GM Free Cymru

BT Brinjal toxicology studies were "woefully inadequate"

BT BRINJAL Event EE1 The Scope and Adequacy of the GEAC Toxicological Risk Assessment Review of Oral Toxicity Studies in Rats

November 14, 2010 Dr Lou M Gallagher, PhD Wellington, New Zealand

See also: http://www.gmfreecymru.org/pivotal_papers/bt_brinjal.html http://www.testbiotech.de/sites/default/files/Report%20Gallagher_2011.pdf

SUMMARY

This evaluation of Bt brinjal studies is based on requirements for a rigorous evaluation of food safety for the people of India and their health. Departures from Indian and international published standards for the 14‐day and 90‐day studies are a cause for concern.

The current food safety studies for Bt brinjal were not conducted in accordance with published standards, did not accurately summarize results, and ignored toxic endpoints for rats fed Bt brinjal: in particular, rats fed Bt brinjal for 78 out of 90 days (only one dose level) experienced:

Major health problems among test animals were ignored in these reports. The single test dose used was lower than recommended by the Indian protocols. Release of Bt brinjal for human consumption cannot be recommended given the current evidence of toxicity to rats in just 90 days and the studies' serious departures from normal scientific standards.

Unanswered concerns regarding the safety assessment of Bt Brinjal

Neurological function, behavioral effects, reproductive performance and biological resilience of test animals were not evaluated in these studies. Further research based on properly conducted and supervised studies is needed to resolve indications that Bt brinjal may have adverse effects on these clinical endpoints.

Dietary equivalence of dried brinjal, dried Bt brinjal and control diets was not addressed. Concentrations of the new insecticide protein Cry1A(c) were not measured in dried brinjal powder. It is important to know how much of this new protein was actually in the dried samples fed to the rats, especially since there is data to suggest that Cry1A(c) is at least partially destroyed in laboratory heating conditions. That omission makes it impossible to compare the test diet with insecticide concentrations expected in cooked human food.

The use of laboratory animals to test food safety, although widely accepted as a toxicological tool, is only an indication of effects that might be expected from human exposure2. It is essential that deviations from standard protocols be evaluated carefully, as these changes can have a profound impact on results. Yet every departure made by INTOX (the laboratory contracted to do the research) from the Indian Department of Biotechnology protocol (1998) has resulted in lower standards being used, with less power to detect changes in rats eating Bt brinjal. These include: skipping important endpoints such as IgE measurement to test for allergenicity, testing only one dose that was lower than human consumption is likely to be, ignorance of toxicological equivalence, lost data, lack of Good Laboratory Practice standards, inadequate observation of animals, a 29% decrease in exposure days in one study (doses were administered 5 days per week instead of 7), etc.

Consequently, the studies submitted by Mahyco are woefully inadequate to determine the safety of Bt brinjal for long‐term human consumption.

((DETAILED ANALYSIS AND DISCUSSION OMITTED HERE -- PLEASE SEE PDF OF FULL REPORT))

CONCLUSIONS

A review of the adequacy of current toxicology studies to address the safety of genetically modified Bt brinjal for commercial release shows that the studies were not conducted according to the published standard, did not accurately summarize results, and ignored toxic endpoints for rats fed Bt brinjal.

For a brief period of time (1998 to 2001) there appeared to be an exemption given to genetically modified foods that showed no signs of toxicity: if a food tested at a dose of 1000 mg/kg‐day produced no toxic effects then further testing was not required. According to OECD 1998 (page 3, item 16) and the 1998 DBT protocol quoted here,

“If a test at one dose of at least 1000 mg/kg body weight (but expected human exposure may indicate the need for a higher dose level) using the procedures described for this study produces no observable toxic effects, then a full study using 3 dose levels may not be necessary.”

Although this apparent exemption is no longer part of GM testing protocol (WHO/FAO 2000, Codex Alimentarius 2003a‐c, EFSA 2008) the 90‐day toxicity study appeared to be conducted at the particular dose of 1000 mg/kg‐day with the expectation of finding no evidence of toxicity.

Were the contract laboratory INTOX PVT LTD and the funder Mahyco uncomfortable with results showing evident toxicity among rats fed Bt brinjal at 1000 mg/kg‐day? Did the researchers write the conclusions for the 14‐day and 90‐day studies themselves or did others write conclusions for them? These questions are of interest since the text does not match the data, the researchers did not sign their reports, and the cover page of the 90‐day report details a completely new report number (R/2183/SOR‐ 90) from that which may be the original, 05.0002.

Not only has the scrutiny of these data provided insight into the substandard and extremely misleading interpretation of results, but it suggests to the reviewer that urgent changes need to be made to ensure that future studies are properly conducted and interpreted.

In particular, current results from these rat feeding studies indicate that rats eating Bt brinjal experienced organ and system damage: ovaries at half their normal weight, enlarged spleens with white blood cell counts at 35 to 40 percent higher than normal (elevated eosinophils in particular) indicating immune function changes possibly due to allergen response, and toxic effects to the liver as demonstrated by elevated bilirubin along with plasma acetylcholinesterase.

Further studies are required to assess the potential outcomes of these indicators of toxicity.

