Safety and efficacy of a feed additive consisting of salinomycin sodium (Sacox®) for rabbits for fattening (Huvepharma N.V.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of the coccidiostat salinomycin sodium (Sacox®) for rabbits for fattening. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the use of salinomycin sodium (SAL-Na) from Sacox® does not raise safety concerns for the target species, consumers, users and the environment with regard to the production strain. In the absence of adequate tolerance studies, the FEEDAP Panel could not conclude on the safety of SAL-Na from Sacox® for rabbits for fattening. The FEEDAP Panel concluded that the additive is safe for the consumer when it is used at the proposed maximum level of 25 mg SAL-Na/kg complete feed for rabbits and a withdrawal period of 1 day is respected. The following maximum residue limits (MRL) are proposed for the marker residue compound salinomycin (SAL): 0.2 and 0.03 mg SAL/kg for liver and kidney, respectively. The additive is not irritant to skin and eyes but should be considered a potential dermal and respiratory sensitiser. A risk for inhalation toxicity could not be excluded. The use of the SAL-Na from Sacox® in feed for rabbits for fattening up to the highest proposed level will not pose a risk for the terrestrial and aquatic compartment and ground water. The risk of secondary poisoning can be excluded for worm-eating birds and mammals, while it cannot be excluded for fish-eating birds and mammals. The FEEDAP Panel concludes that SAL-Na from Sacox® at the minimum concentration of 20 mg SAL-Na/kg complete feed has the potential to control coccidiosis in rabbits for fattening. Development of resistance to SAL-Na of field Eimeria spp. strains isolated from rabbits for fattening should be monitored.

Safety and efficacy of a feed additive consisting of monensin sodium (Coxidin®) for chickens for fattening, chickens reared for laying, turkeys for fattening and turkeys reared for breeding (Huvepharma N.V.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of monensin sodium (Coxidin®) as a coccidiostat for chickens for fattening, chickens reared for laying, turkeys for fattening and turkeys reared for breeding. The additive currently on the market complies with the existing conditions of authorisation. The FEEDAP Panel concluded that Coxidin® remains safe for turkeys for fattening (up to 16 weeks) and extends this conclusion to turkeys reared for breeding (up to 16 weeks). The Panel was not in the position to confirm that the current maximum authorised level of 125 mg monensin sodium/kg complete feed remains safe for chickens for fattening and chickens reared for laying. The use of monensin sodium from Coxidin® at the corresponding maximum authorised/proposed use levels in the target species is safe for the consumer. The existing maximum residue levels (MRLs) for poultry tissues ensure consumer safety. No withdrawal time is necessary. Both formulations of Coxidin® pose a risk by inhalation. The formulation with wheat bran as a carrier was neither irritant to the skin nor a skin sensitiser but it was irritant to the eyes. In the absence of data, no conclusions could be made on the potential of the formulation containing calcium carbonate to be irritant to skin and eyes and to be a skin sensitiser. The use of monensin sodium from Coxidin® in complete feed for the target species poses no risk for the terrestrial compartments and for sediment. No risk for groundwater is expected. For chickens for fattening the risk for aquatic compartment cannot be excluded, but no risks are expected for the other animal categories. There is no risk of secondary poisoning. Coxidin® is efficacious in controlling coccidiosis at a level of 100 mg/kg complete feed for chickens for fattening and at 60 mg/kg complete feed for turkeys for fattening. These conclusions are extended to chickens reared for laying and turkeys reared for breeding. The Panel noted that there are signs of development of resistance of Eimeria spp. to monensin sodium.

