Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Endpoint summary

Description of key information

The substance was found to be neither corrosive nor irritant when tested in an in-vitro skin model. Similarly, the substance was considered to be non-corrosive to the eyes when tested on in-vitro rabbit eyes.
In-vivo, the substance was slightly irritant to skin and produced in eyes minimal conjunctival reactions that cleared within 72 hours.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 September 2012 to 04 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

other: EpiskinTM Model Kit (0.38 cm2 tissues)

Cell source:

other: SkinEthic Laboratories, Lyon, France

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Species: human reconstructed epidermis (tissues).
- Supplier: SkinEthic Laboratories, Lyon, France.
- Selection: at receipt, the medium quality and temperature indicators were checked to ensure the good quality of the tissues before use.
- Storage conditions: the living tissues were kept at room temperature from receipt until the end of the in vitro phase.
- Description: the EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstituted from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.

REMOVAL OF TEST SUBSTANCE
For all treated tissues (test item-treated, positive and negative controls), at the end of the designated incubation period, each tissue insert was removed from the well of the treatment plate and rinsed with Phosphate Buffered Saline Dulbeccos (PBS).
Rinsing was achieved by gently filling and emptying each tissue insert 12 times with 2 mL PBS to gently remove any residual dosage form. As the dose formulation adhered to the walls of the compartment, it was eliminated using a cotton bud.

POSITIVE CONTROL
Name: glacial acetic acid.

NEGATIVE CONTROL
Name: 0.9% NaCl.

SCORING SYSTEM:
- Optical density (OD) was measured at 540 nm:
Relative mean viability (%) = 100 x mean OD(test item) / mean OD(negative control)

DECISION CRITERIA
see table 7.3.1/1 in Any Other Information on Materials and Methods

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Amount/concentration applied
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg +/- 2 mg per tissue.
The test item was spread over the whole tissue surface without damaging the tissue sample.
As the test item was a solid, 100 µL of 0.9% NaCl were applied over the test item to ensure good contact with the epidermis.

Duration of treatment / exposure:

Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes (duplicate tissues were treated with the positive
and negative control materials for an exposure period of 240 minutes).

Duration of post-treatment incubation (if applicable):

MTT-loading after a 3h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure duration: 3 minutes

Value:

86

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure duration: 60 minutes

Value:

79

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure duration: 240 minutes

Value:

104

Following the 3, 60 and 240 minute exposure periods the test material treated tissues appeared blue which was considered to be indicative of viable tissue. 

The relative mean tissue viability for the positive control treated tissues was 3% relative to the negative control treated tissues following the

240-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

The mean OD value for the negative control was 0.232. The negative control acceptance criterion was therefore satisfied.

Table 7.3.1/2 Mean OD Values and viabilities for the negative and positive controls and the test item

Group	Exposure duration	OD570 nm		Viability (%)
		Mean	SD	Mean	SD
Negative control	240 min	0.232	0.000	100	0
Positive control	240 min	0.006	0.001	3	0
Test item	3 min	0.199	0.002	86	1
	60 min	0.184	0.010	79	4
	240 min	0.241	0.002	104	1

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the in vitro study, the test substance was considered to be non-corrosive to the skin.

Executive summary:

The objective of this study was to evaluate the corrosive potential of the test substance using the Episkin reconstituted skin model. This study was conducted in compliance with OECD Guideline No. 431 and the principles of Good Laboratory Practice.

A preliminary test was performed to detect the ability of the test item to directly reduce MTT. Following the preliminary test, the skin corrosion potential of the test item was tested in the main test. The test item, and both negative and positive controls were applied on duplicate tissues and incubated at room temperature as follows: positive and negative controls for 240 minutes (± 5 minutes); test item for 3 minutes (± 5 seconds), 60 minutes (± 5 minutes) and 240 minutes (± 5 minutes). At the end of the designated incubation periods, each tissue was rinsed with phosphate Buffered Saline Dulbeccos. The cell viability was then assessed by means of the colourimetric MTT reduction assay and relative viability (%) calculated for each tissue relative to control tissues.

