Fatty acids, C14-18, C14-18-alkyl esters

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

26 May - 23 Jun 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP-guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (Dec 2012)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 22-25 g (range)
- Housing: animals were housed individually in labelled Makrolon cages (MI type, height 12.5 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd.), Surrey, UK). The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on the third day 3. During the acclimation period the animals were housed in groups in Macrolon cages (MIII type, height 18 cm).
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-23.1
- Humidity (%): 41-68
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 May 2010 To: 23 Jun 2010

Vehicle:

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

Concentration:

25, 50 and 100% (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Irritation: A preliminary irritation study was performed according to the procedure of the main study. Two mice were treated daily for 3 consecutive days with either a 50% solution or 100% test substance. 3-4 hours after the last exposure, the irritation severity on the treated skin site was assessed. The body weight was recorded on Day 1 and 3. Very slight erythema (grade 1 of 4) was observed on both the treated sites in the animal exposed to 100% concentration. None of the animals exhibited signs of toxicity during the study period and the body weight was not affected by the treatment. Based on these results, the highest test substance concentration selected for the main study was 100%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by beta-scintillation counting
- Criteria used to consider a positive response: DMP values will be measured for each animal and for each dose group. The stimulation Index (SI) will be calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥3, the test substance may be regarded as a skin sensitiser, based on the test guidelines and the recommendations done by ICCVAM.

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
During the induction phase (Day 1-3), the dorsal surface of both ears was epidermally treated (25µL/ear) with the vehicle control or 25, 50 and 100% test substance, at approximately the same time each day for 3 consecutive days. On Day 6, all the animals were injected via the tail vein with 0.25 mL sterile phosphate buffered saline (PBS), containing 20 µCi 3H-methyl thymidine. After approximately 5 hours, all the animals were sacrificed by intraperitoneal injection of Euthasol 20% and the draining (auricular) lymph node of both ears was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination, and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS. A single cell suspension of lymph node cells (LNC) was prepared the same day in PBS by gentle separation through a stainless steel gauze (diameter 125 µm).
A single cell suspension of LNCs was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 µm). To precipitate the DNA, the LNCs were exposed to 5% trichloroacetic acid (TCA) and stored in the refrigerator until the next day.
Radioactivity measurements were performed on Day 7, using Ultima Gold cocktail as the scintillation fluid. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

other: A reliability check was performed with alpha-hexylcinnamaldehyde every 6 months to demonstrate that the LLNA test system is reliable and sufficiently sensitive

Positive control results:

A reliability check with a positive control substance was performed every 6 months to demonstrate that the LLNA test system, as used by the test laboratory, is reliable and sufficiently sensitive. In the test performed in April 2010 (project No. 494039), 5, 10 and 25% alpha-hexylcinnamaldehyde, technical grade in acetone/olive oil (4:1 v/v) was used as the positive control. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.7, 2.7 and 8.8 respectively. The SI-value for the vehicle control was 1.0. An EC3 value of 10.7% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 -monthly HCA reliability checks in CBA/J female mice of the recent years were 14.1, 13.8, 13.9, 16.0, 11.9 and 16.9%. Based on the results, it was concluded that the Local Lymph Node Assay in the mouse as supplied by Janvier performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

Parameter:

SI

Value:

1.2

Test group / Remarks:

25% group

Parameter:

SI

Value:

2.1

Test group / Remarks:

100% group

Parameter:

SI

Value:

2

Test group / Remarks:

50% group

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The mean DPM values for the control, 25, 50 and 100% groups were 488, 571, 951 and 1013, respectively (see Table 1). The slight increase in DPM with increasing dose level was not statistically significant.

Effects on the lymph nodes:

The right auricular node was enlarged in 1/5 mice in the highest dose group. As this was a single case, it is not considered to be a sensitivity reaction. No macroscopic abnormalitites were observed in the surrounding area.

Table 1: Individual values for radioactivity measurements (disintegrations per minute) and mean stimulation indices

Group	Animal No.	Concentration (% w/w)	DPM/animal	Stimulation index (mean± SEM)
1	1	0	480	-
	2	0	629	-
	3	0	367	-
	4	0	447	-
	5	0	515	-
Mean ± SEM			488 ± 43	1.0 ± 0.1
2	6	25	514	-
	7	25	516	-
	8	25	519	-
	9	25	817	-
	10	25	491	-
Mean ± SEM			571 ± 62	1.2 ± 0.2
3	11	50	928	-
	12	50	778	-
	13	50	589	-
	14	50	1084	-
	15	50	1376	-
Mean ± SEM			951 ± 134	1.0 ± 0.3
4	16	100	637	-
	17	100	796	-
	18	100	1137	-
	19	100	1013	-
	20	100	1483	-
Mean ± SEM			1013 ± 146	1.1 ± 0.1

DPM = disintegrations per minute

SEM = standard error of the mean

Skin irritation effects:

Slight erythema was observed at the test site on both ears in 5/5 mice exposed to 100% test substance. This was not considered to have affected the activity of the lymph nodes.

