While some animal testing may, in special circumstances, continue to have a need for the determination of safety in pharmaceuticals, strides are being made in replacing many animal tests with laboratory tests. One such area where progress has been made, and where further action is required, is with the case of the general test for toxic products. While this test is described differently in different compendia, it has the same aim: to determine if mice and guinea pigs die once administered with a pharmaceutical product. Such is the crude nature of the test; it can be argued that if this doubt exists the manufacturer of the product should not be in the business of making medicines in the first place.
The test in question is the Biological Reactivity Test (also referred to as: General Safety Test, Abnormal Toxicity Test, or the Innocuity Test) is a test for the detection of extraneous toxic contaminants which is sometimes undertaken for biological products intended for administration to humans. This article sets out the case for its final elimination.
HISTORY OF THE TEST
The test was first introduced at during the early part of the twentieth century, at a time when pharmaceutical products were less controlled than they are now. The test was developed in 1906 due to the presence of phenol in some medicinal products (1, 2). This animal test was developed to ensure the safe and consistent production of serum products, for example, to titrate the preservative level of diphtheria antiserum (3). It was later expanded to a general ‘safety’ test to detect extraneous contaminants (other than, for example, bacterial endotoxins) in biological products. In 1940 it was established that no reliable conclusions could be drawn from abnormal toxicity testing. Furthermore, the test is variable, non-reproducible (in part from variations to animal body weight, species and strain differences, plus the stress levels of the animals) and non-specific (4). In addition, the test lacks explicit acceptance criteria, as no definite endpoint is defined. The test not significantly changed since around 1940. The principle of the test consists of injecting batches of the product into guinea pigs and/or mice. A batch passes the test if no animal shows any signs of illness, relevant body weight changes, or dies within a defined time frame.
The test is outlined in the Code of Federal Regulations:
- Title 21 – Food and Drugs – Chapter 21 – Food and Drug Administration, Department of Health and Human Services, Subchapter F – Biologics, Part 610: General Biological Products and Standards, Subpart B – general provisions. Sec. 610.11 General safety.
The test is also described in the United States Pharmacopeia:
- Chapter <88> Biological Reactivity Tests, in vivo
And by the World Health Organization (innocuity test in WHO nomenclature):
- World Health Organization (1990) Expert Committee on Biological Standardization, Fortieth Report. Technical Report Series, Vol. 800 (Annex 2). WHO, Geneva
A similar test was, until recently, described in the European Pharmacopeia:
- Chapter 2.9.6 Abnormal Toxicity
The requirements for the test are generally similar, and involve (5):
- The test is conducted on a representative sample of the product in the final container from each lot of the product.
- The test uses animals: two healthy guinea pigs weighing less than 400 grams each and two mice weighing less than 22 grams. The animals must not have been used previously for any test purpose.
- Each test animal is weighed, and the individual weights recorded immediately prior to injection and on the last day of the test.
- With liquid products an intraperitoneal injection of 0.5 millilitre of the product is administered into two mice, and 5.0 millilitres of the liquid product or the reconstituted product into two guinea pigs.
- The duration of the test is 7 days for both species.
- The test is considered a pass if all animals live, and where they do not exhibit any response which is not specific for or expected from the product and which may indicate a difference in its quality. Moreover, the test animals must weigh no less at the end of the test period than at the time of injection.
- If an animal dies, or shows an adverse reaction the test may be repeated. A second repeat is permitted provided that no more that 50% of the test animals do not die as a result of the first repeat test.
The differences between the tests are not of significance. The primary differences are due to starting weights of the animals and the permitted weight gain / weight loss, where weight loss is indicative of an unhealthy animal that might be reacting to the product or to another factor, such as illness or as a stress response (studies of weight loss of laboratory animals have assessed the primary reason as physical and physiologic indicators of dehydration) (6). The concern here is that a weight loss could invalidate the test. Other differences are with the number of animals used for repeat tests and with the number of repeat tests that are permitted. With overweight guinea pigs are prone to diseases (such as atherosclerotic lesions) and hence an overweight guinea pig may die of a cause unrelated to the product under test, yet the reason of death may not be elucidated from any post-test autopsy (7). Overweight and obesity related conditions in terms of weight are dependent upon the age of the animals) (8, 9). This challenge is met by animal acclimatization, conditioning, and diet (10).