Safety and efficacy of a feed additive consisting of narasin (Monteban® G100) for chickens for fattening (Elanco GmbH).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of the coccidiostat narasin (Monteban® G100) for chickens for fattening. In a previous opinion, uncertainties remained on the identification and characterisation of the non-genetically modified production strain of the active substance narasin. The Panel could not conclude either on the safety of Monteban® G100 for chickens for fattening or on the efficacy of the additive at the minimum applied concentration. The FEEDAP Panel excluded risks for environment but the risk for sediment compartment could not be assessed. The applicant provided supplementary information to cover the data gaps and substituted the narasin production strain from Streptomyces spp. NRRL 8092 to Streptomyces spp. NRRL B-67771. The information submitted to taxonomically identify the production strain did not allow to assign it to any described microbial species. Based on the information provided, the Panel concluded that the use of Monteban® G100 did not raise safety concerns as regards the production strain for the target animal, consumer, user and environment. The Panel concluded that 70 mg narasin/kg complete feed was safe for chickens for fattening with a margin of safety of 1.4; narasin from Monteban® G100 was unlikely to increase shedding of Salmonella Enteritidis, Salmonella Typhimurium and Campylobacter jejuni. Narasin, when used in chickens for fattening at 70 mg/kg feed, was not expected to pose a risk to the aquatic compartment and to sediment, while a risk for the terrestrial compartment could not be excluded. No risk for groundwater was expected, nor for secondary poisoning via the terrestrial food chain, but the risk of secondary poisoning via the aquatic food chain could not be excluded. The Panel concluded that 60 mg narasin/kg feed was efficacious in controlling coccidiosis in chickens for fattening.

Guidance for the assessment of detoxification processes in feed.

This statement provides scientific guidance on the information needed to support the risk assessment of the detoxification processes applied to products intended for animal feed in line with the acceptability criteria of the Commission Regulation (EU) 2015/786.

Safety and efficacy of a feed additive consisting of benzoic acid (Kalama® Animal Feed Grade Benzoic acid) for weaned piglets and pigs for fattening (Emerald Kalama Chemical, B.V).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of benzoic acid (Kalama®) as a zootechnical feed additive for weaned piglets at a level of 5000 mg/kg complete feed and for pigs for fattening at a minimum content of 5000 mg/kg and a maximum content of 10,000 mg/kg complete feed. The FEEDAP Panel concluded that benzoic acid is safe for weaned piglets at 5000 mg/kg complete feed and for pigs for fattening at 10,000 mg/kg complete feed. The Panel considered the use of benzoic acid under the proposed conditions of use to be of no concern for consumer safety and the environment. Benzoic acid poses a risk by inhalation, it is irritant to skin and corrosive to eyes, but no conclusions can be drawn on dermal sensitisation. The additive, benzoic acid, is efficacious as a zootechnical feed additive for weaned piglets and for pigs for fattening at the proposed conditions of use.

Efficacy of Availa®Cr (chromium chelate of dl-methionine) as a feed additive for dairy cows (Zinpro Animal Nutrition (Europe), Inc).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the efficacy of Availa®Cr (active compound: chromium chelate of dl-methionine) as a feed additive for dairy cows. In a previous opinion, the FEEDAP Panel concluded that Availa®Cr at a maximum recommended use level of 0.5 mg Cr(III) (8 mg Cr from Availa®Cr/cow per day) was safe for dairy cows and the consumers. Additionally, the FEEDAP Panel considered that the additives posed a risk to the user by inhalation, it was not irritant to skin and eyes, and it should be considered a skin sensitiser. The Panel could not conclude on the efficacy of the additive at the proposed conditions of use. Since the new information provided in the current application is lacking sufficient evidence, the FEEDAP Panel is still not in the position to conclude on the efficacy of chromium dl-methionine from Availa®Cr.

Safety and efficacy of a feed additive consisting of phenylcapsaicin (aXiphen) for chickens for fattening (aXichem AB).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of phenylcapsaicin (aXiphen®) as a zootechnical additive (functional group: physiological condition stabilisers) in feed for chickens for fattening. The additive under assessment, phenylcapsaicin, is safe for chickens for fattening up to the maximum proposed use level of 15 mg/kg complete feed. A margin of safety could not be established. Phenylcapsaicin is not genotoxic. The reference point for phenylcapsaicin derived from a 90-day repeated dose oral toxicity study in rats is 37.2 mg/kg body weight (bw) per day, the lowest of the model averaged BMDL20 values for alanine aminotransferase increase in plasma. The metabolic similarity in the laboratory animals and the target species was not demonstrated and the identity of the marker residue could not be established. In the absence of such data, the safety for the consumers could not be evaluated. The inhalation exposure of phenylcapsaicin (as liquid) for the user was considered unlikely. The FEEDAP Panel considered the additive irritant to the eyes but not to the skin and it is not a dermal sensitiser. In the absence of appropriate data, the environmental risk assessment for phenylcapsaicin could not be performed. It is unlikely that phenylcapsaicin bioaccumulates in the environment and the risk of secondary poisoning is considered low. The FEEDAP Panel could not conclude on the efficacy of the additive in chickens for fattening at the proposed conditions of use.

