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Administrative data

Description of key information

skin irritation (OECD 404, OECD 439): not irritating
eye irritation (OECD 405): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 - 24 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 26 Jul 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 Jul 2012
Deviations:
no
GLP compliance:
yes
Species:
human
Strain:
other: EPISKIN-SM™; three-dimensional human epidermis model
Details on test animals and environmental conditions:
TEST SKIN MODEL
- Source: SkinEthic Laboratories, Lyon, France

TEST METHOD
EPISKIN Small Model™ (EPISKIN-SM™):
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
On the day of receipt, tissues were transferred into 12-well plates and preincubated with prewarmed Maintenance Medium for approx. 26 h at 37 °C.

INCUBATION CONDITIONS
- Temperature (°C): 37 ± 1 (acutal range: 36.6 - 37.5)
- CO2 gas concentration (%): 5 ± 0.5
- Humidity (%): 80 - 100 (actual range: 84 - 88)
Type of coverage:
open
Preparation of test site:
other: intact human epidermis model
Vehicle:
unchanged (no vehicle)
Controls:
other: concurrent control tissues treated with PBS served as negative controls, positive controls were exposed to 5% (aq) SDS
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 12.9 to 16.4 mg
The skin was moistened with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue.

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: 5% (aq) sodium dodecyl sulphate (SDS) in PBS
The positive control was re-spread after 7 minutes contact time.
Duration of treatment / exposure:
15 min
Observation period:
not applicable
Number of animals:
not applicable
The test was performed in triplicates for each test or control group.
Details on study design:
TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: Residual test item was washed from the skin surface with phosphate buffered saline.
- Time after start of exposure: 15 min
After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for approximately 42 h at 37°C.

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, after incubating tissues in maintenance medium for 42 h, tissues were transferred into a 12-wells plate and incubated in 2 mL MTT-medium (0.3 mg/mL) for 3 h at 37 °C. Thereafter the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol. Tubes were stored refrigerated and protected from light for approximately 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. The absorption was corrected for the background absorption.

Irritation / corrosion parameter:
other: other: cell viability (% of negative control)
Value:
100
Remarks on result:
other:
Remarks:
Basis: other: mean value of negative controls (PBS). Time point: 15 min. Reversibility: other: not applicable.
Irritation / corrosion parameter:
other: other: cell viability (% of negative control)
Value:
22
Remarks on result:
other:
Remarks:
Basis: other: mean value of positive controls (5% (aq) SDS). Time point: 15 min. Reversibility: other: not applicable.
Irritation / corrosion parameter:
other: other: cell viability (% of negative control)
Value:
93
Remarks on result:
other:
Remarks:
Basis: other: mean value of the test item. Time point: 15 min. Reversibility: other: not applicable.
Other effects / acceptance of results:
The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 93%. Since the mean relative tissue viability for the test substance was above 50% after 15 minutes treatment, the test substance is considered to be non-irritant.

Table 1. Mean absorption in the in vitro skin irritation test 

 

A

(OD570)

B

(OD570)

C

(OD570)

Mean (OD570) ± SD

Mean tissue viability (percentage of control)

Negative control

1.315

0.979

1.114

1.136 ± 0.169

100

Test substance

1.006

0.997

1.152

1.052 ± 0.087

93

Positive control

0.173

0.404

0.186

0.254 ± 0.130

22

OD = optical density

SD = standard deviation

Triplicate exposures are indicated by A, B and C.

The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that the test substance did not interact with MTT.

Interpretation of results:
other:
Remarks:
CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
DSD: not classified
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 - 30 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Version / remarks:
Aug 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Food and Argricultural Materials Inspection Centre (FAMIC), 12 Nohsan, Notification No. 8147, April 2011; including the most recent partial revisions
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Health Care Inspectorate, Ministry of Health, Welfare and Sport, Den Haag, The Netherlands
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L'Arbresle, France
- Age at study initiation: 12 - 24 weeks
- Weight at study initiation: 2.71 - 3.03 kg
- Housing: individually in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters
- Diet: pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad, Italy) approx. 100 g per day; hay and wooden sticks were available
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 May 2014 To: 30 May 2014
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
other: adjacent areas of the untreated skin of each animal served as controls
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 g

