9-Octadecenoic acid (Z)-, reaction products with diethylenetriamine, di-Me sulfate-quaternized

Endpoint summary

Description of key information

There are reliable in vitro skin corrosion, in vitro skin irritation and in vivo skin irritation studies on EC 308-415-1. In addition, there is a reliable in vivo skin irritation study available on each of the analogue substances EC 931-745-8 and EC 937-237-2, which are also provided to support the endpoint requirement for skin irritation. There are reliable in vitro (BCOP) and in vivo eye irritation studies on EC 308-415-1. In addition, there is a reliable in vivo eye irritation study available on each of the analogue substances EC 931-745-8 and EC 937-237-2, which are also provided to support the endpoint requirement for eye irritation.

The substance was not corrosive in a reliable in vitro study performed by means of the Human Skin Model Test with EpiDerm™ tissues models. The substance was also not irritating in a reliable in vitro study performed by means of the Human Skin Model Test according to OECD TG 439.

In a reliable in vivo dermal irritation study performed according to OECD Guideline 404, three young adult New Zealand White rabbits were dermally exposed to 0.5 mL of a structurally similar substance moistened with 0.5 mL purified water for 4 hours under semi-occlusive dressing. Animals then were observed for 14 days. The skin reaction was scored according to OECD Guideline 404.One hour after removal of the dressing a well-defined erythema was observed in all animals which progressed into moderate to severe and persisted as very slight up to 10 or 14 days after treatment. A very slight to moderate oedema was recorded in all animals from the 1-hour up to the 72-hour reading or up to 10 days after treatment. Scaling was present in all animals 7 to 14 days after removal of the application patch. No alterations and no corrosive effects were observed on the treated skin. The substance is considered to be a skin irritant (Category 2).

In a primary skin irritation study according to US Guideline EPA OPPTS 870.2500 (1998), OECD Guideline 404 (1992), EU Guideline B.4 (1992) and Japanese Guideline Nousan No. 6283 (1999), revised Nousan 8628 (2000), three White New Zealand rabbits were semi-occlusive dermally exposed to 0.5 ml of EC 931-745-8 for 4 hours. Animals then were observed for 14 days. Irritation was scored by the method of Draize. The substance induced in all animals moderate to severe erythema and very slight to slight oedema. Focal eschar was noted for two animals on study day 7, and desquamation was noted for all animals on study days 7 through 14. Very slight to slight erythema was still present for all animals at study termination (study day 14). Mean scores from gradings at 24, 48 and 72 hours after removal of the test material for erythema were for all animals >/=2.3 but < 4. Oedema was less severe. Inflammation persisted to the end of the observation period (14 days) in all three animals. The substance is irritating to the skin based on results of the topical semi-occlusive application in rabbits.

In a primary dermal irritation study performed according to OECD Guideline 404 (Apr. 24, 2002), three young adult New Zealand White rabbits were dermally exposed to 0.5 mL of EC 937-237-2 (100 % a.i.) moistened with 0.5 mL purified water for 4 hours under semi-occlusive dressing. Animals then were observed for 14 days. The skin reaction was scored according to OECD Guideline 404. One hour after removal of the dressing a well-defined erythema was observed in all animals which progressed into moderate to severe and persisted as very slight up to 10 or 14 days after treatment. A very slight to moderate oedema was recorded in all animals from the 1-hour up to the 72-hour reading or up to 10 days after treatment. Scaling was present in all animals 7 to 14 days after removal of the application patch. No alterations and no corrosive effects were observed on the treated skin. In this study, the substance is a dermal irritant. The substance is irritating to the skin based on results of the topical semi-occlusive application in rabbits.

An in vitro study was performed to assess the corneal damage potential of the substance by means of the BCOP assay using fresh bovine corneae. The substance was tested as 20% (w/v) suspension. Relative to the negative control, the substance caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 12.91 (threshold for serious eye damage: IVIS > 55). According to OECD 437, no prediction for the damage hazard of the substance to the eye can be made. The substance is not serious eye damaging (CLP/EPA/GHS, Category 1).

A reliable study was performed to assess the irritancy potential of the substance to the eye of the New Zealand White rabbits. A single application of the substance to the non-irrigated eye of three rabbits scattered or diffuse corneal opacity, iridial inflammation and moderate conjunctival irritation. Treated eyes appeared normal at the 7- and 21 -day observations. The substance produced a maximum group mean score of 14 and thus does not meet the criteria for classification as an eye irritant.

