Fatty acids, C10-12, esters with polylactic acid, sodium salts

Endpoint summary

Description of key information

Skin sensitisation (OECD 429, LLNA): sensitising

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:

21 March - 10 April 2012

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:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

National Institute for Quality and Organizational Development in Healthcare and Medicines, Budapest, Hungary

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Dose range finding animals:

- Strain: CRL:NMRI BR mice
- Source: TOXI - COOP ZRT , H-1103, Budapest, Cserkesz u. 90
Justification of strain: The aim of the preliminary screen is to assess the ability of the test item causing irritation and/or systemic toxicity (not the sensitizing effect). CRL: NMRI BR mice were used in the pre-screen test for a better visibility of erythema and oedema.
Number of animals: 4 animals (2 animals/group);
Sex: Female, nulliparous, non pregnant
Age of animals: Young adult mice, 8 weeks old

Main test animals:

- Strain: CBA/Ca mice
- Source: TOXI-COOP ZRT. , H-1103, Budapest, Cserkesz u. 90
- Age at study initiation: Experiment 1: 8-12 weeks, Experiment 2: 9 weeks
- Weight at study initiation: Experiment 1: 15.5-19.6 g, Experiment 2: 17.5-20.4 g
- Housing: 1. During acclimatization period: Group caging in small groups
2. During test phase: Grouped caging (4 animals)
Cage type II, Polypropylene/Polycarbonate
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
Batch No: 565 6537, Expiry Date: 05/2012
Nutrition Values: Crude protein 18.3 %, Total/Crude fat 3.8 %, Crude fibre 4.3 %, Crude ash 6.0 %, N-free substances 55.2 %
- Water (e.g. ad libitum):tap water from municipal supply, as for human consumption, from a bottle ad libitum
- Acclimation period:Experiment 1 : 30 days or 7 days, Experiment 2: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours daily , from 6.00 a.m. to 6 p.m.

Vehicle:

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

Remarks:

AOO

Concentration:

Hexyl cinnamic acid is diluted with AOO to a concentration of 25% (v/v)
Test item diluted in vehicle to 50 % (v/v), 25% (v/v), 10 % (v/v), 5 % (v/v) and 2,5 % (v/v)

No. of animals per dose:

4 female animals

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
The test item was a highly viscous liquid hence application of the 100 % concentration (the undiluted test item) was not possible. The maximum available concentration based on solubility was 50 %. Because of structural characteristics of the test item no higher test concentration was available. The preliminary test was performed with test item concentrations of 50 % (the maximum attainable concentration) and 25% (w/v) in the selected vehicle (AOO). Both formulations were clear solution and appropriately applicable on the ears of animals. Groups of 2 CRL: NMRI BR mice were treated with the 50 % or 25 % formulations.
- Irritation:
All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to the first treatment (Day 1) and prior to termination (Day 6). Both ears of each mouse were observed for erythema and scored. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
- Lymph node proliferation response:
The maximum dose selection was performed according to the OECD 429 guideline. The preliminary irritation/toxicity screen was conducted in a similar experimental manner to the exposure phase of the main test except there was no assessment of lymph node proliferation and fewer animals were used.No mortality or significant effect on the body weights were observed during the preliminary test. Erythema was observed in both treatment groups during the whole test (from Day 1 to Day 6), but it was not significant (the maximum score was 2 in both groups). Increased ear thickness values were observed in both groups, although the maximum value (18.2 % in the 50 % dose group) did not exceed the 25 % value.Based on the preliminary test results, since no signs of systemic toxicity or significant irritation were observed, 50 % was selected as the highest test concentration in the main test. Although according to evaluation criteria of the relevant OECD guideline the observed local effect was not significant, four test concentrations was intended to be examined to ensure that no unexpected adverse effect interferes the evaluation of the test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429: Skin Sensitization : Local Lymph Node Assay
In Vivo Treatment
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance (positive control group) or the vehicle (negative control group) using a pipette, on the dorsal surface of each ear. After the treatments animals returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. This procedure was followed in both Expriments.
Proliferation Assay
On Day 6, the animals were intravenously injected via the tail vein with 250 μL of sterile PBS containing 20 μCi of 3HTdR using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
Five hours (± 30 minutes) after intravenous injection the mice were anesthetized by inhalation of Isofluran CP® and sacrificed by cervical dislocation. The draining auricular lymph nodes were excised. Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and resuspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % trichloracetic acid (TCA) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8°C overnight (approx. 18 hrs), each precipitate was removed and the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and stored at room temperature protected from light until measurement.
For the measurement of incorporated radioactivity the vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expresses the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA.
Instrument used for the measurement:
Name: Packard Tri-Carb 2300 TR Liquid Scintillation Analyzer
Serial Number: 406 117

