Fatty acids, C18-unsatd., trimers, compds. with oleylamine

Endpoint summary

Description of key information

in vitro EPISKIN(TM) 15 min - 42h skin irritation test (OECD439):
The mean tissue viability attained after treatment with WS400151 was 83.7 % and validity of the assay was confirmed, both for the negative and the positive controls. Therefore, WS400151 was predicted to be not irritant to the skin. 
in vitro Bovine Corneal Opacity and Permeability Test (BCOP) irritation test (OECD437):
The test substance did not produce changes indicative of serious eye damage. 
Eye irritation, rabbit with neat WS400151:
Mean irritation score 24, 48 and 72 h for each of 3 animals:
0.0 for corneal opacity; 0.0 for iris lesion; 0.7 to 1.0 for redness of conjunctivae; 0.7 to 1.0 for oedema of conjunctivae (chemosis); Minor findings (conjunctival redness, chemosis and discharge, all up to grade 1 at 1 h post dosing and discharge grade 1 at 24 h) were not relevant.
Conclusion: No requirement of classification regarding eye irritation.
Local lymph node assay, mouse (Endpoint study record "7.4.1 Skin sensitisation - LLNA_mouse_HLS_GAH0023"):
Group Mean Stimulation Index (SI) = 1.0, 8.6, 19.3 and 15.4 at test substance concentrations of 0 % (vehicle control), 10, 25 and 50% w/v, respectively.
Conclusion: Classification and labelling as
"irritant (Xi)" and  "may cause sensitisation by skin contact (R43)" [DIRECTIVE 67/548/EEC] and
“Category 1” (Warning: May cause an allergic skin reaction)" [REGULATION (EC) 1272/2008].

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method) of 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: L’Oreal. In Vitro Skin Irritation Test: Human Epidermis Model EPISKIN, EPISKIN Skin Irritation Test 15min - 42 hours, Standard Operating Procedure: February 2009 Version 1.8.

Deviations:

yes

Remarks:

An error in the cited SOP was not adopted. Instead, correctly, 10 mg test substance + 90 μL water were used for checking the coloring potential of the test substance

GLP compliance:

yes (incl. QA statement)

Amount / concentration applied:

Types of Treatment in the Main Test
- Negative control: Sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium, dose volume 10 µl/tissue sample.
- Test Substance: WS400151, dose 10 ± 2 mg/tissue sample.
As an exception one tissue received < 8 mg due to the stickiness of the test substance, but this was not considered to affect the scientific integrity of the study, as the tissue surface was fully covered with the test substance.
- Positive control: 5% Sodium Dodecyl Sulphate (SDS) in distilled water, dose volume 10 µl/tissue sample.

Duration of treatment / exposure:

15 ± 0.5 minutes with the test substance, negative or positive control at room temperature

Details on study design:

Test System
EPISKIN human epidermis skin constructs consisting of normal, human-derived epidermal keratinocytes and forming a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum (matrix: collagen type 1 coated with type IV collagen).

Principle of the Test – Main Test
Irritant substances are sufficiently cytotoxic to cause cell deaths in the cell layers. Therefore, cell viability of the multilayers was determined by measurement of mitochondrial dehydrogenase activity assessed by reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a soluble, blue coloured, formazan salt. Depending on the percentage of tissue viability attained (compared to negative control viability) a test substance is classified as skin irritating or not skin irritating.

Pre-Tests – Checking for Interference of the Test Substance with the Assay
False negatives were ruled out prior to the main test. It was demonstrated, that the test substance, WS400151, itself did not directly reduce MTT without the involvement of mitochondrial dehydrogenase activity and that the intrinsic colour and the colouring potential of the test substance in contact with water did not interfere with the assay. In addition, the pH of the test substance was estimated to be ca. 7 to 7.5 using pH indicator paper.

Main Test
Each treatment group (test substance, negative/positive controls) comprised 3 tissue samples placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well.

Tissue Processing and Quantitative Determination of Cell Viability
Incubation of the tissues prior to treatment: ≥ 24 h at 37 ± 2° C in the maintenance medium in humidified atmosphere of 5% CO2 in air.
Positive and negative control preparations were dispensed over each tissue using a positive displacement pipette and 7 minutes afterwards positive controls were re-spread with a curved flat spatula. Test material was spread over the tissue using the curved edge of a spatula.

