5,9-dimethyldec-4-enal

Endpoint summary

Description of key information

Skin irritation: Irritating, OECD TG 404, 2016

Skin irritation: non-irritating, OECD TG 439, 2016

Eye irritation: non-rritating, OECD TG 405, 2016

Eye irritation: not-irritating (IVIS = 2.0 and IVIS < 3.0), OECD TG 437, 2016

Respiratory irritation: not classified for respiratory irritation, OECD TG 403, 2016

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01-03-2016 to 15-03-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

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)

Remarks:

inspected: June 2015; signature: September 2015

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: recognised animal supplier
- Age at study initiation: 12 - 52 weeks
- Weight at study initiation: 2.93 or 3.24 kg
- Housing: Individually housed in suspended cages.
- Diet: certified rabbit diet ad libitum
- Water: mains drinking water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod: 12 hours light / 12 hours dark

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5mL
- Concentration (if solution): Test material was used as supplied.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours (initial observation); additional observations are made on Days 7 and 14 to assess the reversibility of skin reactions (as appropriate).

Number of animals:

2; following the guideline sequential testing approach

Details on study design:

TEST SITE
- Area of exposure: dorsal
- Type of wrap if used: semi-occlusive (2.5 cm x 2.5 cm cotton gauze patch secured with surgical adhesive tape)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, any residual test item removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 hours

SCORING SYSTEM:
Erythema and Eschar Formation
No erythema _________________________________________________________________________0
Very slight erythema (barely perceptible) ________________________________________________1
Well-defined erythema ________________________________________________________________2
Moderate to severe erythema __________________________________________________________3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) ________________4

Oedema Formation
No oedema __________________________________________________________________________0
Very slight oedema (barely perceptible) _________________________________________________1
Slight oedema (edges of area well-defined by definite raising) _____________________________2
Moderate oedema (raised approximately 1 millimetre) ____________________________________3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) ___4
Any other skin reactions and clinical signs of toxicity, if present, were also recorded.

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 1h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: mean score (n=2); Glossy skin, slight desquamation and reduced regrowth of fur was seen at day 14

Irritation parameter:

erythema score

Basis:

mean

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: mean score (n=2); see comments above

Irritation parameter:

erythema score

Basis:

mean

Time point:

48 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: mean score (n=2); see comments above

Irritation parameter:

erythema score

Basis:

mean

Time point:

72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: mean score (n=2); see comments above

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 1h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: mean score (n=2); Glossy skin, slight desquamation and reduced regrowth of fur was seen at day 14

Irritation parameter:

edema score

Basis:

mean

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: mean score (n=2); see comments above

Irritation parameter:

edema score

Basis:

mean

Time point:

48 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: mean score (n=2); see comments above

Irritation parameter:

edema score

Basis:

mean

Time point:

72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: mean score (n=2); see comments above

Irritant / corrosive response data:

- Erythema: Well defined erythema (score = 2) and slight edema (score = 2) at treated skin sites were noted immediately, at 1 hour and at 24, 48 and 72 hours after patch removal. The erythema extended approximately 10 mm beyond the test site 1 hour after patch removal and at the 24, 48, 72 Hour and 7 Day observations.
- Edema: and slight edema (score = 2) was noted immediately, at 1 hour and at 24, 48 and 72 hours after patch removal.
- Reversibility of effects:
1. Day 7: Inflammation (erthymea and edema) persisted (score = 2) with light brown discoloration of the epidermis and loss of skin elasticity and flexibility were noted at both treated sites.
2. Day 14: Inflammation (erthymea and edema) had fully reversed (score = 0) in all treated sites; however, glossy skin, slight desquamation and reduced regrowth of fur were noted at both treated skin sites at the 14 Day observation.

Other effects:

Light brown discoloration of the epidermis and loss of skin elasticity and flexibility were noted at both treated skin sites at the 7 Day observation. Glossy skin, slight desquamation and reduced regrowth of fur were noted at both treated skin sites at the 14 Day observation.
Both organisms showed expected body weight gain during the course of the study. There was no clinical signs of toxicity reported during the duration of the test.

