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Description of key information

Skin irritation, in vivo: non-irritating, OECD TG 404, 2016

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

Eye irritation, in vivo: non-irritating, OECD TG 405, 2016

Eye irritation, in vitro: no prediction can be made, OECD TG 438, 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:
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: 3.32 or 4.06 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:
animal #1
Time point:
other: 1h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
other: 1h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: 1h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: 1h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
- Erythema: Very slight erythema (score = 1) at one treated skin site 1 and 24 hours after patch removal; fully reversed within 48 hours
- Edema: No evidence of skin irritation was noted during the study.
- Reversibility of effects: All effects reversed within 48 hours.
Other effects:
One animal showed body weight loss (0.06 Kg) and the other animal showed expected gain (0.02 kg) in body weight during the study period. 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

0

0

1 Hour

0

1

24 Hours

0

1

48 Hours

0

0

72 Hours

0

0

Edema Formation

Immediately

0

0

1 Hour

0

0

24 Hours

0

0

48 Hours

0

0

72 Hours

0

0

 

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

Erythemea/Escar Formation:

1: total = 1.00 ; mean score = 0.33

2: total = 0; mean score = 0

Edema Formation:

1: total = 0; mean score = 0

2. total = 0; mean score = 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 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, and 72 hours. A single 4-Hour, semi occluded application of the test item to the intact skin of two rabbits produced very slight erythema at one treated skin site 1 and 24 hours after patch removal. No corrosive effects were noted. Mean scores for following grading at 24, 48 and 72h were 0.33 and 0 in erythema and eschar and 0 in edema scoring criteria. Under the conditions of the study, the substance is not considered to be a skin irritant.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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: 3.29 - 3.50 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): at least 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:
2 (female). Testing was conducted sequentially. The response in those animals was such that exposure of a third would not affect classification of the test item, therefore, no further testing was needed under guidelines.
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=2
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
other: mean; n=2
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: mean; n=2
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: mean; n=2
Irritant / corrosive response data:
No corneal effects were noted. Iridial inflammation was noted in both treated eyes 1 hour after treatment although fully resolved by 24 Hour observation. Moderate conjunctival irritation was noted in both treated eyes 1 hours after treatment with minimal conjunctival irritation noted at 24 and 72 Hour observations. All treated eyes appeared normal at the 72 hours observation.

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

Organism number

1

2

Time After Treatment

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

-

Mean (24 – 72 h)

 

 

 

0

 

 

 

 

0

 

 

 

 

 

 

 

 

 

 

 

 

Area of Cornea Involved

0

0

0

0

-

0

0

0

0

-

 

 

 

 

 

 

 

 

 

 

 

IRIS

1

0

0

0

-

1

0

0

0

-

Mean (24 – 72 h)

 

 

 

0.00

 

 

 

 

0.00

 

 

 

 

 

 

 

 

 

 

 

 

CONJUNCTIVAE

 

 

 

 

 

 

 

 

 

 

Redness

2

1

1

0

-

2

1

1

0

-

Mean (24 – 72 h)

 

 

 

0.70

 

 

 

 

0.70 

 

 

 

 

 

 

 

 

 

 

 

 

Chemosis

1

1

0

0

-

1

1

0

0

-

Mean (24 – 72 h)

 

 

 

0.33

 

 

 

 

0.33

 

 

 

 

 

 

 

 

 

 

 

 

Discharge

1

0

0

0

-

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 under GLP 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 two animals. The other eye remained untreated and was used for control purposes. The test was conducted in a stepwise manner conducted singularly in accordance with the guideline. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. A single application of the test material to the non-irrigated eye of two rabbits produced no corneal effects. Iridial inflammation was noted in both treated eyes 1 hour after treatment although was absent at 24 Hour observation. Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48 Hour observations. All treated eyes appeared normal at the 72 Hours observation. Under the conditions of this study, the test substance is not considered to be irritating to the eye.

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 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, and 72 hours. A single 4-Hour, semi occluded application of the test item to the intact skin of two rabbits produced very slight erythema at one treated skin site 1 and 24 hours after patch removal. No corrosive effects were noted. Mean scores for following grading at 24, 48 and 72h were 0.33 and 0 in erythema and eschar and 0 in edema scoring criteria. Under the conditions of the study, the substance is not considered to be a skin irritant.

