Allyl phenoxyacetate

Endpoint summary

Description of key information

Skin sensitisation (OECD 429): skin sensitising

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:

07 Mar - 13 Mar 2007

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:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 18.1-23.7 g
- Housing: maximum 4 animals per cage in cages suitable for animals of this strain and weight range, tents and nestlets as cage-enrichment
- Diet: RM1 (Special Diet Services Limited, Witham UK), ad libitum
- Water: mains water, ad libitum
- Acclimation period: at least 5 days

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

Vehicle:

other: ethanol/diethyl phthalate (1:3) (test substance); acetone/olive oil (4:1) (positive control substance)

Concentration:

Test substance: 0.5, 1, 2.5, 5 and 10% (w/v)
Positive control: 5, 10 and 25% (w/v)

No. of animals per dose:

4

Details on study design:

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A stimulation index (SI) was calculated for each group using the activity of each test group divided by the activity of the vehicle control group. The criterion for a positve 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.

TREATMENT PREPARATION AND ADMINISTRATION: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration. The application was repeated daily for 3 consecutive days; local irritation reactions were assessed. Three days after the third application an injection of 250 µl phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (³HTdR) was made into the tail vein of each experimental mouse. Approximately five hours later, following injection of ³HTdR, the mice were euthanized and draining auricular lymph nodes were excised and pooled for each test group. A single cell suspension of pooled lymph node cells was prepared in PBS by gentle separation through a 200-mesh stainless steel gauze. The cell suspensions were washed three times by centrifugation with approximately 10 mL PBS. To precipitate the DNA, the cell suspensions were exposed to 3 mL of 5% trichloroacetic acid and stored at 4 °C overnight.

Positive control substance(s):

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

Statistics:

The calculation of the EC3 value was conducted by linear interpolation according to the equation:
EC3 = [(3-d)/(b-d)] x (a-c) + c
where the data points lying immediately above and below the SI value of 3 on the LLNA dose-response plot 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).

Positive control results:

The SI values calculated for the positive control at concentrations of 5, 10 and 25% (w/v) were 1.7, 2.3 and 6.4 respectively. The application at concentration of 25% resulted in a greater than 3-fold increase in isotope incorporation at this concentration.

Parameter:

SI

Value:

0.8

Test group / Remarks:

0.5% (w/v)

Parameter:

SI

Value:

1.3

Test group / Remarks:

1% (w/v)

Parameter:

SI

Value:

1.6

Test group / Remarks:

2.5% (w/v)

Parameter:

SI

Value:

7.5

Test group / Remarks:

5% (w/v)

Parameter:

SI

Value:

8.1

Test group / Remarks:

10% (w/v)

Parameter:

EC3

Value:

3.1

Cellular proliferation data / Observations:

IRRITATION
The application of the test substance at concentrations of 0.5, 1, 2.5, 5 and 10% (w/v) in ethanol/diethyl phthalate (1:3) did not result in an increase in visual levels of irritancy to the skin on or around the ear area apart from on Day 3, where slight reddening of the ear skin at the 5 and 10% dose groups was observed. This had resolved by Day 6.

CELLULAR PROLIFERATION DATA
The lymph nodes of all animals per group were pooled and DPM values were measured from the pooled lymph node cell suspensions. Treatment with test substance concentrations of 0.5, 1, 2.5, 5 and 10% (w/v) in ethanol/diethyl phthalate (1:3) resulted in DPM values per lymph node of 284, 473, 560, 2642 and 2856, respectively. The DPM value per lymph node of the vehicle control was 353.

EC3 CALCULATIONE: EC1.6 =[(3-d)/(b-d)] x (a-c) + c
a= The dose concentration with SI above 3; b = The SI value of a, c = The dose concentration with SI lower 3; d = The SI value of c
EC3 = 3.1

Interpretation of results:

other: Skin Sens Cat 1B is required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the mouse Local Lymph Node Assay the test substance revealed sensitising properties. The EC3 value was calculated to be 3.1% hence, the test substance was assessed to be a weak sensitiser (Skin Sens. 1B).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitising potential of the test substance was investigated in a Local Lymph Node Assay (LLNA) in mice according to OECD Guideline 429 and in compliance with GLP (2007). Four female CBA/Ca mice per test group were treated with the test substance at concentrations of 0.5, 1, 2.5, 5 or 10% (w/v) in ethanol/diethyl phthalate 1:3 or with vehicle alone for three consecutive days by open application on the ears (25 µL/ear). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and approximately after five hours the draining (auricular) lymph nodes were excised and pooled for each test group. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. Treatment with test substance concentrations of 0.5, 1, 2.5, 5 and 10% (w/v) in ethanol/diethyl phthalate 1:3 resulted in DPM values per lymph node of 284, 473, 560, 2642 and 2856, respectively. The SI values calculated for the substance concentrations 0.5, 1, 2.5, 5 and 10% were 0.8, 1.3, 1.6, 7.5 and 8.1, respectively. The EC3 value was calculated to be 3.1%. The application of the test substance did not result in an increase in visual levels of irritancy to the skin on or around the ear area apart from on Day 3, where slight reddening of the ear skin at the 5 and 10% dose groups was observed. The effect had resolved by Day 6. The reliability check with hexyl cinnamic aldehyde indicated that the LLNA is an appropriate model for testing for contact hypersensitivity. Based on the results, the test substance was regarded as a week skin sensitizer under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on sensitisation of the test substance meet the criteria for Skin Sens. 1B (H317) according to Regulation (EC) 1272/2008.