Assessment of the feed additive consisting of orthophosphoric acid for all animal species for the renewal of its authorisation (Prayon S.A.; Chemische Fabrik Budenheim KG; BK Giulini GmbH).

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the application for renewal of orthophosphoric acid as a technological additive (functional group: preservatives) for all animal species. The applicant has provided evidence that the additive currently on the market complies with the existing conditions of authorisation. There is no evidence that would lead the FEEDAP Panel to reconsider its previous conclusions. Thus, the Panel concluded that the additive remains safe for all animal species provided that the optimal Ca:P ratio is maintained. Additionally, the FEEDAP Panel concluded that orthophosphoric acid remains safe for the consumer and the environment under the authorised conditions of use. Regarding the user safety, orthophosphoric acid is corrosive to the skin and eyes and should be considered as hazardous to the respiratory tract. There is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

Safety and efficacy of a feed additive consisting of monensin sodium (Elancoban® G200) for chickens for fattening, chickens reared for laying and turkeys (Elanco GmbH).

Following a request from the European Commission, EFSA was asked to deliver a new scientific opinion on the coccidiostat monensin sodium (Elancoban® G200) when used as a feed additive for chickens for fattening and turkeys. Based on the new data provided, the Panel updates its previous conclusions as follows: monensin sodium is produced by fermentation by a non-genetically modified strain of Streptomyces sp. NRRL B-67924. Genome analysis suggests the production strain may belong to a new species within the genus Streptomyces. The production strain and its DNA were not detected in the final additive. The product is free of antimicrobial activity other than monensin. The FEEDAP Panel cannot conclude on the safety of monensin sodium from Elancoban® G200 in feed for chickens for fattening and chickens reared for laying at the proposed maximum use level due to a dose-related reduction of the final body weight. The toxicological profile of monensin sodium was evaluated in studies made with the product obtained from the parental strain ATCC 15413. Based on a comparison of the genomes of the two strains, the FEEDAP Panel concludes that toxicological equivalence has been established, thus the conclusions already drawn on Elancoban® G200 are valid for the product obtained with the new production strain concluding that the additive is safe for the consumer and the environment; the production strain does not represent an additional risk when safety for the user is considered. Monensin sodium from Elancoban® G200 is safe for turkeys up to 16 weeks of age at the concentration of 100 mg monensin sodium/kg feed and has the potential to control coccidiosis at the minimum concentration of 60 mg/kg complete feed.

Efficacy of a feed additive consisting of Bacillus velezensisNITE BP-01844 (BA-KING®) for chickens for fattening, chickens reared for laying, turkeys for fattening, turkeys reared for breeding and all avian species for fattening, or rearing to slaughter or point of lay including non-food producing species (Toa Biopharma Co., Ltd.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the efficacy of BA-KING® Bacillus velezensis as a zootechnical feed additive to be used as a gut flora stabiliser for chickens for fattening, chickens reared for laying, turkeys for fattening, turkeys reared for breeding and all avian species for fattening or rearing to slaughter or point of lay including non-food producing species. The product under assessment is based on viable spores of a strain identified as B. velezensis, which is considered suitable for the qualified presumption of safety (QPS) approach to safety assessment. In a previous opinion, the FEEDAP Panel concluded that BA-KING® was safe for the target species, consumers of products derived from animals fed the additive and the environment. Additionally, the additive was not irritant to skin but potentially irritant to eyes and respiratory sensitiser. The Panel could not conclude on the efficacy of the additive for the target species at the proposed conditions of use. In the current application, two additional efficacy trials in chickens for fattening were provided. The results showed an improvement in the performance parameters of chickens when supplemented with BA-KING® at 2.0 × 108 CFU/kg complete feed relative to a control group. Considering the previously submitted studies and the newly submitted studies in chickens for fattening, the Panel concluded that BA-KING®, supplemented at 2.0 × 108 CFU/kg complete feed, has the potential to be efficacious in all avian species for fattening or reared for laying/breeding and non-food-producing avian species at the same physiological stage.

