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Administrative data

Description of key information

Reconstructed human skin corrosivity test (OECD 431) - 3 minutes 81.3% viability; 60 minutes 99.3% viability
Reconstructed human skin irritation test (OECD 439)- viability at 43 2 hours 56.9%
Bovine Corneal Opacity Test (OECD 437): In vitro score 66.1 at 4hrs

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed in a GLP laboratory in accordance with OECD and EU guidelines with minor deviations that were not considered not to affect the purpose or integrity of the study.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
The control groups for this study were shared with study 41402573. The control groups raw data will be filed with study 41402564.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
The control groups for this study were shared with study 41402573. The control groups raw data will be filed with study 41402564.
GLP compliance:
yes (incl. certificate)
Species:
other: SKINETHIC™ Reconstructed Human Epidermis Model
Type of coverage:
open
Preparation of test site:
not specified
Controls:
other: SKINETHIC™ Reconstructed Human Epidermis Model
Amount / concentration applied:
16 mg of the test itemwas applied topically to the corresponding tissues ensuring uniform covering. 10 μL of sterile water was topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis.
Duration of treatment / exposure:
42 minutes
Observation period:
42 hours
Number of animals:
Quadruplicate tissues were treated with test item, negative and positive controls.
Details on study design:
TEST SITE
- Area of exposure: 0.5 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): 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 6-well post exposure incubation plates prefilled with 2.0 mL of growth medium.
- Time after start of exposure: 42 minutes

SCORING SYSTEM:
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
ca. 100
Remarks on result:
other:
Remarks:
Basis: mean Negative control. Time point: 42 minutes exposure. Reversibility: no data. (migrated information)
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
ca. 1.2
Remarks on result:
other:
Remarks:
Basis: mean Positive control. Time point: 42 minutes exposure. Reversibility: no data. (migrated information)
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
ca. 56.9
Remarks on result:
other:
Remarks:
Basis: mean Test item. Time point: 42 minutes exposure. Reversibility: no data. (migrated information)

Table 1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

 

 

Item

 

OD562 of tissues

 

Mean OD562 of triplicatet issues

 

±SD of

OD562

Relative individual tissue viability (%)

 

Relative mean viability (%)

± SD of Relative mean viability (%)

 

Negative

Control

Item

2.166

 

 

 

2.039

 

 

 

0.111

106.2

 

 

100

 

 

 

5.4

 

1.961

 

96.2

 

1.989

 

97.5

 

 

Positive

Control

Item

 

0.029

 

 

 

0.025

 

 

 

0.003

 

1.4

 

 

 

1.2

 

 

 

0.2

 

0.024

 

1.2

 

0.023

 

1.1

 

 

 

TestItem

 

1.118

 

 

 

1.159

 

 

 

0.038

 

54.8

 

 

 

56.9

 

 

 

1.9

 

1.192

 

58.5

 

1.168

 

57.3

 

 

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply:
EU DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the SKINETHICTM reconstructed human epidermis model after a treatment period of 42 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was 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 (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 item treated tissues relative to the negative controls. Histopathological analysis maybe conducted if considered necessary. The concentration of the cytokine or LDH release in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined under certain circumstances.

Quadruplicate tissues were treated with the test item or control items for an exposure period of 42 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 three tissues for each group were taken for MTT-loading. The remaining treated tissues were retained for possible histology. The growth medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading the inserts were removed and immersed in 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 triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 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 56.9% after the 42-Minute exposure period and 42 hour post-exposure incubation period. It was considered unnecessary to proceed with tissue histology or analysis of inflammatory mediators.

The test item was classified as non-irritant. The following classification criteria apply: EU DSD & CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 January 2015 - 16 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed in a GLP lab in accordance with OECD test guidelines with one minor deviation that was considered not to affect the integrity of the study.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
Control groups data and documentation were shared with another study. This deviation was considered not to affect the integrity of the study.
GLP compliance:
yes (incl. certificate)
Species:
other: Cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Local abattoir
- Age at study initiation: 12 to 60 months old

The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
Amount / concentration applied:
0.75 mL of the test substance present at 20% w/v in a 0.9% w/v sodium chloride solution.
Duration of treatment / exposure:
240 minutes followied by three rinces in fresh minimum essential medium (MEM).
Observation period (in vivo):
Opacity readings were taken and coreneas were observed visually following MEM rince. Permeability to sodium fluoresein was als evaluated.
Number of animals or in vitro replicates:
Nine eyes were used during the study, corneas with opacity values close to the median value were used.
Three corneas were issued to each group, test group, positive and negative control group.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: Corenas were rinced three times with fresh MEM.
- Time after start of exposure: 240 minutes


SCORING SYSTEM:

Opacity Measurement:
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability Measurement:
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score:
The following formula was used to determine the In Vitro Irritancy Score: In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value).
In Vitro Irritancy Score: ≤ 3 - No category. Not requiring classification to UN GHS or EU CLP
In Vitro Irritancy Score: > 3; ≤55 - No prediction of eye irritation can be made
In Vitro Irritancy Score > 55 -Category 1. UN GHS or EU CLP Causes serious eye damage

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.