In the preliminary assay, the MTT solution colour did not turn blue/purple when compared to the negative control. The test item was presumed not to directly reduce MTT.

In the main test, the blue discolouration of the test item-treated tissues following the 3 , 60 and 240 minute exposure periods was representative of viable tissue. The relative mean viabilities of the test item-treated tissues were 86% , 79% and 104% of the negative control for the 3, 60 and 240 minutes exposure periods respectively.

The test item was considered to be non corrosive to the skin.

 

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02 October 2012 to 16 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: EpiskinTM Model Kit (0.38 cm2 tissues)

Cell source:

other: SkinEthic Laboratories, Lyon, France

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: several
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: no data
- Wavelength: 540 nm
- Filter: no data
- Filter bandwidth: no data

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
SCORING SYSTEM:
- Optical density (OD) was measured at 540 nm:
Relative mean viability (%) = 100 x mean cOD(test item) / mean cOD(negative control)
where:
- mean cOD Negative Control = mean ODNC – mean ODblank
- mean cOD Test Item = mean ODTI – mean ODblank
- mean cOD Positive Control = mean ODPC - mean ODblank
DECISION CRITERIA
Mean relative viability is = or < 50%: category 2 (H315)
Mean relative viability is > 50% : no category

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Amount/concentration applied
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 +/- 2 mg
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- Concentration (if solution):
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- Concentration (if solution): 5% in distilled water

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

MTT-loading after a 42h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Time point: 15 min exposure + 42h expression

Value:

109

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6%

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: other: concentration of IL 1-alpha (pg/ml)

Run / experiment:

Time point: 15 min exposure + 42h expression

Value:

53.5

Remarks on result:

other:

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

The preliminary assay showed that the test item had neither colouring potential nor direct MTT reducing properties.  

All of the acceptance criteria for the negative and the positive controls were fulfilled, therefore the study was considered to be valid (see table 7.3.1/2).

Table 7.3.1/1 Individual and mean OD Values and tissue viabilities for the negative and positive controls and the test item

Group	Tissue n°	OD measurements		Mean OD	Viability (%)
		1st	2nd
Negative control	1	0.911	0.929	0.920	102
	2	0.888	0.871	0.880	98
	3	0.908	0.897	0.903	100
Positive control	1	0.055	0.053	0.054	6
	2	0.034	0.035	0.035	4
	3	0.065	0.062	0.064	7
Test item	1	0.932	0.936	0.934	104
	2	1.036	1.023	1.030	114
	3	0.987	0.966	0.977	108

Table 7.3.1/2 Mean tissue viability and standard deviations for the negative and positive controls and the test item

Group	OD		Viability (%)
	Mean	SD	Mean	SD
Negative control	0.901	0.020	100	2
Positive control	0.051	0.015	6	2
Test item	0.980	0.048	109	5

Table 7.3.1/3 Determination of IL-1 alpha concentration (pg/ml) in Episkin samples

Sample identification	Il-1a measured concentration (pg/ml)	Mean Il-1a measured concentration (pg/ml) n= 3 tissues	Inter tissue %CV n= 3 tissues
Negative control 1	BLQ	NC	NC
Negative control 2	BLQ
Negative control 3	12.6
Test sample 1	58.2	53.5	34.6
Test sample 2	69.3
Test sample 3	33.1

BLQ: Below limit of Quantification (< 5.00 pg/mL)

NC: Not Calculated

% CV: standard deviation/mean concentration x 100

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study, the test substance was considered to be non-irritant to skin.

Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test substance using the Episkin reconstituted humal epidermis model.The study was conducted in compliance with OECD Guideline No. 439 and the principles of Good Laboratory Practice.