In a primary eye irritation study according to US Guideline EPA OPPTS 870.2400 (1998), OECD Guideline 405 (1987), EU Guideline B.5 (1992) and Japanese Guideline Nousan No. 6283 (1999), revised Nousan 8628 (2000), 0.1 ml of EC 931-745-8 was instilled into the conjunctival sac of three White New Zealand rabbits. The eyes were not rinsed after substance application. Animals then were observed for 21 days. Irritation was scored by the method of Draize. Positive corneal, iridal and conjunctival findings were noted for all animals. Corneal neovascularization was noted for two animals on study days 7 through 14 or 17. Iridal irritation subsided by study day 7, and corneal and conjunctival irritation completely subsided by study termination (study day 21). The substance induced a maximum mean total score of 50.3 (maximum possible mean total score of 110) at 48 hours post-instillation. Mean scores following grading at 24, 48 and 72 hours after instillation of the substance for corneal opacity were >/= 1 but < 3, for iritis >/= 1 but < 1.5 and for conjunctival redness and oedema >/= 2. All effects were completely reversible in all animals within 21 days. The substance is considered to be irritating to eyes.

In a primary eye irritation study according to OECD Guideline 405 (2002), 0.1 mL of EC 937-237-2 (75 % in propylene-glycol) was instilled into the conjunctival sac of three New Zealand White rabbits. The eyes were not rinsed after substance application. Animals then were observed for 7 days. Irritation was scored by the method of Draize. At 24 hours post application the eyes of all animals were further examined with fluorescein for cornea damage. All three animals showed moderate ocular reaction 1 h after application of the test substance (two scores for conjunctivae redness and chemosis). Twenty-four hours after application moderate ocular reactions were still observed in all three animals. Cornea opacity or iris irritation was not observed in any of the three animals. Slight irritation of the conjunctivae persisted until 72 h after treatment in all animals but 7 days after application all three animals were without any sign of ocular irritation. The substance is considered to be not irritating to eyes. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 2017 - 20 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

July 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm™ Kit

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 ± 2 mg (39.7 mg/cm2 according to guideline)

Duration of treatment / exposure:

3 min or 1 hour

Number of replicates:

Duplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

107.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

92.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The acceptance criteria are met:

• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.369 to 1.766)
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (4.8%)
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 2.5% to 9.1%)

Results after treatment with the substance and the controls

Dose Group	Exposure Interval	Mean Absorbance (OD) of 2 Tissues	CV [%]	Mean Rel. Absorbance [%]**
Negative Control	3 minutes	1.388
Positive Control		0.298
Test Item		1.487
Negative Control	60 minutes	1.683
Positive Control		0.080
Test Item		1.553

3 Minutes Treatment Historical Data:

Positive Control		Negative Control
Mean Viability [%]	22.65	Mean OD	1.67
Stand. Dev.	8.84	Stand. Dev.	0.13
Range of Viabilities [%]	4.60 – 39.83	Range of ODs	1.34 – 1.93

60 Minutes Treatment Historical Data:

Positive Control		Negative Control
Mean Viability [%]	6.72	Mean OD	1.64
Stand. Dev.	3.01	Stand. Dev.	0.15
Range of Viabilities [%]	2.30 – 14.77	Range of ODs	1.32 – 1.85

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not considered to be corrosive.

Executive summary:

An in vitro study was performed to assess the corrosive potential of the substance by means of the Human Skin Model Test with EpiDerm™ tissues models. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (21.4%) and for the 1 hour exposure period 4.8%) thus confirming the validity of the test system and the specific batch of tissue models. After 3 minutes exposure to the substance the relative absorbance value was not reduced (107.1%). After 1 hour exposure the relative absorbance value decreased to 92.3%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the substance is not considered to be corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2017 - 13 October 2017

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)

Version / remarks:

July 28, 2015

Deviations:

yes

Remarks:

The substance/water mixture was gently shaken for 60 minutes at room temperature instead of 15 minutes during the test for Direct MTT Reduction and Colour Interference.

GLP compliance:

yes

Specific details on test material used for the study:

Batch: RL55/17
Purity: 100%
Appearance: Yellow to brown paste
Expiry Date: 15 March 2018
Storage Conditions: At room temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Test system recommended by the OECD Test Guideline.

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkin™ Kit

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 10 ± 2 mg

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

The tissues were incubated for about 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Value:

92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the substance’s colour change potential in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the substance after 3 hour incubation with MTT-reagent showed blue colour.