Safety and efficacy of a feed additive consisting of Saccharomyces cerevisiae DBVPG 48 SF (BioCell®) for horses, pigs and ruminants (Mazzoleni S.p.A.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of Saccharomyces cerevisiae DBVPG 48 SF (BioCell®) as a zootechnical feed additive for horses, pigs and ruminants. The product, manufactured in three formulations (microsphere, micropellet and powder), is intended for use in complete feed at a minimum inclusion level of 3 × 109 CFU/kg complete feed for horses, 4 × 108 CFU/kg complete feed for dairy cows and minor dairy species, 4 × 109 CFU/kg complete feed for calves, cattle for fattening, minor growing and fattening ruminants, piglets and pigs for fattening and minor porcine species and 6 × 109 CFU/kg complete feed for sows and minor porcine species for reproduction. Saccharomyces cerevisiae is considered by EFSA to be suitable for the qualified presumption of safety approach to safety assessment. The identity of the strain was conclusively established and, therefore, the use of the additive in animal nutrition is considered safe for the target species, the consumer and the environment. The additive, in any formulation, is not irritant to the eyes and skin but should be considered a respiratory sensitiser. The Panel cannot conclude on the skin sensitisation potential of the additive. The Panel concluded that the additive has the potential to be efficacious at the proposed conditions of use for horses, dairy ruminants and all pigs. However, the Panel was not in the position to conclude on the efficacy of the additive for calves, and neither for cattle for fattening, minor growing and fattening ruminants.

Safety and efficacy of a feed additive consisting of halofuginone hydrobromide (STENOROL®) for chickens for fattening and turkeys for fattening/reared for breeding (Huvepharma N.V.).

Following a request from the European Commission, EFSA was asked to deliver a new scientific opinion on the coccidiostat halofuginone hydrobromide (STENOROL®) when used as a feed additive for chickens for fattening and turkeys for fattening/reared for breeding. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concludes that the safety for turkeys for fattening established in its previous opinion can be extended to turkeys for breeding up to 12 weeks of age. Based on the new data provided on the safety for consumer, environment and efficacy, the Panel updates its previous conclusions as follows: halofuginone hydrobromide is not genotoxic. Applying an uncertainty factor of 100 to the lowest no observed adverse effect level (NOAEL) of 0.03 mg/kg body weight (bw) per day, an acceptable daily intake (ADI) of 0.3 µg halofuginone/kg bw is established. The chronic exposure of consumers to residues of halofuginone would amount to 6-19% of the ADI after 3 days of withdrawal. Therefore, the Panel considers that the additive is safe for the consumer of tissues obtained from chickens for fattening and turkeys for fattening fed the additive at a maximum level of 3 mg/kg complete feed at a 3-day withdrawal time. For control purposes, the Panel recommends the setting of the following maximum residue limits (MRLs): liver, 50 µg/kg; kidney, 40 µg/kg; muscle, 3 µg/kg; skin/fat, 10 µg/kg wet tissue. Based on an updated environmental risk assessment, no concern for groundwater is expected. Halofuginone is unlikely to bioaccumulate and the risk of secondary poisoning is not likely to occur. No safety concerns are expected for terrestrial and aquatic environments. The additive has the potential to control coccidiosis in chickens for fattening and turkeys for fattening/reared for breeding up to 12 weeks of age at a minimum level of 2 mg/kg complete feed.

Assessment of the feed additive consisting of ammonium chloride (Amoklor™) for all ruminants, dogs and cats for the renewal of its authorisation and its extension of use to sows (Latochema Co Ltd).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the assessment of the application for the renewal of authorisation of ammonium chloride (Amoklor™) as a zootechnical additive for all ruminants, dogs and cats and its extension of use in sows for urinary health. The applicant provided evidence that the additive currently in the market complies with the existing conditions of authorisation. There is no new evidence that would lead the FEEDAP Panel to reconsider its previous conclusions. Thus, the Panel concludes that the additive remains safe for ruminants other than lambs for fattening, lambs for fattening, cats and dogs, consumers and the environment under the current authorised conditions of use. Inhalation exposure of the additive is considered very likely. Amoklor™ should be considered a potential respiratory sensitiser but not a skin sensitiser. The additive is not irritant to the skin, but the Panel could not conclude on its eye irritation potential. The present application for renewal of the authorisation does not include a proposal for amending or supplementing the conditions of the original authorisation that would have an impact on the efficacy of the additive. Therefore, there was no need for assessing the efficacy of the additive in the context of the renewal of the authorisation. Regarding the extension of use in sows, the FEEDAP Panel concludes that the additive is safe and efficacious for sows at the inclusion level of 5,000 mg/kg feedingstuffs from week 9th to 11th of gestation and from week 15th of gestation to 1st of lactation.

