reaction products of diglycidyl ether bisphenol F (DGEBF) and oligomeric phenol diglycidyl ethers with acrylic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-07-20 - 2010-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

During the acclimation phase of the animals used for the pre-experiment, the relative humidity in the animal room was between approximately 45 - 76 % for a few hours and the temperature was between 22 + 3°C for a few hours.

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:

reaction products of diglycidyl ether bisphenol F (DGEBF) and oligomeric phenol diglycidyl ethers with acrylic acid

EC Number:

700-487-6

Molecular formula:

Not applicable.

IUPAC Name:

reaction products of diglycidyl ether bisphenol F (DGEBF) and oligomeric phenol diglycidyl ethers with acrylic acid

Details on test material:

Identity: Epoxy half acrylate

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

The test item in the main study was assayed at 10, 25, and 50%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.

No. of animals per dose:

4 females for each test group and 4 females for control group

Details on study design:

Topical Application

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 10, 25, and 50% (w/w) in dimethylformamide. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-Methyl Thymidine

3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytic, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL).
Five days after the first topical application, all mice were administered with 250 µL of 81.8 µCi/mL 3HTdR (corresponds to 20.5 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Incorporated 3HTdR

Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release, WDT, 30827 Garbsen, Germany).
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background 3HTdR levels were also measured in two 1mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Interpretation of Raw Data

The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (Stimulation Index; S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Positive control substance(s):

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

Results and discussion

Positive control results:

Concentration SI
0 % 1.00
5 % 2.04
10 % 3.41
25 % 6.14

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Concentration SI 10 % 3.76 25 % 8.04 50 % 9.54

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Concentration DPM per lymph node 10 % 1679 25 % 3591 50 % 4260

Any other information on results incl. tables

Calculation and Results of Individual Data

(Vehicle: dimethylformamide)

Test item concentration % (w/w)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b^)	S.I.
---	BG I	15	---	---	---	---
---	BG II	25	---	---	---	---
---	1	3593	3573	8	446.6	1.00
10	2	13453	13433	8	1679.1	3.76
25	3	28748	28728	8	3591.0	8.04
50	4	34100	34080	8	4260.0	9.54

BG =  Background (1 ml 5% trichloroacetic acid) in duplicate

1    =  Control Group

2-4=  Test Group

S.I. =  Stimulation Index

^a)   =  The mean value was taken from the figures BG I and BG II

^b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are above 3.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: other: CLP, EU GHS (Regulation (EC) No 1272/2008)

Conclusions:

In this study Stimulation Indices of 3.76, 8.04, and 9.54 were determined with the test item at concentrations of 10, 25, and 50% in dimethylformamide. The EC3 value could not be calculated, since all obtained SI were above 3. The test item Epoxy half acrylate was found to be a skin sensitiser under the described conditions.

Executive summary:

In the study Epoxy half acrylate dissolved in dimethylformamide was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50% (w/w).

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 3.76, 8.04, and 9.54 were determined with the test item at concentrations of 10, 25, and 50% in dimethylformamide, respectively.

The test item Epoxy half acrylate was found to be a skin sensitizer under the test conditions of this study.