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ISO / ISO 10993-23:2021/Amd 1:2025 Biological evaluation of medical devices — Part 23: Tests for irritation — AMENDMENT 1: Additional in vitro reconstructed human epidermis models

ISO 10993-23:2021/Amd 1:2025 Biological evaluation of medical devices — Part 23: Tests for irritation — AMENDMENT 1: Additional in vitro reconstructed human epidermis models

Foreword

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This document was prepared by Technical Committee ISO/TC 194, Biological and clinical evaluation of medical devices, in collaboration with the European Committee for Standardization (CEN) Technical Committee CEN/TC 206, Biological and clinical evaluation of medical devices, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).

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Biological evaluation of medical devices — Part 23: Tests for irritation — AMENDMENT 1: Additional in vitro reconstructed human epidermis models

Introduction, fifth paragraph

Replace the paragraph with the following paragraphs:

Traditionally, tests in small animals have been performed prior to testing on humans to help predict human responses. More recently, in vitro tests as well as human tests have been added as adjuncts or alternatives. For skin irritation testing of neat chemicals, in vitro tests were developed using reconstructed human epidermis (RhE) models[31]. The method was adapted for detection of irritant chemicals in medical device extracts. The results of a large interlaboratory study that tested two types of RhE models showed that these models can also be used to detect the presence of irritant chemicals extracted from polymeric materials [polyvinylchloride (PVC) and silicone] commonly used in the manufacture of medical devices[6]. This method was found to be equally sensitive in the detection of low concentrations of some strong irritant compounds when compared to the human patch testing and intracutaneous rabbit test[14]. Therefore, a stepwise approach for irritant testing can start with the in vitro RhE model.

In 2023, two new type RhE models listed in OECD 439 were adopted for medical devices after they demonstrated equivalent predictive capacity compared to the two other RhE models in interlaboratory studies[42][43][44].

 

Clause 4, second paragraph

Replace the paragraph with the following paragraphs:

Preference for in vitro tests instead of in vivo tests in accordance with ISO 10993-2, shall be considered, with replacement of the latter as new in vitro tests are scientifically validated and qualified for use with medical devices and become reasonably and practicably available. The results of a large interlaboratory study that tested two types of RhE models showed that these models can also be used to detect the presence of irritant chemicals extracted from polymeric materials [polyvinylchloride (PVC) and silicone] commonly used in the manufacture of medical devices[6]. This method was found equally sensitive to detect low concentrations of some strong irritant compounds when compared to the human patch testing and intracutaneous rabbit test[14]. Therefore, the in vitro irritation test shall be performed before animal testing or human patch test is considered.

In 2023, two new type RhE models listed in OECD 439 were adopted for medical devices after they demonstrated equivalent predictive capacity compared to the two other RhE models in catch-up validation studies[42][43][44].

 

6.1, first paragraph

Replace the paragraph with the following paragraphs:

The in vitro method with RhE models for testing irritation was developed specifically to detect skin irritation potential for neat chemicals[3][12] (see OECD 439). The method was adapted and validated with two RhE models for detection of irritant chemicals in medical device extracts[5][6][12][13][17][18][19]. This method was found equally sensitive to detect low concentrations of some strong irritants in extracts from polymeric medical materials (PVC and silicone) when compared to the human patch testing and intracutaneous rabbit test[14]. Hence, the RhE test as described in this document can replace the in vivo rabbit test for irritation by skin exposure and by intracutaneous (intradermal) administration.

Two new type RhE models listed in OECD 439 were adopted for medical devices in 2023 after demonstrating equivalent predictive capacity in interlaboratory studies[42][43][44].

 

6.2.2, eight paragraph

Replace the paragraph with the following:

For known cases of test-compounds requiring specific controls, some chemicals can directly reduce the MTT reagent (e.g. electrophiles, test articles with high pH), while other chemicals can directly colour the tissue or the cells. Such test sample properties can only interfere if sufficient amounts of the chemical are still present on the tissue at the end of the exposure period. In these cases, a special procedure allowing the quantification of the “true” MTT reduction should be applied. A protocol for the determination of possible interactions with MTT is provided in References [20], [21], [42] and [45]. The use of specific and adapted controls enables the calculation of true tissue viability after subtracting the unspecific optical densities (OD) due to direct chemical MTT reduction or chemical residual colour extracted from the tissues, or both.