Safety and efficacy of a feed additive consisting of lasalocid A sodium (Avatec® 150G) for chickens for fattening and chickens reared for laying (Zoetis Belgium SA).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of the coccidiostat lasalocid A sodium (Avatec® 150G) for chickens for fattening and chickens reared for laying. Taking into account the results of all tolerance studies submitted for the re-evaluation of the additive, the FEEDAP Panel considers that lasalocid A sodium from Avatec® 150G is safe at a maximum content of 90 mg/kg complete feed for chickens for fattening. A margin of safety cannot be established. No conclusion on the safety of lasalocid for chickens reared for laying can be made. Three new floor pen studies showed efficacy of 90 mg lasalocid A sodium/kg complete feed reducing the adverse clinical consequences of an Eimeria infection in chickens for fattening. Considering also the previously reported positive floor pen study and the three positive anticoccidial sensitivity tests, the FEEDAP Panel can conclude on an efficient coccidiostatic level of 90 mg lasalocid A sodium/kg complete feed for chickens for fattening. This conclusion is extended to chickens reared for laying.

Safety of a feed additive consisting of halofuginone hydrobromide (STENOROL®) for chickens for fattening and turkeys (Huvepharma N.V.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety for the target species of the coccidiostat halofuginone hydrobromide from STENOROL® when used as a feed additive for chickens for fattening and turkeys. In its previous assessment, the FEEDAP Panel could not conclude on the safety of STENOROL® for the target species at the highest proposed use level of 3 mg halofuginone hydrobromide/kg complete feed. On the basis of the new data provided, the FEEDAP Panel updates its previous conclusions on the safety for the target species as follows: halofuginone hydrobromide from STENOROL® is safe for chickens for fattening and for turkeys up to a maximum of 12 weeks of age at the highest proposed concentration of 3 mg/kg complete feed. For chickens for fattening, a margin of safety of about 1.3 can be established while for turkeys for fattening a margin of safety cannot be established.

Metabolomic Analysis of Wooden Breast Myopathy Shows a Disturbed Lipid Metabolism.

Myopathies have risen strongly in recent years, likely linked to selection for appetite. For white striping (WS), causes have been identified; but for wooden breast (WB), the cause remains speculative. We used metabolomics to study the breast muscle of 51 birds that were scored for both at 35 days of age to better understand potential causes. A partial least square discriminant analysis revealed that WS and WB had distinct metabolic profiles, implying different etiologies. Arginine and proline metabolism were affected in both, although differently: WB increased arginine in breast muscle implying that the birds did not use this pathway to increase tissue blood flow. Antioxidant defenses were impeded as shown by low anserine and beta-alanine. In contrast, GSH and selenium concentrations were increased. Serine, linked to anti-inflammatory properties, was increased. Taurine, which can stabilize the cell's sarcolemma as well as modulate potassium channels and cellular calcium homeostasis, was also increased. Mineral data and depressed phosphatidylethanolamine, cAMP, and creatine-phosphate suggested compromised energy metabolism. WB also had drastically lower diet-derived lipids, suggesting compromised lipid digestion. In conclusion, WB may be caused by impaired lipid digestion triggered by a very high appetite: the ensuing deficiencies may well impair blood flow into muscle resulting in irreparable damage.

Application of Meta-Analysis and Machine Learning Methods to the Prediction of Methane Production from In Vitro Mixed Ruminal Micro-Organism Fermentation.