Systemic effects:

There was no mortality. No clinical signs were observed during the study period and there were no effects on body weight.

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:

Justification for read-across

There are only limited human data available on the skin sensitisation potential of fatty acids, C14-18, C14-18-alkyl esters (CAS 85566-24-1). In order to fulfil the standard information requirements set out in Annex VII, 8.3, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across to avoid the need to test every substance for every endpoint).

The target and source substances are considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information on the source substance is used to predict comparable results for the target substance fatty acids, C14-18, C14-18-alkyl esters (CAS 85566-24-1). The target substance is characterized as an UVCB substance comprised of esters of mainly C14 fatty acid/C14 alcohol, C16 fatty acid/C14 alcohol and C16 fatty acid/C16 alcohol substances. The source substance is structurally similar to the target substance. Decyl oleate (CAS 3687 -46 -5) is a mono-constituent substance consisting of C18:1 oleic acids esterified with decan-1-ol. Thus, target and source substance contain similar structural properties based on common functional groups.

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

Discussion

 

Skin sensitisation

 

CAS 3687-46-5

 

The skin sensitising potential of decyl oleate (CAS 3687-46-5) was evaluated in a local lymph node assay (LLNA) performed according to OECD 429 (Beerens-Heijnen, 2010). 25 µL of a 25, 50 and 100% suspension of test substance in acetone/olive oil (4:1 v/v) was applied to the dorsal surface of both ears of 5 CBA mice/dose for 3 consecutive days. On Day 6, each animal was injected via the tail vein with 0.25 mL sterile phosphate buffered saline containing 20 µCi of 3H-methyl thymidine. After approximately 5 hours, the mice were sacrificed and the draining lymph nodes of the ears were excised. The nodes were pooled for each animal in PBS and the DNA precipitated with 5% TCA at 4 °C overnight. Slight edema (score 1 of 4) was noted on the ears of 5/5 mice treated with the undiluted substance. This is not considered to have had a significant effect on the activity of the lymph nodes. All the nodes of the animals in the control and treatment groups were normal in size, and no macroscopic abnormalities were noted in the surrounding area. The positive control group (hexyl cinnamic aldehyde) was valid. The mean DPM/animal values for the control, 25, 50 and 100% groups were 488, 571, 951 and 1013, respectively. The SI values calculated for the 25, 50 and 100% groups were 1.2, 2.0 and 2.1, respectively. The SI was lower than 3 up to and including 100%, therefore the test substance is considered to be not skin sensitising. 

 

CAS 85566 -24 -1

 

The sensitising potential of fatty acids, C14-18, C14-18-alkyl esters (CAS 85566-24-1) was assessed in a repeated insult patch test (epicutaneous test) on 52 human volunteers (8 males, 42 females) (Croda, 1978). During the induction phase, volunteers were exposed to the test item on the upper back between scapulae, for a total of ten times at an average of 3 times/week. At each occasion, an occlusive patch with 0.15 mL of the test substance, in a 20% dilution, was applied to a surface area of 1.5" x 2" for 24 h. The challenge was performed approximately two weeks after the last induction. The challenge patches were applied to the original and to virgin sites. The volar forearms of each participant served as the virgin test sites. The sensitisation reaction was assessed at 24 and 48 h after patch removal. The repeated cutaneous applications of

fatty acids, C14-18, C14-18-alkyl esters (20% dilution), under occlusive patch, in healthy adult volunteers, did not result in any cutaneous sensitisation reactions.

 

Conclusions for skin sensitisation 

Based on read-across and substance-specific data, sufficient evidence is available to conclude that the substance fatty acids, C14-18, C14-18-alkyl esters (CAS 85566-24-1) is not a skin sensitizer. 

 

Migrated from Short description of key information:
Skin sensitisation (OECD 429): not sensitising

Justification for selection of skin sensitisation endpoint:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is an adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Justification for selection of respiratory sensitisation endpoint:
Study not required according to Annex VII-X of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

Based on read-across following an analogue approach, the available data on skin sensitisation 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.

There are no data available on respiratory sensitisation.