Results after treatment with the substance and controls

Treatment Group	Tissue No.	Mean OD of 2 Wells blank corrected	Mean OD of 3 tissues blank corrected	Rel. Viability [%] Tissue 1, 2 + 3	Relative Standard Deviation [%]	Mean Rel. Viability [%]
Negative Control	1	1.253	1.184	105.9	5.5	100.0
	2	1.173		99.1
	3	1.125		95.1
Positive Control	1	0.221	0.261	18.6	13.5	22.1
	2	0.277		23.4
	3	0.286		24.2
Test Item	1	1.216	1.102	102.7	10.1	92.0
	2	1.096		92.6
	3	0.993		83.9

Interpretation of results:

GHS criteria not met

Conclusions:

After treatment with the substance the mean relative absorbance value decreased to 92.0%. Therefore, the substance is not considered to possess an irritant potential.

Executive summary:

An in vitro study was performed to assess the irritation potential of the substance by means of the Human Skin Model Test according to OECD TG 439. Three tissues of the human skin model EpiSkin™ were treated with the substance, the negative control (PBS) or the positive control (5% sodium lauryl sulfate) for 15 minutes. After treatment with the substance the mean relative absorbance value decreased to 92.0%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential. In conclusion, it can be stated that the substance is not irritant to skin according to UN GHS and EU CLP regulation.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24th May 2019 - 3rd July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

Adopted 28 July 2015.

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

Adopted 28 July 2015.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Skin Irritation (2-1-4), 12 Nousan No. 8147, Agricultural Production Bureau.

Version / remarks:

November 24, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: RM1006673
Intended use: Industrial chemical.
Appearance: Yellow to brown paste.
Storage conditions: In the dark at room temperature (15 to 25°C).
Expiry date: 31 July 2019
Purity: 100%

Species:

rabbit

Strain:

New Zealand White

Remarks:

Male

Details on test animals or test system and environmental conditions:

Animal Information.
Animals for this study were selected from a stock supply of healthy adult rabbits of the New Zealand White strain. They were in the weight range of 3.14 to 3.81 kg and were approximately 17 to 22 weeks of age, prior to treatment (Day 1). All rabbits were acclimatized to the experimental environment for a period of at least 7 weeks prior to the start of the study.

Animal Care and Husbandry.
Each animal was housed individually in a plastic cage with perforated floors and was offered 150 g of a standard laboratory rabbit diet per day; drinking water was provided ad libitum. The batch of diet used for the study is analyzed for nutrients, possible contaminants and micro-organisms likely to be present in the diet and which, if in excess of specified amounts, might have an undesirable effect on the test system. A dietary supplement of hay was offered.
During the acclimatization and study period the animals were given small soft white untreated wood blocks for environmental enrichment. A stainless steel key ring was also attached to the front of each cage as environmental enrichment.
Results of routine physical and chemical examination of drinking water, were reported by the supplier.
Animal room environmental controls were set to maintain temperature within the range 15 to 21°C, and relative humidity within 45 to 70%. These environmental parameters were recorded and the permanent record archived with other departmental raw data. Lighting was controlled by means of a time switch to give 12 hours of artificial light in each 24 hour period.
Each animal was identified by a numbered tag placed through the edge of one ear. Each cage was identified by a label displaying the study number and animal number.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

0.5 mL undiluted

Duration of treatment / exposure:

Single animal received 3 min, 1 hr and 4 hr

Observation period:

The observation was reported post-exposure at the following time points: 0 (3 minutes,1 and 4hr), 24, 48 and 72 hours, 5, 6, 7, 8 ,9, 10, 11, 12, 13, 14 and 15 days.

Number of animals:

3

Details on study design:

Dose Administration.
On the day before application of the substance, hair was removed with clippers from the dorso-lumbar region of each rabbit exposing an appropriate sized area of skin. Approximately 0.5 mL of substance was applied to the treatment site under a 2-ply 25 mm x 25 mm porous gauze pad secured with a single strip of surgical adhesive tape. An additional site was similarly treated with the exception of the test substance and acted as a control. The treatment site was covered with cotton wool and "Tubigrip" elasticated bandage dressing (the posterior end was secured by strip of surgical tape) for the duration of the exposure of one hour or more. The animals were not restrained during the exposure period and returned to their cages immediately after treatment.
At the end of the exposure period the semi-occlusive dressing and gauze pad were removed and the treatment site was washed with warm tap water (30 - 40°C) to remove any residual substance. The treated area was blotted dry with absorbent paper.
A single animal (number 91) received three exposures of three minutes, one or four hours duration in a step-wise manner and acted as a preliminary screen.
If clear evidence of skin irritation was observed after removal of the dressings (i.e. ≥ grade 2 erythema or edema) then no other animal would be treated until the absence of skin corrosion or severe irritation had been confirmed. If a corrosive effect was observed at any time after any of the three exposures, the test would have been immediately terminated and the animal euthanized. In the absence of a severe effect in the preliminary animal two further animals were committed to the study. A minimum period of one week was left between administration to the sentinel animal and any subsequent animal being committed to the study.

