Fatty acids, C18-unsatd, dimers, hydrogenated, diisopropyl esters

Endpoint summary

Description of key information

Skin irritation (OECD 439): not irritating
Eye irritation (OECD 405): not irritating (RA CAS 68334-05-4)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 - 14 Apr 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

(2013)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

(2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Species:

human

Strain:

other: human-derived epidermal keratinocytes (EpiSkin)

Details on test animals or test system and environmental conditions:

TEST METHOD
Reconstructed Human Epidermis tissues are provided as kits (SkinEthic). The EpiSkin model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum. The test item is applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the test item treated tissues relative to the negative control.

ADAPTATION TO CELL CULTURE CONDITIONS
On arrival, the human-derived epidermal keratinocyte tissues were transferred into pre-labeled 12-well plates (one for each culture condition) containing 2 mL of pre-warmed maintenance medium. The tissues were incubated overnight at 37 °C and 5% CO2 in air.

INCUBATION CONDITIONS (INCUBATOR)
Temperature (°C): 37
CO2 gas concentration (%): 5

Type of coverage:

other: in vitro system

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with DPBS served as negative controls, positive controls were exposed to 5% SDS

Amount / concentration applied:

TEST MATERIAL:
Amount(s) applied (volume or weight with unit): 10 μL

NEGATIVE CONTROL SUBSTANCE:
Amount(s) applied (volume or weight with unit): 10 μL DPBS

POSITIVE CONTROL SUBSTANCE:
Positive control substance: 10 μL SDS, 5% (w/v)

Duration of treatment / exposure:

15 min

Observation period:

Post-treatment incubation period: 42 h

Number of animals:

Not applicable. The test was performed in triplicates for each treatment and control group.

Details on study design:

TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Triplicate tissues were treated with the test item and the concurrent positive and negative controls for an exposure period of 15 min. At the end of the exposure period, each tissue was removed from the well and rinsed with DPBS containing calcium and magnesium. The rinsed tissues were transferred to other wells of the same 12-well plate containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C and 5% CO2 in air for 42 hours.
- Time after start of exposure: 15 min

CELL VIABILITY MEASUREMENTS:
For determining alterations in cell viability, MTT reduction assays were performed immediately after the 42 hour incubation period. Therefore, tissues were transferred to new wells of the same 12-well plate containing 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium. The tissues were incubated for 3 h at 37 °C and 5% CO2 in air. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. The epidermis was carefully separated from the collagen matrix and both parts (epidermis and collagen) placed into labeled 1.5 mL tubes containing 500 µL of acidified isopropanol. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues. For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a 96-well plate. The optical density was measured at 562 nm wave length in a microplate reader.

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of negative controls (DPBS). Time point: 15 min . Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

15.3

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls (SDS). Time point: 15 min . Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

108.1

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 15 min . Reversibility: other: not applicable. (migrated information)

Irritant / corrosive response data:

The relative mean viability of the test item treated reconstructed human-derived keratinocyte tissues after a 15 min exposure period was 108.1% compared to the negative control. Therefore, the test item is considered to be a non-irritant.

Table 1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.767	0.767	0.05	100	100*	6.2
	0.719			93.7
	0.814			106.1
Positive Control Item	0.145	0.118	0.06	18.9	15.3	7.7
	0.158			20.6
	0.050			6.5
Test Item	0.813	0.829	0.01	106.0	108.1	1.9
	0.842			109.8
	0.833			108.6

SD: Standard deviation

*: The mean viability of the negative control tissues is set at 100%.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Study period:

19 - 29 Apr 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Fatty acids, C18-unsatd., dimers, 2-ethylhexyl esters (CAS No. 68334-05-4). In accordance 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 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan, Belton, Leics, England
- Age at study initiation: at least 7 - 9 weeks
- Weight at study initiation: 1572 - 1809g
- Housing: animals were individually housed in labelled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm)
- Diet: pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least three times a week
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 (actual range: 18.6 – 19.3)
- Humidity (%): 40 - 70 (actual range: 43 - 67)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye of the same animal served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

24h

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance.

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0)

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Irritation parameter:

iris score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility: not applicable

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Irritant / corrosive response data:

Instillation of 0.1 mL of Fatty acids, C18-unsaturated, dimers, 2-ethylhexyl esters into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness and discharge. The irritation completely resolved within 48 hours in all animals.
No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. There was no evidence of ocular corrosion.