- Criteria used to consider a positive response:
Since no systemic toxicity or obvious sign of irritation was observed in any of the Experiments performed the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.
The test item is considered as a skin sensitizer, if the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
Using this EC3 value according to the published data for classification of contact allergens.
Table 4: Classification of Relative Skin-Sensitization Potency
EC3 value (%)
Potency classification
≥ 10- ≤ 100 Weak
≥ 1- < 10 Moderate
≥ 0.1- < 1 Strong
< 0.1 Extreme

Note: classification is performed using the local lymph node assay EC3 values

Positive control substance(s):

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

Statistics:

Significance of the dose-response was evaluated by linear regression using SI values obtained from both Experiments. Although the response observed was dose-related, it proved to be not statistically significant: the r value was 0.82, the p value was 0.09.

Positive control results:

No mortality, signs of toxicity or local effects were observed in the positive control group.
Significant lymphoproliferative response (SI ≥ 3) was noted for HCA with a stimulation index value of 11.7 in Experiment 1 and 6.2 in Experiment 2

Parameter:

SI

Remarks:

Experiment 1

Value:

1

Test group / Remarks:

Negative control

Parameter:

SI

Remarks:

Experiment 1

Value:

11.7

Test group / Remarks:

Positive control

Key result

Parameter:

SI

Remarks:

Experiment 1

Value:

6.1

Test group / Remarks:

Test item / 25%

Parameter:

SI

Remarks:

Experiment 1

Value:

2.4

Test group / Remarks:

Test item / 10%

Parameter:

SI

Remarks:

Experiment 1

Value:

2.6

Test group / Remarks:

Test item / 5%

Parameter:

SI

Remarks:

Experiment 1

Value:

2.1

Test group / Remarks:

Test item / 2.5%

Parameter:

SI

Remarks:

Experiment 2

Value:

1

Test group / Remarks:

Negative control

Parameter:

SI

Remarks:

Experiment 2

Value:

6.2

Test group / Remarks:

Positive control

Key result

Parameter:

SI

Remarks:

Experiment 2

Value:

5.4

Test group / Remarks:

Test item / 50%

Cellular proliferation data / Observations:

Clinical observations
No mortality or signs of systemic toxicity was observed during the test (in any Experiment). No significant irritation indicated by an erythema core ≥ 3 was observed in any treatment group in any Experiment. Although erythema was observed in Experiment 1 in the 25% and in the 10% test item treated groups (from Day 2 to Day 4) and in Experiment 2 in the 50% test item treated group (from Day 2 to Day 5), it was not excessive (the maximum erythema score was 2).

Table 1: DPM, DPN and Stimulation Index Values for all Groups in Experiment 1

Test Group Name	MeasuredDPM/group	Group*DPM	DPN (DPM/Node)	Stimulation Index Values
Negative (vehicle) control: AOO	5559	5497.0	687.1	1.0
Positive control: 25 % HCA in AOO	64314	64252.0	8031.5	11.7
dermosoft®decalact 25 % in AOO	33769	33707.0	4213.4	6.1
dermosoft®decalact 10 % in AOO	13432	13370.0	1671.3	2.4
dermosoft®decalact 5 % in AOO	14478	14416.0	1802.0	2.6
dermosoft®decalact 2.5 % in AOO	11794	11732.0	1466.5	2.1

