Device FAQs

Q: What are the differences between the latest version of ISO 10993-5(2009) and 10993-1999?

A: The main change is in section 8.5.1, which states that quantitative evaluation is preferable and qualitative means are appropriate for screening purposes. Qualitative means represent the traditional MEM elution on Agar. This version of the guideline gives four examples of quantitative tests: Neutral Red Uptake NRU, Colony formation assay, MTT, and XTT. The overall process is similar, with the cells being exposed to the test article extract and with an evaluation of the cytotoxicity after 24 to 72 hours.  The colony formation assay is similar to the classic Japanese cytotoxicity on V79. The main difference between the traditional MEM and these quantitative assays reside in the scoring method. The viability of the cells is judged via the capacity of uptaking neutral red or reducing MTT (or XTT), while the traditional MEM was relying on microscopic observation of morphologic changes with a rough estimate of the proportion of cells affected. With the use of an optical density measurement, these new tests allow to obtain an unbiased value associated with the viability of the cells. The guideline defines 70 percent as the minimum viability after exposure to the test article to be considered non-cytotoxic. The guideline confirms the preference for using serum-supplemented medium as extraction vehicle, at 37C for 24 hours (the 37°C for 72 hours is not an option). The traditional MEM is still a valid test, fully described in this current version of the guideline, but recommended for screening purposes. As part of a biocompatibility battery of test for regulatory submission, Nelson Labs Europe is recommending the MTT cytotoxocity test.  For non-GLP and not for submission tests, an MEM is probably still fine.

 

Q: How does the change of cytotoxicity testing guideline affect the studies already performed using the MEM Elution test?

A:The FDA and other notifying bodies are the entities ultimately deciding on the validity of the tests submitted. The MEM elution test has been considered an accepted standard for cytotoxicity evaluation for 30 years and has proven its reliability. It is still described in ISO_10993-5 and in the US Pharmacopeia.  As such it is a valuable test. The FDA may however decide to follow the new recommendation of ISO 10993-5.2009 and require a quantitative cytotoxicity evaluation.  According to the category of the medical device and the grade of the MEM Elution test, a Sponsor may want to consider repeating the test with a quantitative mean, or take the risk of submitting the MEM Elution test results.

 

Q: I could use some guidance on the extraction media.  I have the same questions on historical data for the positive and negative controls as in the marrow test.  The significance level is not shown for the evaluation of the results.

 

A: We recommend choosing 1 vehicle from each group

 

Extract Group 1       

1.  Fischer’s Cell Culture Medium (Serum-Free)       

2.  USP 0.9% Sodium Chloride for Injection (NaCl)

3.  Sponsor-specified medium

 

Extract Group 2

1. Polyethylene Glycol (PEG)

2. Dimethyl Sulfoxide (DMSO)

3.  Ethanol (EtOH)

4.  Sponsor-specified medium

 

The group 1 is a list of polar vehicle. The group 2 is a list of alternative vehicle. We cannot use a purely non-polar vehicle like oil; as it would not mix properly with the cell culture medium that is necessary for the cells. For the group 1, the Fischer’s medium can be dosed at 100 percent, but cannot be heated to more than 50°C. NaCl can go up to 70°C, but will be dosed only at 50 percent. From the group 2, the most classic is DMSO if the test article can support it. Ethanol and PEG are alternatives.

The historical data are not necessary as we run the negative control for each study.  However, we have the historical data as we run this test quite frequently and have been doing so for years.

The number of cells with aberration obtained after exposure to the test article is compared to the negative control. If there is no statistically significant difference, the test article is considered non- genotoxic.

 

Q: A material caused a statistically significant increase in C3a level as compared to the negative control and the untreated control C3a levels yet passed the overall test. Why?

 

A: The criteria for a test article to fail are if it fails both C3a and SC5b-9. C5 is downstream of the complement activation cascade; an actual activation of C3 would lead to an activation of C5 and therefore an increase in concentration of SC5b-9. Real complement activation would result in the concomitant activation of C3 and C5. An increase of C3a, not associated with an increase of C5b, can potentially be attributed to some non-specific interference on the test system by the test article or its extract.

Q: Does CH50 complement Activation test add some info compared to only C3a and sC5b-9?