After the 15 ± 0.5 minutes treatment period, residual test substance or positive control substance was removed by rinsing each tissue with 25 mL sterile DPBS and gently swabbing as appropriate. Inserts were blotted on absorbent paper to remove remaining DPBS.

This was followed by incubation of each insert at 37 ± 2° C in humidified atmosphere of 5% CO2 first for 42 ± 1 h in wells each containing 2 mL maintenance medium and then for 3 hours ± 5 minutes in wells each containing 2 mL of 0.3 mg/mL MTT. Finally the tissue samples were processed further and formazan was extracted by vortexing and storage in acidic isopropanol, 500 µL/sample, at 2-8°C in the dark over 70 hours. The absorbance was quantitatively determined at 540 nm with acidified isopropanol (0.04 N HCl final concentration) as a blank.

Irritation / corrosion parameter:

other: other: Negative Control Tissue viability as % of OD of negative controls

Value:

100

Remarks on result:

other:

Remarks:

Basis: mean. Time point: Following 42 ± 1 h incubation in fresh maintenance medium, 3 h incubation in MTT solution and 70 h formazan extraction. Max. score: 100.0. Reversibility: other: not applicable to the EPISKIN skin irritation assay. Remarks: Mean OD of replicate blanks has been accounted for by subtraction. (migrated information)

Irritation / corrosion parameter:

other: other: WS400151 treated tissue viability as % of OD of negative controls

Value:

83.7

Remarks on result:

other:

Remarks:

Basis: mean. Time point: Following 42 ± 1 h incubation in fresh maintenance medium, 3 h incubation in MTT solution and 70 h formazan extraction. Max. score: 100.0. Reversibility: other: not applicable to the EPISKIN skin irritation assay. Remarks: Mean OD of replicate blanks has been accounted for by subtraction. (migrated information)

Irritation / corrosion parameter:

other: other: Positive Control Tissue viability as % of OD of negative controls

Value:

19.9

Remarks on result:

other:

Remarks:

Basis: mean. Time point: Following 42 ± 1 h incubation in fresh maintenance medium, 3 h incubation in MTT solution and 70 h formazan extraction. Max. score: 100.0. Reversibility: other: not applicable to the EPISKIN skin irritation assay. Remarks: Mean OD of replicate blanks has been accounted for by subtraction. (migrated information)

Irritant / corrosive response data:

See Table 1

Other effects:

The pre-tests gave no indication of interference of the test substance with the present in vitro EPISKIN skin irritation assay.

 

Tabelle 1:  Results of in vitro EpiSkin (TM) Skin Irritation Test
Exposure Period: 15 ± 0.5 minutes
	OD 540 Tissue 1	OD 540 Tissue 2	OD 540 Tissue 3	OD 540 Mean of Tissue 1, 2 + 3	Tissue Viability [% of Negative Control ± s.d.]
Negative Control	0.798	0.780	0.778	0.785 ± 0.011	100.0 ± 1.4
WS400151	0.639	0.706	0.627	0.658 ± 0.043	83.7 ± 5.4
Positive Control	0.152	0.223	0.095	0.157 ± 0.064	19.9 ± 8.2

 

OD 540 of individual tissues = Mean Optical Density [wavelength 540 nm] of 2 measurements minus Mean OD of six blanks

 

Mean OD of six blanks ± standard deviation (s.d.) = 0.123 ± 0.004

 

Hence assay validity was confirmed both, for the negative and the positive controls, and the test substance, WS400151,

was predicted to be not irritating to the skin. 

 

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: criteria specified in the OECD 439 test guideline.

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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

of 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

of 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

of 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF Test Data for Registration of Agricultural Chemicals, Eye Irritation (2-1-5), 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS

- Stock supply of healthy adult rabbits
- Number and Sex: 3 females
- Age on day of dosing (Day 1): 23 - 34 weeks
- Weight prior to dosing (Day 1): Minimum 4.09 kg, maximum 4.82 kg
- Housing: Individual housing in plastic cages with peforated floor
- Environmental enrichment: Small soft white untreated wood blocks
- Diet : Standard laboratory rabbit diet (125 g/animal/day). In addition, dietary supplement of hay
replaced by a supplement of wholemeal bread as from 2 days prior to dose instillation.
- Water: Drinking water, ad libitum
- Acclimation period: 7 or 8 weeks prior to study start under laboratory conditions.

