Registration Dossier

Administrative data

Description of key information

Skin Irritation/Corrosion (read-across from structurally similar substance)

Non-irritant, reconstituted human epidermis (EPISKIN), eq. OECD 439, Warren (2008).

Eye Irritation/Corrosion (read-across from structurally similar substance)

Irritating, rabbit, OECD 405, Zelenák (2013).

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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
A RAAF report will shortly be provided.
Reason / purpose for cross-reference:
read-across source
Irritation / corrosion parameter:
other: Assessment of direct test material production of MTT (tissue viability)
Value:
76.3
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Max. score: 100.0. Reversibility: no data. (migrated information)
Irritation / corrosion parameter:
other: Absorbance (540 nm)
Value:
0.598
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Max. score: 0.783. Reversibility: no data. (migrated information)
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 10 June 2008 and 16 June 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Remarks:
Study conducted on read-across material
Justification for type of information:
A RAAF report will shortly be provided.
Reason / purpose for cross-reference:
other: read across target
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(pre-dates inception of guideline)
Deviations:
yes
Remarks:
Longer incubation time
Principles of method if other than guideline:
Performed using the protocol supplied with the in vitro model (EpiSkin-SM Model supplied by SkinEthic Laboratories, Nice, France)
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007
Details on test animals or test system and environmental conditions:
An in vitro study, therefore no animals used.
Type of coverage:
other: not applicable for this type of study
Preparation of test site:
other: not applicable for this type of study
Vehicle:
unchanged (no vehicle)
Controls:
other: Triplicate tissues treated with 10 µl of Phosphate Buffered Saline Dulbeccos (PBS) were used to serve as negative controls. Triplicate tissues treated with 10 µl of SDS 5% w/v were used to serve as positive controls.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg of the test material was applied to the epidermis surface.
- Concentration (if solution): not applicable


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:
Exposure period of 15 minutes.
Observation period:
42-hour post-exposure incubation period
Number of animals:
Triplicate tissues were treated with the test material
Details on study design:
TEST SITE
- Area of exposure: total exposure
- % coverage: 100 %
- Type of wrap if used: not applicable


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


SCORING SYSTEM: The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.
Irritation / corrosion parameter:
other: other: Assessment of direct test material production of MTT (tissue viability)
Value:
76.3
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Max. score: 100.0. Reversibility: no data. (migrated information)
Irritation / corrosion parameter:
other: other: Absorbance (540 nm)
Value:
0.598
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Max. score: 0.783. Reversibility: no data. (migrated information)

Direct MTT Reduction

The MTT solution containing the test material did not turn blue/purple which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD540 values, standard deviations and tissue viabilities for the negative control, test material and positive control are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 76.3% after a 15‑minute exposure.

The qualitative evaluation of tissue viability is given in Table 2.

Following the 15-minute exposure two of the test material treated tissues appeared blue which was considered indicative of viable tissue and one of the test material treated tissues appeared blue/white which was considered indicative of semi-viable tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues and the Standard Deviation (SD) value of the % viability was ≤20%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was ≥0.6 and the SD value of the % viability was ≤20%. The negative control acceptance criterion was therefore satisfied.

Table1. Mean OD540 Values and % Viabilities for the Negative Control Material, Positive Control Material and Test Material.

Material

OD540of tissues

Mean OD540of triplicate tissues

± SD of OD540

Relative individual tissue viability

Relative mean % viability

± SD of % viability

Negative
Control MaterialÄ

0.799

0.783

0.014

102.0

100*

1.81

0.772

98.6

0.777

99.2

Positive
Control MaterialÄ

0.107

0.093

0.026

13.7

11.9

3.35

0.109

13.9

0.063

8.0

Test Material

0.339

0.598

0.238

43.3

76.3

30.3

0.648

82.8

0.806

102.9

Ä= Control group shared with another study

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

Table2.Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation).

Material

Tissue 1

Tissue 2

Tissue 3

Negative Control MaterialÄ

-

-

-

Positive Control MaterialÄ

+

+

+

Test Material

+

-

-

MTT visual scoring scheme
-
              =             blue tissue (viable)
+
             =             blue/white tissue (semi-viable)
++
          =             tissue is completely white (dead)

Ä= Control group shared with SPL Project number 1071/0025

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was considered to be Non-Irritant.
Executive summary:

Introduction. 

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end‑point will be used to either confirm a non-irritant result or will be used to override the non‑irritant result.

