Registration Dossier

Administrative data

Description of key information

Skin irritation, in vivo: non-irritating, OECD TG 404, 2014

Skin irritation, in vitro: non-irritating (mean relative viability 104.4%), OECD TG 439, 2014

Eye irritation, in vivo: non-eye irritating, OECD TG 405, 2015

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25-02-2014 to 10-10-2014
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:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2012; signature: November 2012
Species:
rabbit
Strain:
New Zealand White
Remarks:
Hsdlf: NZW
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised Supplier
- Age at study initiation: 12 to 20 weeks
- Weight at study initiation: 2.72 to 2.86 g
- Housing: Individually housed in cages with perforated floors, with shelters and cage enrichment
- Diet (ad libitum): Global Diet 2930C (Recognised Supplier); provided ad libitum
- Water (ad libitum): mains drinking water, ad libitum
- Acclimation period: at least 5 days

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

IN-LIFE DATES: From: 25-02-2014 To: 28-02-2014
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 mL.
- Concentration (if solution): Not applicable.

VEHICLE
- Amount applied: Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.
Duration of treatment / exposure:
4 hours
Observation period:
72 hours (initial observation); additional observations are typically made daily up to Days 7 and 14 to assess the reversibility of skin reactions (as appropriate).
Number of animals:
3 males
Details on study design:
TEST SITE
- Area of exposure: Dorsal (2.5 cm x 2.5 cm cotton gauze patch secured with adhesive tape)
- % coverage: Not reported
- Type of wrap if used: cotton gauze patch secured with adhesive tape elastic corset

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes
- Time after start of exposure: Four hours after the application, the dressing was removed by gentle swabbing with cotton wool soaked in distilled water.

OBSERVATION TIME POINTS
(indicate if minutes, hours or days): 1 hour, 4 hours, 24 hours, 48 hours and 72 hours. Additional observations daily up to 7 or 14 days, as appropriate

SCORING SYSTEM: consistent with Draize Scale:
- Method of calculation:
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) *.......................................................................................... 4
*. to eschar formation preventing grading of erythema
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 millimeter) ........................................................... 3
Severe oedema (raised more than 1 millimeter 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
Remarks:
n = 3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
mean
Remarks:
n = 3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
Very slight erythema and very slight edema were noted at all treated skin sites immediately after patch removal (at 1 hour). All treated skin sites appeared normal one hour after patch removal.
Other effects:
- Other adverse local effects: None.
- Other adverse systemic effects: All males showed expected gain in body weight during the study.

Table 1.0 : Individual Scores and Mean Scores following 4-hour exposure

Skin Reaction

Reading (hours)

1# Male

#2 Male

3# Male

Erythema/Escar Formation

24

0

0

0

 

48

0

0

0

 

72

0

0

0

 

Total

0.0

0.0

0.0

 

Mean

0.0

0.0

0.0

Oedema Formation

24

0

0

0

 

48

0

0

0

 

72

0

0

0

 

Total

0.0

0.0

0.0

 

Mean

0.0

0.0

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 item is not considered to be irritating to the skin.
Executive summary:

The study was performed to OECD TG 404 and EU Method B.4 guidelines in accordance with GLP to assess the primary skin irritancy potential of the test item in New Zealand White rabbits. Three rabbits were exposed for 4-hour by semi-occluded application of the test item to the intact clipped skin with 0.5 mL test item introduced under a 2.5 cm x 2.5 cm semi-occlusive patch. The patch was secured in position with adhesive tape and elasticated corset. After exposure to the test item the patches were removed and individual dose sites were scored at approximately 1, 24, 48, and 72 hours. Very slight erythema (score = 1) and very slight oedema (score = 1) was observed in the treated skin at 1 hour. Mean scores following grading at 24, 48 and 72h were zero in all scoring criteria. Under the conditions of the study, the test item is not considered to be a skin irritant.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22-01-2014 to 27-01-2014
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2012 ; signature: November 2012
Test system:
human skin model
Remarks:
EPISKIN TM Small Model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiSkin RHE Model (Lot no.: 14-EKIN-001). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt. to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. As a complimentary endpoint the concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert. 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Application of test item and rinsing:
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

The tissues were subsequently placed into was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination. Following 42 hour post exposure incubation the treated plates were then tested for MTT formazan extraction.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µl
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl
- Concentration (if solution): Not applicable.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl
- Concentration (if solution): 5% w/v aqueous solution.
Duration of treatment / exposure:
Tissues were treated with the test item for 15 minutes and then washed with DPBS with Ca++ and Mg++ to remove residual test item.
Positive control: SDS (5% w/v) was respread after 7 minutes contact time to maintain distribution of the SDS - for the remainder of the contact period. This was not deemed necessary in the negative control or test item definitive test group.
Duration of post-treatment incubation (if applicable):
Subsequently the skin tissues were incubated for 42 hours at 37°C.
Number of replicates:
Triplicate; treatment and concurrent negative control and positive control groups
Details on study design:
TEST SITE
- Area of exposure: 10 μl of the undiluted test item was added topically into 12-well plates on top of the skin tissues. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 15 minutes