Other effects:

Remnants of the test substance were present on the outside of the eyelids on Day 1. No staining of (peri) ocular tissues by the test substance was observed. No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for grouping of substances and read-across

There are only limited data available on eye irritation of Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3). In order to fulfil the standard information requirements set out in Annex VII and VIII, 8.2., 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). Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Overview of skin and eye irritation

CAS	Chemical name	Molecular weight [g/mol]	Skin irritation	Eye irritation
103213-20-3 (a)	Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters	645	Experimental result: not irritating	RA: CAS 68334-05-4
68334-05-4 (b)	Fatty acids, C18-unsaturated, dimers, 2-ethylhexyl esters	673	Experimental result: not irritating	Experimental result: not irritating

(a) The substance subject to registration is indicated in bold font.

(b) Reference (read-across) substances are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

The above mentioned substances are considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information is used to predict the same endpoints for Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Skin irritation

Beside a non-reliable skin irritation study (CIR, 2003; RL4) indicating no irritation to skin in rabbits a reliable skin irritation study is available with Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3). In the in vitro key skin irritation study performed according to OECD TG 439 and in compliance with GLP human-derived epidermal keratinocytes (EpiSkin) were exposed to 10 µL of the neat test material for 15 minutes followed by a post-treatment incubation period of 42 hours (Warren, 2015a). The irritation potential of the test material was predicted from the relative mean cell viabilities compared to the mean viability of the negative control using the MTT assay. Positive (5% SDS) and negative (DPBS) controls were included in the study and gave the expected results. The relative mean cell viability for the test material was calculated to be 108.1% when compared to the negative control after an incubation period of 42 hours. Based on the result of the study and according to EU classification criteria, the test substance is considered to be not-irritating.

 

Eye irritation

Beside a non-reliable eye irritation study performed in rabbits (CIR, 2003; RL4) and a supporting in vitro eye irritation study performed in human corneal epithelial cells (pre-validated HCE model, Warren, 2015b), both indicating no irritation to eyes, no reliable data is available with Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3). Therefore, read across from the structurally analogue substance Fatty acids, C18-unsaturated, dimers, 2-ethylhexyl esters (CAS 68334-05-4) was applied.

CAS 68334-05-4

In the key eye irritation study with Fatty acids, C18-unsaturated, dimers, 2-ethylhexyl esters (CAS 68334-05-4) performed according to OECD TG 405 and in compliance with GLP (Stitzinger, 2010a) 0.1 mL of the neat test material was instilled in the eye of three New Zealand White rabbits. The eyes were examined and the changes were graded according to the Draize scoring system 1, 24, 48 and 72 hours post-application. No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred during the course of the study. Neither iridial irritation nor corneal opacity and chemosis were observed in any animal during the course of the study. All animal showed conjunctivae irritation (grade 0.33) which was fully reversible within 48 hours. Based on the result of the study and according to EU classification criteria, the test substance is considered to be not-irritating to eyes.

CAS 103213-20-3

In the supporting skin irritation study with Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3) performed according to “Prevalidation of a new in vitro reconstructed human corneal model to assess the eye irritating potential of chemicals” (Van Goethem et al, 2006) and in compliance with GLP SkinEthic tissues (Human corneal epithelial cells) were treated with 30 µL of the neat test material for 10 minutes (Warren, 2015b). After exposure with the test material, all tissues were rinsed and cell viability was assessed using the MTT assay. Positive (2% SDS) and negative (Solution A: 1 mM Na2HPO4, 10 mM glucose, 30 mM HEPES, 3 mM KCl, 130 mM NaCl) controls were included in the study and gave the expected results. The relative mean tissue viability (percentage of the negative control) was calculated to be 92.5%. Based on the result of the study and according to expert judgement, the test substance is considered to be not-irritating.

Taken together, the target substance Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3) is not expected to be irritating to skin or eyes.

Justification for selection of skin irritation / corrosion endpoint:
The reliable GLP compliant OECD Guideline study was choosen.

Justification for selection of eye irritation endpoint:
The reliable GLP compliant OECD Guideline study was choosen.

Justification for classification or non-classification

The available data on skin and eye irritation of Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.