Clinical Observations.
All animals were observed daily for signs of ill health or toxicity.

Dermal Responses.
Examination of the treated skin was made immediately before administration and approximately 1, 24, 48 and 72 hours after the removal of the dressings and until Day 15.
Local dermal irritation was assessed according to the guideline.

Irritation parameter:

erythema score

Remarks:

4 h exposure (mean score)

Basis:

animal #1

Remarks:

91 M

Time point:

other: 48/72/5 days

Score:

ca. 2.3

Reversibility:

fully reversible

Remarks:

No signs of erythema reported by day 14. Although, desquamation has been observed.

Remarks on result:

probability of moderate irritation

Irritation parameter:

erythema score

Remarks:

4 h exposure (mean score)

Basis:

animal #2

Remarks:

92M

Time point:

other: 6,7 and 8 days

Score:

ca. 2

Reversibility:

fully reversible

Remarks:

On day 6, 7 and 8 exhibited loss of flexibility, and by day 14 and 15 desquamation was observed.

Remarks on result:

probability of mild irritation

Irritation parameter:

erythema score

Remarks:

4 h exposure (mean score)

Basis:

animal #3

Remarks:

93M

Time point:

24/48/72 h

Score:

ca. 2

Reversibility:

not fully reversible within: 15 days

Remarks:

veryslight erythema (barely perceptible)

Remarks on result:

probability of mild irritation

Irritation parameter:

edema score

Remarks:

4 h exposure (mean score)

Basis:

animal #1

Remarks:

91M

Time point:

other: 48/72/5days

Score:

ca. 1

Reversibility:

fully reversible within: 11 days

Remarks on result:

probability of weak irritation

Irritation parameter:

edema score

Remarks:

4 h exposure (mean score)

Basis:

animal #2

Remarks:

92M

Time point:

other: 6, 7 and 8 days

Score:

ca. 0

Reversibility:

other: No edema observed

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Remarks:

4 h exposure (mean score)

Basis:

animal #3

Remarks:

93M

Time point:

24/48/72 h

Score:

ca. 0.7

Reversibility:

fully reversible within: 10 days

Remarks on result:

probability of weak irritation

Irritation parameter:

primary dermal irritation index (PDII)

Remarks:

4 h exposure

Basis:

animal #1

Remarks:

91M

Time point:

24/48/72 h

Score:

ca. 2.7

Remarks on result:

probability of mild irritation

Irritation parameter:

primary dermal irritation index (PDII)

Remarks:

4 h exposure

Basis:

animal #2

Remarks:

92M

Time point:

24/48/72 h

Score:

ca. 1.3

Remarks on result:

probability of weak irritation

Irritation parameter:

primary dermal irritation index (PDII)

Remarks:

4 h exposure

Basis:

animal #3

Remarks:

93M

Time point:

24/48/72 h

Score:

ca. 2.7

Remarks on result:

probability of weak irritation

Other effects:

Loss of elasticity and desquamation were also observed (see information on results below)

Individual values for erythema andedema

Animal number	Type of Response Site	Score after removal of dressings
		0 hours		1 hour
		Test sites		Test site	Control site
		1	2	3	4
1 #	Erythema	0	0	1	0
	Edema	0	0	0	0

Animal number	Type of Response Site	Score after removal of dressings
		24 hours				48 hours				72 hours
		Test sites			Control site	Test sites			Control site	Test sites			Control site
		1	2	3	4	1	2	3	4	1	2	3	4
1 #	Erythema	1	0	1	1	2	2	3	0	2	2	2	0
	Edema	1	0	0	1	0	0	1	0	0	0	1	0