SCORING SYSTEM: Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean tissue viability
Value:
104.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minute exposure. Reversibility: no data. Remarks: n=3; SD = 16.7% ; Score in terms of percentage of negative control.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test facility has validated the OECD TG 439 assay.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Concurrent controls within the OECD TG 439 guideline acceptance ranges. Therefore the test system performed adequately.

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test item

Item

OD562 of tissues

Mean OD562 of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

1.030

0.980

0.046

105.1

100*

4.7

0.970

99.0

0.939

95.8

Positive Control Item

0.050

0.054

0.004

5.1

5.5

0.4

0.058

5.9

0.053

5.4

Test Item

0.973

1.023

0.164

99.3

104.4

16.7

0.890

90.8

1.206

123.1

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

 

The relative mean tissue viability for the positive control treated tissues was 5.5% relative to the negative control treated tissues and the standard deviation value of the viability was 0.4%. The mean OD562 for the negative control treated tissues was 0.980 and the standard deviation value of the viability was 4.7%. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 16.7%. All assay acceptance criteria were met.

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this in vitro study, the test item is considered to be not irritating to skin.
Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item 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 104.4% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The standard deviation between three replicates was 16.7%. The concurrent positive control relative mean viability was 5.5% and the standard deviation was 0.4%. The standard deviation in the negative control tissues was 4.7%. All assay acceptability criteria were met. Under the conditions of this study, the test item is considered to be not irritating to the skin.

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:
31-03-2014 to 10-04-2014
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:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: March 2014; signature: May 2014
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Recognised Supplier
- Age at study initiation: 12 to 20 weeks
- Weight at study initiation: 2.35 to 2.60 g
- Housing: Individually housed in cages with perforated floors, with shelters and cage enrichment
- Diet (ad libitum): Global Diet 2930C (Recognised Supplier); provided ad libitum
- Water (ad libitum): mains drinking water, ad libitum
- Acclimation period: at least 5 days

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

IN-LIFE DATES: From: 31-03-2014 To: 10-04-2014
Vehicle:
unchanged (no vehicle)
Controls:
other: one eye remained untreated
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL.
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.
Duration of treatment / exposure:
A volume of 0.1 mL of the test item, 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 item, and then released. The other eye remained untreated and was used for control purposes. Irrigation of the eye with distilled water or saline, after 1 hour was deemed not necessary or was not reported during the study.
Observation period (in vivo):
Ocular assessment was conducted at approximately 1, 24, 48 and 72 hours after instillation of the test item, according to numerical evaluation.
Number of animals or in vitro replicates:
3 (male). Testing was conducted sequentially following testing with a sentinel.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not applicable
- Time after start of exposure:

SCORING SYSTEM:
The irritation was assessed consistent 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
Remarks:
n = 3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
n = 3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
n = 3
Time point:
24/48/72 h
Score:
0.57
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
n = 3
Time point:
24/48/72 h
Score:
0.47
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritant / corrosive response data:
No corneal effects were noted during the study. Iridial inflammation was noted in two treated eyes one hour after treatment. Moderate conjunctival irritation was noted in all treated eyes one hour after treatment.
At 24 hours no corneal effects were noted, all scores were zero. No iridial effects were noted, all scores were zero. Minimal conjunctival irritation (score = 1) was noted in all treated eyes 24 h and two treated eyes at 48 h after treatment. Chemosis was minimal (score = 1) in two treated eyes at the 24 h through 48 h observation. In all treated eyes and all effects had reversed (score = 0) at the 72 h observation.
Other effects:
- Lesions and clinical observations: Not applicable.
- Ophthalmoscopic findings: Not applicable.
- Histopathological findings: Not applicable.
- Effects of rinsing or washing: Not applicable.
- Other observations: All animals showed expected gain in bodyweight during the study.