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 (± 1) minutes. At the end of the treatment

period, each tissue was rinsed with Phosphate Buffered saline Dubelccos and incubated for 42 (± 1) hours at 37°C. The cell viability was then assessed by means of the colourimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability). Also, the concentration of the inflammatory mediator IL-1a was evaluated in the culture medium retained following the 42 hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

In the preliminary test, the test item was found not to have a colouring potential and no direct MTT reducing properties. In the main assay, the relative mean viability of the tissues treated with the test item was 109% with a standard deviation of 5% following a 15 minute exposure and a 42 hour recovery period.

As the mean relative viability was > 50% after the MTT reduction, culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The mean concentration of IL-1a within the retained medium was 53.5 pg/mL . Due to this value being below 60 pg/mL, the test item was considered to be non-irritant to skin.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 October 2012 to 05 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CEGAV, Argenvilliers, France
- Age at study initiation: 2 to 4 months old on the day of treatment
- Mean body weight at study initiation: 2788 g (range: 2700 g to 2945 g)
- Fasting period before study: No
- Housing: The animals were individually housed in noryl cages
- Diet: 110 pelleted diet (free access)
- Water: Tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: At least 5 d before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3°C
- Humidity: 50 ± 20%
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: From 23 October 2012 to 05 November 2012.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: each animal served as its own control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: a quantity of 0.5 g/flank was used.


DOSE FORMULATION PREPARATION
- The test substance was used in its original form. The pH of the test substance was measured with pH paper in order to confirm that the pH value is > 2 and < 11.5.
- Dose formulations preparations were prepared by the testing laboratory extemporaneously on the day of each administration.
- The dose formulation preparations were stored at room temperature and delivered to the study room in paper bags.

Duration of treatment / exposure:

3 min, 1 h, 4 h

Observation period:

1, 24, 48 and 72 h after removal of the dressing; if relevant, daily until reversibility of reactions

Number of animals:

Three males

Details on study design:

TEST SITE
- Area of exposure: Right and left anterior and/or posterior flanks of the animals
- % coverage: 6 cm2
- Type of wrap if used: gauze pad held in place by a non-irritation semi-occlusive dressing and a restraining bandage.

REMOVAL OF TEST SUBSTANCE
- Washing:using a moistened cotton pad
- Time after start of exposure: at removal of each dressing (see Duration of exposure)

SCORING SYSTEM: according to OECD guideline 404

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.7

Max. score:

2

Reversibility:

fully reversible within: Day 7

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: Day 3

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible within: Day 4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

CUTANEOUS REACTIONS:
- After a 3 minute exposure to animal 1, no cutaneous reactions were observed.
- After a 1 hour exposure, a well-defined erythema (grade 2) was noted in animal 1 from Day 1 to Day 3, followed by a very slight erythema (grade 1) on Days 4 and 5. In addition, a very slight edema (grade 1) was observed on day 1.
- After a 4 hour exposure, a well-defined erythema (grade 2) was noted from Day 1 until Day 2 in 2/3 animals (i.e., in animal 2 and 3) and until Day 3 in 1/3 of animals (i.e., in animal 1), followed by a very slight erythema (grade 1) up to Day 3 in animal 2, Day 4 in animal 3 or Day 7 in animal 1.
- The mean scores calculated for each animal over 24, 48 and 72 hours were as follows:
- erythema: 1.7, 1.0, 1.3; showing no significant inflammation,
- edema: 0.0, 0.0; 0.0; showing no significant inflammation.

Other effects:

MORTALITY: No unscheduled deaths occurred during the study.
CLINICAL SIGNS: No clinical signs indicative of systemic toxicity were observed in any animals.
BODY WEIGHT: The body weight of the animals was unaffected by the test substance treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

The substance was slightly irritant when applied to the rabbit skin.

Executive summary:

The potential of the test item to induce skin irritation was assessed in 3 rabbits according to OECD Guideline 404 in compliance with Good Laboratory Practices.