Table 2: DPM, DPN and Stimulation Index Values for all Groups in Experiment 2

Test Group Name	MeasuredDPM/group	Group*DPM	DPN (DPM/Node)	Stimulation Index Values
Negative (vehicle) control: AOO	5632	5585.0	698.1	1.0
Positive control: 25 % HCA in AOO	34738	34691.0	4336.4	6.2
dermosoft®decalact 50 % in AOO	30313	30266.0	3783.3	5.4

PC = Positive control

HCA = Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

*Group DPM = measured DPMgroup - average DPMbackground

Average DPMbackground = 47.0

Interpretation of results:

other: Skin Sens. 1B, H317. Classification according to Regulation (EC) No. 1272/2008 (CLP/EU GHS).

Conclusions:

Under the conditions of the present Local Lymph Node Assay the test item, tested at the maximum attainable concentration of 50% and at concentrations of 25 %, 10%, 5% and 2.5% as homogenous formulations (solutions) in a suitable vehicle (Acetone: Olive oil 4:1 (v/v)), was shown to have sensitisation potential. Based on the EC3 values calculated using the dose-response and the regression curve according to the published data, the test item was classified as a weak to moderate sensitiser in this LLNA.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of the test item following dermal exposure in the Local Lymph Node Assay. The maximum dose selection was performed according to the OECD guideline 429 and based on results of a preliminary formulation evaluation and also on results of a preliminary irritation/toxicity test. The test item was a highly viscous liquid and adequately miscible with Acetone: Olive oil 4:1 (v/v) mixture (AOO). Since this vehicle is the most preferred one by the relevant guidelines, no other vehicles were evaluated. The maximum attainable concentration based on solubility was 50% in AOO. Because of the structural characteristic of the test item no higher test concentration was available. A preliminary irritation/toxicity test was performed with test item concentrations of 50% and 25% (w/v) in the selected vehicle. The appearance of the formulations in AOO was a clear solution. Based on the results of a preliminary irritation/toxicity test 50% was selected as the highest test concentration in the main test.

No mortality or signs of systemic toxicity were observed during the study (either in Experiment 1 or in Experiment 2). No significant, treatment related effect on body weight was observed in any treatment group in any of the Experiments performed. No significant irritation (indicated by an erythema score ≥ 3) was observed during the study (in any of the Experiments).

Since no obvious signs of irritation or systemic toxicity were observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

Larger lymph nodes than the control was observed in the positive control groups (in both Experiments) and in the 50% dose group in Experiment 2. In spite of the normal visual appearance of the lymph nodes in the 25% test item treated group, significant lymphoprolipheration (SI ≥ 3) was observed at concentration of 25% in Experiment 1 and at 50% in Experiment 2. The stimulation index values were 5.4, 6.1, 2.4, 2.6 and 2.1 at test item concentrations of 50%, 25%, 10%, 5% and 2.5%, respectively. Dose-related response was observed, but it was not statistically significant (p = 0.09; significance of the dose-response was evaluated by linear regression using SI values obtained from both experiments). Although no strong dose-response relationship was observed, the significant (SI ≥ 3) proliferation values observed at 50% and 25% concentrations was considered to be an evidence, that the test item has sensitisation potential. EC₃ calculation was conducted by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method . The calculated EC₃ value of the test item was 12.4 % in this LLNA. Using this EC₃ value according to the published data for classification of contact allergens, the test item can be ranked among weak sensitisers (10 ≤ EC₃ ≤ 100) in this LLNA. Additionally EC₃ value based on the equation of the regression curve was also calculated: the relevant value was 9.3 in this LLNA. Using the EC₃ value based on the regression curve the test item can be ranked among moderate sensitisers (1 ≤ EC₃ < 10 ) in this LLNA.

