Eriksson et al (2008): This was a population-based case-control study of exposure to pesticides as a risk factor for non-Hodgkin lymphoma (NHL), consisting of 910 cases and 1016 controls. The subjects were men and women aged 18 – 74 years living in Sweden, diagnosed with NHL between December 1999 and April 2002. All cases were diagnosed and classified histopathalogically according to WHO criteria. Controls were selected from the national population registry.
Exposure assessment was performed by a questionnaire which included work history, exposure to pesticides, organic solvents and several other (unidentified) chemicals. For dose-response analysis of pesticides, information was collected on the number of years, days per year and hours per day of exposure. The questionnaire also included smoking habit, medications, leisure activities and residential proximity to industrial installations, but data on these variables were not included in the review. Supplementary phone interviews were conducted if necessary. All exposures of less than a full day, or occurring during the same calendar year as the diagnosis or one year prior, were disregarded.
Data were analysed by unconditional logistic regression (univariate and multivariate) adjusted for age, sex and year of diagnosis or enrolment. In the univariate analysis, different pesticides were analysed separately, and the unexposed category consisted of subjects who were not exposed to any of the included pesticides. All controls were used in the analyses of NHL subgroups. In the dose-response calculations made for agents with at least 20 exposed subjects, the median number of days of exposure among controls was used as a cut-off. Latency period calculations and multivariate analyses (performed because most pesticide exposures involved more than one chemical) included agents with statistically significantly increased ORs, or with an OR >1.50 and at least 10 exposed subjects.
Univariate analysis adjusted for age, sex and year of diagnosis or enrolment revealed a significant association between NHL and exposure to glyphosate (29 cases and 18 controls; OR = 2.02; 95% CI = 1.10 – 3.71), exposure to glyphosate with a latency of >10 years before diagnosis (unstated no. of cases and controls; OR = 2.26; 95% CI = 1.16 – 4.40) and exposure to glyphosate for >10 days (17 cases and 9 controls; OR = 2.36; 95% CI = 1.04 – 5.37). However, NHL was not associated with exposure to glyphosate with a latency of 1 – 10 years before diagnosis (unstated no. of cases and controls; OR = 1.11; 95% CI = 0.24 – 5.08) or exposure to glyphosate for <10 days (12 cases and 9 controls; OR = 1.69; 95% CI = 0.70 – 4.07). Multivariate analysis adjusting for exposure to other chemicals yielded a low and statistically non-significant risk estimate for glyphosate (OR = 1.51; 95% CI = 0.77 – 2.94).
When the different sub-types of NHL were analysed separately, exposure to glyphosate was associated with a significantly enhanced risk of small lymphocytic lymphoma / chronic lymphocytic leukaemia (195 cases; OR = 3.35; 95% CI = 1.42 – 7.89) and unspecified NHL (38 cases; OR = 5.63; 95% CI = 1.44 – 22.0). Odds ratios for other types of lymphoma were not statistically significant.
The same research group have published a previous (Hardell et al, 2002) epidemiology study on the association between pesticide exposure and NHL, in which univariate analysis found a significant association with glyphosate (OR = 3.04; 95% CI = 1.08 – 8.52) based on 8 cases and 8 controls. Noting the small sample size and the broad CI, the Australian DoHA (2005) concluded that strength of association was questionable, and it was equivocal whether glyphosate was indeed a risk factor for NHL.
The current follow-up study improves on its predecessor in several respects, as it was based on a larger population (910 vs 515 cases), had larger sample sizes, included both men and women, and collected exposure data from living individuals only.21 The follow-up would therefore have increased statistical power and diminished recall bias. Compared with the 2002 study, the risk estimate was lower (OR of 2.02 vs 3.04) but the association between glyphosate exposure and NHL was strengthened, as evidenced by the narrower 95% CI (1.10 – 3.71 vs 1.08 – 8.52). However, the 2008 and 2002 studies failed to demonstrate associations by multivariate analysis, which yielded ORs of only 1.51 and 1.85, with 95% CIs that had lower bounds of less than 1.0 (0.77 – 2.94 and 0.55 – 6.20). Eriksson et al (2008) noted that many glyphosate users had previously been exposed to MCPA, and suggested this as an explanation for why neither chemical showed a significant OR when subjected to multivariate analysis.
