Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine

Endpoint summary

Description of key information

Supporting in vitro studies and a key in vivo irritation study conducted in rabbits, demonstrated that the registered substance is not irritating to skin.

In vitro studies with the pure substance indicated that the substance might be irritating for eye, which was confirmed by in vivo testing, leading to classifcation as category 2 according to CLP regulation, but not classified according to DSD. The 15% formulation was further tested in vivo in rabbits, and was demonstrated to be not irritating, therefore 15% can be considered as a concentration limit for non-classification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at dosing: Approx. 12.5 – 17.5 months
- Weight at dosing:
Animal no. 1: 2.8 kg
Animal no. 2: 2.9 kg
Animal no. 3: 2.9 kg
- Housing: Before and after the 4-hour exposure period, the animals were kept singly in cages measuring 380 mm x 425 mm x 600 mm (manufacturer: Dipl. Ing. W. EHRET GmbH, 16352 Schön¬walde, Germany). During the exposure period, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn. The cages excluded irritation of the skin by excrements and urine.
- Diet (e.g. ad libitum): ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. The food was available ad libitum before and after the exposure period.
- Water (e.g. ad libitum): Drinking water was offered ad libitum before and after the exposure period.
- Acclimation period: At least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3°C (maximum range)
- Humidity (%): 30% - 70% (maximum range)
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: May 6, 2013 To: May 18, 2013

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: The surrounding untreated skin served as a control.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg test item

Duration of treatment / exposure:

Initial test (1 rabbit) : 4 hours
Confirmatory test (2 rabbits): 4 hours

Observation period:

72 hours

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: Approximately 24 hours before the test, the fur was removed by closely clipping the dorsal area of the trunk of the animals. Care was taken to avoid abrading the skin. Only animals with healthy intact skin were used.
- % coverage: area: approx. 6 cm2
- Type of wrap if used: The test item was applied to the test site and then covered with a gauze patch, which was held in place with non-irritating tape for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure time no residual test item had to be removed.

SCORING SYSTEM:
Erythema and eschar formation
0 : no erythema
1 : very slight erythema (barely perceptible)
2 : well-defined erythema
3 : moderate to severe erythema
4 : severe erythema (beef redness) to eschar formation preventing grading of erythema
Oedema formation
0 : no oedema
1 : very slight oedema (barely perceptible)
2 : slight oedema (edges of area well defined by definite raising)
3 : moderate oedema (raised approx. 1 mm)
4 : severe oedema (raised more than 1 mm and extending beyond the area of exposure)

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24 h, 48 h, 72 h

Score:

0.33

Max. score:

4

Reversibility:

not fully reversible within: 48 h

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 24 h, 48 h, 72 h)

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 24 h, 48 h, 72 h

Score:

0.33

Max. score:

4

Reversibility:

not fully reversible within: 48 h

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 24 h, 48 h, 72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24 h, 48 h, 72 h

Score:

0

Max. score:

4

Table 1. Acute dermal irritation/corrosion test (patch test) of Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine in rabbits

	Skin irritation scores
	Animal no.
	1 E/Oe	2 E/Oe	3 E/Oe
Time after removal of the patch (4-hour exposure)
60 minutes	0/0	1/0	1/0
24 hours	1/0	1/0	1/0
48 hours	0/0	0/0	0/0
72 hours	0/0	0/0	0/0

0 no pathological findings

E erythema and eschar formations

Oe oedeama

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine is non-irritating to skin, hence, no labelling is required.

Executive summary:

Acute dermal irritation/corrosion test was tested by means of a semi-occlusive patch test in rabbits according to EC method B.4. (Regulation (EC) No. 440/2008) and OECD test guidance 404 (2002). 500 mg Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine were applied to the closely clipped dorsal area of the trunk of the animals. As it was expected that the test item would not produce any severe irritancy or corrosion, the test was started using at first only one animal, receiving a single patch for an exposure period of 4 hours.

As neither a corrosive effect nor a severe irritant effect was observed after a four-hour exposure, the test was completed using two additional animals, each with one patch only, for an exposure period of 4 hours. After the 4 hour exposure period the patch was removed and the skin sites were evaluated.Scores were taken 60 minutes, 24, 48 and 72 hours after patch removal.