 

6.3.1, second paragraph

Add the following sentence at the end of the paragraph:

Two new additional RhE models, KeraSkin™ 3) and LabCyte EPI-MODEL24 4) were evaluated in a same method as the international interlaboratory study[42][43][44]. The specific protocol is available as supplementary material[42][45].

Add the following footnotes:

3)   KeraSkin™ is a trademark of a product supplied by Biosolution Co. Ltd. (Seoul, Korea). This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results.

4)   LabCyte EPI-MODEL24 is a product supplied by Japan Tissue Engineering Co. Ltd. (Aichi, Japan). This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results.

 

6.3.1, third paragraph

Replace the first sentence with the following:

These RhE models have been validated by European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM) for determining skin irritation of chemicals and are included in OECD 439 and EU Guideline B.46[41].

 

6.4.2, NOTE

Replace NOTE with the following:

  NOTE In the interlaboratory studies the incubation time was (18 ± 1) h for EpiDerm™ tissues (EPI-200), (24 ± 2) h for the SkinEthic™ RHE model, (18 ± 1) h for KeraSkin™ RhE and (18 ± 1) h for LabCyte EPI-MODEL24. However, all models except LabCyte EPI-MODEL24 show similar outcomes at either 18 h or 24 h exposure [13][19][17][42].

 

6.6.3, first indent

Replace first sentence with the following:

  Apply (100 ± 2) μl of the undiluted test samples (i.e. medical device extracts), VCs, NC or PC on the surface of three single tissues each.

 

6.6.3, NOTE 2

Number all three NOTES in this subclause and replace NOTE 2 with the following:

  NOTE 2 In the interlaboratory studies, the incubation time was (18 ± 1) h for EpiDerm™ tissues EPI-200 model and (24 ± 2) h for the SkinEthic™ RHE model, (18 ± 1) h for KeraSkin™ RhE and (18 ± 1) h for LabCyte EPI-MODEL24.

 

6.7.1, after the second indent

Add the following NOTE:

  NOTE For KeraSkin™ RhE, 200 µl of a MTT solution (0,5 mg/ml) can be used to prefill the plate then add 100 µl of the MTT solution inside insert[42].

 

6.7.2, first indent

Replace the list item with the following:

 

— Transfer per tissue 1 to 3 × 200 µl aliquots (which equals to 1 to 3 wells per tissue) from each test well into a 96-well flat bottom microtitre plate (labelled appropriately) depending on the instructions of the RhE supplier. A single 96-well plate shall be used per measurement.

 

6.7.2, second indent

Number all NOTES in this subclause and add NOTE 3, as follows:

  NOTE 3 In the case of LabCyte EPI-MODEL24, reading of the OD can be done also at 650 nm as indicated in Reference [45].

 

6.10, second paragraph

Replace the paragraph with the following:

For a medical device or medical device component extracted with both polar (e.g. saline) and non-polar (e.g. sesame oil) solvents, if at least one of the extracts shows a positive effect on the tissues (viability ≤ 50 % and SD ≤ 20 %) the device or the device component tested shall be considered to have irritant activity (see Table 1). If the result is non-irritant (viability of more than 50 %) with the two solvents, the device or the device component shall be considered a non-irritant.

 

Annex C, PLATE CONFIGURATION FOR READING

Replace the first paragraph with the following:

Record the positions of samples on 96-well plate. Strictly adhere to the fixed plate design as indicated in the instructions of the manufacturer. Examples of a plate layout as used in the medical device interlaboratory studies for irritation testing are presented in Table C.5, Table C.6[5], Table C.7[42] and Table C.8[43][44].

 

Annex C, Table C.6 key

Replace “SkinEthic RHE” in Table C.6 key with “SkinEthicTM RHE”.

 

Annex C, PLATE CONFIGURATION FOR READING

Add the following new Tables C.7 and C.8:

Table C.7
KeraSkinTM RhE model
  1 2 3 4 5 6 7 8 9 10 11 12  
A bckg bckg bckg empty empty empty empty empty empty empty empty empty  
B NC PC VC1 V1T1 V1T2 V1T3 V1T4 V1T5 V1T6 V1T7 V1T8 V1T9 Tissue1
C NC PC VC1 V1T1 V1T2 V1T3 V1T4 V1T5 V1T6 V1T7 V1T8 V1T9 Tissue2
D NC PC VC1 V1T1 V1T2 V1T3 V1T4 V1T5 V1T6 V1T7 V1T8 V1T9 Tissue3
E empty empty VC2 V2T1 V2T2 V2T3 V2T4 V2T5 V2T6 V2T7 V2T8 V2T9 Tissue1
F empty empty VC2 V2T1 V2T2 V2T3 V2T4 V2T5 V2T6 V2T7 V2T8 V2T9 Tissue2
G empty empty VC2 V2T1 V2T2 V2T3 V2T4 V2T5 V2T6 V2T7 V2T8 V2T9 Tissue3

