Fatty acids, C16-18, esters with diethylene glycol

Endpoint summary

Description of key information

Skin irritation: not irritating

Eye irritation: not irritating

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Basic data given (no details on test substance and test design (type of coverage, exposure duration))

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted Jul 2015

Deviations:

yes

Remarks:

(no details on test substance and test design (type of coverage, exposure duration))

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

not specified

Controls:

not required

Duration of treatment / exposure:

no data

Observation period:

5 days
Reading time points: 1, 24, 48 and 72 h

Number of animals:

3 males

Irritation parameter:

erythema score

Basis:

animal: #1, #2 and #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 5 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 5 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritant / corrosive response data:

Very slight erythema (graded with score 1) were observed in all test animals starting 1 h after patch removal which were stable up to the 72 h reading time point. Moreover, slight edema (scored with 2) were determined in 1/3 animals 1 h after application which was still visible as very slight edema (score 1) up to the 72 h reading time point. The remaining animals revealed very slight edema (score 1) at the 1 h reading time point which were reversible 24 h later. Latest on Day 5 after test substance application, the skin reactions were fully reversible.

Table 1. Individual skin reaction scores

Observation time	Rabbit no.
	1		2		3
	Erythema	Edema	Erythema	Edema	Erythema	Edema
1 h	1	2	1	1	1	1
24 h	1	1	1	0	1	0
48 h	1	1	1	0	1	0
72 h	1	1	1	0	1	0
Mean value 24 + 48 + 72 h	1,00	1,00	1,00	0,00	1,00	0,00
Mean value 24 + 48 + 72 h all animals	1,00	0,33

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23 - 24 Jul 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The eye irritation potential was determined using the SkinEthic reconstructed Human Corneal Epithelium model following a 10 min exposure period. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

The test method was designed to be compatible with the prevalidation protocol published by Van Goethem et al., 2006 (Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals). The method follows forms part of the currently ongoing Colipa/ECVAM Eye Irritation Validation Study. However, as the test item was a solid, adaptions were necessary during the course of the test in regard to test substance application. In this study, appropriate sized discs of the test item were prepared for direct application to the tissue culture surface. The exact weight of each disc was irrelevant as adequate contact between the test item and tissue culture surface was considered the critical factor. The methodology for the application of solid test items was followed as closely as possible.

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Species:

human

Strain:

other: transformed corneal epithelium cells (cell line HCE)

Details on test animals or tissues and environmental conditions:

TEST System
- Source: SkinEthic Laboratories, Lyon, France
- Date received: 23 July 2015
- Batch number: 15-HCE-039
- Maintainance medium lot number: 15-MIMA-029

Principles of the test system
The SkinEthic HCE model consists of transformed human corneal epithelial cells of the HCE cell line. The cells form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling histologically the mucosa of the human eye. The model represents an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium.
The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death. In the assay, cytotoxicity was determined by the reduction of MTT to formazan (quantitative measurement of cell viability).


Adaption to cell culture conditions
On arrival, the SkinEthic HCE tissues (Day 6 cultures), were transferred into 24-well plates containing 300 µL maintenance medium overnight 37.5 °C, 5% CO2).

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37.5
- CO2 air concentration (%): 5

Vehicle:

unchanged (no vehicle)

Controls:

other: yes, negative control: Solution A (supplied by SkinEthic); positive control: sodium dodecyl sulphate (2% in sterile water)

Amount / concentration applied:

Appropriate sized discs of the test item were prepared for direct application to the tissue culture surface. Adequate contact between the test item and tissue culture surface was considered as critical factor.

Duration of treatment / exposure:

10 min

Observation period (in vivo):

Not applicable.

Number of animals or in vitro replicates:

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

Details on study design:

PREPARATION OF TEST SUBSTANCE
The test substance was warmed to approx. 40°C to produce a liquid. Prior to application, the test item was allowed to cool down to room temperature prior to application.

SOLUTION A
Na2HPO4 (0.142 g/L)
Glucose (1.802 g/L)
HEPES (7.149 g/L)
KCl (0.224 g/L)
NaCl (7.597 g/L)


REMOVAL OF TEST SUBSTANCE:
At the end of the exposure period, each tissue was rinsed with Dulbecco's Phosphate Buffered Saline (DPBS) without Ca 2+ and Mg 2+. Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper.

CELL VIABILITY MEASUREMENTS:
Rinsed tissues were loaded with 300 µL MTT (0.5 mg/mL solved in maintenance medium) for approx. 3 hrs. After MTT-loading the tissues were immersed in isopropanol for extraction of formazan crystals. After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 562 nm.


TEST ACCEPTAMNCE CRITERIA:
The assay meets the acceptance criterion if the relative mean tissue viability for the positive control treated tissues is <60% relative to the negative control treated tissues.

Irritation parameter:

mean percent tissue viability 

Run / experiment:

10 min exposure

Value:

95.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

tissue viability: 100%

Positive controls validity:

valid

Remarks:

tissue viability: 38.1%

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: no, pre-guideline protocol

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 2: Viability of HCE tissues - Individual data

	Individual OD562	Mean OD562	Relative mean viability (%)
Negative control^1	1.249	1.214	100
	1.210
	1.184
Test item	1.204	1.162	95.7
	1.122
	1.159
Positive control^2	0.528	0.463	38.1
	0.403
	0.458

1: Solution A (supplied by SkinEthic)

2: sodium dodecyl sulphate (2% in sterile water)

Interpretation of results:

study cannot be used for classification

Conclusions:

No indication of eye irritation potential was noted, but the study can not be used for classification and labelling purposes alone and requires further evaluation and/or data generation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

 

Skin irritation

The target substance Fatty acids, C16-18, esters with diethylene glycol was tested for its potential to induce skin irritation similar to OECD 404. Due to the limited data available, read across to the analogue substance ethylene distearate (CAS 627-83-8) is considered additionally to allow hazard evaluation based on a weight-of-evidence approach.