Under the present test conditions, rabbits exposed for 4 hours to 500 mg Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine/animal showed slight skin reactions expressed as erythema at 60 minutes and/or 24 hours of observation. Mean 24-48h scores were 0.33 for erythema and 0.00 for oedema. The findings were reversible by 48 hours observation period and the scores were below the trigger value for classification.There were no systemic intolerance reactions.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable, however in isolation of other studies cannot be used for classification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Justification for test system used:

Skin corrosion refers to the production of irreversible tissue damage in the skin following the application of a test item [as defined by the Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS)]. The OECD Guideline 431 does not require the use of live animals or animal tissue for the assessment of skin corrosivity.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS-1001
- Tissue batch number(s): 100-AC0650-1; CellSytems® Biotechnology GmbH, 53842 Troisdorf, Germany
- Production date: April 8, 2013
- Shipping date: Not provided
- Delivery date: Not provided
- Date of initiation of testing: March 11, 2013 start of experimental phase

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Not provided
- Temperature of post-treatment incubation (if applicable): Not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: solution of 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Not specified
- Wavelength: 540 nm
- Filter:
- Filter bandwidth:
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The magnitude of viability was quantified by using MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue ). In this case the optical density (OD) of the extracted (solubilised) dye from the negative control tissue was at least 20-fold greater than the OD of the extraction solvent alone. The negative control tissue has been shown to be stable in culture (provide similar viability measurements) for the duration of the test exposure period.
- Barrier function: The stratum corneum has been shown to be sufficiently robust to resist the rapid penetration of certain cytotoxic marker chemicals (e.g. 1% Triton X-100). This property was estimated by the exposure time required to reduce cell viability by 50% (ET50) (for the epiCS-1001 model this is >2 hours). Each batch of the epidermal model used meets defined production release criteria, set by the supplier, among which those for viability and for barrier function are the most relevant (MTT, 2 hours Triton X-100: target > 50%). The barrier properties of the tissues were verified by the supplier.
- Morphology: Human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells were present under a functional stratum corneum. The skin model also had a stromal component layer. Stratum corneum was multi-layered with the necessary lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic markers. The containment properties of the model prevented the passage of material around the stratum corneum to the viable tissue. Passage of test chemicals around the stratum corneum would lead to poor modelling of the exposure to skin.
- Contamination: The skin model was free of contamination with bacteria (including mycoplasma) or fungi.
- Reproducibility: The tissue employed has been shown to demonstrate reproducibility over time between laboratories. Moreover it has been shown to be capable of predicting the corrosive potential of the reference chemicals when used in the testing protocol selected.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
Cell viability measurements were carried out at the end of the exposure period (1st period: 3 min; 2nd period: 60 min). The measurements were made for each of the three tissues in duplicate.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin
if the viability after 3 minutes exposure is less than 50%, or
if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL of test item were applied to the skin model with a surface area of 0.6 cm2 to uniformly cover the skin surface.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL D-PBS (Dulbecco's phosphate buffered saline)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8 N KOH

Duration of treatment / exposure:

3 minutes and 60 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

test group 3 minutes

Value:

89.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

3% mean tissue viability

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

test group 1 hour

Value:

92.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

< 0.1% mean tissue viability

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
- Acceptance criteria met for positive control: The mean viability of cells treated with the positive reference item 8 N KOH were 3.0% (3-minute incubation) and <0.1% (1-hour incubation) of the negative control and were below the cut-off values. Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline:

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions the test item tested at two exposure times of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to determine cytotoxic properties to skin cells which might lead to corrosion by the test item to human skin, in an experiment with an artificial three-dimensional model of human skin. The epiCS-1001 model was employed.
The test item was applied to the skin surface. Dulbecco’s phosphate buffered saline (D-PBS) was used as the negative control. 8 N KOH was used as the positive reference item. Two exposure times of 3 minutes or 1 hour were employed.
In comparison to the negative controls, the mean viability of cells exposed to the test item was 89.3% after a 3-minute exposure period and 92.8% after a 1-hour exposure. These values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.
The mean viability of cells treated with the positive reference item 8 N KOH were 3.0% (3-minute incubation) and <0.1% (1-hour incubation) of the negative control and were below the cut-off values. Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.
Under the present test conditions the test item tested at two exposure times of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is conducted according to GLP and standard methods, therefore it is considered to be relevant, adequate and reliable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