Methods.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 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 540 nm.

Data are presented in the form of % viability (MTT reduction in the test material treated tissues relative to negative control tissues).


Results. 

The relative mean viability of the test material treated tissues was 76.3% after a 15‑minute exposure.

Quality criteria.

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion. 

The test material was considered to be non-irritant. 

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 08 July 2008 and 10 July 2008.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in line with good scientific principles and reported to a high standard using a non-validated in vitro method.
Remarks:
Study conducted on read-across material
Justification for type of information:
A RAAF report will shortly be provided.
Reason / purpose for cross-reference:
other: read across target
Qualifier:
no guideline followed
Principles of method if other than guideline:
Performed using the standard protocol with the SkinEthic Model (SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France))
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007
Details on test animals or tissues and environmental conditions:
In-vitro study, so no test animals used.
Vehicle:
unchanged (no vehicle)
Controls:
other: Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of 1% w/v Sodium Dodecyl Sulphate served as the positive control.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
- Concentration (if solution): not applicable


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:
10 minutes
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Triplicate SkinEthic tissues
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Rinsed with water.
- Time after start of exposure: 10 minutes


SCORING SYSTEM: relative mean viability


TOOL USED TO ASSESS SCORE: optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader.
Irritation parameter:
other: Relative mean tissue viability
Run / experiment:
Mean
Value:
ca. 68.8
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Assessment of Eye Irritation Potential – Viability of HCE Tissues

Material

Individual Tissue Viability

Mean OD540

% Viability

Negative ControlÅ

1.034

1.008

100*

0.981

Positive ControlÅ

0.525

0.488

48.4

0.451

Test Material

0.793

0.693

68.8

0.593

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


Å = Control group shared with another study

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

Interpretation of results:
GHS criteria not met
Conclusions:
According to the protocol followed the test material was considered to be a Non-Irritant (NI).
Executive summary:

Introduction. 

The purpose of this study was to determine the eye irritation potential of the test materials using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Methods. 

The experimental design of the study consists of a test for Direct Reduction of MTT by the test materials, followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test material for 10 minutes. Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of 1% w/v Sodium Dodecyl Sulphate served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured (OD540). Data are presented in the form of % viability (MTT conversion relative to negative controls).

The test material was classified according to the following criteria:

i)                   If the % relative mean tissue viability was ≥ 60% the test material was considered to be Non-Irritant (NI).

ii)                  If the % relative mean tissue viability was < 60% the test material was considered to be Irritant (I).

Results.

The relative mean viability of the test material treated tissues after a 10 minute exposure was 68.8%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criteria.

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion.

According to the protocol followed the test material was considered to be a Non-Irritant (NI).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
A RAAF report will shortly be provided.
Reason / purpose for cross-reference:
read-across source
Irritation parameter:
other: relative mean tissue viability
Run / experiment:
Mean
Value:
68.8
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 February 2013 to 26 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Remarks:
Study conducted on read-across material
Justification for type of information:
A RAAF report will shortly be provided.
Reason / purpose for cross-reference:
other: read across target
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 14 weeks
- Weight at study initiation: 3491 and 3551 g
- Housing: Individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbits in adjoining cages
- Diet: ad libitum
- Water: municipal tap water ad libitum
- Acclimation period: 27 days

ENVIRONMENTAL CONDITIONS
- Temperature: 16.8 to 20.2 °C
- Humidity: 24 to 68 % (relative)
- Air changes: 15 to 20 changes per hour
- Photoperiod: 12 hour light/dark cycle (light from 06:00 to 18:00)
Vehicle:
unchanged (no vehicle)
Controls:
other: The untreated contralateral eye served as the control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g
- pH: The pH of the test material was determined prior to dosing. The test material was found to be acceptable for use in the test (pH 5)

- TOPICAL ANAESTHETIC AND SYSTEMIC ANALGESIC
- Schedule: Sixty minutes (60 ± 10 minutes) prior to administration, a systemic opiate analgesic (Buprenorphine 0.01 mg/kg) was administered by subcutaneous injection. Five minutes (5 ± 1.5 minutes) prior to administration, a topical ocular anaesthetic (Humacain (oxybuprocaine) one-two drops per eye) was applied to each eye (including the control eye to ensure direct comparison of any ocular observations). Eight hours (8 to 9 hours) after test material application, Buprenorphine 0.01 mg/kg and a nonsteroidal anti-inflammatory drug (NSAID) (Meloxicam 0.5 mg/kg) were administered by subcutaneous injection. The systemic opiate analgesic was injected approximately every 12 hours and the NSAID every approximately every 24 hours until the ocular lesions were resolved and no clinical signs of pain or distress were present.
Duration of treatment / exposure:
A single dose was administered
Observation period (in vivo):
3 weeks (21 days)
Number of animals or in vitro replicates:
2
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): physiological saline
- Time after start of exposure: At the 1 and 24 hour observation point in one animal and at the 1 hour observation point only in the second animal.