Routine analysis of the batch of diet used for nutrients, possible contaminants and microorganisms and of the drinking water were conducted or available at the testing facility.


ENVIRONMENTAL CONDITIONS

Air conditioned animal room set at:
- Temperature (°C): 18 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod: 12 hrs artificial light per 24 hrs

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.1 g of undiluted liquid test substance was administered into the conjunctival sac of one eye per rabbit (right eye). The contralateral eye (left eye) remained untreated to serve as a control. (The test substance was too viscous to be drawn into a syringe, therefore 0.1 g aliquots were weighed and administered.)

Duration of treatment / exposure:

There was no mentioning in the report that eyes were rinsed after treatment. Consequently the treatment/exposure period was equivalent to the entire observation period following instillation or lasted until removal of the test material by physiological mechanisms, whichever happened sooner.

Observation period (in vivo):

14 days in one animal (sentinel) and 72 hours in the other two animals

Number of animals or in vitro replicates:

3 adult female rabbits

Details on study design:

EYE EVALUATION:

Before treatment start both eyes of each animal were investigated to ensure that there was no pre-existing corneal damage, iridial inflammation or conjunctival irritation.

One animal (the sentinel) was initially treated and in the absence of a severe effect the remaining two animals were committed to the study.

Eyes were evaluated in all animals at approximately 1, 24, 48 and 72 hours after test substance instillation adopting the numerical scoring system listed in Table 1 in the field below. Grades attained at 24, 48 and 72 hours after instillation were included in the mean gradings of ocular lesions and used for evaluation of the necessity of eye irritation/corrosion classification. In addition, eyes of the sentinel animal were checked 7 and 14 days after treatment (Days 8 and 15, respectively) for possible delayed effects.

Equipment used for eye evaluation: Ophthalmoscope or pencil beam torch

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

4

Reversibility:

other: Corneal ulceration or opacity were not evident

Irritation parameter:

other: Area of corneal opacity

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

4

Reversibility:

other: Corneal ulceration or opacity were not evident

Irritation parameter:

iris score

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

2

Reversibility:

other: Iridic changes were not evident

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0.9

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0.9

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

other: discharge

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0.2

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

other: discharge

Basis:

animal #1

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

other: discharge

Basis:

animal #2

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

other: discharge

Basis:

animal #3

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritant / corrosive response data:

Corneal and iridic lesions were not evident throughout the study. Conjunctival redness grade 1 or 2 was seen in all treated eyes at 1, 24 and 48 hours after instillation and was associated with chemosis grade 1 or 2 in two animals at all of these time points and in the third animal at 24 and 48 hours after instillation. In addition, discharge grade 1 was seen in all animals at 1 and/or 24 hours after instillation. All findings were completely reversible, discharge having disappeared by 24 or 48 hours after instillation in individual animals and conjunctival redness and chemosis having completely disappeared by 72 hours after instillation. Throughout the study no other signs of ocular irritation were evident.

Other effects:

Observation of the animals for defined behavioural criteria led to the conclusion that instillation of the test substance induced no initial pain response. Any other signs, of systemic toxicity or ill health were not evident.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The outcome of the present study does not necessitate any labelling regarding eye irritation according to EU classification rules [DIRECTIVE 67/548/EEC and REGULATION (EC) 1272/2008]. The minor findings noted are within the category “not irritating to eyes”. They were fully reversible.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In view of the absence of any relevant effects during the skin and eye irritation studies with WS400151, the attained results do not necessitate any classification and labelling regarding skin or eye irritation according to EU classification rules [DIRECTIVE 67/548/EEC and REGULATION (EC) 1272/2008].

 

However, the positive sensitisation response elicited at three concentration levels of WS400151 in a local lymph node assay*in mice necessitates classification and labelling as "irritant (Xi)" and "May cause sensitisation by skin contact (R43)” according to DIRECTIVE 67/548/EEC and as “Category 1” (Warning: May cause an allergic skin reaction) according to REGULATION (EC) 1272/2008.

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*Endpoint study record "7.4.1 Skin sensitisation - LLNA_mouse_HLS_GAH0023”