-bromo--2'-deoxy-uridine (BrdU), in Dulbecco's Phosphate Buffered Saline (DPBS) at a dose of 200 µl per mouse (approximately 150 mg/kg (15 mg/ml). The BrdU was administered by intraperitoneal injection using a 27-gauge needle. This thymidine analog becomes incorporated into the DNA of proliferating cells, including proliferating nodal lymphocytes. The mice were sacrificed using CO2 asphyxiation, the auricular lymph nodes were collected and the jugular vein was opened. The auricular lymph nodes were combined and single-cell suspensions were generated in Fetal Bovine Serum (FBS) and fixed with 85 % ethanol. The cell suspensions were used to determine BrdU incorporation into the lymphocyte DNA (percentage of proliferating BrdU+ Lymph Node Cells (% BrdU+ LNC)) and the total number of cells in the nodes, for each individual animal.
 
FLOW CYTOMETRY: flow cytometric analyses were conducted using a FAC Scan flow cytometer equipped with an Omnichrome argon laser emitting at 488 nm with 15 mW of power. For DNA and/or BrdU determinations, clumps of nuclei were excluded from analysis using gates set on integrated red fluorescence signals. The histograms generated were analyzed using Cell Quest Software.
 
DETERMINATION OF STIMULATION INDEX
- Proliferation of lymphocytes (No. of BrdU+ cells): measured aliquots of fixed cells were washed and resuspended in 1 N HCl containing 0.5 % TritonX-100. This acid denaturation step allows the BrdU antibody to quantitatively interact with BrdU that has been incorporated into the cellular DNA. Following a one-hour denaturation period, the samples were neutralized by washing with sodium tetraborate. The cell nuclei were then washed with a staining buffer (1.0 % Bovine Serum Albumin, 0.03 % sodium azide and 0.5 % Tween 20 in Phosphate buffered saline (PBS) and incubated with the BrdU-specific (fluorescein conjugated) antibody. The nuclei were again washed with the staining buffer, resuspended in OPBS containing the DNA-specific dye propidium iodide and the percentage of nuclei staining positive for BrdU (i.e., proliferating cells in "S" phase) was determined using flow cytometry.
- Cell number detrmination: to determine total number of cells in the nodes for each animal, a measured aliquot of fixed cells was transferred to a tube containing propidium iodide, papain and Tween 20. The number of cells in each aliquot (representing a1/500 dilution of the LNC suspension) was determined by flow cytometry. The total number of cells in the lymph node was determined by sampling an aliquot that represented 1/50 (dilution factor = 500) of the total number of cells in the node (Equation 1). To determine the number of BrdU+ cells in the lymph node, the total number of LNCs counted in an aliquot of the cell suspension was multiplied by the percentage of LNCs that incorporated BrdU (Equation 2).
Equation 1: (Mean no. of cells in aliquot) X (Dilution Factor) = Total no. of cells in lymph node
Equation 2: (Total no. of cells in node) X (BrdU+ Cells) %/100 % = No. of proliferating lymphocytes
 
ANALYSIS OF STIMULATION INDEX DATA
- Calculation of Stimulation Index (SI): for each animal, lymph node cell proliferation as measured by the number of Proliferating Lymphocytes was determined by flow cytometry and the mean ± SD was then calculated for each group. The number of Proliferating Lymphocytes in each animal was then divided by the mean number of Proliferating Lymphocytes in the Vehicle Control group (for the 100 % test substance group, the mean number of Proliferating Lymphocytes was divided by the mean number of BrdU+ LNC in the Naive Control group.) This “Test / Control Ratio” is the "Stimulation Index" (SI) and was calculated:
No. of proliferating lymphocytes per animal/ Mean no. of proliferating lymphocytes of vehicle control group = Stimulation Index (SI) per animal (Equation 3).
 
INTERPRETATION OF DATA: a test substance is considered to have a sensitizing potential if treatment results in a 3-fold or greater increase in the number of proliferating lymphocytes (Equation 3) relative to that obtained for the vehicle control. Therefore, test substance that yield a SI ≥ 3 are characterized as potential sensitizing substances.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

SI values were calculated with the mean group SI and standard deviation and ANOVA followed by the Students' t-Test

Results and discussion

Positive control results:

The SI of the positive control group, 25 % HCA, was 4.2 therefore it is characterised as a sensitising substance

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.9

Test group / Remarks:

100 % v/v

Results 2

Parameter:

SI

Value:

1.5

Test group / Remarks:

50 % v/v

Results 3

Parameter:

SI

Value:

1.2

Test group / Remarks:

25 % v/v

Cellular proliferation data / Observations:

Topical application of the test substance at 25 and 50 % (v/v) in the DMF vehicle, and at 100 %, (v/v) resulted in SI values less than 3 (SI < 3.0). Therefore, the test substance is not a skin sensitizer in the LLNA. None of the test concentrations tested resulted in dermal irritation. The positive control had a greater than 25 % increase in ear thickness on day 6 which is consistent with historical results in the literature.

DETAILS ON STIMULATION INDEX CALCULATION
SI were calculated by dividing the total number of proliferating lymphocytes for each animal by the mean number of proliferating lymphocytes of vehicle-exposed group. For both the naive control group and the 100 % test article group, the total lymphocyte proliferation was divded by that of the naive control group

CLINICAL OBSERVATIONS:
- Preliminary dermal irritation test: the test article treatment did not result in increases in ear thickness of 25 % or more so the test substance was not considered irritating.
- All animals survived the in-life phase of the study.
- One mouse in the 100 % test dose group had few faeces on Day 4 and 5 of the main study and appeared emaciated on Day 5, but recovered by Day 6. All other animals were observed to be normal.

TEST SUBSTANCE FORMULATION AND DOSING
The test article formed homogenous solution in the vehicle, DMF. No difficulties were experiences with the application of the test articles to the ears or with the retention of test articles by esr surface. Test article residue was observed at the dose sites on all animals treated with the test substance in both the screen day (Day 4) and the main test (starting on Day 3). Test substance residue at the dose sites was observed on all animals in the 25 % HCA Positive Control group beginning on Day 3 of the main test. This residue did not affect ear measurement.

BODY WEIGHTS:
- Losses were noted but were insignificant (<2 grams).

Applicant's summary and conclusion

Interpretation of results:

other: not classified for skin sensitisation according to the CLP Regulation (EC) No.1272/2008

Conclusions:

Non-skin sensitiser

Executive summary:

The study was performed to determine the sensitizing potential of the topically applied test substance according to the OECD guideline 429 and EPA OPPTS 870.2600 in compliance with GLP.

The test substance concentrations used in the main LLNA study were chosen such that the maximum concentration tested was the highest achievable solution of the test substance in the vehicle, while avoiding both overt systemic toxicity and excessive local dermal-irritation. Concentrations of 25, 50 and 100 % (v/v) were chosen. Five animals per group were treated by topical application of the test substance concentrations, vehicle or positive control in the same manner as in the screen. A naive group of five animals was sham-treated. DMF was chosen as the vehicle, based on solubility. 

Under the test conditions, topical application of the test substances a 25 and 50 %(v/v) in the DMF vehicle, and at 100 % (v/v), resulted in SI values less than 3 (SI < 3.0). Therefore, the test substance is not a skin sensitizer in the LLNA.