Registration Dossier

Administrative data

Description of key information

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:
22 Nov - 18 Dec 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study. Conclusions were drawn without discussion of results in terms of no clear dose-relationship and possible and potentially confounding irritating effects.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
conclusions were drawn without discussion of results in terms of no clear dose-relationship and possible and potentially confounding irritating effects.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
conclusions were drawn without discussion of results in terms of no clear dose-relationship and possible and potentially confounding irritating effects.
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
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 or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Very slight erythema (Draize score 1) was noted on both ears on Days 3 and 4.
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".
The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3-fold increase in ³HTdR incorporation. The equation used for the calculation of EC3 is:
EC3= c + [[(3−d)/(b−d)] x (a−c)]
where:
a = lowest concentration giving stimulation index > 3
b = actual stimulation index caused by ‘a’
c = highest concentration failing to produce a stimulation index of 3
d = actual stimulation index caused by ‘c’

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 6.29, thus meeting the reliability criteria for the LLNA (SI > 3).
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
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%: 2.99 50%: 4.97 100%: 2.24 EC3 value: 25.1% (using SI values for 25 and 50% for calculation) EC3 value: 86.1% (using SI values for 50 and 100% for calculation)
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 1732.94, 5187.56, 8607.26 and 3882.91, respectively.

- Clinical Observations, Skin Irritation and Mortality Data

Sticky coloured residual test item on the ears was noted in all test animals. Very slight erythema on both ears was noted on Days 2 to 4, in animals treated with the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1 (Table 1). Thickening of the ears, possibly due to the residual test item, was noted in all test animals (Table 2). A greater than 25% increase in mean ear thickness was noted in animals treated with the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1, but was considered to be due to residual test item on the ears rather than excessive irritation. No irritation indicated by an equal to or greater than 25% increase in mean ear thickness was noted in animals treated with the test item at a concentration of 25% v/v in acetone/olive oil 4:1.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

 

Table 1. Local Skin Irritation – Main Test

Concentration

Animal No.

Local skin irritation (erythema score)

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

Vehicle

1-1

0

0

0

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

0

0

0

25%

2-1

0

0

0

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

0

0

0

50%

3-1

0

0

1

1

1

1

1

1

0

0

0

0

3-2

0

0

1

1

1

1

1

1

0

0

0

0

3-3

0

0

1

1

1

1

1

1

0

0

0

0

3-4

0

0

1

1

1

1

1

1

0

0

0

0

100%

4-1

0

0

1

1

1

1

1

1

0

0

0

0

4-2

0

0

1

1

1

1

1

1

0

0

0

0

4-3

0

0

1

1

1

1

1

1

0

0

0

0

4-4

0

0

1

1

1

1

1

1

0

0

0

0

 

Table 2. Mean Ear Thickness Changes

Concentration

Ear thickness (mm)

Mean Day 1 pre-dose (n =4)

Mean Day 3 post-dose (n =4)

Mean change Day 1-3 (%)

Mean Day 6 post-dose (n =4)

Mean change Day 1-6 (%)

Vehicle

0.209

0.213

2.096

0.224

7.186

25%

0.213

0.233*

9.706

0.236

11.176

50%

0.214

0.271*

26.608**

0.239

11.696

100%

0.210

0.297*

41.369**

0.258

22.619

* Possibly due to residual test item on the ears

** > 25% increase considered to be due to residual test item on ears

 

- Body weight

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 except for one animal treated with the undiluted test item which showed a greater than expected bodyweight gain (+23.5%).

 

- 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 3.

 

Table 3. Disintegrations per Minute (dpm), Disintegrations per Minute/Node and Stimulation Index

Concentration

dpm

dpm/Node*

Stimulation Index

Result

Vehicle

13863.51

1732.94

na

na

25%

41500.47

5187.56

2.99

Negative

50%

68858.08

8607.26

4.97

Positive

100%

31063.26

3882.91

2.24

Negative

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

na = not applicable

- Calculation of EC3 value

EC3= c + [[(3−d)/(b−d)] x (a−c)]

a = 50

b = 4.97

c = 25 or 100

d = 2.99 or 2.24

EC3= 25 + [[(3−2.99)/(4.97−2.99)] x (50−25)] = 25.1 (using SI values for 25% and 50% for calculation)

EC3= 100 + [[(3−2.24)/(4.97−2.24)] x (50−10)] = 86.1 (using SI values for 50% and 100% for calculation)

The concentration of test item expected to cause a 3-fold increase in ³HTdR incorporation (EC3 value) was calculated to be 25.1 or 86.1%.