Himalayan

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at dosing: Approx. 8 - 9 months
- Weight at dosing:
Animal no. 1: 3.3 kg
Animal no. 2: 2.9 kg
Animal no. 3: 3.5 kg
- Fasting period before study:
- Housing: For 8 hours following test item application, the animals are kept singly in restrainers which allow free movement of the head but prevent a complete body turn, wiping of the eyes by the paws and exclude irritation of the eye by excrements and urine.
During the acclimatisation period and after the 8-hour period in restrainers, the animals are kept singly in cages with dimensions of 380 mm x 425 mm x 600 mm (manufacturer: Dipl.Ing. W. EHRET GmbH, 16352 Schönwalde, Germany).
- Diet (e.g. ad libitum): Ssniff® K-H (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. The food was available ad libitum.
- Water (e.g. ad libitum): Tap water was offered ad libitum.
- Acclimation period: At least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.9°C – 21.7°C (20°C ± 3°C (maximum range))
- Humidity (%): 39.7 – 84.1%. A malfunctioning of the air conditioning system, caused by servicing, resulted in relative humidity values that exceeded the maximum range of 30% - 70% on five test days.
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: May 24, 2013 To: June 04, 2013

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of the test item were administered into one eye each of three animals.

VEHICLE: No vehicle

Duration of treatment / exposure:

Single instillation into the conjunctival sac

Observation period (in vivo):

8 days

Number of animals or in vitro replicates:

3: Initial test 1; Confirmatory test 2

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: No

SCORING SYSTEM:
Cornea
Opacity: degree of density ( area most densetaken for reading)
No ulceration or opacity: 0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible: 1
Easily discernible translucent area, details of iris slightly obscured: 2
Nacreous area, no details of iris visible, size of pupil barely discernible: 3
Opaque cornea, iris not discernible through the opacity : 4
Iris
Normal: 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, iris reacting to light (a sluggish reaction is considered to be an effect): 1
Haemorrhage, gross destruction, or no reaction to light: 2
Conjunctivae
Redness: (refers to palpebral and bulbar conjunctivae, excluding cornea and iris)
Normal: 0
Some blood vessels hyperaemic (injected): 1
Diffuse, crimson colour, individual vessels not easily discernible: 2
Diffuse beefy red: 3
Chemosis
Swelling (refers to lids and/or nictitating membranes)
Normal: 0
Some swelling above normal: 1
Obvious swelling, with partial eversion of lids: 2
Swelling with lids about half closed: 3
Swelling, with lids more than half closed: 4

Any further lesions are listed.

Fluorescein-Test:
DEGREE OF STAINING
0: no staining
1: scattered or diffuse colouration, details of iris clearly visible
2: easily discernible translucent area, details of iris slightly obscured
3: opalescent areas, details of iris not discernible, extent of pupil difficultly
Determinable
4: opaque cornea, iris not discernible through the opacity
INVOLVED AREA OF CORNEA
0: none
1: up to 1/4 of the surface
2: 1/4 to 1/2 of the surface
3: 1/2 to 3/4 of the surface
4: 3/4 to whole surface.

TOOL USED TO ASSESS SCORE: hand-slit lamp / fluorescein
The eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48, 72 hours and 4 and 5 days after the administration (animal nos. 1 and 3) and prior to the administration and 1, 24, 48, 72 hours and 4 to 8 days after the administration (animal no. 2). The eye reactions were observed and registered.
24 hours after administration, fluorescein was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions. The fluorescein test was repeated on each day of observation.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 8 days (animal no.2)

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

2

Remarks on result:

other: The irises were not affected by instillation of the test item.