The test substance was ground to a powder using a pestle and mortar prior to weighing and the test substance was moistened with 0.3 mL water (Elix, Millipore S.A.S., Molsheim, France), immediately before application, to ensure close contact with the animal's skin.
Duration of treatment / exposure:
4 h
Observation period:
72 h
Reading time points: 1, 24, 48 and 72 h
Number of animals:
3 males
Details on study design:
TEST SITE
- Area of exposure: dorsal area of the trunk (10x15 cm)
- Type of wrap if used: The test substance was applied using a metalline patch (2x3 cm). The patch was mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: The skin was cleaned of residual test substance using tap water.
- Time after start of exposure: 4 h

SCORING SYSTEM: Draize scoring system
Irritation parameter:
erythema score
Basis:
mean
Remarks:
out of all 3 animals
Time point:
other: mean over 24, 48 and 72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
out of all 3 animals
Time point:
other: mean over 24, 48 and 72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritant / corrosive response data:
Exposure to the test substance for 4 h resulted in very slight erythema (grade 1) in the treated skin areas of two rabbits at the 1 h reading time point. The skin irritation resolved within 24 hours after exposure in both animals. No edema was found.
There was no evidence of a corrosive effect on the skin.
Other effects:
No staining of the treated skin by the test substance was observed and no test substance remnants were seen.

Table 1. Results of skin irritation study.

Observation time

Rabbit no.

1

2

3

Erythema

Edema

Erythema

Edema

Erythema

Edema

1 h

1

0

1 r

0

0

0

24 h

0

0

0

0

0

0

48 h

0

0

0

0

0

0

72 h

0

0

0

0

0

0

Mean value

24 + 48 + 72 h

0

0

0

0

0

0

r: scattered erythema

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:
other:
Remarks:
CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
DSD: not classified
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:
25 Mar - 08 Apr 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
Aug 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Food and Argricultural Materials Inspection Centre (FAMIC), 12 Nohsan, Notification No. 8147, April 2011; including the most recent partial revisions
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Health Care Inspectorate, Ministry of Health, Welfare and Sport, Den Haag, The Netherlands
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L'Arbresle, France
- Age at study initiation: 12-13 weeks
- Weight at study initiation: 2.59 kg - 2.92 kg
- Housing: individually in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters
- Diet: pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad, Italy) approx. 100 g per day; hay and wooden sticks were available
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Mar 2014 To: 08 Apr 2014
Vehicle:
unchanged (no vehicle)
Controls:
other: the untreated eye served as control
Amount / concentration applied:
TEST MATERIAL
- the test material was ground to a powder
- Amount applied: 31.9 mg (range 31.8 - 32.1 mg) (equivalent to a volume of approx. 0.1 mL)


Duration of treatment / exposure:
single application without washing
Observation period (in vivo):
7 days
Reading time points: 1, 24, 48 and 72 h and 7 days
Number of animals or in vitro replicates:
3 males
Details on study design:
SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: solution of 2% fluorescein in water
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
out of all 3 animals
Time point:
other: mean over 24, 48 and 72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility: not applicable
Irritation parameter:
iris score
Basis:
mean
Remarks:
out of all 3 animals
Time point:
other: mean over 24, 48 and 72 h
Score:
0
Max. score:
2
Reversibility:
other: reversibility: not applicable
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: mean over 24, 48 and 72 h
Score:
1.7
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: mean over 24, 48 and 72 h
Score:
1.7
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: mean over 24, 48 and 72 h
Score:
1.7
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: mean over 24, 48 and 72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: mean over 24, 48 and 72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: mean over 24, 48 and 72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
There was no evidence of ocular corrosion. Instillation of the test substance into one eye of each of three rabbits resulted in effects on the iris and conjunctivae. Iridial irritation grade 1 was observed in two animals and resolved within 24 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in all animals.
No corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.
Other effects:
No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen.

Table 1. Results of eye irritation study

Rabbit #

Time [h]

conjunctivae

iris

cornea

redness

swelling

1

1

2

2

0

0

24

2

1

0

0

48

2

0

0

0

72

1

0

0

0

7d

0

0

0

0

average
24+48+72

1,7

0,3

0,0

0,0

2

1

2

2

1

0

24

2

1

0

0

48

2

1

0

0

72

1

0

0

0

7d

0

0

0

0

average
24+48+72

1,7

0,7

0,0

0,0

3

1

2

2

1

0

24

2

1

0

0

48

2

0

0

0

72

1

0

0

0

7d

0

0

0

0

average
24+48+72

1,7

0,3

0,0

0,0

 

 

Time [h]

conjunctivae 

iris

 

cornea

 

 

redness

swelling

average
score

1

2,00

2,00

0,67

0,00

24

2,00

1,00

0,00

0,00

48

2,00

0,33

0,00

0,00

72

1,00

0,00

0,00

0,00

7d

0,00

0,00

0,00

0,00

24+48+72

1,67

0,44

0,00

0,00

No mortality occurred and no signs of systemic toxicity were observed in the animals during the test period.

