Fatty acids, C16-18 and C18-unsatd., Me esters, distn. residues

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:

From July 30,2010 to September 09,2010

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)

Deviations:

yes

Remarks:

Please see Deviation from protocol remarks here below

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Other name: Fatty acids, C16-18 and C18-unsaturated, methyl esters, distillation residues- Physical state: black , brown liquid- Analytical purity:NO DATA- Storage condition of test material: room temperature

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent, UK
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: 15.1 to 18.8 g
- Housing: The animals were housed in groups of 2 or 3 in cages (dimensions 36.5 x 20.7 x 14 cm) with a stainless steel grid top and an integrated food hopper.
- Diet : wooden chewstick , ad libitum.
- Water : ad libitum
- Acclimation period: 8 days
- Justification: The mouse is the species that this test has been validated in.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20 to 21oC
- Humidity (%): 42 to 64%.
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light):12 h light/dark cycle was in operation (light hours 0700 to 1900 h)
- Monitoring: The environment is monitored throughout the day and recordings are made every 15 min.

Vehicle:

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

Concentration:

25 µL , 12.5 µL , 6.25 µL

No. of animals per dose:

5 female rats for 3 concentrations

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:PRE-SCREEN TESTS:
Two females were identified, allocated to the dose group and treated. For 3 consecutive days (Days 1 to 3) animals received an open application of 25 μL of
undiluted test item onto the dorsum of each ear.
Observations were conducted frequently on each day of dosing (predose, immediately post dose and approximately 1 and 2 h after dosing) and once daily thereafter until the kill, by exposure to a rising concentration of carbon dioxide followed by cervical dislocation, on Day 6. The animals were then discarded.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
For 3 consecutive days (Days 1 to 3) animals received an open application of 25 μL of the appropriate formulation onto the dorsum of each ear. There was no treatment on Days 4 and 5. On Day 6 each animal received an intravenous injection (250 μL) of phosphate buffered saline (PBS) containing approximately 20 μCi of [methyl-3H] thymidine into the lateral tail vein. Approximately 5 h after intravenous administration all animals were killed by exposure to a
rising concentration of carbon dioxide and the major blood vessels were severed to exsanguinate. Each pair of draining auricular lymph nodes was collected from each animal and the animal was then discarded. A single cell suspension of lymph node cells from each paired sample was prepared by gentle disaggregation through a 200 μm mesh stainless steelgauze. The lymph nodes cells were washed in excess of PBS (approximately 1 mL) and thencentrifuged at approximately 1300 g for 10 min at 4°C. The supernatant was drawn off, the mesh was discarded and the pellet was washed a second time with approximately 1 mL PBS and then centrifuged (also at approximately 1300 g for 10 min at 4°C). Following centrifugation, the supernatant was discarded and the pellet was precipitated with approximately 1 mL 5% trichloroacetic acid at 2 to 8oC for approximately 18½ h. The pellet was again centrifuged at approximately 1300 g for 10 min at 4°C and the supernatant
discarded. The pellet was then re-suspended in 200 μL ‘Solvable’ and the suspension transferred to a vial containing 10 mL scintillation fluid (Aquasafe 500 plus liquid, Zinsser Analytic, Maidenhead, UK). Incorporation of tritiated thymidine was measured by β-scintillation counting and was expressed as disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

No formal statistical analysis was carried out.

Positive control results:

hexylcinnamicaldehyde at concentrations of 5%, 10% and 25%
The samples from 3 animals receiving 25% HCA (Group 4) were spilled and the DPM values for these individuals are excluded from the group mean DPM and stimulation index calculations. Although the DPM value obtained from Animal l9 was greater than that obtained from Animal 16, which was included in the calculations, its accuracy is equivocal and its inclusion in the calculations is not considered to be merited.
Conclusion
A stimulation index equal to or greater than 3, which is necessary for a test item to be labelled a sensitizer, was achieved in this study. Therefore, HCA was considered to be a sensitizer in CBA/Ca mice. This provided evidence that the procedures used at Charles River, Edinburgh are valid. .
The stimulation indices in a recent positive control study were 2.3, 3.6 and 8.8 for formulations of hexylcinnamicaldehyde prepared at concentration of 5%, 10% and 25%, respectively.

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

gruop 1

Remarks on result:

other: see Remark

Remarks:

Reults were corrected for background radiation and expressed as the Stimulation Index (SI). This was obtained by dividing the mean DPM obtained from each group by the mean DPM for the control group. The SI for the control group, therefore, is one. A positive response is indicated by an SI ≥3, together with consideration of dose-response and, where appropriate, statistical significance. As there was no SI value ≥3 recorded for any group treated with the distillation residues, it was not possible to calculate the estimated concentration of test item that would produce a 3-fold increase in draining lymph node cell proliferation (the EC3 value). GRUOP STIMULATION INDEX 1 1 2 0.8 3 1.1 4 1.8

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

group 2

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

group 3

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

group 4

Deviation from protocol:

The daily mean humidity in the animal room was within the range of 40 to 70% that was stated in the protocol. However, there were several days when values were intermittently outside the range. The minimum recorded humidity was approximately 36% and the

maximum was approximately 78%. Since there was no effect on the animals’ health, these deviations are not considered to have had any impact on the integrity or outcome of the study.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Under the conditions of the study, since treatment with Fatty acids, C16-18 and C18-
unsaturated, methyl esters, distillation residues at concentrations of up to 100% did not
achieve a stimulation index of ≥3, it was considered that the test item does not have the
potential to cause sensitisation.

Executive summary:

This study investigated the delayed contact hypersensitivity potential of the test item, Fatty

acids, C16-18 and C18-unsaturated, methyl esters, distillation residues, using CBA/Ca mice.

For the purpose of this report the test item will be referred to as the distillation residues.

A preliminary test was conducted using 2 females. Each mouse received an open application

of 25 μL of undiluted distillation residues onto the dorsum of each ear on 3 consecutive days.

There was no evidence of systemic toxicity or local irritation and, as a result of these findings,

formulation concentrations were selected for the main study. Three groups, each consisting

of 5 females, were treated in the same manner as the preliminary test mice with

concentrations prepared at 25%, 50% or 100% (ie undiluted distillation residues),

respectively, also for 3 consecutive days. The vehicle was acetone:olive oil (4:1 v/v) and one

group of 5 females received only this and acted as controls. Three days after the final

application each animal from the main study received an intravenous injection of [methyl-3H]

thymidine into the lateral tail vein. Approximately 4½ h later the draining lymph nodes were

collected in order that incorporation of tritiated thymidine could be assessed by scintillation

counting.

There were no systemic signs of toxicity in any animal during the observation period and

body weight changes were considered to be acceptable for mice of this age and strain.

The stimulation indices (SI) values for the mice treated with the distillation residues at

concentrations of 25%, 50% or 100%, when compared with the control group, were 0.8, 1.0

and 0.9, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Under the conditions of the study, since treatment with Fatty acids, C16 -18 and C16 -18 unsaturated, methyl esters, distillation residues at concentrations of up to 100% did not achieve a stimulation index of ≥3, it was considered that the test item does not have the potential to cause sensitisation.

Migrated from Short description of key information:
Not sensitising

Justification for classification or non-classification

The substance is considered not sensitizing, therefore no classification is warranted under 67/548/EEC or Regulation 1272/2008.