Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acrylate

Endpoint summary

Description of key information

positive in a LLNA (without possibility for determination of the potency by EC3 value)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-30 - 2012-07-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(adopted 24 April 2002)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Isobornyl acrylate
- Substance type: organic
- Physical state at room temperature: liquid

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8 -9 weeks (first pretest) and 10 - 11 weeks (2nd and 3 rd pretest and main study)
- Housing: single
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Lab., Horst, The Netherlands)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
- Acclimation period: At least 5 days. Under test conditions after health examination. Only animals without any visible signs of
illness were used for the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2 °C
- Humidity (%): relative humidity: 45-65 %
-
- Photoperiod (hrs dark / hrs light): artifical light: 6.00 a.m. - 6.00 p.m

Vehicle:

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

Concentration:

Test concentrations (main study): 0 (vehicle group), 5, 10 and 25 %
Test concentrations (non-GLP) pre-tests: 5, 10, 25, 50 and 100%

No. of animals per dose:

Main study: 20 females (nulliparous and non-pregnant), 3 dose groups and 1 vehicle group of 5 animals, respectively.
Pre-tests (non-GLP): 3 x 2 females

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 different test item concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1 v/v). 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 (day 6) 250 µL of phosphate-buffered saline (PBS) containing 20.2 µCi of 3HTdR (equivalent to 3HTdR 80.6 µCi/mL) were injected into each test and control mouse via the tail vein.
Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Sodium (Release, WDT, 30827 Garbsen, Germany).
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). 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 -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 -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Interpretation of Raw Data
The proliferative response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal 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.
Observations
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (systemic toxicity or local skin irritation) were recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables and for the DPM values
(group mean DPM ± standard deviation).
The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together.

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitisation properties in CBA/CaOlaHsd mice. The periodic positive control experiment was performed with -Hexyl cinnamaldehyde in acetone:olive oil (4+1 v/v) using CBA/CaOlaHsd mice in April 2012.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see: Remarks on results including tables and figures (results of Individual data)

Parameter:

SI

Value:

4.07

Test group / Remarks:

5%

Parameter:

SI

Value:

14.07

Test group / Remarks:

10%

Parameter:

SI

Value:

22.84

Test group / Remarks:

25%

Table 1: Calculation and Results of Individual Data

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

Test item concentration			DPM values measured	DPM-BG per animal (2 lymph nodes)^a)	S.I.^b)
% (w/w)	Group no.	Animal no.
---	---	BG I	21	---	---
---	---	BG II	23	---	---
0	1	1	442	420	---
0	1	2	1174	1152	---
0	1	3	369	347	---
0	1	4	1073	1051	---
0	1	5	794	772	---
5	2	6	2308	2286	3.1
5	2	7	3784	3762	5.0
5	2	8	3546	3524	4.7
5	2	9	2909	2887	3.9
5	2	10	2779	2757	3.7
10	3	11	6640	6618	8.8
10	3	12	10884	10862	14.5
10	3	13	7731	7709	10.3
10	3	14	12319	12297	16.4
10	3	15	15193	15171	20.3
25	4	16	16576	16554	22.1
25	4	17	22879	22857	30.5
25	4	18	16657	16635	22.2
25	4	19	15713	15691	21.0
25	4	20	13734	13712	18.3

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

1    =  Control Group

2-4 =  Test Groups

S.I. =  Stimulation Index

^a)   =  values corrected for mean background value (BGI and BGII).

^b)          =   Stimulation Indices relative to the mean of the control group (Group 1)

Table 2: Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD^b)	S.I.
Vehicle Control Group (acetone:olive oil (4+1 v/v))	748.4	361.9	1.00
5% Isobornyl acrylate	3043.2	597.4	4.07
10% Isobornyl acrylate	10531.4	3465.3	14.07
25% Isobornyl acrylate	17089.8	3432.2	22.84

a)      Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

b)      Standard Deviation

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

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item was found to be a skin sensitiser under the described conditions.

Executive summary:

In the study the test item Isobornyl acrylate dissolved in acetone:olive oil (4+1 v/v) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

A test item is regarded as a sensitizer in the LLNA if exposure to one or more test item concentrations results in a 3-fold or greater

increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 4.07, 14.07 and 22.84 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1 v/v), respectively. A clear dose response was observed. Since the S.I. values of all treatment groups were above the threshold value of 3, the EC3 value could not be calculated.

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In a Local Lymph Node Assay in mice according to OECD TG 429 with GLP, Isobornyl acrylate revealed skin sensitizing properties.

Short description of key information:
Isobornyl acrylate is considered as skin sensitizer due to the results of a valid OECD 429 GLP study.

In the study the test item Isobornyl acrylate was dissolved in acetone:olive oil (4+1 v/v) in concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.A test item is regarded as a sensitizer in the LLNA if exposure to one or more test item concentrations results in a 3-fold or greater

increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 4.07, 14.07 and 22.84 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1 v/v), respectively. A clear dose response was observed. Since the S.I. values of all treatment groups were above the threshold value of 3, the EC3 value could not be calculated.

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

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Due to the results of a valid key study available, Isobornyl acrylate is classified as skin sensitiser category 1 according to Regulation 1272/2008/EC or UN-GHS. A potency could not be calculated based on available data.