At best, the association between glyphosate and NHL in this study is equivocal, remains potentially confounded by established risk factors such as immunosuppression and Epstein-Barr virus (as noted previously by the Australian DoHA, 2005), and could also have been affected by recall, exposure measurement and information bias if NHL cases or their interviewers believed that their disease may be related to pesticides (Mink, unpublished). Mink has also observed that, by excluding 88 potential cases who died before they could be interviewed, the study population did not represent those cases with more aggressive disease. Furthermore, the dose-response analysis may have been confounded by exposure to other herbicides, and was based on unequal cut-off points for glyphosate (<10 days or >10 days) and “other” herbicides (<32 days or >32 days) (Mink, unpublished).
APPENDIX 5: PHARMACOKINETICS OF GLYPHOSATE AND ITS METABOLITE AMPA IN RATS
Anadon et al (2009): Laboratory grade glyphosate (Sigma Chemical Co, St Louis, MO, USA; purity 95%) was administered to male Wistar rats (Charles River Inc, Margate, Kent, UK; bw 200 – 210 g) at 100 mg/kg bw IV (in 0.1 mL glycerol formal) or 400 mg/kg PO (gavage to fasted animals in 0.5 mL corn oil). Groups of 8 rats were killed and exsanguinated at 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h post-dosing, and the concentrations of glyphosate and aminomethyl phosphonic acid (AMPA) were measured in plasma by HPLC with fluorescence detection.
Glyphosate, IV administration: Following an initial peak concentration (Cmax) of 166 µg/mL plasma pharmacokinetics were biphasic, consistent with a two-compartment open model, with rapid distribution and gradual elimination. The volume of distribution at steady state was 2.99 L/kg, suggesting extensive diffusion into the tissues. Clearance was 0.995 L/h/kg. The elimination half-life from plasma was 9.99 h and the area under the concentration vs time curve (AUC) was 100 mg.h/L.
Glyphosate, PO administration: Absorption from the GIT was gradual, with a Cmax of 4.62 µg/mL occurring in plasma at 5.2 h. Oral bioavailability was poor (23.2%). Clearance was the same as following IV administration and the AUC was similar (at 93.3 mg.h/L), but the elimination half-life from plasma was appreciably more prolonged (14.4 h).
AMPA: The metabolite first appeared in plasma within 0.25 h of PO dosing, and had similar pharmacokinetic behaviour to glyphosate. The Cmax (0.42 µg/mL) occurred at 2.4 h. An AUC of 6.1 mg.h/L was attained, ca 6.5% of glyphosate’s AUC in plasma. The elimination half-life of 15.1 h was similar to that of the parent chemical after PO administration.
th Human health risk assessments are performed for the APVMA by the Department of Health and Ageing (DoHA).
2 According to the OECD (2008) Guidance Document on Mammalian Reproductive Toxicity Testing and Assessment, there is no generally accepted classification of malformations (permanent structural changes that may adversely affect survival, development or function) and variations (divergence beyond the usual range of structural constitution, which may not adversely affect survival or health). The nomenclature used by study laboratories and regulatory agencies may therefore vary, in part because there is a continuum between normal and abnormal development, because some observations are classified as malformations in one species and variations in another, or due to the use of different nomenclature conventions by different organisations. The highly authoritative DevTox website (http://www.DevTox.org), whose terminology and classification system was developed by a series of international harmonisation workshops, does not classify sternebral unossification as either a malformation or variation.
3 Besides identifying the effects of the test compound on animals, the major purpose of regulatory toxicology studies is to establish the doses at which the effects do or do not occur. This is most commonly done by comparing findings from groups of animals treated over a range of doses with those from an untreated group of the same species and genetic background, housed under the same conditions as the test groups. These untreated animals are usually referred to as “study” or “concurrent” controls. In addition to presenting data from the test groups and study controls, reports may also include “historical control” (HC) data from other studies performed in animals from the same supplier and genetic background at the same laboratory. HC mean values and ranges are sometimes used during evaluation to clarify the biological significance of differences between the study controls and groups of animals treated with the test compound. HC data can also provide information about whether a study control group’s results are atypical compared with those observed in other control groups. The use of HC data generated within a five-year span around the study under review is accepted internationally under the OECD (2008) guidelines.
4 POEA (also known as polyoxyethylene tallow amine and polyoxyethyleneamine; CAS Registry no. 61791-26-2) is a mixture of polyethoxylated long chain alkylamines synthesised from animal-derived fatty acids (Williams et al, 2000).
