Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic, n-heptanoic, n-octanoic and n-decanoic acids

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-06-18 to 2013-07-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

Principles of method if other than guideline:

Ear thickness measurements were not taken.

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:

Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester

Cas Number:

65870-94-2

Molecular formula:

C33H62O6

IUPAC Name:

Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester

Test material form:

liquid

Details on test material:

Description: clear colourless liquid
Expiry date: 29 June 2013
Storage conditions: 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

IN-LIFE DATES: From: 2013-06-18 To: 2013-07-02

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary test: 100 %
Main test: 25 and 50 % v/v in acetone/olive oil 4:1 and 100 %

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 undiluted test item for three consecutive days (Days 1, 2 and 3) showed no signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness. Based on this information the undiluted test item and the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1 were selected for the main test.

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, 50 and 100 % 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 7.33 and classified as a sensitiser.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.26

Test group / Remarks:

25%

Results 2

Parameter:

SI

Value:

1.28

Test group / Remarks:

50%

Results 3

Parameter:

SI

Value:

1.48

Test group / Remarks:

100%

Results 4

Parameter:

EC3

Remarks on result:

not determinable

Any other information on results incl. tables

Table 7.4.1/1: Results of skin sensitization

Concentration (% v/v) in acetone/olive oil 4:1	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	8967.55	1120.94	na	na
25	11288.98	1411.12	1.26	Negative
50	11444.45	1430.56	1.28	Negative
100	13314.84	1664.36	1.48	Negative

dpm = Disintegrations per minute

a= Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total

number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

Clinical observations and mortality data: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body Weight: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

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:

Test Guidance

OECD Guideline 429 and EC Method B.42 (Skin Sensitisation: Local Lymph Node Assay).

Method and materials

Groups of CBA/Ca (CBA/CaOlaHsd) mice (4 females/dose) were topically applied with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50 % or 25 % v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone. On Day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3HTdR). Five hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3HTdR. The results were expressed as disintegrations per minute (dpm) per group; dpm/node and the obtained values were used to calculate Stimulation Indices (SI). Animals were observed for mortality, clinical signs and body weight during the study. The test concentrations for the main study were determined from a preliminary study using one female mouse at the concentration of 100 %.

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. DPM per group for vehicle, 25, 50 and 100 % were 8967.55, 11288.98, 11444.45 and 13314.84, respectively. DPM per node for vehicle, 25, 50 and 100 % were 1120.94, 1411.12, 1430.56 and 1664.36, respectively. Stimulation index for 25, 50 and 100 % were 1.26, 1.28 and 1.48, respectively. Positive control (α-hexylcinnamaldehyde, 25%) exhibited evidence of sensitisation indicating the validity of the study. In this study, the substance is not a skin sensitizer in mice.

Conclusion

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