SCORING SYSTEM: Draize (1977) and OECD 405.
The eyes were examined at in the first animal 1, 24, 48 and 72 hours, then 1, 2 and 3 weeks after treatment. The eyes were examined in the second animal 1, 24, 48 and 72 hours, then 1 and 2 weeks after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: Mean of 24, 48 and 72 hour observations
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 21 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: Mean of 24, 48 and 72 hour observations
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 21 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
other: Mean of 24, 48 and 72 hour observations
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 21 days
Irritation parameter:
conjunctivae score
Remarks:
discharge
Basis:
animal #1
Time point:
other: Mean of 24, 48 and 72 hour observations
Score:
2.67
Max. score:
3
Reversibility:
fully reversible within: 21 days
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: Mean of 24, 48 and 72 hour observations
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: Mean of 24, 48 and 72 hour observations
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: Mean of 24, 48 and 72 hour observations
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
other: Mean of 24, 48 and 72 hour observations
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Remarks:
discharge
Basis:
animal #2
Time point:
other: Mean of 24, 48 and 72 hour observations
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: Mean of 24, 48 and 72 hour observations
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritant / corrosive response data:
For the irritant response of each animal, the mean score of the 24, 48 and 72 hour observations were calculated for each effect observed.
Other effects:
There was no mortality during the test. One animal showed slight bodyweight loss during the treatment period. The second rabbit was found to be within the normal range. There were no clinical signs observed that could be related to treatment.
No initial pain reaction was observed in either animal.

Table 1: Results for Animal No. 01973

Time

Irritation Score

IPR/

PR

Conjunctivae

Cornea

Iris

Control eye

R

CH

D

OD

OE

0 hours*

0

0

0

0

0

0

0

0

1 hour

2

2

3

0

0

0

0

0

24 hours

2

2

3

1

3

0

0

0

48 hours

1

1

3

1

3

0

0

0

72 hours

1

1

2

1

3

0

0

0

7 days

1

1

1

1

3

0

0

0

14 days

1

1

1

1

3

0

0

0

21 days

0

0

0

0

0

0

0

0

* Pre-treatment

IPR/PR - Initial Pain Reaction/Pain Response

R – Redness

CH- Chemosis

D – Discharge

OD –Opacity (degree of density)

OE – Area of opacity

Table 2: Results for Animal No. 01950

Time

Irritation Score

IPR/PR

Conjunctivae

Cornea

Iris

Control eye

R

CH

D

OD

OE

0 hours*

0

0

0

0

0

0

0

0

1 hour

2

2

3

1

4

0

0

0

24 hours

2

1

2

1

4

0

0

0

48 hours

2

1

1

1

3

0

0

0

72 hours

2

1

1

1

3

0

0

0

7 days

1

0

1

0

0

0

0

0

14 days

0

0

0

0

0

0

0

0

* Pre-treatment

IPR/PR - Initial Pain Reaction/Pain Response

R – Redness

CH- Chemosis

D – Discharge

OD –Opacity (degree of density)

OE – Area of opacity

Table 3: Results of Bodyweight Observations

Time point

Bodyweight (g)

Bodyweight gain (g)

Animal no. 01973

Prior to treatment

3491

325

21 February 2013 (16 days)

3752

Termination of the study

3816

Animal no. 01950

Before treatment

3551

-1

Before euthanasia

3550

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Under the conditions of the study, the test material caused eye irritation in two New Zealand White rabbits. The effects were found to be reversible within 21 days.
Executive summary:

The eye irritation potential of the test material was investigated in New Zealand White rabbits in a study conducted in accordance with the standardised guideline OECD 405 under GLP conditions.