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 8 days (animal no.2)

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0.78

Max. score:

4

Reversibility:

fully reversible within: 8 days (animal no. 2)

Irritant / corrosive response data:

Cornea opacity (grade 1) was observed in all animals 60 minutes to 4 days, in animal no. 2 until 7 days after instillation.
Conjunctival redness (grade 1) was observed in all animals 60 minutes to 4 days, in animal no. 2 until 7 days after instillation.
Chemosis was observed in all animals:
- animal no. 1: 60 minutes (grade 2) and 24 to 72 hours (grade 1) after instillation;
- animal no. 2: 60 minutes (grade 2) and 24 hours to 4 days (grade 1) after instillation;
- animal no. 3: 60 minutes (grade 2) and 24 hours (grade 1) after instillation.
The fluorescein tests performed on each day of observation revealed corneal staining (grade 1) in all animals up to 3/4 of the surface.
All findings observed had disappeared in animal nos. 1 and 3 until 5 days, in animals no. 2 until 8 days after instillation.
The irises were not affected by instillation of the test item.
There were no systemic intolerance reactions concerning behaviour, body weight and food consumption.

Table 1. Examination of the treated eye

 

Time after administration	Cornea	Iris	Conjunctivae		Fluorescein Test
	Opacity		Redness*	Chemosis**	Grade	Area
Animal no.: 1/2/3
Right eye: 0.1 mL Test item/animal
24 hours	1/1/1	0/0/0	1/1/1	1/1/1	1/1/1	3/3/3
48 hours	1/1/1	0/0/0	1/1/1	1/1/0	1/1/1	3/3/3
72 hours	1/1/1	0/0/0	1/1/1	1/1/0	1/1/1	3/3/3
24-72 hours	1/1/1	0/0/0	1/1/1	1/1/0.33	1/1/1	3/3/3
Mean 24-72 h	1	0	1	0.78	1	3

* refers to palpebral and bulbar conjunctivae; excluding cornea and iris

** swelling: refers to lids and/or nictitating membrane

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

According to the EC-Commission directive 67 /548/EEC and its subsequent amendments on the approximation of the laws, regulations and administrative provision relating to the classification, packaging and labelling of dangerous substances and the results obtained under the present test conditions, Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine is non- irritating to eyes, hence, no labelling is required.
According to the EC Regulation 1272/2008 and subsequent regulations, the test item required labelling with 'Warning' and 'H319: Causes serious eye irritation'. According to the classification of the Globally Harmonized Classification System (GHS) the test item should be assigned to the hazard category 2A and labelled with an exclamation mark, 'Warning' and 'Causes serious eye irritation'.

Executive summary:

Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine was tested in an acute eye irritation/corrosion test in rabbits according to EC method B.5. (Regulation (EC) No. 440/2008) and OECD guideline 405 (2002). A single instillation of 0.1 mLper animal into the conjunctival sac of the right eye of three rabbits caused the following changes:
Cornea opacity (grade 1) was observed in all animals 60 minutes to 4 days, in animal no. 2 until 7 days after instillation.

Conjunctival redness (grade 1) was observed in all animals 60 minutes to 4 days, in animal no. 2 until 7 days after instillation.

Chemosis was observed in all animals:

- animal no. 1: 60 minutes (grade 2) and 24 to 72 hours (grade 1) after instillation;

- animal no. 2: 60 minutes (grade 2) and 24 hours to 4 days (grade 1) after instillation;

- animal no. 3: 60 minutes (grade 2) and 24 hours (grade 1) after instillation.

Mean 24 -72h scores for cornea, iris, conjuctiva and chemosis were 1/4, 0/2, 1/3 and 0.78/4 respectively.

The fluorescein tests performed on each day of observation revealed corneal staining (grade 1) in all animals up to 3/4 of the surface.

All findings observed had disappeared in animal nos. 1 and 3 until 5 days, in animals no. 2 until 8 days after instillation.

The irises were not affected by instillation of the test item.

There were no systemic intolerance reactions concerning behaviour, body weight and food consumption.