The European pharmacopeial (Ph. Eur.) test is no longer current for any pharmaceutical. In 2018, during its 159th plenary session, the European Pharmacopoeia Commission endorsed the complete suppression of the test for abnormal toxicity from the European Pharmacopoeia (Ph. Eur.) (11). As part of this exercise, 49 monographs revised to remove the test for abnormal toxicity were adopted by the Commission; notably, these included 36 monographs on vaccines for human use. In addition, as the general chapter Abnormal Toxicity (2.6.9) will no longer be referenced in any monograph, it was subsequently be rendered obsolete and will also be deleted from the Ph. Eur.The decision formed part of Ph. Eur. Commission’s commitment to the reduction of animal use wherever possible in pharmacopoeia testing, in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The decision to suppress the test for abnormal toxicity at the 159th session of the Ph. Eur. Commission is a strong illustration of this commitment.
This position is in keeping with that of the World Health Organisation, where the WHO Expert Committee on Biological Harmonisation has indicated: “noted that, in one region of the world [Europe], the abnormal toxicity test had been deleted for most products. This was linked to the implementation of, and compliance with, good manufacturing practices and, where this occurred, there was abundant evidence that the abnormal toxicity test did not provide additional assurances of the quality of the product.” (12)
U.S. Lags Behind
While the U.S. has made progress in suspending the test for biologics, it has not eliminated the test completely. Here, there is no case for continuing to have the test appear within the pages of the United States Pharmacopeia. Although the test is long established, the test has limited value as a quality control test; it is variable, non-reproducible and non-specific (13). Due to advances in the production of medicines, the test has gradually been phased out in Europe. In 1997, the requirement to run the test for blood products was removed for all products distributed within Europe.
The most significant change in the U.S. occurred in 2015, when the Federal Register was updated stating the requirement for conducting the General Safety Test could be removed for biologics, provided the BLA is revised and accepted by FDA. This was as per:
FDA (2015) Revocation of General Safety Test Regulations That Are Duplicative of Requirements in Biologics License Applications, Federal Register, Vol. 80, No. 127, p37971 (14).
Here the Register states:
“The Food and Drug Administration (FDA) is proposing to amend the biologics regulations by removing the general safety test (GST) requirements for biological products. FDA is proposing this action because the existing codified GST regulations are duplicative of requirements that are also specified in biologics licenses, or are no longer necessary or appropriate to help ensure the safety, purity, and potency of licensed biological products. FDA is taking this action as part of its retrospective review of its regulations to promote improvement and innovation, in response to an Executive order.”
A proposal can be made to discontinue the test where it is “no longer necessary or appropriate to help ensure the safety, purity, and potency of licensed biological products.”
For remaining products, the test serves little value and it should be possible for a pharmaceutical manufacturer to establish a review of potential risks of toxicity and to put in place appropriate controls and alternative tests.
In summary, this is based on an assessment of the following primary risks, these are:
|Contamination risk||Measure to verify absence of the contaminant|
In-process bioburden testing
End-product endotoxin in place
|Leachables and extractables (15)||Plastics having product contact are assessed|
|Residual contaminants||Assessed through product shelf-life studies|
|Other contamination events||Avoided through validated processes and use of modern manufacturing GMPs.
Shown not to occur.
It should the case that of the above is a low risk. Pharmaceutical compounds are tested extensively with regards to their safety/toxicity profile. With regards to processing, abnormal product contamination is extremely unlikely if the validated manufacturing process is followed. In the event that anything untoward happened during manufacturing, there should be in place a series of product-specific tests: physical, chemical, and biological that would alert the Quality Department of any potential problems. For example, appropriate analytical methods, as defined in product specifications, are capable of detecting contamination and ensure batch-to-batch consistency. Any concerns with specific toxins would be assessed using in vitro or in chimico tests.
In addition, controls need to be in place to minimise the risk of bioburden contaminating the products as they are manufactured. A summary of the process steps and the points of bioburden monitoring are outlined below. This takes the form of key manufacturing steps against the microbiological risk. This is undertaken through individual process assessment. In summary, such assessments show:
|Process step||The manufacturing flow diagram, and consideration of the key process steps. This assesses the risk of any additional chemical, microbiological, or particle contamination.|
|Room||The cleanroom in which each key process step takes place are identified. These rooms are under environmental control.|
|Equipment||The key pieces of equipment associated with each key process step and in each cleanroom have been identified. These items have been qualified.|
|Potential failure modes, causes and controls||
From the examination of teach activity, the following questions are asked, primarily in terms of microbiological, chemical or particulate contamination:
Failure modes: what could go wrong and where?;
Causes: where contamination could potential arise what would be the key cause(s)?;
Where a possible failure or root cause of contamination existed, what control measures were in place?