Table 1. Individual skin reactions

Skin Reaction	Observation Time (following patch removal)	Individual Scores
		Number and Sex
		#1 (Female)	#2 (Female)
Erythema/Eschar Formation	Immediately	2	2
	1 Hour	2R	2R
	24 Hours	2R	2R
	48 Hours	2R	2R
	72 Hours	2R	2R
	7 Days	2RBrLeLf	2RBrLeLf
	14 Days	0GDFr	0GDFr
Edema Formation	Immediately	2	2
	1 Hour	2	2
	24 Hours	2	2
	48 Hours	2	2
	72 Hours	2	2
	7 Days	2	2
	14 Days	0	0

R = Reaction extended approximately 10 mm beyond the test site

Br = Light brown discoloration of the epidermis

Le = Loss of skin elasticity

Lf = Loss of skin flexibility

G = Glossy skin

D = Slight desquamation

Fr = Reduced regrowth of fur

 

Mean scores per organism at 24, 48 and 72h:

Erythemea/Escar Formation:

1: total = 2.0 ; mean score = 2.0

2: total = 2.0; mean score = 2.0

Edema Formation:

1: total = 2.0; mean score = 2.0

2. total = 2.0; mean score = 2.0

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test material is considered to be irritating.

Executive summary:

The study was performed to OECD TG 404, EU Method B.4 and the Japanese MAFF (2000) and US EPA OPPTS 870.2500 to assess the primary skin irritancy potential of the test substance in accordance with GLP in New Zealand White rabbits. Following single 4-Hour, semi-occluded applications to the intact rabbit skin. 0.5 ml of the test material was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the clipped skin to assess the irritancy potential of the test item. The patch was secured in position with a strip of surgical adhesive tape. After 4 hours of exposure to the test substance, the patches were removed and individual dose sites were scored at approximately 1, 24, 48, 72 hours and 7 and 14 days, respectively. A single 4-Hour, semi occluded application of the test item to the intact skin of two rabbits produced well-defined erythema and slight edema at both skin sites at 1 hour, 24 through 72 hours after patch removal. No corrosive effects were noted. Other skin reactions noted were light brown discoloration of the epidermis, loss of skin elasticity and flexibility, glossy skin, slight desquamation and reduced regrowth of fur. Mean scores for following grading at 24, 48 and 72h were 2.0 in erythema and eschar and 2.0 in edema scoring criteria. At day 14, mean scores were zero for erythema and edema, respectively. Glossy skin, slight desquamation and reduced fur growth persisted. Under the conditions of the study, the substance is considered to be a skin irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

07-03-2016 to 11-04-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: June 2015; signature: September 2015

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: 12-52 weeks old
- Weight at study initiation: 3.04 - 3.61 kg
- Housing: individually housed in suspended metal cages; with environment enrichment
- Diet: certified rabbit food ad libitum
- Water: mains water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

A volume of 0.1 mL of the test material, was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test material, and then released. The left eye remained untreated and was used for control purposes.

Observation period (in vivo):

Ocular assessment was conducted at approximately 1, 24, 48 and 72 hours after instillation of the test substance, according to numerical evaluation.

Number of animals or in vitro replicates:

3 (2 m/ 1 f). Testing was conducted sequentially.

Details on study design:

The study was performed in a stepwise manner and was started by treatment of a single rabbit. The other animals were treated in a similar manner after considering the degree of eye irritation observed in the first and/or second animal.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

SCORING SYSTEM:
The irritation was assessed according to Draize (1977) numerical scoring system. At each observation period, the highest scores given were recorded. Any other ocular effects were also noted.

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:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: mean; n=3

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0.1

Max. score:

2

Reversibility:

fully reversible within: 48-hours

Remarks on result:

other: mean; n=3

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

1.8

Max. score:

3

Reversibility:

fully reversible within: 7-days

Remarks on result:

other: mean; n=3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7-days

Remarks on result:

other: mean; n=3

Irritant / corrosive response data:

No corneal effects were noted. Iridial inflammation was noted in one treated eye 1 hour and 24 hours after treatment, respectively. Moderate conjunctival irritation was noted in treated eyes 1 and 24 hours after treatment with minimal conjunctival irritation noted at the 48 and 72 Hour observations. All treated eyes appeared normal at the 7-day observation.