According to the EC-Commission directive 67/548/EEC and its subsequent amendments on the approximation of the laws, regulations and administrative provision relating to the classification, packaging and labelling of dangerous substances and the results obtained under the present test conditions, Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine is non- irritating to eyes, hence, no labelling is required.

According to the EC Regulation 1272/2008 and subsequent regulations, the test item required labelling with 'Warning' and 'H319: Causes serious eye irritation'. According to the classification of the Globally Harmonized Classification System (GHS) the test item should be assigned to the hazard category 2A and labelled with an exclamation mark, 'Warning' and 'Causes serious eye irritation'.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Guideline is based on Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) BCOP test method, 2007

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Note for Guidance on Non-Clinical Tolerance Testing of Medicinal Products, CPMP/SWP/2145/00, adopted March 1, 2001.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine eyes from cattle in the age range of 6 to 12 months were obtained from Hubert Bahlmann GmbH & Co., Lindern.

Details on test animals or tissues and environmental conditions:

Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control item: 0.9% NaCI solution; Positive control item: 1% NaOH solution

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750µL of the test or control items were added to completely cover the cornea’s epithelium in the anterior chamber.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

After rinsing, the corneas were incubated at 32 ± 1 °C for two hours. After this post-exposure incubation period, the corneas were examined.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse . To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing 1% Penicillin/Streptomycin . Upon arrival at the laboratory the eyes were examined for defects such as but not limited to increased opacity, scratches and neovascularisation. Only corneas from eyes free of defects were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM) , while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer (BASF OP-3.0). A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative (or solvent) control corneas. The remaining corneas were then distributed into treatment and positive control groups.

NUMBER OF REPLICATES: 3
Three corneas were used for each treatment group (test item, negative and positive controls).

NEGATIVE CONTROL USED
0.9% NaCl solution

POSITIVE CONTROL USED
1% NaOH solution

APPLICATION DOSE AND EXPOSURE TIME: 750 µL, 10 minutes
750 µL of the test or control items were added to completely cover the cornea’s epithelium in the anterior chamber.
After the exposure period of 10 minutes the exposure solution was removed from each chamber and the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: yes. 2 hours.
After rinsing, the corneas were incubated at 32±1°C for two hours. After this post-exposure incubation period, the corneas were examined

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.
- POST-EXPOSURE INCUBATION: 2 hours at 32±1°C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry. Measurements at 492 nm were recorded as optical density (OD492) or absorbance values. The fluorescein permeability values were determined using OD492 values based upon a visible light spectrophotometer using a standard 1 cm path length.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD492 value)

DECISION CRITERIA:
A test item that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.
As stated in the OECD guideline, if the test substance is not identified as an ocular corrosive or severe irritant, additional testing should be conducted for classification and labelling purposes. The BCOP test method has an overall accuracy of 79% (113/143) to 81% (119/147), a false positive rate of 19% (20/103) to 21% (22/103), and a false negative rate of 16% (7/43) to 25% (10/40), when compared to in vivo rabbit eye test method data classified according to the EPA, EU, or GHS classification systems. When substances within certain chemical (i.e., alcohols, ketones) or physical (i.e., solids) classes are excluded from the database, the accuracy of BCOP across the EU, EPA, and GHS classification systems ranges from 87% (72/83) to 92% (78/85), the false positive rates range from 12% (7/58) to 16% (9/56), and the false negative rates range from 0% (0/27) to 12% (3/26).

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

test substance

Value:

5.76

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

mean IVIS 107.362

Remarks on result:

other: A test item that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The negative or solvent/vehicle control responses should result in opacity and permeability values, that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.
- Acceptance criteria met for positive control: yes. A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
The corneas treated with the positive control item 1% NaOH solution revealed an opacity value of 74.617 and a permeability value of 2.183 compared to the negative control. The IVIS value of 107.362 was well above the cut-off value of 55.1 and, hence, the acceptance criteria for the test were fulfilled. 1% NaOH solution was a severe irritant and corrosive to eyes.