Interpretation of results:
other:
Remarks:
CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
DSD: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a human skin model and by a primary skin irritation/corrosion study in the rabbit.

In an in vitro skin irritation test according to OECD Guideline 439 and in compliance with GLP 12.9 to 16.4 mg test substance (equivalent to 34 - 43 mg/cm²) were applied topically to moistened human skin tissues (EPISKIN-SM) for 15 min (Tosoh Corporation 2014a). After a 42 h post-incubation period, the cytotoxic (irritancy) effect was assessed. Cytotoxicity was determined as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT. The relative mean tissue viability obtained after 15 min treatment with the test substance compared to the negative control tissues was 93%. Since the mean relative tissue viability for the test substance was above 50% after 15 min treatment the test substance is considered to be non-irritant. The positive control 5% (aq) sodium dodecyl sulphate revealed a mean cell viability of 22% after 15 min exposure. The absolute mean OD 570 (optical density at 570 nm) of the negative control tissues treated with PBS was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 15%, indicating that the test system functioned properly.

In a primary skin/corrosion study according to OECD Guideline 404 and in compliance with GLP 0.5 g test substance moistened with water was applied sequentially to the clipped skin of 3 male New Zealand White rabbits under semi-occlusive conditions for 4 h (Tosoh Corporation, 2014b). Scoring of skin reactions (erythema and edema) was performed 1, 24, 48 and 72 h after removal of the patch. Exposure to the test substance resulted in very slight erythema (grade 1) in two animals at the 1 h reading time point. The erythema had resolved within 24 h. No edema was found and no staining of the treated skin by the test substance was noted. The overall mean irritation scores over 24, 48 and 72 h for erythema and edema were 0. No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred. Based on the results, the test substance was not irritating to the skin under the conditions of the test.

 

In conclusion, the test substance is non-irritant in the in vitro skin irritation test and in the in vivo primary skin/corrosion study in rabbits under the experimental conditions used.

Eye

The eye irritation potential of the test substance was determined in a tiered testing strategy. Firstly a bovine corneal opacity and permeability test (BCOP test) was performed followed by an acute eye irritation study in the rabbit.

The BCOP test with the test substance was performed according to OECD Guideline 437 and in compliance with GLP (Tosoh Corporation, 2014c).The non-surfactant solid test substance was applied as a 20% solution in physiol. saline to the epithelial surface of three cattle corneas for 4 h ± 10 min at 32 ± 1 °C. After exposure the corneas were washed and the damage by the test substance was assessed directly by quantitative measurement of changes in corneal opacity with an opacitometer. In addition the permeability of the corneas was measured with an ultraviolet/visible spectrophotometer after incubation of the corneas with sodium fluorescein solution for 90 min. The results of the opacity and permeability measurement were used to calculate an in vitro irritation score (IVIS) of 22. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control physiol. saline did not induce irritancy on the corneas.The mean in vitro irritation score of the positive control (20% (w/v) imidazole) was 117 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Since (1,4-diazabicyclo[2.2.2]octane-2-yl)methanol induced an IVIS of > 3 ≤ 55, no prediction on the classification could be made and additional testing was required.

 

In an acute eye irritation study according to OECD Guideline 405 and in compliance with GLP samples of 31.9 mg test substance (corresponds to a volume of approx. 0.1 mL) were instilled into one eye of each of 3 male New Zealand White rabbits (Tosoh Corporation, 2014d). Eye reactions were scored 1, 24, 48 and 72 hours and 7 days after instillation. Instillation of the test substance resulted in effects on the iris and conjunctivae. Iridial irritation grade 1 was observed in two animals and resolved within 24 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in all animals. The overall mean irritation scores over 24, 48 and 72 h for conjunctival redness, chemosis, iris and corneal effects were 1.67, 0.44, 0 and 0, respectively. No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred. Based on the results, the test substance was not irritating to the eyes under the conditions of the test.

Justification for classification or non-classification

The available data on skin and eye irritation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.