Table 1. Individual scores and mean scores for 24, 48 and 72 hours

Organism number	1					2					3
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	1 Hour	24 Hours	48 Hours	72 Hours	7 Days
CORNEA
Degree of Opacity	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Mean (24-72 h)				0.0					0.0					0.0
Area of Cornea Involved	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
IRIS	0	0	0	0	0	0	1	0	0	0	1	0	0	0	0
Mean (24-72 h)				0.0					0.3					0.0
CONJUNCTIVAE
Redness	2	2	2	1	0	2	2	2	2	0	2	2	2	1	0
Mean (24-72 h)				1.7					2.0					1.70
Chemosis	2	2	1	1	0	2	2	1	1	0	2	2	1	1	0
Mean (24-72 h)				1.3					1.3					1.3
Discharge	2	2	0	0	0	2	1	0	0	0	2	1	0	0	0

 

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test material is not irritating to the eye.

Executive summary:

The study was performed to OECD TG 405 and EU Method B.5 to assess the irritancy potential of the test material to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test material was placed into the conjunctival sac of one eye of three animals. The other eye remained untreated and was used for control purposes. The test was conducted in a stepwise manner conducted singularly. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Further observation was made at 7-days. A single application of the test material to the non-irrigated eye of three rabbits produced no corneal effects. Iridial inflammation was noted in one treated eye 1 hour and 24 hours after treatment, respectively. Moderate conjunctival irritation was noted in treated eyes 1 and 24 hours after treatment with minimal conjunctival irritation noted at the 48 and 72 Hour observations. All treated eyes appeared normal at the 7-day observation. Under the conditions of this study, the test substance is not considered to be irritating to the eye.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-01-2016 to 21-01-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: June 2015; signature: September 2015

Species:

other: bovine

Strain:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: negative (0.9% w/v sodium chloride solution) and positive (ethanol; > 99.8% purity) controls were used in this study.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

10 minutes at 32 ± 1ºC.

Duration of post- treatment incubation (in vitro):

A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

Number of animals or in vitro replicates:

Three (3) per test item, or negative or positive controls, respectively.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. Following mounting: the anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically. Additionally, only corneas with opacity < 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: 0.9% w/v Sodium chloride solution

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: Ethanol; > 99.8% purity

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: Yes. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the
anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A posttreatment opacity reading was taken and each cornea was visually observed.
- POST-EXPOSURE INCUBATION: Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The mean opacity and mean permeability values (OD492) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD492 value). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (n=3)

Value:

2

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:

OTHER EFFECTS:
- Visible damage on test system: No. The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: 1. Ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the current historical control data (HCD) mean of for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 27.24 to 53.42. ACTUAL: PC IVIS = 47.4
2. sodium chloride 0.9% w/v solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values for bovine corneas treated with the respective negative control of the current historical control data (HCD). When testing liquids the negative control limit for opacity should be ≤2.76 and for permeability ≤0.12. ACTUAL: PC IVIS = 1.7, opacity ≤ 2.0 and permeability ≤ 0.039.

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	5	2	2	4	2	-	0.010	-	-
	18	2	3	3	1	-	0.039	-	-
	19	2	2	3	1	-	0.030	-	-
	-	-	-	-	1.3 #1	-	0.026 #2	-	1.7
Positive Control	22	2	36	35	33	31.7	0.901	0.875	-
	23	1	30	31	30	28.7	1.136	1.110	-
	24	1	28	31	30	28.7	1.582	1.556	-
	-	-	-	-	-	29.7 #3	-	1.180 #3	47.4
Test Item	26	2	2	5	3	1.7	0.069	0.043	-
	27	3	4	7	4	2.7	0.059	0.033	-
	28	2	2	4	2	0.7	0.023	0.000	-
	-	-	-	-	-	1.7 #3	-	0.025 #3	2.0

OD = Optical Density

#1 = Mean of post-incubation – pre-treatment values

#2 = Mean permeability

#3 = Mean corrected value

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test substance is not considered to be irritant in the in vitro eye corrosion/irritation test using Bovine Corneal Opacity and Permeability model. In vitro irritancy score (IVIS) was < 3.0 in the prediction model.