Table 1. In vitro irritancy score (IVIS)

	Cornea No.	Opacity	Permeability	IVIS
				Per Cornea		Per Group
						Mean	SD
NaCl 0.9%	1	-1.081	0.007	-0.976		-0.319	0.601
	2	0.157	0.003	0.202
	3	-0.259	0.005	-0.184
NaOH 1%	4	64.245	2.209	97.380		107.362	8.715
	5	80.170	2.219	113.455
	6	79.436	2.121	111.251
Test item	7	5.789	0.027	6.194		5.756	3.235
	8	7.580	0.078	8.750
	9	1.755	0.038	2.325
NC: negative control
SD: standard deviation

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the present test conditions Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine, tested in the in vitro BCOP test method, had an IVIS value of < 55.1 and consequently it is not classified as a severe irritant and is not corrosive.

Executive summary:

The purpose of this study was to determine if the test item can be classified as “ocular corrosive and severe irritant” employing an in vitro system. The BCOP test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability in isolated corneas from bovine eyes. The corneas of the eyes were dissected and the remaining sclera was mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

Three corneas were used for each treatment group (test item, negative and positive controls). The test item was used as supplied. 0.9% NaCl solution was used as the negative control and 1% NaOH solution as the positive control item. The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder for an exposure time of 10 minutes.

The optical density (OD₄₉₂) or absorbance values were measured at a wavelength of 492 nm. An opacity value of 5.041 and a permeability value of 0.048 compared to the negative control were determined. An IVIS 5.76 was calculated. Hence, the test item was not classified as a severe irritant and not corrosive, based on the results of this test.

The corneas treated with the positive control item 1% NaOH solution revealed an opacity value of 74.617 and a permeability value of 2.183 compared to the negative control. The IVIS value of 107.362 was well above the cut-off value of 55.1 and, hence, the acceptance criteria for the test were fulfilled. 1% NaOH solution was a severe irritant and corrosive to eyes.

Under the present test conditions Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine, tested in the in vitro BCOP test method, had an IVIS value of < 55.1 and consequently it is not classified as a severe irritant and is not corrosive.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

- In a first supporting in vitro study, the test substance was applied to a three-dimensional model (epiCS-1001) of human skin to determine cytotoxic properties to skin cells, which might lead to corrosion by the test item to human skin (Flügge, 2013a). Two exposure times of 3 minutes or 1 hour were employed. In comparison to the negative controls, the mean viability of cells exposed to the test item was 89.3% after a 3-minute exposure period and 92.8% after a 1-hour exposure. These values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this skin model and was predicted to be non-corrosive to human skin.

- In a second supporting in vitro study, the test substance was applied to a three-dimensional model (epiCS-1001) of human skin to determine cytotoxic properties to skin cells, which might lead to irritation by the test item to human skin (Flügge, 2013b). The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm of vital dye MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) after exposure of the treated cells. An exposure time of 20 minutes was employed following a 42 hours post-treatment incubation period in fresh medium. The mean viability of cells exposed to the test item was 17.6% of the negative controls and, hence, below the 50% cut-off value. The results of the latter study were very variable, showing a standard deviation for optical density of 0.103 upon 0.115 (or variation coefficient of 90%). Therefore the reliability was questionable and in agreement with the Sponsor it was decided not to use these results for classification purpose, but to perform an in vivo test instead.

- In a key in vivo study, the test substance was applied to rabbit skin according to OECD 404 method (Hansen, 2013a).

500 mg Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine were applied to the closely clipped dorsal area of the trunk of the animals by a semi-occlusive patch. After the 4 hour exposure period the patch was removed and the skin sites were evaluated. Scores were taken 60 minutes, 24, 48 and 72 hours after patch removal. Under the present test conditions, rabbits exposed for 4 hours showed slight skin reactions expressed as erythema at 60 minutes and/or 24 hours of observation. Mean 24-48h scores were 0.33 for erythema and 0.00 for oedema. The findings were reversible by 48 hours observation period and the scores were below the trigger value for classification.

There were no systemic intolerance reactions.

- In conclusion the pure substance was not irritating to skin based on in vitro testing on the three-dimensional skin model and in vivo testing in rabbits, therefore no classification is needed for skin irritation.

