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Endpoint summary

Description of key information

Skin irritation: In vitro skin irritation (OECD 439, GLP)

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential dermal irritation of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after exposure to the test article. The protocol was based upon the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439).

 

According to the prediction model presented in OECD TG 439, the test article was predicted to be non-Irritant (GHS No Category) since the treated tissues resulted in a mean tissue viability of 103.25%.

 

Eye irritation: In vitro eye irritation (OECD 437, GLP)

 

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test article to isolated bovine corneas. Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test article. An In Vitro Score was determined for the test article based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas. The methods and procedures used in this assay were consistent with OECD Test Guideline 437: Bovine Corneal Opacity and Permeability Test Method for
Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2020).

 

The In Vitro Score of the test article was 0.8. According to the prediction model presented in OECD TG 437, the test article was predicted to not require classification or labelling for ocular irritation (GHS No Category).

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

Study period:

From 28 January to 30 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

yes

Remarks:

See 'Any other information on materials and methods incl. tables' section

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDerm'M Model (EPl-200) (MatTek Corporation, Ashland, USA) consists of normal, humanderived epidermal keratinocytes which have been cultured to form a multilayered, highly
differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm'M Model incorporates several features which make it advantageous in the study of potential dermal toxicity. First, the test system uses a serum-free medium which eliminates the possibility of serum protein and test article interaction (Shopsis and Eng, 1988). Secondly, the target cells are epithelial, derived from human skin. Third, since the tissue has a functional stratum corneum, the test materials are applied directly to the tissue surface, at air interface, so that undiluted and/or end use dilutions can be tested directly. Prior to use, each 6-, 24- and 96-well plate will be uniquely identified with a number written in permanent marker with the test article identification or control treatment group.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Negative control: Neat (undiluted)
Positive control: 5%
Test item: Neat (undiluted)

Duration of treatment / exposure:

60 +/- 1 minutes

Duration of post-treatment incubation (if applicable):

42 +/- 2 hours post-exposure incubation period

Number of replicates:

Negative control: 3
Positive control: 3
Test item: 3

Irritation / corrosion parameter:

% tissue viability

Value:

103.25

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The assay was accepted since the following criteria were met:
1) the positive control (5% SDS) resulted in a mean tissue viability <20% (2.63%);
2) the mean OD570 value of the negative control tissues (1.637) was >= 0.8 and <= 2.8;
and 3) the standard deviation of the positive and negative control calculated from individual percent tissue viabilities of the three identically treated replicates were <= 18% (6.63% for the negative control and 0.19% for the positive control).

The skin irritancy potential of the test article was evaluated as described in OECD TG 439:

In VitroResult	GHS Category
mean tissue viability ≤ 50%	Category 1 or 2*
mean tissue viability > 50%	No Category

* - Additional testing would be warranted to resolve between GHS Category 1 (Severe/Corrosive) and GHS Category 2 (Irritant)

 

Test results:

Test Article Number	Sponsor’s Designation	Conc. (w/v)	pH	Mean Viability (%)	Skin Irritation Prediction
22AA20	GR-87-6331 (Scentaurus Melrose)	Neat	5.0	103.25 +/- 8.93	Non-Irritant
Positive Control	SDS	5%	NA	2.63 +/- 0.19	Irritant

Interpretation of results:

GHS criteria not met

Conclusions:

According to the prediction model presented in OECD TG 439, the test article was predicted to be non-Irritant (GHS No Category) since the treated tissues resulted in a mean tissue viability of
103.25%.

Executive summary:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential dermal irritation of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after exposure to the test article. The protocol was based upon the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439).

 

According to the prediction model presented in OECD TG 439, the test article was predicted to be non-Irritant (GHS No Category) since the treated tissues resulted in a mean tissue viability of 103.25%.

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:

From 25 January to 03 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Isolated bovine cornea

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

Bovine eyes will be obtained from the abattoir of J.W. TREUTH & SONS, Inc., Baltimore, MD. The eyes will be excised by an abattoir employee (as soon after slaughter as possible) and held in HBSS on ice. Once the required number of eyes has been obtained, the eyes will be transported to IIVS. Immediately upon receipt of the eyes into the laboratory, preparation of the corneas will be initiated .

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

100% for test material and controls

Duration of treatment / exposure:

10 minutes for test material and controls

Duration of post- treatment incubation (in vitro):

2 hours for test material and controls

Number of animals or in vitro replicates:

Test material: 3
Positive control: 3
Negative control: 3

Irritation parameter:

in vitro irritation score

Value:

0.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The BCOP assay was accepted when the positive control, ethanol, produced an In Vitro Score that fell within 2 standard deviations of the historical mean. The current acceptance range for ethanol is 38.0 – 63.1.

Evaluation of Test Results for BCOP:

The following prediction models were used to evaluate the ocular irritancy potential as described by Sina et al. (1995)2 for non-regulatory purposes or as described in OECD TG 437 (2017).

The prediction model according to Sina et. al based on a wide range of materials (note: While this classification system provides a good initial guide to interpretation of these in vitro data, these specific ranges may not be applicable to all classes of materials or other exposure times. Whenever possible, results should be compared to “benchmark” materials tested under similar exposure conditions.):

 

In Vitro Score	Sina et al Prediction
≤ 25	Mild irritant
> 25; ≤ 55	Moderate irritant
> 55	Severe irritant

 

Prediction model outlined in OECD TG 437:

 

In Vitro Irritation Score (IVIS)	UN GHS
≤3	No Category
>3; ≤55	No prediction can be made*
>55	Category 1

* - Additional testing would be required for a definitive classification of ocular irritation potential according to GHS

Interpretation of results:

GHS criteria not met

Conclusions:

The In Vitro Score of the test article was 0.8. According to the prediction model presented in OECD TG 437, the test article was predicted to not require classification or labelling for ocular irritation (GHS No Category).

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test article to isolated bovine corneas. Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test article. An In Vitro Score was determined for the test article based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas. The methods and procedures used in this assay were consistent with OECD Test Guideline 437: Bovine Corneal Opacity and Permeability Test Method for
Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2020).

 

The In Vitro Score of the test article was 0.8. According to the prediction model presented in OECD TG 437, the test article was predicted to not require classification or labelling for ocular irritation (GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Under the experimental conditions of this in vitro skin irritation study, the test item is not classified as skin irritant according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS (result > 50%).

 

According to the in vitro eye irritation study and under the experimental conditions reported, the test item is not corrosive/irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) ( IVIS ≤ 3 ).