Executive summary:

The study was performed according to OECD TG 437 and EU Method B.47 to assess the eye irritancy potential in accordance with GLP of the test material in isolated bovine corneas. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (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 is assessed by quantitative measurements of changes in corneal opacity and permeability. The ocular irritancy of the test substance was tested through topical application for 10 ± 1 minutes. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol), was 47.4 and was within the historical positive control data range (27.24 to 53.42). The test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score (IVIS) of 2.0 after 10 minutes of treatment. Since the IVIS was < 3.0 the test substance was predicated as not irritating to the eye. Under the conditions of this study the test material is not considered to be an irritant or corrosive in the Bovine Corneal Opacity and Permeability test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin Irritation:

Key study : in vitro, OECD TG 439, 2016 : The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test substance in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).The relative mean viability of the test item treated tissues was 115.3% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The inflammatory mediator IL 1α in the retained culture medium was measured. The mean concentration of inflammatory mediator IL 1α in the culture medium retained from the test item treated tissues was 17.736 pg/mL. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 18.4%. This exceeded the acceptance criterion for an acceptable test of ≤18%. It was considered that this would not invalidate the results. An unequivocal negative result was obtained for the three identically test item treated tissues and the negative and positive control tissues met the appropriate acceptance criteria. It was considered that the results of the study are acceptable on the basis that the test system proved sensitive to the positive control and the test item did not induce a response close to the irritation cut off of ≤50% for any of the three identically treated tissues. Under the conditions of this study, the test substance is considered to be not irritating to the skin.

 

Key study : In vivo, OECD TG 404, 2016 : The study was performed to OECD TG 404, EU Method B.4 and the Japanese MAFF (2000) and US EPA OPPTS 870.2500 to assess the primary skin irritancy potential of the test substance in accordance with GLP in New Zealand White rabbits. Following single 4-Hour, semi-occluded applications to the intact rabbit skin. 0.5 ml of the test material was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the clipped skin to assess the irritancy potential of the test item. The patch was secured in position with a strip of surgical adhesive tape. After 4 hours of exposure to the test substance, the patches were removed and individual dose sites were scored at approximately 1, 24, 48, 72 hours and 7 and 14 days, respectively. A single 4-Hour, semi occluded application of the test item to the intact skin of two rabbits produced well-defined erythema and slight edema at both skin sites at 1 hour, 24 through 72 hours after patch removal. No corrosive effects were noted. Other skin reactions noted were light brown discoloration of the epidermis, loss of skin elasticity and flexibility, glossy skin, slight desquamation and reduced regrowth of fur. Mean scores for following grading at 24, 48 and 72h were 2.0 in erythema and eschar and 2.0 in edema scoring criteria. At day 14, mean scores were zero for erythema and edema, respectively. Glossy skin, slight desquamation and reduced fur growth persisted. Under the conditions of the study, the substance is considered to be a skin irritant.

 

Eye Irritation:

Key study : In vitro, OECD TG 437, 2016 : The study was performed according to OECD TG 437 and EU Method B.47 to assess the eye irritancy potential in accordance with GLP of the test material in isolated bovine corneas. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (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 is assessed by quantitative measurements of changes in corneal opacity and permeability. The ocular irritancy of the test substance was tested through topical application for 10 ± 1 minutes. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol), was 47.4 and was within the historical positive control data range (27.24 to 53.42). The test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score (IVIS) of 2.0 after 10 minutes of treatment. Since the IVIS was < 3.0 the test substance was predicated as not irritating to the eye. Under the conditions of this study the test material is not considered to be an irritant or corrosive in the Bovine Corneal Opacity and Permeability test.

