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Description of key information

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:
26 Oct 2005 - 15 Nov 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study conducted in compliance with GLP regulations.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Interfauna UK Limited, UK
- Strain: CBA/Ca/Ola/Hsd
- Diet (e.g. ad libitum): Diet (RM1), supplied by Special Diet Services Limited, UK
- Water (e.g. ad libitum): Mains water
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12/12


Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1, 2.5, 5, 10 or 25 % w/v
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
A preliminary sighting study was conducted on one animal per dose group for 2.5, 10 and 50 % w/v concentrations to determine the acceptable toxicity and lymph node activation levels.


MAIN STUDY
Groups of four female mice were used for the main LLNA study. Approximately 25μl of a 1, 2.5, 5, 10 or 25 % w/v preparation of the test substance in acetone in olive oil (4:1) was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone in olive oil (4:1) alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all the animals were injected, via the tail vein, with
approximately 250μl of phosphate buffered saline (PBS) containing 20 μCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.
A single cell suspension was prepared by mechanical disaggregation of lymph nodes through 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10ml of PBS. Approximately 3ml of 5% w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1ml of TCA.
The lymph node suspensions were transferred to scintillation vials and 10ml of scintillant
(Optiphase) was added prior to β-scintillation counting using a Packard Tri-Carb 3100TR Liquid Scintillation Counter.

Clinical observations: Animals were checked at least once daily for signs of systemic toxicity.
Bodyweights: The bodyweight of each animal was recorded prior to dosing on day 1 and prior to injection of 3H-methyl thymidine on day 6 for LLNA study mice or prior to termination for sighting study mice.

Positive control study: Approximately 25μl of a 5%, 10% or 25% w/v preparation of hexylcinnamaldehyde in acetone in olive oil was applied, and a vehicle control group was similarly treated using acetone in olive oil alone (4:1).

Data evaluation:
The results are expressed as disintegrations per minute (dpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle control group to give a test:control ratio known as the stimulation index (SI), for each concentration. The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

EC3 calculations
The estimated concentration of the test substance required to produce a 3-fold increase in the draining lymph node cell proliferative activity was calculated (EC3).
The EC3 value was derived by interpolating between two points on the Stimulation Index (SI) axis, one immediately above and the other immediately below the SI value of 3 (vehicle-treated control values [SI=1] not being used for the latter). Where the data points lying immediately above and below the SI value of three have the co-ordinates a (the concentration giving the SI immediately above 3), b (the SI of a), c (the concentration giving the SI immediately below 3) and d (the SI of c), the EC3 value was calculated using the following equation: EC3 = [(3-d)/(b-d)] x (a-c) + c
The quantity applied per square centimetre was derived from this value, assuming that the area of the mouse ear is 1cm2 and that 1μl is equivalent to 1mg.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The application of hexylcinnamaldehyde at concentrations of 5%, 10% and 25% w/v in acetone in olive oil (4:1) resulted in a greater than 3-fold increase in isotope incorporation at the 10 and 25 % w/v concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.
Parameter:
SI
Remarks on result:
other: 8.7
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: At 25 % w/v 51892 dpm.

Concentration of

test substance

(% w/v)

Number of lymph

nodes assayed

Disintegrations

per minute

(dpm)

dpm per

lymph node

Test : control

ratio (SI)

0 (vehicle only)

8

5964

746

N/A

1

8

4722

590

0.8

2.5

8

7956

995

1.3

5

8

8213

1027

1.4

10

8

15161

1895

2.5

25

8

51892

6487

8.7

EC3

Calculated to be 11.2 % (2800 µg/cm2)

N/A- not applicable

The application of the test substance at concentrations of 1, 2.5, 5, 10 and 25 % w/v in acetone in olive oil (4:1) resulted in an increase in isotope incorporation which was greater than 3-fold at the 25 % w/v concentration. Consequently, the test substance was shown to be a potential skin sensitiser. The concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was calculated to be 11.2 % w/v (2800 μg/cm2), indicative of a sensitiser of weak potency.