 

Eye irritation:

- In a first supporting in vitro study, severe eye irritancy potential and corrosivity potential of the test item containing >95% active ingredient was tested by means of the BCOP test method (Leuschner, 2013). Three corneas were used for each treatment group (test item, negative and positive controls). The test item was used undiluted. 0.9% NaCl solution was used as the negative control and 1% NaOH solution as the positive control item. An IVIS score of 5.76 was calculated. Hence, the test item could not be classified as a severe irritant and not corrosive, based on the results of this test. Nor could it be classified as non-irritant as the IVIS score was higher than 3.

-In a second suppporting in vitro study, the eye irritancy potential of test item containing >95% active ingredient was tested by means of the EpiOcular^TM Model (Flügge, 2013c). In this test, viability of the human keratinocyte cells was measured after topical exposure to three construct tissues for test substance and concurrent controls. The tissue viability was determined by photometrical measurement of formazan production as a result of enzymatic reduction of the vital dye MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide) versus negative control-treated constructs. 50 mg of the test item, 50 µL of the concurrent negative or positive control were administered by topical application onto the construct on cultures for 6 hours, followed by a post-incubation for 18 hours. After the 3-hour incubation with MTT assay and subsequent extraction with isopropanol the cell viability of the test item exposed cells was 34.6% of the mean negative control value, which was below the cut-off of < 60%., by which it was considered to be cytotoxic and predicted to be irritant. However the viability of cells treated with the positive reference item, methyl acetate, was only 7.7% of the negative controls and far below the 60% cut-off value, by which the study was considered questionable; therefore in vivo testing was decided.

- In a key in vivo study, the pure substance was tested in an acute eye irritation/corrosion test in rabbits according to EC method B.5 and OECD guideline 405 (Hansen, 2013b). A single instillation of 0.1 mLper animal into the conjunctival sac of the right eye of three rabbits caused cornea opacity and redness (both grade 1 in all animals up to 7 days after instillation) and chemosis (grade 2 after 60 minutes and grade 1 in all animals up to day 4 after instillation). Mean 24 -78h scores for cornea, iris, conjuctiva and chemosis were 1/4, 0/2, 1/3 and 0.78/4 respectively. The fluorescein tests performed on each day of observation revealed corneal staining (grade 1) in all animals up to 3/4 of the surface. All findings observed had disappeared in animal nos. 1 and 3 until 5 days, in animals no. 2 until 8 days after instillation. The irises were not affected by instillation of the test item. There were no systemic intolerance reactions concerning behaviour, body weight andfood consumption.

- In a support in vivo study, aute eye irritation/corrosion of Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine 15% formulation in fatty esterswas tested in rabbits according to EC method B.5 or OECD guideline 405 (Hansen, 2013c). A single instillation of 0.1 mL of the 15% formulation per animal into the conjunctival sac of the right eye of three rabbits did not cause any changes. The corneae, irises and conjunctivae were not affected by instillaton of the test item. Mean 24 -78h scores for cornea, iris, conjuctiva and chemosis were 0/4, 0/2, 0/3 and 0/4 respectively.There was no fluorescein staining on the cornea.

There were no systemic intolerance reactions concerning behaviour, body weight and food consumption.

- In conclusion, the in vitro studies with the pure substance were considered to be supporting for the in vivo study with the pure substance, which has to be classified as category 2 according to CLP regulation, but not classified according to DSD. The 15% formulation was further tested in vivo in rabbits, and was demonstrated to be not irritating, therefore 15% can be considered as a concentration limit for non-classification.

Justification for classification or non-classification

According to the EC-Commission directive 67/548/EEC and EC Regulation No. 1272/2008 relating to the classification, packaging and labelling of dangerous substances and the results obtained under the present test conditions, Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine is non-irritating to skin, hence, no labelling is required.

The pure substance needs not to be classified to the Directive 67/548/EEC, whereas according to CLP regulation 1272/2008, the pure substance is classified as Category 2, with signal word 'Warning' and hazard statement: H319 -Causes serious eye irritation. The 15% formulation however was not irritating and does not need to be classified under Directive 67/548/EEC and under CLP regulation 1272/2008. The 15% can be considered as a concentration limit for non-classification.