Animal number	Type of Response  Site	Score after removal of dressings
		Day 5				Day 6				Day 7				Day 8
		Test sites			Control site	Test sites			Control site	Test sites			Control site	Test sites			Control site
		1	2	3	4	1	2	3	4	1	2	3	4	1	2	3	4
1 #	Erythema	1	2	2LE	0	0	2	2LE	0	1	1	2LE	0	0	1	1LE	0
	Edema	0	0	1	0	0	0	1	0	0	0	1	0	0	1	1	0
		Day 9				Day 10				Day 11				Day 12
	Site	1	2	3	4	1	2	3	4	1	2	3	4	1	2	3	4
	Erythema	0	1	1	0	~	~	~	~	0	1	1	0	1	0	0	0
	Edema	0	0D	1D	0	~	~	~	~	0	0	0	0	0	0	0	0
		Day 13				Day 14				Day 15
	Site	1	2	3	4	1	2	3	4	1	2	3	4
	Erythema	0	1D	1D	0	0	0	1D	0	0	0	1D	0
	Edema	0	0	0	0	0	0	0	0	0	0	0	0

#           Sentinel animal

Site 1:    3 minutesexposure

Site 2:    1 hourexposure

Sites 3 & 4: 4 hours exposure

LE         Loss ofelasticity

D           Desquamation

~           Data notrecorded

Animal number	Type of Response Site	Score after removal of dressings
		1 hour		24 hours		48 hours		72 hours
		Test site	Control site	Test site	Control site	Test site	Control site	Test site	Control site
		1	2	1	2	1	2	1	2
2	Erythema	1	0	1	0	1	0	1	0
	Edema	0	0	0	0	0	0	0	0
3	Erythema	2	0	2	0	2	0	2	0
	Edema	1	0	1	0	0	0	1	0

Animal number	Type of Response Site	Score after removal of dressings
		Day 5		Day 6		Day 7		Day 8
		Test sites	Control site	Test sites	Control site	Test sites	Control site	Test sites	Control site
		1	2	1	2	1	2	1	2
2	Erythema	1	0	2LF	0	2LF	0	2LF	0
	Edema	0	0	0	0	0	0	0	0
3	Erythema	2	0	2	0	2	0	2	0
	Edema	0	0	0	0	0	0	1	0
		Day 9		Day 10		Day 11		Day 12
	Site	1	2	1	2	1	2	1	2
2	Erythema	2LF	0	1LF	0	1LF	0	1LF	0
	Edema	0	0	0	0	0	0	0	0
3	Erythema	2	0	2LF	0	2LF	0	2LF	0
	Edema	1	0	0	0	0	0	0	0
		Day 13		Day 14		Day 15
	Site	1	2	1	2	1	2
2	Erythema	1LF	0	1D	0	1D	0
	Edema	0	0	0	0	0	0
3	Erythema	2LF	0	1	0	1	0
	Edema	0	0	0	0	0	0

Sites 1 & 2: 4 hours exposure

LF         Loss offlexibility

D           Desquamation

Primary Irritation Index - 3 min and 1 h exposure (Scores from 24, 48 and 72 hours after bandage removal)

Animal number and (exposure time)	Sum of Erythema	Sum of Edema	PII
1 (3 min)	5	1	2.0
1 (1 h)	4	0	1.3

Primary Irritation Index - 4 h exposure (Scores from 24, 48 and 72 hours after bandage removal)

Animal number	Sum of Erythema	Sum of Edema	PII
1	6	2	2.7
2	3	1	1.3
3	6	2	2.7
Total	15	5	2.22

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The substance was classified as ‘moderately irritating’ following four hour exposure conducted according to the OECD 404 guideline.

Executive summary:

In a reliable in vivo irritation study conducted according to OECD 404 guideline, 3 male rabbits were topically exposed to 0.5 mL of the undiluted substance, a single 4 h, exposure under semi-occlusive conditions and were observed for 15 days.

Very slight or well-defined erythema was apparent in all animals throughout the observation period; moderate to severe erythema was evident in the sentinel animal 48 h after bandage removal. Very slight oedema was apparent in two animals for the majority of the first nine days after bandage removal and in the third animal at the 24 h assessment. Loss of elasticity was evident in the sentinel animal from day 5 to 8 and loss of flexibility was apparent in one of the other animals from day 6 to 13 and in the third animal from day 10 to 13. Exfoliation was apparent in one animal on days 9 and 13 and in two animals on days 14 and 15. The Primary Irritation Index was calculated to be 2.22; under the criteria of ECETOC, the substance was classified as ‘moderately irritating’ following 4 h exposure.Under the criteria of the Globally Harmonised System as adopted by the European Commission Regulation 12572/2008, the substance was assigned to Category 2 for skin irritation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2017 - 12 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

July, 2013

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

GLP compliance:

yes

Specific details on test material used for the study:

