Fatty acids, C14-18 and C16-18-unsatd., maleated, reaction products with oleylamine

Endpoint summary

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: WS400107, dose 10 µl/tissue sample.
- 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
It was demonstrated, that the intrinsic colour and the colouring potential of the test substance, WS400107, in contact with water did not interfere with the assay. However, direct reduction of MTT by WS400107 itself without the involvement of mitochondrial dehydrogenase activity could not be ruled out, as there was a change of colour in a WS4000107/MTT solution during the pre-test. Therefore in the main test, water killed tissues (which have no metabolic activity but absorb and bind the test substance like viable tissues) were included in addition to the live tissues. In addition, the pH of the test substance was estimatedto be approximately 7.0 using pH indicator paper.

Main Test
Each treatment group (test substance, negative/positive controls) comprised 3 live (viable) tissue samples placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. In addition test substance treated and untreated water killed tissues, each in triplicate, were included in the main test and processed further in a similar manner as the live tissue samples.

Tissue Processing and Quantitative Determination of Cell Viability
Incubation of the tissues prior to treatment in maintenance medium:
- Live tissue samples: ≥ 24 h at 37 ± 2° C in humidified atmosphere of 5% CO2 in air
- Thawed, water killed tissues: ≥ 1 h at room temperature

Positive and negative control preparations and the test substance were dispensed over each assigned tissue sample 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 thus making contact with the whole surface of the tissue.

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. 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 a minimum of 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: WS400107 treated tissue viability as % of OD of negative controls

Value:

112.7

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. Reversibility: other: not applicable to the EPISKIN skin irritation assay. Remarks: Mean OD of replicate blanks and of water killed tissues has been accounted for by subtraction. Therefore, a maximum possible score is not applicable.. (migrated information)

Irritation / corrosion parameter:

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

Value:

12.8

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. Reversibility: other: not applicable to the EPISKIN skin irritation assay. Remarks: Mean OD of replicate blanks has been accounted for by subtraction. An accurate maximum possible score is not applicable.. (migrated information)

Irritant / corrosive response data:

See Table 1

Other effects:

In a pre-test, direct reduction of MTT by WS400107 itself without the involvement of mitochondrial dehydrogenase activity was disclosed. This was accounted for in the main test by inclusion of water killed tissues (which have no metabolic activity but absorb and bind the test substance like viable tissues) in addition to the live tissues.

 

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	0.725	0.689	0.698	0.704 ± 0.019	100.0 ± 2.7
WS400107	0.701	0.756	0.740	0.732 ± 0.028	112.7 ± 4.0**
Positive Control	0.043	0.090	0.138	0.090 ± 0.047	12.8 ± 6.7

 

*  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.) = 0.138 ± 0.004

 

** Derived from “true MTT reduction” OD 540 values, thus accounting for possible direct reduction of MTT by the test substance

 

 

Hence assay validity was confirmed both, for the negative and the positive controls, and the test material, WS400107, proved to be not irritating.

 

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): 40 - 41 weeks
- Weight prior to dosing (Day 1): Minimum 3.84 kg, maximum 4.18 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: 29 or 30 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 mL 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.

Duration of treatment / exposure:

The eyes were not rinsed after treatment. Therefore, 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):

8 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 seven days after treatment (Day 8) 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:

0.1

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible within: 24 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible within: 24h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

chemosis score

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:

chemosis score

Basis:

animal #2

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:

chemosis score

Basis:

animal #3

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:

mean

Remarks:

over 3 animals and 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 #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

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

other: Discharge

Basis:

animal #3

Remarks:

mean over 3 time points

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritant / corrosive response data:

Corneal and iridic lesions were not evident throughout the study. Conjunctival redness (grade 1) was seen in all treated eyes at 1 hour and in one of them also at 24 hours after instillation having completely disappeared in two animals by 24 hours and in the other animal by 48 hours after instillation. In addition, chemosis and discharge (both grade 1) were recorded in all animals at 24 hours after instillation and had completely disappeared by 48 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 practically 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 in vivo 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 WS400107, 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].