The positive control item (25 % HCA in AOO) induced the appropriate stimulation over the control in both Experiments, thus confirming the validity of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In vivo data (LLNA)

A local lymph node assay (LLNA) according to OECD guideline 429 was conducted with fatty acids, C10-12, esters with polylactic acid, sodium salts (Dermosoft decalact, CAS No. 1312021-45-6, EC No. 700-937-1) in order to investigate its ability to induce skin sensitisation. The test was performed observing GLP provisions (Penzes , 2012). The test item was a highly viscous liquid and adequately miscible with Acetone:Olive oil 4:1 (v/v) mixture (AOO). The maximum attainable concentration based on solubility was 50% in AOO. A preliminary irritation/toxicity test was performed with test item concentrations of 50% and 25% (w/v). Based on the results of the preliminary irritation/toxicity test 50% was selected as the highest test concentration in the main test. The main test consisted of two independent experiments. In experiment 1 four groups of female CBA/Ca mice received the test item at 25%, 10% 5% or 2.5%. In experiment 2 one group received the test item at 50%. In both experiments the negative control group received the vehicle AOO only, and the positive control group received 25% α-hexylcinnamaldehyde (HCA).The test substance was applied on the external surface of each ear (25 µL / ear) of the animals for three consecutive days. There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected with tritiated methyl thymidine (3HTdR) and sacrificed approx. 5 h after the injection. Auricural lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI).

No mortality, no signs of systemic toxicity and no significant irritation (indicated by an erythema score ≥ 3) were observed during the study in either experiment. Moreover, no significant, treatment related effect on body weight was observed in any treatment group in any of the experiments. Larger lymph nodes than the control were apparent in the positive control groups and in the 50% dose group in experiment 2. In spite of the normal visual appearance of the lymph nodes in the 25% test item treated group, significant lymphoprolipheration (SI ≥ 3) was observed at the concentration of 25% in experiment 1 and at 50% in experiment 2. The stimulation index values were 5.4, 6.1, 2.4, 2.6 and 2.1 at test item concentrations of 50%, 25%, 10%, 5% and 2.5%, respectively. Although no strong dose-response relationship was observed, the significant (SI ≥ 3) proliferation values observed at 50% and 25% concentrations were considered to be an evidence for a skin sensitisation potential of the test item. The positive control item induced the appropriate stimulation over the control in both experiments, thus confirming the validity of the test.

EC3 calculation was conducted by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve. The calculated EC3 value of the test item was 12.4%. Using this EC3 value according to the published data for classification of contact allergens, the test item, therefore, can be considered as weak sensitiser (10 ≤ EC3 ≤ 100). In addition, the EC3 value based on the equation of the regression curve was also calculated. The relevant value was 9.3 in this LLNA. Using the EC3 value based on the regression curve, the test item can be ranked among moderate sensitisers (1 ≤ EC3 < 10) in this LLNA.

Human data

The skin sensitisation potential of Dermosoft decalact was also investigated by two human repeat insult patch tests (HRIPT) under semi-occlusive conditions. While in one HRIPT a concentration of 25% of the registered substance in glycerol was tested (Tatsene, 2012a ), the concentration in the second HRIPT was 6% (Tatsene, 2012b ). Summaries of the studies are provided in IUCLID section 7.10.4. Groups of 50 volunteers were exposed to the test substance for a series of nine consecutive 24 h exposures. Volunteers were then given a 10 - 14 day rest period after which a challenge dose was applied once to a previously unexposed test site. The challenge dose was equivalent to any one of the original nine exposures. Skin reactions were scored 24 and 48 h after application of the challenge. In both studies no adverse reactions of any kind were noted. The test item diluted in glycerol when tested under semi-occlusion did not induce irritant and sensitisation to the skin.

Conclusion on skin sensitisation

The two HRIPT have not been performed according to OECD harmonised guidelines under GLP conditions and there is no general regulatory acceptance of this kind of investigations. In consequence, the results of the HRIPT are considered as supporting information only and they are not deemed appropriate to disregard the clearly positive indication of skin sensitisation in the LLNA.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available in vivo data on skin sensitisation (LLNA) obtained with fatty acids, C10-12, esters with polylactic acid, sodium salts (Dermosoft decalact, CAS No. 1312021-45-6, EC No. 700-937-1) meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP). The registered substance, therefore, is classified as moderate skin sensitiser (Skin Sens. 1B, H317). The negative results of two human repeat insult patch tests (HRIPT) are considered not sufficient to disregard the positive outcome of the LLNA.