Batch: RL55/17
Purity: 100%
Appearance: Yellow to brown paste
Expiry Date: 15 March 2018
Storage Conditions: At room temperature

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Vehicle:

physiological saline

Remarks:

20% suspension (w/v)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

Triplicate

Irritation parameter:

in vitro irritation score

Value:

12.91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

For the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.36).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =126.28) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Results after 240 Minutes Treatment Time

Test Group	Opacity value = Difference (t240-t0) of Opacity	Permeability at 490 nm (OD490)	IVIS	Mean IVIS	Proposed in vitro Irritancy Score
Negative Control	Mean = 0.33	Mean = 0.068		1.36	Not categorized
Positive Control	121.67*	0.187*	124.47	126.28	Category 1
	116.67*	0.133*	118.66
	131.67*	0.271*	135.73
Substance	9.67*	0.197*	12.62	12.91	No prediction can be made
	14.67*	0.122*	16.49
	6.67*	0.197*	9.62

Interpretation of results:

other: No prediction can be made.

Conclusions:

The substance is not serious eye damaging (CLP/EPA/GHS (Cat 1), but a prediction for the damage hazard cannot be made (GHS).

Executive summary:

An in vitro study was performed to assess the corneal damage potential of the substance by means of the BCOP assay using fresh bovine corneae. The 20% (w/v) suspension in saline of the substance, the positive, and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1 °C. For the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.36). The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =126.28) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The substance was tested as suspension. Relative to the negative control, the substance caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 12.91 (threshold for serious eye damage: IVIS > 55). According to OECD 437, no prediction for the damage hazard of the substance to the eye can be made. The substance is not serious eye damaging (CLP/EPA/GHS (Cat 1).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August 2017 - 17 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2 October 2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch: RL55/17

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

At the start of the study the animals weighed 3.49 - 3.8 kg and were 12 to 52 weeks old.
Acclimitisation period: 5 days.
Drinking water and food - ad libitum.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL (equivalent to 96.3 mg).

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

1 hour, 24, 48 and 72 hours, 7 days and 21 days.

Number of animals or in vitro replicates:

Three

Irritation parameter:

maximum mean total score (MMTS)

Basis:

animal: all

Time point:

other: 1 hour

Score:

14

Max. score:

14

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

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:

5

Reversibility:

fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 1 hour

Score:

5

Reversibility:

fully reversible within: 24 hours

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:

5

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #3

Time point:

other: 1 hour

Score:

5

Reversibility:

fully reversible within: 24 hours

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: 1 hour

Score:

10

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24 h

Score:

6

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

48 h

Score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

72 h

Score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: 1 hour

Score:

12

Reversibility:

fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24 h

Score:

14

Reversibility:

fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

48 h

Score:

12

Reversibility:

fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

72 h

Score:

12

Reversibility:

fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

7 d

Score:

12

Reversibility:

fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: 1 hour

Score:

10

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24 h

Score:

8

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

48 h

Score:

6

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

72 h

Score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

7 d

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

7 d

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.25

Reversibility:

fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.75

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Scattered or diffuse corneal opacity was noted in one treated eye at the 24, 48, 72-hour and 7-days observations. Iridial inflammation was noted in two treated eyes one hour after treatment and in the other treated eye at the 24, 48 and 72-hour observations. Moderate conjunctival irritation was noted in all treated eyes one after treatment. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation in one treated eye at the 24-hour observation. Moderate conjunctival irritation persisted in one treated eye with minimal conjunctival irritation noted in two treated eyes at the 48- and 72-hour observations. Minimal conjunctival irritation was noted in one treated eye at the 7- and 14-day observations. Two treated eyes appeared normal at the 7-day observation and the remaining treated eye appeared normal at the 21-day observation.

Interpretation of results:

GHS criteria not met

Conclusions:

The substance produced a maximum group mean score of 14 and thus is classified as a mild eye irritant (not classified) based on in vivo testing in rabbits.

Executive summary:

A study was performed to assess the irritancy potential of the substance to the eye of the New Zealand White rabbits. A single application of the substance to the non-irrigated eye of three rabbits scattered or diffuse corneal opacity, iridial inflammation and moderate conjunctival irritation. Treated eyes appeared normal at the 7- and 21 -day observations. The substance produced a maximum group mean score of 14 and thus is classified as a mild eye irritant based on in vivo testing in rabbits.

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

Based on reliable studies conducted on the substance and supported by studies conducted on analogues, the substance is classified as a skin irritant (Category 2) but not classified for eye irritation.