Two animals were exposed to the test material. 0.1 g of the test material was instilled into the conjunctival sac of the eye of each rabbit. The eyes were rinsed at the 1 and 24 hour examination points with physiological saline in one animal, and at the 1 hour observation point only in the second animal. Prior to dosing and during the observation period, a pain management regimen was administered (topical anaesthetic and systemic analgesic) which was continued until all eye effects had resolved. The animals were observed for up to 21 days, and any effects noted were evaluated in accordance with the Draize Scale (1977).

The animal individual mean scores (considering readings taken at 24, 48 and 72 hours after the treatment) were as follows:

Chemosis: 1.33 and 1.00

Discharge: 2.67 and 1.33

Redness: 1.33 and 2.00

Corneal opacity: 1.00 and 1.00

Iris: 0.00 and 0.00

There were no observed pain reactions related to the test material. One animal was reported to have a reduced bodyweight during the study.

Under the conditions of the study, the test material caused eye irritation in two New Zealand White rabbits. The effects were found to be reversible within 21 days.

Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
A RAAF report will shortly be provided.
Reason / purpose for cross-reference:
read-across source
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 21 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 21 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 21 days
Irritation parameter:
conjunctivae score
Remarks:
discharge
Basis:
animal #1
Time point:
24/48/72 h
Score:
2.67
Max. score:
3
Reversibility:
fully reversible within: 21 days
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Remarks:
discharge
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation/Corrosion

In the key study (Warren 2008), the skin irritation potential of the read-across material was evaluated in a GLP compliant study performed to a method equivalent to the adopted OECD guideline No. 439. The study was assigned a reliability score of 1 in accordance with the principles for assessing data quality as described in Klimisch et al. (1997).

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™ reconstituted human epidermis model (performed in triplicate) after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours.  Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. 

The relative mean viability of the test material treated tissues was 76.3 % after a 15minute exposure.

Under the conditions of the test, the test material was considered to be non-irritant. 

In accordance with column 2, point 8.1.1 of Annex VIII of the Regulation (EC) No. 1907/2006 (REACH), the in vivo test does not need to be performed if an acute dermal toxicity test does not indicate skin irritation up to the limit dose (2000 mg/kg). As no irritation was observed in the acute dermal toxicity study submitted to support this registration, in vivo testing for this endpoint is therefore omitted.

Furthermore, the in vitro test fulfils the requirements set out in point 1.4 of Annex XI of the same Regulation for in vitro tests; therefore confirmation of the negative result is not required.

 

Eye Irritation/Corrosion

In the key study (Zelenák 2013), the eye irritation potential of the read-across material was investigated in New Zealand White rabbits in a study conducted in accordance with the standardised guideline OECD 405 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the principles for assessing data quality as described in Klimisch et al. (1997).

Two animals were exposed to the test material. 0.1 g of the test material was instilled into the conjunctival sac of the eye of each rabbit. The eyes were rinsed at the 1 and 24 hour examination points with physiological saline in one animal, and at the 1 hour observation point only in the second animal. Prior to dosing and during the observation period, a pain management regimen was administered (topical anaesthetic and systemic analgesic) which was continued until all eye effects had resolved. The animals were observed for up to 21 days, and any effects noted were evaluated in accordance with the Draize Scale (1977).

The animal individual mean scores (considering readings taken at 24, 48 and 72 hours after the treatment) were as follows:

Chemosis: 1.33 and 1.00

Discharge: 2.67 and 1.33

Redness: 1.33 and 2.00

Corneal opacity: 1.00 and 1.00

Iris: 0.00 and 0.00

There were no observed pain reactions related to the test material. One animal was reported to have a reduced bodyweight during the study.

Under the conditions of the study, the test material caused eye irritation in two New Zealand White rabbits. The effects were found to be reversible within 21 days.

 

In the supporting study (Warren 2008), the eye irritation potential of the read-across material was evaluated using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death. The study was performed under GLP conditions, in line with good scientific principles but to a non-validated method. The study was therefore assigned a reliability score of 2 in accordance with the principles for assessing data quality as described in Klimisch et al. (1997).

The experimental design of the study consisted of a test for Direct Reduction of MTT by the test material, followed by the main test. For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test material for 10 minutes. Negative and positive controls were performed concurrently in triplicate.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured (OD540). 

The relative mean viability of the test material treated tissues after a 10 minute exposure was 68.8 %.

According to the protocol followed the test material was considered to be a Non-Irritant (NI).

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not meet the criteria for classification for skin irritation.

 

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance should be classified for eye irritation as Category 2 (H319: Causes serious eye irritation) with the signal word warning and the pictogram GHS07.