 

Key study : In vivo, OECD TG 405, 2016 : The study was performed to OECD TG 405 and EU Method B.5 to assess the irritancy potential of the test material to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test material was placed into the conjunctival sac of one eye of three animals. The other eye remained untreated and was used for control purposes. The test was conducted in a stepwise manner conducted singularly. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Further observation was made at 7-days. A single application of the test material to the non-irrigated eye of three rabbits produced no corneal effects. Iridial inflammation was noted in one treated eye 1 hour and 24 hours after treatment, respectively. Moderate conjunctival irritation was noted in treated eyes 1 and 24 hours after treatment with minimal conjunctival irritation noted at the 48 and 72 Hour observations. All treated eyes appeared normal at the 7-day observation. Under the conditions of this study, the test substance is not considered to be irritating to the eye.

 

Respiratory Irritation:

Key study : INHALATION : In vivo, OECD TG 403, 2016 : The study was performed according to OECD TG 403 and EU Method B.2 in accordance with GLP to assess the acute inhalation toxicity of the test item. Three groups of ten RccHanTM : WIST strain rats (five males and five females) were exposed to an aerosol atmosphere of the test item. The groups were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period (group 1 and 2) or a twenty-eight day extended observation period (group 3). The mean achieved atmosphere concentrations were as follows: Group 1: 5.00 mg/L, Group 2: 1.04 mg/L and Group 3: 3.05 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction <4 μm: Group 1: 2.25 μm and 76.7%; Group 2: 2.19 μm and 77.8% and Group 3: 2.17 μm and 78.2%. The Geometric Standard Deviation was 2.21 in all groups, respectively. There was 5 male and 5 female mortalities in Group 1, no mortalities in Group 2 and 2 male and 3 female mortalities in Group 3. Within Group 2, all males and females exhibited body weight losses or showed no body weight gain on Day 1 post-exposure. All males exhibited body weight gains during the remainder of the recovery period with the exception of two males which showed a slightly reduced gain from Days 1 to 3 post-exposure. These were in the ranges expected and considered normal for this type of study. One female exhibited a body weight loss from Days 3 to 7 post-exposure and two females exhibited body weight losses during the final week of the recovery period. Applicant assessment indicated these were not significant as they were not combined with clinical signs or abnormal necropsy. Within Group 3, all surviving males and females exhibited body weight losses on Day 1 post-exposure. All male survivors exhibited body weight gains during the remainder of the exposure period, with the exception of one male that showed a slightly reduced gain from days 3 to 7 of the recovery period. These were in the ranges expected and considered normal for this type of study. One female exhibited a body weight loss from Days 3 to 7 post-exposure. Both surviving females exhibited body weight losses or showed no body weight gain from Days 7 to 14 post-exposure. One of these females exhibited a further body weight loss from Days 14 to 21 post-exposure. Both surviving females exhibited body weight gains during the final week of the extended recovery period. In Group 2 clinical observations for all males and females appeared normal at day 2 post exposure. In Group 3 survivors (male/female) recovered such that no significant observations were apparent on Day 5 post-exposure. Three surviving males indicated a deterioration in condition exhibiting observations again from Day 6, subsequently, the surviving males appeared normal on Day 9 post-exposure, however two of these males deteriorated slightly on Day 20 (decreased respiratory rate) but subsequently appeared normal again on Day 26. Two surviving females appeared normal from Days 8 or 9 post-exposure but showed a deterioration in condition on Day 11 with observations persisting in one of these females until the end of the extended recovery period (decreased respiratory rate and laboured respiration). One other surviving female appeared normal for one day (Day 14) and then on Days 27 and 28 post-exposure. During necropsy in Group 2 no macroscopic abnormalities were seen in three females; dark patches in lungs was seen in five males and two females. In Group 3, no macroscopic abnormalities were observed in in three male survivors dark patches in lungs was seen in two female survivors. In the mortalities, gaseous distension of the stomach, small and/or large intestine and/or dark patches in lung was observed. Under the conditions of this study, the inhalation LC50 (male/female) was: 3.05 (C.I. 2.90 – 3.20) mg/L; LC50 (male): 3.09 (C.I. 0.78 – 5.41) mg/L and LC50 (female): 3.00 (C.I. 1.50 – 4.50) mg/L within the RCCHan WIST rat. The test item was classified as Acute Inhalation Toxicity: Category 4 according to Regulation (EC) 1272/2008.

