Reaction products of slack wax (petroleum), oxidized, esterified with n-butanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2015 to 13 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

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 slack wax (petroleum), oxidized, esterified with n-butanol

EC Number:

945-889-4

Molecular formula:

Too Complex

IUPAC Name:

Reaction products of slack wax (petroleum), oxidized, esterified with n-butanol

Test material form:

other: waxy solid

Details on test material:

- Physical state: brown waxy solid
- Analytical purity:100%
- Expiration date of the lot/batch: 13 July 2017
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 changes per hour
- Photoperiod: 12 h dark / 12 h light

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary test: 50 and 25%
Main test: 25, 2.5 and 0.25% v/v in acetone/olive oil 4:1

No. of animals per dose:

Preliminary screening test: 1 female/dose
Main test: 4 females/dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Test item was used undiluted and freshly prepared as a solution at the concentrations of 25 and 50% in acetone/olive oil 4:1
- Irritation: Mouse treated with diluted test item for three consecutive days (Days 1, 2 and 3) showed no signs of systemic toxicity. Very slight erythema on both ears and a greater than 25% increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 50% w/w in acetone/olive oil 4:1. No visual skin irritation or irritation idicated by an equal to or greater than 25% increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 25% w/w in acetone/olive oil 4:1. Based on this information the dose levels selected for the main test were 25%, 2.5% and 0.25% w/w in acetone/olive oil 4:1.

MAIN STUDY
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION: The mice were treated by daily application of 25 µL of test material at concentrations of 0 (vehicle control), 25, 2.5 and 0.25% to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). On Day 6, all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse. Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. Lymph node cells were washed with PBS and precipitated with 5 % (w/v) trichloroacetic acid (TCA) for radioactive material at 4 °C. Pellets were re-suspended in 1 mL TCA and transferred to 10 mL of scintillation fluid (Optiphase ‘Trisafe’). 3HTdR incorporation was measured by β -scintillation counting. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Positive control substance(s):

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

Statistics:

No data

Results and discussion

Positive control results:

Stimulation index for α-hexylcinnamaldehyde at 25 % v/v in acetone/olive oil 4:1 was 9.96 and classified as a sensitiser.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.36

Test group / Remarks:

0.25%

Results 2

Key result

Parameter:

SI

Value:

1.62

Test group / Remarks:

2.5%

Results 3

Key result

Parameter:

SI

Value:

1.85

Test group / Remarks:

25%

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under these test conditions,the substance is not classified as sensitizing to the skin according to CLP Regulation (EC) No. 1272/2008

Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear, according to the OECD Guideline 429 and EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay).

Method

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/w, this concentration was selected as the highest dose level to be investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 uL (25 uL per ear)) of the test item as a solution in acetone/olive oil 4:1 at cocnentrations of 0.025, 2.5 and 25% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using α-

hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

Results

No mortality and no clinical signs were observed during the observation period. Body weight of the animals was unaffected by the test item treatment. The stimulation index for 0.25, 2.5 and 25% were 1.36, 1.62 and 1.85, respectively. The positive control (α-hexylcinnamaldehyde, 25%) exhibited evidence of sensitisation (SI = 9.96) indicating the validity of the study. In this study, the substance is not a skin sensitizer in mice.

Conclusion

The test item was considered to be a non-sensitizer under the conditions of the test.