The test item was first applied for periods of 3 minutes, 1 hour and 4 hours to a single New Zealand White rabbit. After the 4-hour application, since the mean value from grading at 24, 48 and 72 hours after patch removal was < 2.3 for erythema or for edema, the test item was applied on the skin of two other animals for 4 hours.

A quantity of 0.5 g/flank was applied to a skin area of approximately 6 cm² under a semi-occlusive dressing for 4 hours. Skin reactions were observed 1, 24, 48, and 72 hours after removal of the dressing. The mean values of the scores for erythema and oedema were calculated for each animal. Each animal was observed once a day for mortality and clinical signs. The body weight was recorded at the beginning and the end of the observation period.

No unscheduled deaths occurred during the study and no clinical signs indicative of systemic toxicity were noted in any animals. The body weight of the animals was unaffected by the test item treatment.

After a 4 hour exposure, a well-defined erythema was noted from Day 1 until Day 2 in 2 animals or until Day 3 in 1 animal, followed by a very slight erythema up to Days 3, 4 or 7. These reactions were fully reversible within 7 days.

The mean scores calculated for each animal over 24, 48 and 72 hours were 1.7, 1.0, 1.3 for erythema and 0.0, 0.0; 0.0 for oedema.

It was concluded that the test item was slightly irritant when applied topically to rabbits.

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:

From 24 May 2012 to 01 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

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
- Source: breeder: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age/weight at study initiation: the animals were 12 to 20 weeks old at the beginning of the study and their body weight ranged from 2.62 to 3.01 kg.
- Fasting period before study: no
- Housing: the animals were individually housed in suspended cages
- Diet: 2930C Teklad Global Rabbit diet (free access)
- Water: free access
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 24 May 2012 to 01 June 2012.

Vehicle:

unchanged (no vehicle)

Controls:

other: the other untreated eye served as a control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of the test substance (which was found to weigh approximately 66 mg)
- Concentration: Undiluted

VEHICLE
-none

Duration of treatment / exposure:

Not applicable: single application not followed by rinsing.

Observation period (in vivo):

The eyes were examined for any ocular reaction approximately 1, 24, 48, and 72 h after the treatment.

Number of animals or in vitro replicates:

Two

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: No

SCORING SYSTEM:
- Draize scale for scoring ocular Irritation (Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49).

TOOL USED TO ASSESS SCORE:
- Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.66

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritant / corrosive response data:

- No corneal or iridial effects were noted during the study.
- Minimal conjunctival irritation was noted in both the animals 1 and 24 hours after treatment and in one animal at the 48 hour observation. However, these effects were reversible within 48 hours in one animal and within 72 hours in the other animal.

Other effects:

No bodyweight gain was noted in one animal and the other animal showed expected gain in bodyweight during the study.

Table 7.3.2/1 Individual Scores and individual total Scores for ocular irritation

Rabbit Number and Sex	72058 Male				72080 Male
Time after treatment	1 h	24 h	48 h	72 h	1 h	24 h	48 h	72 h
CORNEA
E = Degree of opacity	0	0	0	0	0	0	0	0
F = Area of cornea involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	1	1	0	0	1	1	1	0
B = Chemosis	1	0	0	0	1	0	0	0
C = Discharge	1	0	0	0	1	0	0	0
Score (A + B + C) x 2	6	2	0	0	6	2	2	0
Total Score	6	2	0	0	6	2	2	0

Table 7.3.2/2 Individual Total Scores and Group Mean Scores for ocular irritation

Rabbit Number and Sex	Individual total scores at:
	1 h	24 h	48 h	72 h
72058 Male	6	2	0	0
72080 Male	6	2	2	0
Group Total	12	4	2	0
Group Mean Score	6.0	2.0	1.0	0

Interpretation of results:

GHS criteria not met

Conclusions:

The substance produced minimal conjunctival irritation when applied into rabbit eye.

Executive summary:

The potential of the test item to induce eye irritation was assessed in rabbits according to OECD Guideline 404 in compliance with Good Laboratory Practice.