 

CAS 85116-97-8

Fatty acids, C16-18, esters with diethylene glycol was tested in a study similar to OECD guideline 404 (EViC-CEBA, 1994). The results of the conducted study were reported in a summary with limited information. However, as basic data are provided sufficient for hazard assessment, the study was considered as reliable (RL 2).The test substance was topically applied to the skin of 3 male New Zealand White rabbits. The treated skin was observed and evaluated at 1, 24, 48 and 72 h post-application. The animals were observed for a time period of 5 days. Very slight erythema (score 1) were observed in all test animals starting 1 h after patch removal which were stable up to the 72 h reading time point. Moreover, slight edema (score 2) were determined in 1/3 animals 1 h after application which was still visible as very slight edema (score 1) up to the 72 h reading time point. The remaining animals revealed very slight edema (score 1) at the 1 h reading time point which were reversible 24 h later. Latest on Day 5 after test substance application, the skin reactions were fully reversible. Based on severity of local reactions and the reversibility of skin reactions, the test substance Fatty acids, C16-18, esters with diethylene glycol is not considered to exhibit skin irritating properties.

 

CAS 627-83-8

The skin irritation properties of ethylene distearate have been investigated in several studies (CAS 627-83-8).

The skin irritation properties of ethylene distearate were tested in a study equivalent to OECD guideline 404 (Product Safety Labs 1991a). In the study, 3 male and 3 female New Zealand White rabbits were exposed to 0.5 g of the unchanged test substance, applied to the shaved and abraded skin for 25 h using an occlusive dressing. The treated skin was observed and evaluated at 25 and 72 h post-application. No erythema or edema was observed in any animal during the study period on intact shaved skin. On abraded skin, slight erythema in 2 animals was observed at 25 h, being fully reversible within 72 h. No further local or systemic effects were apparent in any animal during the study period.

In an equivalently conducted study from the same laboratory (Product Safety Labs, 1991b), the results of the above discussed study were confirmed. No erythema and no edema formation were observed in the test animals on intact skin and erythema on abraded skin was fully reversible within 72 h. No other local or systemic effects were described.

 

 

Eye irritation

The target substance Fatty acids, C16-18, esters with diethylene glycol was tested for its potential to induce eye irritation in a reconstructed Human Corneal Epithelium model. As the applied model system is not formally validated, read across to the analogue substance ethylene distearate (CAS 627-83-8) is considered additionally to allow hazard evaluation based on a weight-of-evidence approach.

 

CAS 85116-97-8

Triplicates of reconstructed Human Corneal Epithelium (HCE) tissues were treated with the test substance. The used SkinEthic HCE model is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death. The test method was designed to be compatible with the prevalidation protocol published by Van Goethem et al., 2006 (Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals). The method follows parts of the currently ongoing Colipa/ECVAM Eye Irritation Validation Study.

In this study, appropriate sized discs of the test item were prepared for direct application to the tissue culture surface to enable adequate contact between the test substance and the tissue culture surface. The discs stayed in contact with the SkinEthic HCE tissues for 10 min. Afterwards, each tissue was rinsed with Dulbecco's Phosphate Buffered Saline without Ca 2+ and Mg 2+. Rinsed tissues were loaded with MTT solved in maintenance medium for approx. 3 hrs. After MTT-loading the tissues were immersed in isopropanol for extraction of formazan crystals. After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured at 562 nm. The relative mean viability of test substance treated tissues was 95.7%. The relative mean tissue viability for the positive control treated tissues was 38.1% relative to the negative control treated tissues thereby indicating validity of the conducted test. As incubation with the test substance did not reduce relative tissue viability below the defined cut off level of 60%, the test substance is not considered to exhibit eye irritating properties based on the results of the conducted test.

 

CAS 627-83-8

The eye irritation properties of ethylene distearate were tested in a study similarly performed to OECD guideline 405 (Product Safety Labs, 1991). In a group of 6 New Zealand white rabbits, 0.1 g of the test substance was applied into the eye. Observation of the eyes was performed 24, 48 and 72 h after application. No effects on cornea and iris were noted in any animal during the study period. However, mild conjunctival redness was observed in 4/6 animals after 24 h and in 2/6 animals moderate redness was apparent. In 2 animals the mild redness was still persistent after 72 h with a mean score of 0.78 over 24, 48 and 72 h out of all 6 animals. Chemosis was observed in 1/6 animals after 24 h being fully reversible within 48 h. No chemosis was observed in the remaining 5 animals during the study period. No further systemic effects or abnormal behaviour were observed in any animal during the study period.

 

Conclusion on skin and eye irritation properties

The available studies on irritation performed with the target and an analogue substance indicate no irritating properties towards the skin or eyes for the tested substances. Therefore, based on these data, Fatty acids, C16-18, esters with diethylene glycol is not considered as skin or eye irritant.

 

Justification for classification or non-classification

Based on test substance-specific data and on analogue read-across approach, the available data on skin and eye irritation do not meet the classification critera according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.