bckg, isopropanol solution for background measurement; NC, negative control [DPBS]; PC, positive control [1 % SDS saline]; VC, vehicle control background [e.g. saline (VC1) and sesame oil (VC2)]; V1T1 … V2T9, test samples 1 to 9 in VC1 or VC2. For the KeraSkinTM RhE model the OD is determined in singulo (1 × 200 µl) according to the manufacturer.

Table C.8
LabCyte EPI-MODEL24 RhE model
  1 2 3 4 5 6 7 8 9 10 11 12  
A bckg bckg bckg bckg bckg bckg empty empty empty empty empty empty  
B NC PC VC1 VC2 V1T1 V1T2 V1T3 V1T4 V2T1 V2T2 V2T3 V2T4 Tissue1
C NC PC VC1 VC2 V1T1 V1T2 V1T3 V1T4 V2T1 V2T2 V2T3 V2T4 Tissue1
D NC PC VC1 VC2 V1T1 V1T2 V1T3 V1T4 V2T1 V2T2 V2T3 V2T4 Tissue2
E NC PC VC1 VC2 V1T1 V1T2 V1T3 V1T4 V2T1 V2T2 V2T3 V2T4 Tissue2
F NC PC VC1 VC2 V1T1 V1T2 V1T3 V1T4 V2T1 V2T2 V2T3 V2T4 Tissue3
G NC PC VC1 VC2 V1T1 V1T2 V1T3 V1T4 V2T1 V2T2 V2T3 V2T4 Tissue3

bckg, isopropanol solution for background measurement; NC, negative control [DPBS]; PC, positive control [1 % SDS saline]; VC, vehicle control background [e.g. saline (VC1) and sesame oil (VC2)]; V1T1 … V2T4, test samples 1 to 4 in VC1 or VC2. For the LabCyte EPI-MODEL24 RhE model the OD is determined in duplo (2 × 200 µl) according to the manufacturer.

ANNEX C, Check the wave length

In the first sentence, replace “Table C.7” with “Table C.9”.

Replace the previous Table C.7 with:

Table C.9
Photometer setting
Wavelength (550 nm to 570 nm) 570 nm  
Reference wavelength

(when using the LabCyte EPI-MODEL24)

 650 nm  
   ID/Date:

 

ANNEX C, CHARACTERIZATION OF TEST SAMPLES (see Table C.8)

Replace “(see Table C.8)” by “(see Table C.10)”.

Renumber Table C.8 as Table C.10.

 

Bibliography

Replace Reference [21] with the following:

[21] Method to Detect Skin Irritation of Medical Device Extracts using Reconstructed human Epidermis (RhE). 2023. MK-24-007-0121. Available at, https://www.mattek.com/wp-content/uploads/EPI-200-SIT-Medical-Devices-Testing-Protocol-MK-24-007-0121_08_28_2023.pdf

 

Add the following references at the end of the Bibliography:

[42] Kim J, Kang NH, Kim JS, Kim IS, Kim Y. Round Robin Study for Evaluation an in vitro skin irritation test for medical device extracts using KeraSkinTM in Korea. Food and Chemical Toxicology. 2024 Oct;192:114942. doi: 10.1016/j.fct.2024.114942.

[43] Kato R., Miyajima A., Komoriya K., Haishima Y., Novel cytokine marker available for skin irritation testing of medical devices using reconstructed human epidermis models. Toxicol. In vitro. 2020, 68, DOI: 10.1016/j.tiv.2020.104919

[44] Round robin study report to evaluate LabCyte EPI-MODEL24 SIT as an in vitro irritation test for detection of irritant activity in medical device extracts,2024, available at https://www.jacvam.jp/files/list/02/02_04_LabCyte_EPI-MODEL24_RRS_Report.pdf

[45] SOP of LabCyte EPI-MODEL24 Skin Irritation test method (LabCyte EPI-MODEL24 SIT) for medical device. J-TEC, Aichi (Japan), available at https://jacvam.jp/files/list/02/02_04_LabCyteEM_MDSIT_SOP.pdf