A volume of 0.1 mL of the test substance was placed into the conjunctival sac of the right eye of two New Zealand White rabbits without irrigation. The left eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made 1, 24, 48 and 72 hours following treatment according to the Draize scoring method.

No corneal or iridial effects were noted during the study. Minimal conjunctival irritation was seen in both animals 1 and 24 hours after treatment and in one animal at the 48 hour observation. These effects were reversible within 48 hours in one animal and within 72 hours in the other.

It was concluded that the test item produced minimal conjunctival irritation when applied into rabbit eye.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 21, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted study, meets acceptable scientific principles and in compliance with GLP.

Qualifier:

no guideline available

Principles of method if other than guideline:

The rabbit enucleated eye test was designed to assess the ocular irritancy potential of the test substance in the rabbit following application onto the cornea of the enucleated eye. The assay has undergone inter-laboratory validation and has been shown to reliably detect test substances that are negligible, or moderate to severe ocular irritants.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: yes, two enucleated eyes treated with saline (0.9% Sodium chloride)

Amount / concentration applied:

TEST MATERIAL
- Amount applied : 0.1 mL of the test substance which was found to weigh approximately 48mg (as measured by gently compacting the required volume into an adaptated syringe)

VEHICLE
- none

Duration of treatment / exposure:

10 sec

Observation period (in vivo):

60, 120, 180 and 240 minutes following treatment.

Number of animals or in vitro replicates:

Three enucleated eyes for the test group and two enucleated eyes for the control group.

Details on study design:

PRE-TEST PROCEDURES
Superfusion chamber:
The water heating circulator, was adjusted so that the temperature of the water flowing through the water jacket of the superfusion apparatus, gave a stable temperature, of 32 ±1.5°C, within the chambers of the apparatus. A peristaltic pump was used to supply saline solution at a flow rate of 0.15 to 0.4 mL/minute (at approximately 32°C) into the rear of each chamber of the apparatus in order to irrigate the surface of the cornea.

Selection of eyes:
Prior to enucleation, the eyes of the provisionally selected rabbits were examined for evidence of ocular irritation or defect, following application of Fluorescein Sodium drops BP (1 % w/v). Examination was aided with the Kowa SL-5 slit-lamp biomicroscope. Corneal thickness values were also recorded using the DGH-1000 Ultrasonic pachymeter. Only animals whose eyes showed no evidence of ocular irritation or defect were used for testing purposes.

Enucleation of eyes:
The donor rabbits were sacrificed by intravenous administration of an overdose of sodium 'pentobarbitone. Immediately afterwards, two to three drops of saline solution (approximately 32°C) were applied to the cornea to prevent desiccation during excision. The eye was then carefully removed, positioned in a perspex clamp and placed within the chamber of the superfusion apparatus, with the saline drip at the rear of the chamber adjusted so that saline solution was allowed to irrigate the surface of the cornea. The eyes were then allowed to equilibrate for approximately thirty minutes. Following the equilibration period, the eyes were re-examined to ensure they had not been damaged during excision. Corneal thickness was also measured using the ultrasonic pachymeter. Any eyes in which the corneal swelling was greater than 100/0 relative to the preenucleation measurement, or in which the cornea was stained with fluorescein, were rejected. The post equilibration corneal thickness values for each eye were recorded.

PROCEDURE

Test substance administration:
Three eyes were treated with test substance, two additional eyes remained untreated for control purposes. The treatment eye was removed from the superfusion apparatus whilst still being held in the perspex clamp. The clamp/eye was then placed horizontally into a petri dish.
A volume of 0.1 mL of the test substance, which was found to weigh approximately 48 mg (as measured by gently compacting the required volume into an adapted syringe) was sprinkled as evenly as possible over the surface of the cornea. After ten seconds the test substance was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32°C).
Immediately following washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drip repositioned to irrigate the eye.
The untreated eyes were similarly washed and used for control purposes.