In vitro gas production systems are utilized to screen feed ingredients for inclusion in ruminant diets. However, not all in vitro systems are set up to measure methane (CH4) production, nor do all publications report in vitro CH4. Therefore, the objective of this study was to develop models to predict in vitro CH4 production from total gas and volatile fatty acid (VFA) production data and to identify the major drivers of CH4 production in these systems. Meta-analysis and machine learning (ML) methodologies were applied to a database of 354 data points from 11 studies to predict CH4 production from total gas production, apparent DM digestibility (DMD), final pH, feed type (forage or concentrate), and acetate, propionate, butyrate and valerate production. Model evaluation was performed on an internal dataset of 107 data points. Meta-analysis results indicate that equations containing DMD, total VFA production, propionate, feed type and valerate resulted in best predictability of CH4 on the internal evaluation dataset. The ML models far exceeded the predictability achieved using meta-analysis, but further evaluation on an external database would be required to assess generalization ability on unrelated data. Between the ML methodologies assessed, artificial neural networks and support vector regression resulted in very similar predictability, but differed in fitting, as assessed by behaviour analysis. The models developed can be utilized to estimate CH4 emissions in vitro.

Unraveling the cause of white striping in broilers using metabolomics.

White striping (WS) is a major problem affecting the broiler industry. Fillets affected by this myopathy present pathologies that compromise the quality of the meat, and most importantly, make the fillets more prone to rejection by the consumer. The exact etiology is still unknown, which is why a metabolomics analysis was performed on breast samples of broilers. The overall objective was to identify biological pathways involved in the pathogenesis of WS. The analysis was performed on a total of 51 muscle samples and distinction was made between normal (n = 19), moderately affected (n = 24) and severely affected (n = 8) breast fillets. Samples were analyzed using gas chromatographic mass spectral analysis and liquid chromatography quadrupole time-of-flight mass spectrometry. Data were subsequently standardized, normalized and analyzed using various multivariate statistical procedures. Metabolomics allowed for the identification of several pathways that were altered in white striped breast fillets. The tricarboxylic acid cycle exhibited opposing directionalities. This is described in literature as the backflux and enables the TCA cycle to produce high-energy phosphates through matrix-level phosphorylation and, therefore, produce energy under conditions of hypoxia. Mitochondrial fatty acid oxidation was limited due to disturbances in especially cis-5-14:1 carnitine (log2 FC of 2, P < 0.01). Because of this, accumulation of harmful fatty acids took place, especially long-chain ones, which damages cell structures. Conversion of arginine to citrulline increased presumably to produce nitric oxide, which enhances blood flow under conditions of hypoxia. Nitric oxide however also increases oxidative damage. Increases in taurine (log2 FC of 1.2, P < 0.05) suggests stabilization of the sarcolemma under hypoxic conditions. Lastly, organic osmolytes (sorbitol, taurine, and alanine) increased (P < 0.05) in severely affected birds; likely this disrupts cell volume maintenance. Based on the results of this study, hypoxia was the most likely cause/initiator of WS in broilers. We speculate that birds suffering from WS have a vascular support system in muscle that is borderline adequate to support growth, but triggers like activity results in local hypoxia that damages tissue.

Inhibition of verocytotoxigenic Escherichia coli in model broth and rumen systems by carvacrol and thymol.

The antimicrobial activities of thymol and carvacrol were assessed against a selection of verocytotoxigenic Escherichia coli (VTEC) strains (n=11) and other bacterial species and spoilage bacteria (n=7) using a model broth system. The effects of pH, temperature, water activity, sodium chloride concentrations, inoculum size and the presence of competing microflora on the activities of thymol and carvacrol against E. coli O157:H7 strain 380-94 were also determined. The minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) and numbers of surviving E. coli O157:H7 were determined following incubation. The mean numbers of VTEC surviving exposure to thymol or carvacrol at concentrations of >/=500mug/ml were between 2.0 and 7.8log cfu/ml less than the numbers in the corresponding controls. The susceptibility of E. coli O157:H7 to carvacrol or thymol was found to increase with decreasing storage temperature, water activity, pH and E. coli O157:H7 inoculum size. Sodium chloride (0.5-2.5%) and the presence of a microflora cocktail did not significantly (p>0.05) affect the antimicrobial activities of thymol or carvacrol against E. coli O157:H7. The antimicrobial activity of carvacrol against E. coli O157:H7 was also tested in a model rumen system. A MIC of 500mug/ml carvacrol reduced E. coli O157:H7 inoculated at levels of 10(3) and 10(6)cfu/ml to undetectable levels in the system after 24h incubation. This concentration of carvacrol significantly (p<0.05) decreased the total gas production and volatile fatty acid concentrations in the model rumen assay.