Unanswered concerns regarding the safety assessment of Bt brinjal

Nutritional and toxicological equivalence of dried Bt brinjal samples

Are dried brinjal samples equivalent to cooked brinjal as it is prepared for human consumption, or do dried samples differ in their concentrations of Cry1A(c) and other important proteins, carbohydrate, fat and micronutrients? Would the toxicity profile of Bt brinjal also change as a result of cooking and home processing? Notably, Codex recommends:

“The potential effects of food processing, including home preparation, on foods derived from recombinant-DNA plants should also be considered. For example, alterations could occur in the heat stability of an endogenous toxicant or the bioavailability of an important nutrient after processing. Information should therefore be provided describing the processing conditions used in the production of a food ingredient from the plant. For example, in the case of vegetable oil, information should be provided on the extraction process and any subsequent refining steps.” paragraph 47

Dietary equivalence for brinjal‐fed rats, Bt brinjal‐fed rats and vehicle control rats was not addressed.

Inhalation exposure to Bt brinjal

Oral ingestion of Bt brinjal does not address the issue of inhalation exposures to people who grow Bt brinjal or live near Bt brinjal crops in the ground. Toxicological responses to proteins that reach the lining of the lungs and nasal cavity, previously found to be of concern for agricultural workers, have not been addressed.

Toxicity testing standards

The main reason for conducting the toxicology studies is to have an objective assessment of whether or not the new food is safe for humans to eat. This needs to be a careful and objective assessment since millions of people with varying nutritional status, age and biological resilience will be exposed in the event of commercial release.

Neither of the 90‐day toxicity testing protocols released by the Department of Biotechnology (1998 and 2008) are as methodologically strong as accepted international standards (see Appendix 1). This makes India an “easy target” for developers since the requirements to conduct toxicology studies are less stringent those in the European Union.

The use of laboratory animals to test food safety for humans is already a significant departure from species‐specific testing. Deviations and omissions from accepted protocols need to be checked. Yet every departure made by INTOX on behalf of Mahyco has resulted in lower standards with less power to detect changes in rats eating Bt brinjal. These include leaving out important endpoints such as IgE measurement to test for allergenicity, using only one dose group that is smaller than human consumption is likely to be, ignorance of toxicological equivalence, lost data, lack of Good Laboratory

Practices standards, inadequate observations of animals, a 29% decrease in exposure days (doses were administered 5 days per week instead of 7), failure to quantify Cry1A(c) concentrations in dried fruit powder, etc.

The real risk here is that potential health problems attributable to Bt brinjal will be ignored as masses of people eat the very food their government thought was safe. In the long run, it is the people of India who could pay the price for bad science!

Further research studies

The logical next steps for describing the risk profile of Bt brinjal are to:

• Meet international published standards for conducting tests with scientific rigor in independent testing, following Good Laboratory Practice described in Council Directive 2004/10/EC (EC 2004) with quality assurance checks.

• Conduct dietary equivalence tests with quantitative measurement of Cry1A(c) protein before and after processing for administration to test animals according to ILSI 2004 and 2007 and Section 4 of EFSA 2006 to ensure that: nutritional requirements of animals are met equally between dose groups and that concentrations of Cry1A(c) and other proteins at the time of administration to animals are accurately represented..

• Conduct proper exposure assessment prior to laboratory studies so that future doses of Bt brinjal reflect the maximum exposure expected in the human population: 1000 mg/kg‐day is not enough for an upper limit.

• Conduct the 90‐day sub‐chronic feeding study according to OECD guidelines, following Codex Alimentarius (2003 a‐c) recommendations. That is, use at least three dose groups with doses given on all 90 days (not 5 days per week), include IgE measurements, perform daily observations on animals and include behavioral tests on individuals, include appropriate statistical analyses comparing the Bt brinjal group with appropriate controls and report results accordingly.

• Complete chronic (2‐year) rodent feeding studies and multigenerational studies as suggested by the European Food Safety Authority (2008) to assess reproductive performance, neurological function and behavioral effects.

• Reduced resilience in circumstances of infection or other adverse events needs to be addressed as a potential risk factor for Bt brinjal consumers.

The Bt brinjal EC II Report recommending the commercial approval of Bt brinjal cannot be upheld. Scientifically rigorous safety assessment is needed to dislodge a trust deficit (held by the Indian public) created by the EC II Report. Furthermore, adoption of and adherence to a stronger safety testing protocol in India than the current DBT standard from 2008 is prudent.

Oral ingestion of Bt brinjal does not address the issue of inhalation exposures to people who grow Bt brinjal or live near Bt brinjal crops in the ground. Toxicological responses to proteins that reach the lining of the lungs and nasal cavity, previously found to be of concern for agricultural workers, have not been addressed.

Toxicity testing standards

The main reason for conducting the toxicology studies is to have an objective assessment of whether or not the new food is safe for humans to eat. This needs to be a careful and objective assessment since millions of people with varying nutritional status, age and biological resilience will be exposed in the event of commercial release.

Neither of the 90‐day toxicity testing protocols released by the Department of Biotechnology (1998 and 2008) are as methodologically strong as accepted international standards (see Appendix 1). This makes India an “easy target” for developers since the requirements to conduct toxicology studies are less stringent those in the European Union.

The use of laboratory animals to test food safety for humans is already a significant departure from species‐specific testing. Deviations and omissions from accepted protocols need to be checked. Yet every departure made by INTOX on behalf of Mahyco has resulted in lower standards with less power to detect changes in rats eating Bt brinjal. These include leaving out important endpoints such as IgE measurement to test for allergenicity, using only one dose group that is smaller than human consumption is likely to be, ignorance of toxicological equivalence, lost data, lack of Good Laboratory Practices standards, inadequate observations of animals, a 29% decrease in exposure days (doses were administered 5 days per week instead of 7), failure to quantify Cry1A(c) concentrations in dried fruit powder, etc.

The real risk here is that potential health problems attributable to Bt brinjal will be ignored as masses of people eat the very food their government thought was safe. In the long run, it is the people of India who could pay the price for bad science.