Table 1.0 : Individual and Mean Scores for Cornea, Iris and Conjunctivae

Number and Sex

Time After Treatment

Corneal Opacity

Iridial Inflammation

Conjunctival Redness

Conjunctival Chemosis

1# Male

24 Hours

0

0

1

1

48 Hours

0

0

0

1

72 Hours

0

0

0

0

Total

0

0

1

2

Mean

0.0

0.0

0.3 #

0.7 #

#2 Male

24 Hours

0

0

1

1

48 Hours

0

0

1

1

72 Hours

0

0

0

0

Total

0

0

2

2

Mean

0.0

0.0

0.7 #

0.7 #

#3 Male

24 Hours

0

0

1

1

48 Hours

0

0

1

1

72 Hours

0

0

0

0

Total

0

0

2

2

Mean

0.0

0.0

0.7 #

0.7 #

# All effects reversed by 72 hours.

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item is not considered to be irritating to the eye.
Executive summary:

The study was performed to OECD TG 405 and EU Method B.5 guidelines in accordance with GLP to assess the irritancy potential of the test item to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test item was placed into the conjunctival sac of one eye of three rabbits. The other eye remained untreated and was used for control purposes. The test was conducted in a stepwise manner conducted singularly and then on a further two rabbits 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 item to the non-irrigated eye of three rabbits produced at the one-hour time point, Iridial inflammation (score = 1) noted in two treated eyes and moderate conjunctival irritation (score = 2). At the 24-hour time point, no corneal opacity, no iridial inflammation and minimal conjunctival irritation (redness, score = 1) and minimal chemosis (score = 1) at 24 hours through 48 hours. All treated eyes appeared normal at the 72 hour observation (score = 0). Under the conditions of this study, the test item 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, 2014 : The study was performed to OECD TG 404 and EU Method B.4 guidelines in accordance with GLP to assess the primary skin irritancy potential of the test item in New Zealand White rabbits. Three rabbits were exposed for 4-hour by semi-occluded application of the test item to the intact clipped skin with 0.5 mL test item introduced under a 2.5 cm x 2.5 cm semi-occlusive patch. The patch was secured in position with adhesive tape and elasticated corset. After exposure to the test item the patches were removed and individual dose sites were scored at approximately 1, 24, 48, and 72 hours. Very slight erythema (score = 1) and very slight oedema (score = 1) was observed in the treated skin at 1 hour. Mean scores following grading at 24, 48 and 72h were zero in all scoring criteria. Under the conditions of the study, the test item is not considered to be a skin irritant.

 

Key Study : In vitro, OECD TG 439, 2014 : The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item 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 104.4% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The standard deviation between three replicates was 16.7%. The concurrent positive control relative mean viability was 5.5% and the standard deviation was 0.4%. The standard deviation in the negative control tissues was 4.7%. All assay acceptability criteria were met. Under the conditions of this study, the test item is considered to be not irritating to the skin.

 

Eye Irritation:

Key Study : In vivo, OECD TG 405, 2015 : The study was performed to OECD TG 405 and EU Method B.5 guidelines in accordance with GLP to assess the irritancy potential of the test item to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test item was placed into the conjunctival sac of one eye of three rabbits. The other eye remained untreated and was used for control purposes. The test was conducted in a stepwise manner conducted singularly and then on a further two rabbits 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 item to the non-irrigated eye of three rabbits produced at the one-hour time point, Iridial inflammation (score = 1) noted in two treated eyes and moderate conjunctival irritation (score = 2). At the 24-hour time point, no corneal opacity, no iridial inflammation and minimal conjunctival irritation (redness, score = 1) and minimal chemosis (score = 1) at 24 hours through 48 hours. All treated eyes appeared normal at the 72 hour observation (score = 0). Under the conditions of this study, the test item is not considered to be irritating to the eye.

 

Respiratory Irritation:

Key Study : INHALATION: OECD TG 403, 2013 :

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. A group of ten RccHanTM : WIST strain rats (five males and five females) were exposed to an aerosol atmosphere of the test item. The groups was 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: 5.14 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction <4 μm: Group 1: 1.85 μm and 77.7% . The Geometric Standard Deviation was Group 1: 2.78. There were no male or female mortalities. Seven animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. Three animals showed no body weight gains from Days 1 to 3 post-exposure and two animals showed slight body weight losses from Days 3 to 7 post-exposure. Body weight gains were noted in all animals during the final week of recovery, with the exception of one animal which exhibited a slight body weight loss. Increased respiratory rate, hunched posture, pilo-erection and wet fur were noted in all animals. All recovered and appeared normal on Day 5 post-exposure. No macroscopic abnormalities were detected amongst animals at necropsy. Under the conditions of this study, the inhalation LC50 (male/female) was > 5.14 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 vivo assay (OECD TG 404).

 

For eye irritation, the weight of evidence indicates that the substance has the potential to cause transient irritating effects to the eye but which are not sufficient for classification based on 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 and iritis are low to non-existent and conjunctival effects are moderate to low which fully reversed within 72 hours; the overall evidence is indicative of moderate but transient and reversible effects on the eye.

References:

1. Guidance on Application of the CLP Criteria, ECHA, version 5.0, July 2017