Interpretation of results:
sensitising
Remarks:
Migrated information
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitising potential of butyl acrylate was investigated in a Local Lymph Node Assay (LLNA) conducted according to OECD guideline 429 and GLP regulations (BAMM 2006). The application of the test substance at concentrations of 1, 2.5, 5, 10, or 25 % w/v in acetone : olive oil (4:1) resulted in an increase in isotope incorporation which was greater than 3-fold at the 25 % w/v concentration. Consequently, the test substance was shown to be a potential skin sensitiser. The concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was calculated to be 11.2 % w/v (2800 μg/cm2), indicative of a sensitiser of weak potency.

Butyl acrylate was tested in guinea pigs and mice using a variety of methods and gave a positive response in most of the tests. Butyl acrylate (purity > 99%) was positive in the Guinea Pig Maximization test. Positive results were noted in 7 out of 10 animals at challenge and re-challenge (Van der Walle, 1982). Butyl acrylate was tested by Parker and Turk (1983) in guinea pigs using a variety of methods and gave a positive response in all three tests.

The Mouse Ear Swelling Test in female B6C3F 1 mice did not indicate n-butyl acrylate as a sensitizer. Sensitization with concentrations of 10, 20 or 30 % butyl acrylate, followed by a challenge with 30 % butyl acrylate, did not show a significant change in percent ear swelling at either 24 or 48 hours post challenge, as compared to the 30 % challenge only group (NTP, 1994). The same concentrations (10, 20 and 30 %) were chosen for the Local Lymph Node Assay (LLNA) in B6C3F1 mice. Significant increases in lymph node proliferation were detected with the LLNA at concentrations of 20 % (p 0.05) and 30 % (p 0.01) butyl acrylate, whereas no effect was observed at 10 % (NTP, 1994).

A combination of several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization 1 as defined by OECD, has been conducted to assess the skin sensitizing potential of n-Butyl Acrylate (n-BA).

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (MUSST).

ln this report, the results of the individual studies are summarized and evaluated to predict the presence or absence of skin sensitizing potential of n-Butyl Acrylate (nBA). The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al., 2012 2. Based on the performance standards of the OECD test guideline no. 429 (Local Lymph Node Assay, LLNA, OECD 201 03) , the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95% compared to results in humans (for comparison: for the same data set the LLNA yielded an overall accuracy of 86%). A skin sensitizing (quantitative) potency assessment using the reported results was not validated at the time of writing of this report.

Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/ h-CLAT assays.

DPRA

LuSens/

KeratinoSensTM

MUSST/

h-CLAT

Test battery evaluation

positive

positive

positive

sensitizer

positive

positive

negative

sensitizer

positive

negative

positive

sensitizer

positive

negative

negative

non-sensitizer

negative

positive

positive

sensitizer

negative

positive

negative

non-sensitizer

negative

negative

positive

non-sensitizer

negative

negative

negative

non-sensitizer

64V0348/03A0 18: Direct Reactivity (DPRA) Peptide Assay; 95.8% mean peptide depletion (100.0% cysteine peptide depletion; 91.7% lysine peptide depletion). Positive

66V0348/03A0 17: Keratinocyte Activation AssayLuSens;lnat least two independent experiments ARE-dependent luciferase activityinduction above 1.5-fold attestsubstance concentrationsthatdid not reduce cell viability below 70%was observed. Positive

65V0348/03A016: Dendritic Cell Line Activation Assay Myeloid U937 Skin Sensitization Test (MUSST);lnatleast two independentexperiments an induction of the expression of CD 86 above 1.2-fold was observed at sufficiently noncytotoxic (cell viability >=:70%) concentration. Positive

Based on the results and applying the evaluation criteria, n-Butyl Acrylate (n-BA) is peptide reactive, activates keratinocytes and activates dendritic cells. Applying the evaluation criteria n-Butyl Acrylate (n-BA) is predicted to be a skin sensitizer.

After 24 hours of exposure to test substance (h-CLAT), CD 86 expression was induced in THP-1 cells at a concentration between 46.8 μg/ml and 56.1 μg/ml (experiment 1-2) and for CD54 at a concentration from 39.0 μg/ml to 56.1 μg/ml (experiment 1-2) affording at least 50% viability. 2 from 3 experiments were positive. From this it has to be concluded that test substance does induce dendritic cell activation.

For risk assessment purposes and DNEL derivation, the LLNA (BAMM 2006) is the most appropriate study. Based on all the presented data, Butyl acrylate is considered to be a skin sensitizer of weak potency.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

CLP classification (Regulation (EC) No 1272/2008):

- Skin Sensitization Category 1B