Interpretation of results:
ambiguous
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: inconclusive
DSD: inconclusive
Endpoint conclusion
Additional information:

The skin sensitisation potential of Fatty acids, C18-unsaturated, trimers, hydrogenated was assessed in a Local Lymph Node Assay (LLNA) performed according to OECD guideline 429 and in compliance with GLP (Bradshaw, 2013). 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 toxicologically relevant changes in body weight were observed during the study period.

Sticky coloured residual test item on the ears was noted in all test animals. Very slight erythema on both ears was noted on Days 2 to 4, in animals treated with the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1. Thickening of the ears, possibly due to the residual test item, was noted in all test animals. A greater than 25% increase in mean ear thickness was noted in animals treated with the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1, but was considered to be due to residual test item on the ears rather than excessive irritation. No irritation indicated by an equal to or greater than 25% increase in mean ear thickness was noted in animals treated with the test item at a concentration of 25% v/v in acetone/olive oil 4:1.

Treatment with vehicle and test material at 25, 50 and 100% resulted in DPM/node values of 1732.94, 5187.56, 8607.26 and 3882.91, respectively. The corresponding SI values for the low-, mid- and high-concentrations were 2.99, 4.97 and 2.24. 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).

According to the study director, the test substance was considered to be a sensitiser under the conditions of the study based on a positive response (stimulation index > 3) at a test concentration of 50%. However, no dose-response relationship was observed in the induced stimulation indices at 25, 50 and 100%, the mean stimulation index of animals treated with the undiluted test substance being even less than that of the 25% group. It is conceivable that the positive result at 50% may have been due to an outlier(s). Due to the use of the pooled treatment group approach, individual animal data are not available for statistical analysis.

The EC3 value was calculated using the equation:

EC3= c + [[(3−d)/(b−d)] x (a−c)]

where:

a = lowest concentration giving stimulation index > 3
b = actual stimulation index caused by ‘a’
c = highest concentration failing to produce a stimulation index of 3
d = actual stimulation index caused by ‘c’

In the study report, an EC3 value of 25.1% was calculated using the SI values for the test concentrations 25 and 50%. Thereby, 25% was used as value for ‘c’. However, ‘c’ is defined as the “highest concentration failing to produce a stimulation index of 3”. In the study, the highest test concentration of 100% resulted in an SI value lower than 3. Thus, an EC3 value of 86.1% could likewise be calculated applying the equation above.

Due to the lack of a clear dose-response relationship, both EC3 values are questionable, thus supporting the notion of a “false positive” result at 50%, leading to an overall ambiguous study result.

Furthermore, some irritating effects (in particular ear swelling) were observed which could have influenced the outcome of the lymph node proliferation measurements. However, due to presence of residual test item on the ears of the animals, the irritating effects cannot be undoubtedly evaluated.

Overall, due to the lack of a clear dose-response relationship and potentially confounding irritating effects, the results of the study are considered to be ambiguous and therefore inconclusive for the purpose of classification and labelling.

Conclusions for skin sensitisation

The substance Fatty acids, C18-unsaturated, trimers, hydrogenated has been tested for skin sensitisation in a Local Lymph Node Assay in mice. The stimulation indices after treatment with the test substance at 25, 50 and 100% were 2.99, 4.97 and 2.24, respectively. Some irritating effects were observed: very slight erythema on Days 2-4 in the 50 and 100% groups and ear swelling on Days 3 (possibly due to residual test item on the ears) and 6 in all treatment groups.

Based on the lack of a clear dose-response relationship and potentially confounding irritating effects, the results of the study are considered to be ambiguous.


Migrated from Short description of key information:
Skin sensitisation: ambiguous (OECD 429, 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 sensitising potential of the substance are ambiguous, i.e. neither meet the classification criteria according to Regulation (EC) No 1272/2008 or Directive 67/548/EEC nor are sufficient for non-classification, and are therefore inconclusive.