Fatty acids, C18-unsatd., dimers, di-Me esters, hydrogenated

Endpoint summary

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:

31 Jul - 15 Aug 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: 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: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: 2014C Teklad Global Rodent diet (Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: mains tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

Preliminary screening test: 100%
Main test: 25, 50 and 100% (v/v)

No. of animals per dose:

Preliminary screening test: 1
Main test: 4

Details on study design:

RANGE FINDING TESTS: A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the Draize scoring system. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post-dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.
- Compound solubility: The test item was soluble in in acetone/olive oil (4:1 v/v) at 50% and the solution considered suitable for dosing.
- Clinical signs and Irritation: 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 were noted.
Based on the results of the preliminary screening test, the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-Methylthymidine incorporation determined by β-scintillation.
- Criteria used to consider a positive response: 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).
A test item was regarded as a sensitiser if at least one concentration of the test item resulted 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 was classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
Animals were treated with the undiluted test item, the test item at concentrations of 25% and 50% (v/v) in acetone/olive oil (4:1 v/v) or the vehicle alone by daily application of 25 µL of the appropriate solution to the dorsal surface of each ear for three consecutive days (Days 1-3). Five days following the first topical application of the test item or vehicle (Day 6), each animal was injected with 250 µL of phosphate buffered saline (PBS) containing 20 µCi ³H-TdR (concentration: 80 µCi/mL, specific activity: 2.0 Ci/mmol) via the tail vein. Five hours following administration of ³H-TdR, animals were sacrificed and the draining auricular lymph nodes were excised and pooled for each experimental group (total: 8 nodes/group). A single cell suspension of pooled lymph node cells was prepared in PBS by gentle mechanical disaggregation through 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish and each lymph node cell suspension was transferred into a centrifuge tube. The petri dish was washed with PBS to remove all remaining lymph node cells, and pooled lymph node cells were pelleted by centrifugation. The pellet was resuspended in PBS and re-pelleted. To precipitate macromolecules incorporating radioactive material, the pellet was resuspended in 5% trichloroacetic acid (TCA). After approx. 18 h incubation at 4°C, the precipitates were recovered by centrifugation, resuspended in TCA and transferred to scintillation fluid. ³H-TdR incorporation was measured by β-scintillation counting.

Positive control substance(s):

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

Positive control results:

Treatment with the current positive control substance α-Hexylcinnamaldehyde, technical product (85%) at 25% in acetone:olive oil (4:1 v/v) resulted in a stimulation index (SI) of 5.76, thus meeting the reliability criteria for the LLNA (SI > 3).

Parameter:

SI

Remarks on result:

other: The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were as follows: 25%: 2.94 50%: 2.87 100%: 2.34

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The lymph nodes of all animals per group (8 nodes in total) were pooled and DPM values were measured from the pooled lymph node cell suspensions. Treatment with vehicle, 25, 50 and 100% test item resulted in DPM/node values of 2754.34, 8107.32, 7917.96 and 6444.62, respectively.

- 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.

- Bodyweight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

- Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 1.

 

Table 1. Disintegrations per Minute (dpm), Disintegrations per Minute/Node and Stimulation Index

Concentration (% v/v) in acetone/olive oil 4:1	dpm	dpm/Node*	Stimulation Index	Result
Vehicle	22034.74	2754.34	na	na
25	64858.52	8107.32	2.94	Negative
50	63343.66	7917.96	2.87	Negative
100	51556.96	6444.62	2.34	Negative

 

* Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

na: not applicable

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitisation potential of Fatty acids, C18-unsatd., dimers, di-Me esters, hydrogenated was assessed in a Local Lymph Node Assay (LLNA) performed according to OECD guideline 429 and in compliance with GLP (Bradshaw, 2012). Groups of four female mice per concentration level were treated with 50 µL (25 µL per ear) of the vehicle (acetone/olive oil 4:1) or the test material at 25, 50 and 100% for 3 consecutive days. Five days after the first application, animals were injected with ³H-methylthymidine (³HTdR) at 20 µCi/mouse. The draining auricular lymph nodes from all mice were excised 5 h after injection and pooled for each treatment group. Single cell suspensions of pooled lymph node cells were prepared for determination of ³HTdR incorporation by β-scintillation counting. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/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, SI).

No mortality occurred, no clinical signs of systemic toxicity and no changes in body weight were observed in any animal during the study period.

Treatment with vehicle and test material at 25, 50 and 100% resulted in DPM/node values of 2754.34, 8107.32, 7917.96 and 6444.62, respectively. The corresponding SI values for the low-, mid- and high-concentrations were 2.94, 2.87 and 2.34. The reported historical positive control data, including the most recent test with α-hexylcinnamaldehyde, tech., 85%, fulfilled the reliability criteria for the LLNA (SI > 3).

The test material is thus considered to be not sensitising under the conditions of the test.

Conclusions for skin sensitisation

The substance Fatty acids, C18-unsatd., dimers, di-Me esters, hydrogenated has been tested for the induction of skin sensitisation in a Local Lymph Node Assay in mice. Treatment did not result in a stimulation index greater than 3 at any test substance concentration.

Based on the available data, the substance is considered to be not skin sensitising.

Migrated from Short description of key information:
Skin sensitisation: not sensitising (OECD 429/EU Method B.42, GLP)

Justification for selection of skin sensitisation endpoint:
There is only one study available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on the skin sensitisation potential of Fatty acids, C18-unsatd., dimers, di-Me esters, hydrogenated do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.