Examination:
Examination of the eye was facilitated by use of a slit-lamp biomicroscope. The thickness of the cornea was measured using an ultrasonic pachymeter. For each enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea. A mean value for corneal thickness was then calculated. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
The condition of the corneal epithelium was assessed approximately 60, 120, 180 and 240 minutes following treatment. Assessment was facilitated by the use of the slit-lamp biomicroscope.
The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 240 minutes following treatment, according to the numerical evaluation (i.e., McDonald - Shadduck Score System). This was carried out using the cobalt blue filter of the slit-lamp biomicroscope, following application of Fluorescein Sodium drops.

Irritation parameter:

cornea opacity score

Run / experiment:

mean after 240 minutes of exposure

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: similar to control eyes

Irritation parameter:

fluorescein leakage

Run / experiment:

mean after 240 minutes of exposure

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: similar to control eyes

Irritation parameter:

other: corneal thickness (µm)

Run / experiment:

mean after 240 minutes of exposure

Value:

395.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value in control eyes: 388.7 µm

Irritation parameter:

percent corneal swelling

Run / experiment:

mean after 240 minutes of exposure

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value in control eyes: 2.5%

Table 7.3.2/2 Maximal ocular irritation observations

Corneal opacity	Fluorescein uptake	Corneal Swelling (%)						Condition of Corneal Epithelium
		Test Eyes^a			Control Eyes^b
Cldy x Area	Int x Area	60 mins	120 mins	240 mins	60 mins	120 mins	240 mins
0	0	10.4	8.6	4.0	7.7	5.6	2.5	normal

a = For each time point the swelling recorded is the mean of three eyes

b = For each time point the swelling recorded is the mean of two eyes

Cldy = Corneal cloudiness

Int = Intensity of fluorescein uptake

mins = Minutes following treatment

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was considered unlikely to have the potential to cause severe ocular irritancy in vivo.

Executive summary:

An in vitro study was performed to assess the ocular irritancy potential of the test substance in rabbit following application onto the cornea of enucleated eyes, in compliance with the principles of Good Laboratory Practice.

A volume of 0.1 mL of the test substance was applied onto the cornea of each of the three enucleated rabbit eyes which had been maintained at a temperature of 32 ± 1.5°C within a superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9% sodium chloride). Effects on corneal opacity, the condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling) were observed 60, 120, 180 and 240 minutes after treatment.

No corneal effects (opacity) were noted in the test eyes or control eyes during the study period (i.e. 240 minutes after treatment). Corneal swelling of the test eyes during the study period was comparable to that observed in the control eyes over the same period. No fluorescein uptake was seen in test and control eyes and the condition of the corneal epithelium in the test eyes or controls appeared normal during the study period.

Under the study conditions, the test substance was therefore considered unlikely to have the potential to cause severe ocular irritancy in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The potential of the test substance to induce skin irritation was assessed using a sequential testing strategy. The purpose of this strategy was to avoid the unneccesary use of animals and to minimise any testing that is likely to produce severe responses in animals.

The objective of the first study which was performed in-vitro was to determine whether or not the test item was corrosive.

The study was conducted in compliance with OECD Guideline No. 431 and the principles of Good Laboratory Practice. The Episkin reconstituted skin model was the in-vitro model selected. The test item, and both negative and positive controls, were applied on duplicate tissues and incubated at room temperature as follows: positive and negative controls for 4 hours; test item for 3 minutes, 60 minutes and 4 hours. At the end of the designated incubation periods, the cell viability was assessed by means of the colourimetric MTT reduction assay. The relative mean viabilities of the test item-treated tissues were 86% , 79% and 104% of the negative control for the 3, 60 and 240 minute exposure periods respectively. Since the mean viability was above the cut-off value of 35% whatever the exposure duration, the test item was considered to be non corrosive to the skin (Gerbeix 2012a).

In the second in-vitro study, the potential of the test item to induce irritation was assessed.