 

References:

1. OECD TG 403 (2009)

2. OECD 39 (2009)

 

Applicant assessment indicates: no significant changes were observed at 1.04 mg/L dose level in the upper respiratory tract (such as clinical signs: dyspnoea or rhinitis and/or histopathological effects like hyperemia, oedema and inflammation) or lower respiratory tract, which may be post-mortality or termination related effects and not indicative of function changes or toxicologically relevant morphological change in the absence of mortality. There are no marked changes reported indicative of organ dysfunction sufficient for classification under Regulation (EC) 1272/2008 as STOT-SE. This conclusion is particularly in relation to no significant effects being observed in the absence of mortality at any dose level and due to the application of Acute Inhalation Toxicity: Category 4 according to Regulation (EC) 1272/2008, to the substance.

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for dermal irritation category 2: H315

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for Specific Organ Toxicity – Single Exposure category 3 (respiratory tract irritation)

 

For skin irritation, further in vitro skin corrosion testing does not need to be conducted based on the available information allowing a definitive conclusion on the classification of the substance. In accordance with section 1.2 of REACH Regulation (EC) No. 1907/2006 Annex XI the weight of evidence indicates that the substance is not skin corrosive and therefore in vitro testing may be omitted. An available in vitro (OECD TG 439) skin irritation assay indicates that the substance is not irritating to the skin (based on > 60% MTT viability and IL-1a release endpoints; with a note that standard deviation in MTT viability was 18.4%). Available data in other endpoints (such as skin sensitisation and/or acute dermal toxicity) indicates that the substance is not skin corrosive and is a definitive conclusion on the classification can be made. These other studies are suggestive the substance is not significantly irritating. However, an available in vivo irritation study (rabbit: OECD TG 404, 2016) indicates slight desquamation at day-14 post exposure and whilst the erythema and edema scores were not sufficient for classification (< 2.3 mean scoring 24 to 72 hours in 2/3 organisms) and are suggestive of Globally Harmonised System (revision 4): Skin Irritation category 3 - mild irritant, a precautionary classification for CLP Regulation (EC) 1272/2008: skin irritating category 2 is applied.

 

For eye irritation, the available in vitro assay (BCOP, OECD TG 437) was suggestive of a not irritating to the eye (IVIS < 3.0). Based on an available in vivo study (OECD TG 405, 2016) where the mean scoring and evaluation of the results where two of three organisms demonstrated that the EU criteria had not been met: the substance has the potential to cause transient mild irritating effects to the eye but which are insufficient for classification. Effects in vivo on corneal opacity are non-existent and iritis and conjunctival effects are low to moderate which fully reversed within 7-days; the overall evidence is indicative of transient and reversible effects on the eye.

 

For respiratory tract irritation, no human data is available. Based on the results of an available in vivo, acute inhalation study (OECD TG 403, 2016) applicant assessment indicates: no significant changes were observed at 1.04 mg/L dose level in the upper respiratory tract (such as clinical signs: dyspnoea or rhinitis and/or histopathological effects like hyperemia, oedema and inflammation) or lower respiratory tract, which may be post-mortality or termination related effects and not indicative of function changes or toxicologically relevant morphological change in the absence of mortality. There are no marked changes reported indicative of organ dysfunction sufficient for classification under Regulation (EC) 1272/2008 as STOT-SE. This conclusion is particularly in relation to no significant effects being observed in the absence of mortality at any dose level and due to the application of Acute Inhalation Toxicity: Category 4 according to Regulation (EC) 1272/2008, to the substance.

 

References:

1. ECHA Guidance on Application on the CLP Criteria, section 3.4.2.2 (v5.0, July 2017)