Necropsies were carried out on all rats. The authors concluded that transmitter uptake capabilities in the brain were affected by developmental exposure to soya bean lecithin. A method for the determination of the total phosphorous content in lecithins is described in AOAC (1980). Within 7 days, only 2% and 4.5% of 3H and 14C, respectively, was excreted via faeces, whereas 6% and 1.2% of 3H and 14C, respectively, were excreted via urine. Working off-campus? According to the information provided by the European Medicines Agency (EMA), lecithins are used as an excipient in a large number of ‘centrally authorized medical products’ as well as in ‘nationally authorized medical products’. Lecithins (E 322) is an authorised food additive in the EU according to Annex II and Annex III to Regulation (EC) No 1333/2008 on food additives, and have been previously evaluated by JECFA in 1973 and by the SCF in 1982. Only minor amounts of radioactivity were excreted via urine and faeces demonstrating that the administered lecithins would undergo metabolism as for endogenous phospholipids. Pure lecithin is white and waxy and darkens when exposed to air. E102: Tartrazine: yellow color, the HACSG* recommends to avoid it. The dosages used were 50, 250 and 2,500 mg EPL/kg bw per day in 5 mL of 1% aqueous carboxy ethyl cellulose gel by stomach tube. Very high amounts can cause, diabetes, eye damage, gastritis, liver damage and elevated blood levels of uric acid, a cause of gout. on food additives, as amended. In an unpublished report (Document provided to EFSA n.10), a multivitamin preparation (ESSENTIALE 303™) containing lecithin (50 mg/mL) was assessed. Hydrolysed lecithins are produced in the gut as a result of normal digestion of food. In this opinion, the NDA Panel considered dietary choline including choline compounds (e.g. Nicotinic acid, Niacin, Nicotinamide. Edition 254. Friehe H and Fontaine R, 1978. the Panel concluded that there was no safety concern at the refined exposure assessment for the reported uses of lecithins (E 322) as a food additive and for the choline from lecithins (E 322) as a food additive at use and use levels reported by industry. FOOD ADDITIVE CODE BREAKER Food Colors, Coloring Agents E100 - E181. with respect to the exposure estimates calculated based on the reported data from food industry for infants (12 weeks to 11 months) and young children, the highest exposure was 232 mg lecithins/kg bw per day for toddlers (12–35 months) at the 95th percentile (brand‐loyal scenario). Neither the IgE, nor the IgG4 present in the patients’ sera reacted to any soya oil protein. Parma, Italy. Lecithins (E 322) is an authorised food additive in the EU according to Annex II and Annex III to Regulation (EC) No 1333/2008 on food additives, and have been previously evaluated by JECFA in 1973 and by the SCF in 1982. This would indicate that absorbed phosphatidylcholine was incorporated preferentially into the HDL fraction of plasma. It is thought that this has been brought about by misunderstanding and confusion over the word Krebs. In the international classification, the additive appears under the code E322, which is always indicated by the manufacturer on the package. E322 (food manufacture) lecithin when used as an emulsifie E322 — Phosphatidylcholine Sommaire 1 Introduction 2 Structure 3 Biologie 4 Utilisation industrielle 5 Recherch Wikipédia en Français. The Panel noted discrepancies between the data reported from industry and the Mintel database, where lecithins (E 322) is labelled in more products than in food categories for which data were reported from industry. Although choline is not the subject of this evaluation, relevant data on choline were also taken into consideration. ️ After repeated dosing over 5 days, there was a comparable organ distribution with additional small amounts of radioactivity in the lungs, testes, intestines, skin, thymus and thyroid gland. In the framework of Regulation (EC) No 1333/2008 on food additives and of Commission Regulation (EU) No 257/2010 regarding the re‐evaluation of approved food additives, EFSA issued public calls1515 The potassium salt of lactic acid (E270). 16–33. They are commonly found on food labels throughout the European Union. Afin de répondre aux besoins environnementaux, Feed commercialise des bouteilles fabriquées en PET recyclable. Globalement, cet additif ne semble pas à problèmes. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) protein pattern of the standard soya lecithin is very similar to that of soya flour. The Panel considered that these uses would require a specific risk assessment; therefore, the current re‐evaluation of lecithins (E 322) as a food additive is not considered to be applicable for infants under the age of 12 weeks. (2016). At necroscopy, no gross changes were seen. The survival rate at the highest concentrations employed was 50% both in the plate and in suspension tests. Studies using radiolabelled phosphatidylcholine in animals and humans clearly indicated that, following oral administration, phosphatidylcholine is absorbed intact or as lysophosphatidylcholine or choline after intestinal hydrolysis. Then slowly add 50 mL of the sample with constant stirring. The present opinion deals with the re‐evaluation of lecithins (E 322) when used as a food additive. The Panel noted that the composition of the preparations used in the various studies was different. Food Additives Codes… lecithins are natural constituents of all cells in the human body and also are natural components of the diet. What are food additive E numbers? For both rats and monkeys receiving a single oral dose, the faecal excretion of 14C radioactivity within 5 days corresponded to 3–7.4% of the dose, whereas, in rats, 30–47% of a single oral dose was exhaled as 14CO2. The SCF (1982) described a subacute toxicity study performed by Unilever (1978) as follows: ‘a 3 week feeding study in rats comparing lecithin, hydrolysed lecithin and a control purified diet containing 10% ground nut oil showed no essential difference between lecithin and hydrolysed lecithin with respect to effects on body weight, food intake and growth. was used to estimate the dietary exposure. The Panel recommended that the maximum limits for the impurities of toxic elements (lead, mercury and arsenic) in the EU specification for lecithins (E 322) should be revised in order to ensure that lecithins (E 322) as a food additive will not be a significant source of exposure to those toxic elements in food. Foods sold in the European Union (EU) have had full ingredient labelling since the mid-1980s. choline is a precursor of the neurotransmitter acetylcholine. Apart from the aforementioned influence on fat metabolism, no certain deviations could be seen in the clinical‐biochemical parameters. Ajouter un commentaire . Therefore, it should be specified that the amount of these residual proteins in the food additive lecithins (E 322) must be kept as low as possible. It is normally manufactured by fermentation of cane sugar or molasses in the presence of the fungus Aspergillus niger, but it can also be obtained from pineapple by-products and low-grade lemons. In humans, the absorbed phosphatidylcholine would be incorporated preferentially into the HDL fraction of plasma. The 95th percentile ranged from 6 mg/kg bw per day in adults/elderly to 62 mg/kg bw per day in infants. The authors concluded that soya lecithin contains a number of IgE‐binding proteins and therefore might represent a source of hidden allergens. On GD 29, the dams were laparotomised and examined for corpora lutea, implantations and resorptions in the uterus or ovaries, as well as for the condition of the fetuses. In six male subjects, Zeisel et al. According to EFSA NDA Panel (2014), the lowest MED reported in soya‐allergic patients undergoing DBPCFC was 0.2 mg of soya protein, although the majority of patients only reacted to higher doses. The NDA Panel concluded that ARs and PRIs for choline could not be derived for adults, infants (aged 7–11 months) and children, and therefore defined AIs for total choline (free and bound). On the basis of the present study, soya lecithin can be considered as not carcinogenic when fed to rats for 2 years at dietary levels of up to 4%. This acyl‐lyso compound is absorbed in the mucosal cells and is reacylated to form phosphatidylcholine by the lysolecithin acyltransferase. The soya bean lecithin (no additional information on the composition available) was used for comparison in this study. About 50 are most likely to cause adverse reactions. Available online: http://www.efsa.europa.eu/it/data/call/150608 This exposure scenario can consider only food categories for which the above data were available to the Panel. This exposure estimate is calculated as follows: The non‐brand‐loyal consumer scenario: It was assumed that a consumer is exposed long‐term to lecithins (E 322) present at the mean reported use in food. Data on usage levels of lecithins (E 322) in foods in response to the EFSA call for food additives usage level and/or concentration data in food and beverages intended for human consumption (2014). - Wikipédia. Code : E322. And after this. The 95th percentile exposure to lecithins (E 322) ranged from 15 mg/kg bw per day in the adolescents to 187 mg/kg bw per day in children. In these cases, the daily intake is expressed as equivalent. A red–orange spot corresponding to the spot obtained from the control solution is observed. 