This study was conducted in compliance with OECD Guideline No. 439 and the principles of Good Laboratory Practice. The Episkin reconstituted human epidermis was used as the skin model. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, tissues were rinsed and incubated for 42 hours at 37°C. The cell viability was then assessed by means of the colourimetric MTT reduction assay. The relative mean viability of the tissues treated with the test item was 109% with a standard deviation of 5%. Since the mean relative viability was above 50%, the concentration of the inflammatory mediator IL-1a in the culture medium was determined by ELISA.

The mean concentration of IL-1a was 53.5 pg/mL . Due to this value being below the cut-off value of 60 pg/mL, the test item was predicted to be non-irritant to skin (Gerbeix 2012b).

The result obtained in-vitro was then confirmed in an in-vivo test using only one animal at the beginning. This key study was conducted in compliance with OECD Guideline 404 and the principles of Good Laboratory Practice. The test item was applied for 3 minutes on the skin of a single animal. Since no full thickness destruction of skin tissue was observed in the first hour after removal of the pad, the test item was applied on another treatment site for a further 1 hour. Since no full thickness destruction of skin tissue was observed in the first hour after removal of the pad, the test item was applied on another treatment site for 4 hours.   After the 4 hour application on the animal, since the mean value from grading at 24, 48 and 72 hours after patch removal was below 2.3 for erythema or for oedema, the test item was applied to the skin of two other animals for 4 hours.

No unscheduled deaths occurred during the study and no clinical signs indicative of systemic toxicity were noted in any animals. The bodyweight of the animals was unaffected by the test item treatment. After the 4 hour exposure, a well-defined erythema was noted from Day 1 until Day 2 in 2 animals or until Day 3 in 1 animal, followed by a very slight erythema up to Days 3, 4 or 7. These reactions were fully reversible within 7 days.

The mean scores calculated for each animal over 24, 48 and 72 hours were 1.7, 1.0, 1.3 for erythema and 0.0, 0.0; 0.0 for oedema.

It was concluded that the test item was slightly irritant when applied topically to the rabbits (Gerbeix 2012c).

Eye irritation

An in-vitro study was performed to assess the ocular irritancy potential of the test substance in rabbits following application onto the cornea of enucleated eyes. The study was performed in compliance with the principles of Good Laboratory Practice.

A volume of 0.1 mL of test substance was applied onto the cornea of each of three enucleated rabbit eyes which had been maintained at a temperature of 32 ± 1.5°C within a superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution. Effects on corneal opacity, the condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling) were observed at 60, 120, 180 and 240 minutes after treatment. No corneal effects (opacity) were noted in the test eyes or control eyes during the study period. Corneal swelling of the test eyes during the study period was comparable to that observed in the control eyes over the same period. No fluorescein uptake was seen in test and control eyes and the condition of the corneal epithelium in the test eyes or controls appeared normal during the study period. Under the study conditions, the test substance was therefore considered unlikely to have the potential to cause severe ocular irritancy in-vivo (Sanders, 2012a).

A follow-up in-vivo study was conducted to evaluate the acute eye irritation potential of the test substance in New Zealand White rabbits. This key study was conducted according to the EU Method B.5 and OECD Guideline 405 in compliance with the principles of Good Laboratory Practice.

A volume of 0.1 mL of the test substance was placed into the conjunctival sac of one eye of two rabbits without irrigation. The other eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1, 24, 48 and 72 hours following treatment according to the Draize scoring method. No corneal or iridial effects were noted during the study. Minimal conjunctival irritation was seen in both animals 1 and 24 hours after treatment and in one animal at the 48 hour observation. These effects were reversible within 48 hours in one animal and within 72 hours in the other. In conclusion, under the conditions of the study, the test substance produced minimal conjunctival irritation in rabbits which was reversible within 72 h (Sanders, 2012b).

Justification for classification or non-classification

Based on the results from in vitro and in vivo studies, the substance is not classified as a skin or eye irritant according to regulation EC No. 1272/2008

and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.