TOOL USED TO ASSESS SCORE: fluorescein permeability
Irritation parameter:
cornea opacity score
Remarks:
Negative Control
Basis:
mean
Time point:
other: 240 minutes
Score:
ca. 2.2
Reversibility:
not specified
Irritation parameter:
cornea opacity score
Remarks:
Positive Control
Basis:
mean
Time point:
other: 240 minutes
Score:
ca. 97.7
Reversibility:
not specified
Irritation parameter:
cornea opacity score
Remarks:
Test substance
Basis:
mean
Time point:
other: 240 minutes
Score:
ca. 66.1
Reversibility:
not specified
Irritant / corrosive response data:
The In Vitro irritancy scores are summarized as follows:
Test Item: 66.1
Negative Control: 2.2
Positive Control: 97.7
Other effects:
The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment Cornea
Number
Opacity Permeability (OD) InVitroIrritancyScore
Pre-Treatment Post-Treatment Post-Treatment−
Pre-Treatment
Corrected
Value
  Corrected
Value
Negative
Control⊕
12 3 6 3   0.009    
18 3 5 2   0.012    
19 3 4 1   0.014    
      2.0*   0.012♦   2.2
Positive
Control⊕
1 6 85 79 77 1.505 1.493  
3 2 77 75 73 1.393 1.381  
5 4 82 78 76 1.601 1.589  
        75.3•   1.488• 97.7
Test Item 14 1 69 68 66 0.042 0.03  
16 1 67 66 64 0.047 0.035  
20 2 71 69 67 0.04 0.028  
        65.7•   0.031• 66.1

OD = Optical density

* = Mean of the post-treatment − pre-treatment values

♦ = Mean permeability

• = Mean corrected value

⊕ = Control group shared with Harlan study number 41500024

Table 2: Corneal Epithelium Condition Post Treatment

Treatment Cornea Number Observation
Post Treatment
NegativeControl⊕ 12 Clear
18 Clear
19 Clear
PositiveControl⊕ 1 Cloudy
3 Cloudy
5 Cloudy
Test Item 14 Cloudy
16 Cloudy
20 Cloudy

⊕ = Control group shared with Harlan study number 41500024

Interpretation of results:
irritating
Remarks:
Migrated information Category 1 Criteria used for interpretation of results: EU
Conclusions:
Category 1. UN GHS or EU CLP Causes serious eye damage.
Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The test item is classified according to the prediction model below:

IVIS

CLASSIFICATION

≤ 3

No category. Not requiring classification toUNGHS or EUCLP

>3; ≤55

No prediction of eye irritation can be made

>55

Category 1.UNGHS or EUCLP Causes serious eye damage


 

The In-Vitro irritancy scores are summarized as follows:

Treatment

InVitroIrritancy Score

Test Item

66.1

Negative Control

2.2

Positive Control

97.7

 

 

The test Item was concluded to be an Eye Irritant, Category 1. UNGHS or EUCLP Causes serious eye damage.


Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The substance has been assessed for skin irritation potential using both in vivo and in vitro approaches. A historical in vivo study (non-guideline, non-GLP) was conducted in which the skin irritant potential of the substance was assessed in female guinea pigs. The substance was applied at a concentration of 50% w/w in soft paraffin under occlusive conditions to clipped and chemically depliated skin in 3 animals and clipped, depilated and abraded in skin in a further 3 animals for 21 hours. The irritant reactions were assessed at 24 and 48 hours and the following mean irritancy scores were obtained: intact skin 3.33; abraded skin 4.33 which the researchers rated as severe. Based upon modern approaches, the skin irritant potential of the substance cannot be determined from this study due to deficiencies in the study design; namely the use of a chemical depilatory cream in preparing the skin, the use of an occlusive vehicle (soft paraffin) and the occlusive dressing (polythene sheet) all of which could substantially decrease the barrier function of the skin through prior chemical damage and stratum corneal hydration.

During the acute dermal toxicity study conducted according to OECD402 and GLP the following signs of dermal irritation were noted: very slight to well-defined erythema, very slight edema, crust formation, light brown discoloration of the epidermis, small superficial scattered scabs and scab lifting to reveal glossy skin. The maximal erythema/oedema score was 2 and these had resolved by day 6, leading in some instances to scabbing and crust formation; all lesions had resolved by day 12.

Further to this the substance has been tested in two OECD guideline, GLP in vitro studies as part of the integrated testing strategy for skin effects. The substance did not result in significant reductions in the viability of reconstructed human epidermis when tested in the in vitro skin corrosion assay. Further testing of the substance in the in vitro skin irritation assay did result in a significant reduction in tissue viability when measured by the MTT assay (56.9%, standard deviation 1.9% ), however this did not meet the threshold for classification as Skin Irritant Category 2 (=<50%) as defined for this study.

This result, taken in conjunction with the effects observed in the in vivo guinea pig study and the acute dermal toxicity study would suggest that the test substance is irritant, however it does not fulfill the criteria for classification as a skin irritant according to CLP.

The substance has been tested in two ex-vivo eye irritation studies to assess the potential for ocular damage following exposure. The substance was found to elicit a strong positive reaction (In vitro score of 66.1) in the Bovine Corneal Opacity Study conducted in accordance with OECD guideline 437 and GLP. This is sufficient to classify the substance as Category 1 Serious eye damage according to the CLP classification criteria. No further testing is required for this endpoint.


Justification for selection of skin irritation / corrosion endpoint:
the potential for the test substance to cause skin effects has been tested through the integrated testing strategy. The in vitro skin irritation assay was selected as it is the final test performed in a series of negative assays. A historical guinea pig study conducted on the substance, which resulted in a positive reaction is not deemed to be relevant due to study design deficiencies.

Justification for selection of eye irritation endpoint:
The assay is a Klimisch 1 study which shows positive (serious eye damage) effects in the test system.

Effects on eye irritation: corrosive

Justification for classification or non-classification

Based upon the negative results obtained in two in vitro studies performed in accordance with the integrated testing strategy for assessing skin effects, the substance does not fulfill the criteria for classification as either skin corrosive or skin irritant within the meaning of CLP.

The substance was found to elicit a strong positive reaction (In vitro score of 66.1) in the Bovine Corneal Opacity Study conducted in accordance with OECD guideline 437 and GLP. This is sufficient to classify the substance as Category 1 Serious eye damage according to the CLP classification criteria.