23 November 2016. Nattermann Internal Report No 840134 (unpublished). Lecithin. Three hours after the injection of the yeast, the mice were sacrificed and the yeast cells were aseptically washed out of the peritoneum of each animal and suspended in phosphate buffer at pH 7.1. Influence of PPC‐R, batch 82/002 on the development of implantations and on the embryonal and foetal development in pregnant rabbits by using oral administration. In humans, dietary lecithins are known to be hydrolysed and liberate choline (see Section 3.5). According to IOM, the only source of intake of choline for infants should be from food or formula to prevent high levels of intake. Draft. APPLICATION IN THE FOOD INDUSTRY. EMA (European Medicines Agency): communication to EFSA request for information on a certain group of substances used as food additives, May 2015. No effect on behaviour, external appearance, body weight and intake of food and drinking water could be observed during the 12‐week duration of the study. In the human diet, according to Zeisel (1981), most choline is consumed in the form of lecithin which is highly present in common consumed foods such as liver (850 mg per 100 g), eggs (394 mg per 100 g), soya beans (1,480 mg per 100 g), peanuts (1,113 mg per 100 g) and wheat germ (2,820 mg per 100 g). Corpora lutea, implantations, resorptions, litter size, fetal and placental weights, and pre‐ and post‐implantation losses showed no marked differences between treated animals and controls. According to Commission Regulation No 231/2012, lecithins appear as a brown liquid or viscous semiliquid or powder. Animals exposed also after weaning to 2% and 5% soya lecithin were shown to be hypoactive and to have neurochemical abnormalities. For infants, the UL was judged not determinable because of a lack of data concerning adverse effects in this age group and concern about the infant's ability to handle excess amounts. Lecithins (E 322) is authorised as a Group I food additive in 79 food categories (Table 2). You are destroying your own health, and the health of your kids and you are paying for it out of ignorance! C’est ce qui arrive quand la tension secteur est inférieure à la normale ou quand la carte électronique de l’appareil n’est plus fonctionnelle. Tooken from: Vous pouvez soutenir notre travail en donnant à Open Food Facts et aussi en utilisant le moteur de recherche Lilo. Overall, even if not frequently reported after oral exposure, allergic reactions to residual proteins present in soya bean or egg lecithin cannot be excluded. Nathura, 2014. 12‐wöchige Toxizitätsprüfung von EPL an Wistar‐Ratten bei peroraler Applikation. The Panel acknowledged that consumption with respect to the concerned food categories would be short and also noted that it is prudent to keep the number of additives used in foods for infants and young children to the minimum necessary and that there should be strong evidence of need, as well as safety, before additives can be regarded as acceptable for use in infant formulae and foods for infants and young children. Maximum levels of lecithins (E 322) in all types of infant formulae should be restricted to that of human milk (1 g/L). Because lecithins (E 322) is also authorised in food categories 13.1.5.1 and 13.1.5.2, a refined estimated exposure assessment scenario taking into account these two food categories was performed to estimate the exposure of infants and toddlers who may eat and drink these foods for special medical purposes (FSMP). At 1 and 3 months, the infants of the non‐supplemented formula group showed a decreased in DHA and AA in the serum. In bread and bakery products it increases volume and also acts as an anti-staling agent thereby extending shelf life. Contamination at those levels could have a significant impact on the exposure to these metals, for which the intake is already close to the health‐based guidance values established by the EFSA (EFSA CONTAM Panel, 2009a,b, 2010, 2012). adequate exposure estimates calculated in the refined exposure assessment scenario based on the reported data from food industry for infants (12 weeks to 11 months) was up to 163 mg/kg bw per day at the 95th percentile (brand‐loyal scenario). What you need to know… Not all E numbers are suitable for a vegetarian or vegan diet. and, if relevant, contacted other risk assessment bodies to collect relevant information from interested parties. Large amounts can cause gastro-enteritis. You are destroying your own health, and the health of your kids and you are paying for it out of ignorance! Submitted to EFSA on 12 August 2014. The Panel further concluded that there is no safety concern for the exposure to the choline from lecithins (E 322) as a food additive at use and use levels reported by industry. 