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 89.0% 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 1.805 pg/mL. All assay acceptability criteria were met. Under the conditions of this study, the test substance is considered to be not irritating to the skin.

 

Eye Irritation:

In vivo, OECD TG 405, 2016 : The study was performed to OECD TG 405 and EU Method B.5 under GLP 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 two animals. The other eye remained untreated and was used for control purposes. The test was conducted in a stepwise manner conducted singularly in accordance with the guideline. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. A single application of the test material to the non-irrigated eye of two rabbits produced no corneal effects. Iridial inflammation was noted in both treated eyes 1 hour after treatment although was absent at 24 Hour observation. Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48 Hour observations. All treated eyes appeared normal at the 72 Hours observation. Under the conditions of this study, the test substance is not considered to be irritating to the eye.

 

Key study: In vitro, OECD TG 438, 2016 : The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test material in isolated chicken eyes. The method involves evaluation of the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) (iii) a quantitative measurement of increased thickness (swelling) and (iv) a qualitative evaluation of macroscopic morphological damage to the surface. Also to identify substances not requiring UN GHS classification. 0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes remained untreated for control purposes. The mean corneal opacity was 2.0 (ICE Class III). The mean fluorescein retention was 0.7 (ICE Class II) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class III with maximal corneal swelling 12.94%. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class I) across all categories. The positive control gave a prediction of GHS Category 1 (ICE Class IV) across all categories signifying that the test system performed adequately. Under the conditions of this study, no prediction for eye irritation can be made following assessment of the data for all endpoints.

Respiratory Irritation:

Key study: 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. The mean achieved atmosphere concentrations were as follows: Group 1: 5.17 mg/L, Group 2: 0.95 mg/L and Group 3: 2.60 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction <4 μm: Group 1: 3.48 μm and 56.9%; Group 2: 2.59 μm and 75.1% and Group 3: 2.46 μm and 74.5%. The Geometric Standard Deviation was Group 1: 2.25, Group 2: 1.91 and Group 3: 2.10, respectively. There was 4 male and 5 female mortalities in Group 1, no mortalities in Group 2 and 1 male and 2 female mortalities in Group 3. In Group 1, the surviving male indicated body weight gain during the recovery period. Within Group 2, all males and one female exhibited body weight losses or showed no body weight gain on Day 1 post-exposure. Three females exhibited slight body weight losses from Days 3 to 7 post-exposure and one male exhibited a slight body weight loss during the final week of the recovery period. However, in Group 2 all males and females gained weight during the recovery period. Within Group 3, All surviving males and one out of three surviving females exhibited body weight losses on Day 1 post-exposure. Body weight gains were noted for all survivors during the remainder of the recovery period. These in general, where in the ranges expected and considered normal for this type of study. In Group 2 clinical observations for all males and females appeared normal at day 2 post exposure. All survivors in Group 3 appeared normal at day 4 and 5 post exposure. During necropsy in surviving Group 1 male and two males and three females of Group 2: 0.95 mg/L there was evidence of dark patches in the lungs. No macroscopic abnormalities was observed in three males and two females in Group 2. No macroscopic abnormalities were observed in four males and three female survivors in Group 3: 2.60 mg/L. Under the conditions of this study, the inhalation LC50 (male/female) was: 3.18 (C.I. 2.40 – 3.96) mg/L; LC50 (male): 3.67 (C.I. 2.35 – 4.99) mg/L and LC50 (female): 2.72 (C.I. 1.88 – 3.55) mg/L within the RCCHan WIST rat. Under the conditions of this study, there were no indications of respiratory irritation.

 

References:

1. OECD TG 403 (2009)

2. OECD 39 (2009)

Justification for classification or non-classification

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

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye 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. The substance does not demonstrate significant skin irritation potential necessary for classification and labelling within an available skin irritation in vitro assay (OECD TG 439) and an available in vivo assay (OECD TG 404).

 

For eye irritation, the weight of evidence indicates that the substance has the potential to cause transient mild irritating effects to the eye but which are insufficient for classification based on the applicants recalculation of the mean scoring and evaluation of the results in three organisms demonstrating that the EU criteria had not been met. Effects in vivo on corneal opacity are non-existent and iritis and conjunctival effects are very low which fully reversed within 72 hours; the overall evidence is indicative of transient and reversible effects on the eye. The available in vitro assay (ICE, OECD TG 438) was unable to provide a prediction in relation to the potential for classification and labelling.

 

References:

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

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