 

A: We have compared functional and immunochemical measurements of complement components with assays measuring the generation of activation fragments, for the assessment of classical pathway activation in vitro and in vivo. The generation of the C3a, C3b/C3bi cleavage fragments of C3, and of the C4d cleavage fragment of C4 measured by ELISA and RIA, was correlated with the decrease in total complement hemolytic activity (CH50) and in functional activity of C3 and C4 in normal human serum in which the classical pathway had been activated with aggregated IgG. The decrease in CH50 in in vitro activated serum was also correlated with the generation of C5a and soluble SC5b-9 complexes. In contrast, no or little increase in the concentration of C3a, C3b/C3bi and C4d was observed in plasma samples from patients with low CH50 and with low levels of immunochemical C3 and C4, indicating that fragment quantitation assays provide no information on the presence and extent of complement activation in vivo. Analysis of samples from patients expressing the four C4 genes and patients having one or two C4 null alleles indicated that a ratio of hemolytic C4 to C2 > or = 1 was indicative of complement activation without C4 deficiency, whereas a ratio below 1 was indicative of C4 deficiency with or without classical pathway consumption. Classical pathway activation and C4 deficiency in clinical samples are best predicted by the concomitant assessment of immunochemical levels of C3 and C4 and hemolytic levels of C4, C2 and C3.

Q: What’s the difference between ASTM and “ISO” hemolysis?

 

A: The Autian ("ISO") hemolysis may not be enough to please the FDA fully. ISO 10993-4 cites ATM F756 as one potential guideline for performing hemolysis, but does not impose it.

Nelson Labs Europe offers 2 different tests aiming at analyzing the hemolytic potential of Medical Devices: “ASTM” and “ISO”. The method we refer to as “ISO” is based on the Autian guideline similar to the one referred as NIH.

Even though the outcome of the test following either of these two methods, is unlikely to be different, these are two different tests.  Our “ISO” hemolysis test does not guarantee a compatibility with the ASTM guideline. This test has however been submitted hundreds of times to the FDA with no problem. We just recently started to hear that the agency may favor the ASTM method. We would now recommend that hemolysis studies intended to be submitted to the FDA, be performed according to ASTM standard.

ASTM hemolysis is better than what we call "ISO.” We do not need to do both. ASTM is actually the method recommended by ISO 10993-4.

 

Parameters

Autian

ASTM

Vehicle

NaCl

PBS

Species

Rabbit or Human

Rabbit

Number of Donors

1

3

Incubation time

1

3

Reference

Positive = 100%

Standard curve of hemoglobin

Method

Direct spectrophotometric absorbance of supernatant

Conversion of all isoforms of hemoglobin to methemoglobin using Drabkin reagent

Direct or Indirect

Direct, Indirect, or both

Direct, Indirect, or both

Ratio

Direct Contact: 5g/10mL or 0.5g/10mL (if floats)

Indirect: ISO 10993-12

Direct or Indirect: 3cm2/mL or 0.2-05g/mL. We can also follow ISO 10993-12

 

Q: Has Nelson Labs Europe done testing on adhesives that cure in-situ following the latest ISO 10993-12 guidelines? 

A: Yes, we have done in-situ cured extractions.  Essentially, we will fill a test tube with 20 ml of vehicle and put in the adhesive and let it cure. The goal of this is to capture any products or monomers that may outgas if they are bench cured. The problem is some won't polymerize in a vehicle, some will.  It depends on what drives the polymerization.  As far as the direct tests, like an Agar Diffusion, implant or a toxicology study we can literally put it at the dosing site and let it polymerize and then proceed with the test.  Each situation is unique.  You may find that you have to test it both ways because of incomplete polymerizations in the vehicles.

 

Q: How can we test multiple products at the same time?

A: Option for testing multiple related compounds. (Example for 3 compounds differing only by their content of 2 components A and B.)

Option

total # of test

individual ratio

total ratio

Cost

Advantage

Inconvenient

Comments

A

1

2g of each version 10mL extract

0.6g/mL

1X

Cheap. Compliant at the individual level

may end up being positive (toxic)

 

B

3

2g of one version 10mL extract

0.2g/mL

3X

The cleanest way of testing.

expensive

 

C

1

0.67g of each version in 10mL extract (2g total in 10 mL)

0.2g/mL

1X

most likely to pass (non-toxic)

does not test appropriately the potential toxicity of any individual version

FDA may refuse it

D

2

2g of the material with the highest concentration of chemical A, and 2g of the material with the highest concentration of material B in separate 10mL extracts

0.2g/mL

2X

test appropriately the two most extreme versions.

Fairly cheap

twice the price.  The intermediate versions are not tested

The intermediate versions do not contain more of any components A and B than the extreme versions have.  Their absence of testing may be justified

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Nelson Labs Europe | Device FAQs