|Microbiological samples||Where a failure mode exists and no adequate controls are in place, or there was a poor sample history, a microbiological sample is taken and trend analysis takes place.|
The risk should be based on:
- The probability of occurrence
- The severity of an over-action count
- Probability of detection
Using probability of occurrence and severity, a qualitative assessment has been made based on the table below. An assessment using the table requires two questions to be answered:
- What is the likelihood / probability of contamination occurring and being passed through the process?
- What is the impact / severity on the process if the process stage code was contaminated?
With sterile products, there will be additional measures in place where final products are filled under controlled conditions, within an ISO 14644 class 5 / EU GMP Grade A environment. The environment is continuously monitored for inert particles and viable counts. This monitoring would indicate if there was a risk of any microorganisms being transferred by air or surface contact into the product. Products not conforming to acceptable environmental standards would not be released. In addition, all finished batches will be examined for:
- Sterility, using the pharmacopeia test for sterility. The acceptance criteria is no microbial growth.
- Bacterial endotoxin, using the Limulus amebocyte lysate (LAL) method. The acceptance criteria is <0.5 EU/mL.
In addition to the test for bacterial endotoxin, depyrogenation controls (such as for glassware) need to be in place as part of the production process.
This article makes the case for not operating the General Safety Test / Abnormal Toxicity Test as part of the final product release. This is based on:
- The test being non-specific and of limited value. More robust control is achieved through in-process bioburden and other quality controls through the manufacturing process.
- The absence of any customer complaints that have concluded toxicity as the root cause.
- The final product release tests that check for contamination. For sterile products, these will be based around sterility and endotoxin.
- The changes in approach by pharmacopeia (Europe) and FDA (through the Federal Register), which indicate that the test is not required provided that sufficient control measures are in place.
In addition, there is also the consideration of animal welfare. Based on the substantial number of laboratory animals used for this test, the test cannot be justified in view of its unproven and questionable suitability to detect contaminants and increase the product safety. This is especially so in light of other control measures in place.
Therefore, because appropriate quality control measures are in place and because GMP manufacturing rules are adhered to prevent any risk of contamination, contaminants of the nature that would cause a General Safety Test failure are appropriately controlled by complying with the validated manufacturing process and the batch release test to confirm batch-to-batch consistency.
- Otto R. 1906. The state control of immunosera. In Work from the Royal Institute for Experimental Therapy. In Frankfurt M; Ehrlich P.(Eds). Vol. 2. Jena, Germany: Gustav Fischer
- Marxer A. 1915. Technology of vaccines and immunosera. Braunschweig, Germany: Friedr. Vieweg & Sohn
- Schwanig, M., M. Nagel, K. Duchow, & B. Kraemer (1997) Elimination of abnormal toxicity test for sera and certain vaccines in the European Pharmacopoeia. Vaccine 15
- Kraemer B, Nagel M, Duchow K, Schwanig M, Cussler K. 1996. Is the abnormal toxicity test still relevant for the safety of vaccines, sera and immunoglobulins? ALTEX 13(1):7–16
- Sandle, T. (2016) Calling time on the General Safety Test, GMP Review 14(4):11-13
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- World Health Organization (2002) Expert Committee on Biological Standardization, Fortieth Report. Technical Report Series, Vol. 904. WHO, Geneva
- Kraemer, B., M. Nagel, K. Duchow, M. Schwanig & K. Cussler (1996) Is the abnormal toxicity test still relevant for the safety of vaccines, sera and immunoglobulins? ALTEX 13 (1); Duchow, K. & B. Kramer (1994) Abnormal toxicity - a relevant safety test under GLP- and GMP-conditions in the production of vaccines? ALTEX 11 (5).
- For the update see: Federal Register / Vol. 80, No. 127 / Thursday, July 2, 2015 / Rules and Regulations - http://www.gpo.gov/fdsys/pkg/FR-2015-07-02/pdf/2015-16206.pdf
- This followed a proposal made in Volume 79, Number 163, pages 49727-49731. See: http://www.gpo.gov/fdsys/pkg/FR-2014-08-22/html/2014-19888.htm
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