5 Fecundability is the probability that conception will occur in a given population of couples during a specific time interval.
6 Conditional fecundability is the probability of conception per unit time conditional on a woman being susceptible at the beginning of that time interval.
7 The ratio of conditional fecundability of the exposed and unexposed groups. A CFR <1.0 indicates a reduced probability of conception in the exposed group.
8 As described by Derelanko (2002), the development of a single cell into malignant tumours is believed to occur in three stages, the first of which is initiation (a normal cell changes irreversibly – usually by genetic alteration – in a way that allows unrestricted division; however, initiated cells may remain latent for months or years, during which they are indistinguishable from normal). The subsequent stage, promotion, involves prolonged and repeated exposure to a promoting agent which causes the initiated cell to undergo clonal expansion and form a pre-cancerous focus. Promoters, which do not interact directly with DNA, are believed to act via a variety of mechanisms most often resulting in increased cellular replication. The final step is progression, in which the pre-cancerous focus becomes transformed into a malignant tumour, a process characterised by changes in the number and arrangement of chromosomes, an increased rate of replication, and invasiveness.
9 Label Safety Directions for liquid glyphosate-based professional strength products require users to wear PPE including coveralls and gloves, consistent with recommendations in the Handbook of First Aid Instructions and Safety Directions (DoHA, 2012).
10 De Roos et al reduced the dataset from 32 to 22 MM cases by excluding subjects with missing data for several variables (Sorahan, 2012).
11 Pathology scores relating to the skeletal development of the hands and feet.
12 Kimmel et al do not report the numbers of foetuses Bhide and Patil (1989) examined at each dose, so the incidences of septal defects in all seven rabbit studies combined are unknown.
13 The discrepancy between the BVL’s conclusions for the EU and JMPR reviews occurred because the JMPR assigns No Observed Adverse Effect Levels to toxicology studies, as opposed to No Observed Effect Levels (as assigned by the EU and Australia). By JMPR criteria, the histological abnormalities in the salivary glands at 3000 ppm were not classified as an adverse effect.
-10 Cellular viability was not quantified, so it could not be confirmed that the “LD50” actually corresponded to the death of half the population of exposed cells.
14 HUVEC cells were chosen because in vivo, they form a permeable barrier between the blood and the underlying tissues and would be exposed directly to circulating chemicals, for which they may be a target.
15 Cellular viability was not quantified, so it could not be confirmed that the “LD50” actually corresponded to the death of half the population of exposed cells.
17 The doses are believed to have been based on glyphosate acid technical because Dallegrave et al stated that the dosing regimen was chosen by reference to a NOAEL for glyphosate of 1000 mg/kg bw/d for maternal and foetal effects in a developmental toxicity study in rats.
18 In Holson (1990), rat dams gavaged with POEA over GD 6 – 15 showed clinical signs and decreased food consumption at 100 mg /kg bw/d, together with mortality and decreased bodyweight gain at 300 mg/kg. However, there were no foetal effects at 300 mg/kg bw/d, the highest dose administered (Williams et al, 2012).
19 Endocrine disruptor screening program Test Guideline OPPTS 890.1500: Pubertal development and thyroid function in intact juvenile/peripubertal male rats. EPA 740-C-09-004, October 2009.
rd Endocrine disruptor screening program Test Guideline OPPTS 890.1500: Pubertal development and thyroid function in intact juvenile/peripubertal male rats. EPA 740-C-09-004, October 2009.
20 According to the study authors, Translation eF1A1 is responsible for binding aminoacyl-tRNA to ribosomes during polypeptide synthesis and its increased expression is directly proportional to cellular proliferation, oncogenic transformation, apoptosis and delayed cell senescence; Carbonic anhydrase III is involved in the cellular response to oxidative stress; VEGF is involved in angiogenesis (a pre-requisite for neoplastic growth); Stefin A3 plays a role in skin growth and its induction by TPA leads to keratinocyte differentiation and proliferation; Annexin II is up-regulated in several human cancers; Peroxyredoxin-2 is over-expressed in some cancers; and Calcyclin and Calgranulin-B are implicated in cell cycle progression, differentiation, cancer development and metastasis.
21 In Hardell et al (2002), the next-of-kin provided information for deceased individuals.