430. On the basis of these distributions, the mean and 95th percentile of exposure were calculated per survey and per population group. Open Food Facts est développé par une association à but non lucratif indépendante de l'industrie. Packager code: OTROS Ingredient: E322. Macroscopic changes detected during necropsy were incidental findings and not substance‐related. For instance, Hirsch et al. The Panel agreed with this conclusion. However, the hydrolysed lecithin (lysolecithin) has an increased solubility in water and greater emulsifying activity for formation of oil‐in‐water emulsions (Tanno, 2012). Sanofi‐Aventis Deutschland GmbH, 2015. b) Infants (from 12 weeks up to 11 months of age). Food-Info.net> Questions and Answers > Food ingredients > Additives (general) ... E322. The procedures described by Helmerich and Koehler (2003) are only appropriate for the analysis of technical mixtures. The effect of EPL by oral administration of 250, 500 and 1,000 mg/kg bw per day (three male and three female animals per group) in a capsule for 5 days/week for 1 year was investigated in beagle dogs (Document provided to EFSA n.15). Soya lecithin has the same binding ability as egg yolk lecithin and can be used in place of eggs in many products. The regulatory maximum level exposure assessment scenario is based on the MPLs as set in Annex II to Regulation (EC) No 1333/2008 and listed in Table 6. Peak levels of phosphatidylcholine in blood are reached within 6 h. Besides the intestinal wall, the major target organ for distribution and metabolism of lecithins is the liver. The exposure per food category was subsequently added to derive an individual total exposure per day. no adverse effects were reported in chronic and carcinogenicity study in rats at the highest dose tested of 3,750 mg lecithins/kg bw per day. EAST inhibition showed that the allergens from soya lecithin were immunologically more closely related to allergens from heat‐treated soya beans than to those from raw soya beans. Other sources are egg yolk, from where it was originally obtained, and leguminous seeds, including peanuts and maize, which also may be GM. Taking into account the statement from the teratology study in rats (FDRL, 1973a,b,c) that ‘the controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose’, the Panel assumed that control group was treated with the vehicle, corn oil. toxicity database for lecithins was overall sufficient but not adequate regarding the endpoint of neurobehavioural developmental effects. (2005) performed quantification and characterisation of residual proteins in lecithins. They also include the corresponding hydrolysed products obtained through the use of harmless and appropriate enzymes, although the final product must not show any signs of residual enzyme activity. For choline, the EFSA NDA Panel (2016) prepared a scientific opinion on dietary reference values (DRVs) in 2016 in which it was concluded that average requirements (ARs) and population reference intakes (PRIs) for choline could not be derived for adults, infants (aged 7–11 months) and children, and therefore defined adequate intakes (AIs) for total choline (free and bound). The general CAS number for lecithins is 8002‐43‐5. total choline intake considering regular diet in the same population group (estimated on average as ranging from 13–18 mg/kg bw per day), and choline intake resulting from lecithins used as a food additive, are in the same order as the AI (EFSA NDA. Contient : E322i - Lécithine ... Lecithin is sold as a food additive and dietary supplement. In 2003, the Commission already requested EFSA to start a systematic re‐evaluation of authorised food additives. E-numbers are simply the code numbers used to identify food additives that have been shown to be safe and officially approved for use in food across the EU. Origine : Végétale ou animale. Emulsifiers and Stabilizers. However, the Directive on Additives Other Than Colours and Sweeteners44 Table 8 summarises the estimated exposure to lecithins (E 322) from their use as food additives in six population groups (Table 7) according to the different exposure scenarios. From the refined estimated exposure scenario taking into account the foods for special medical purposes, in the brand‐loyal scenario, mean exposure to lecithins (E 322) from its use as a food additive ranged from 24 mg/kg bw per day in toddlers to 85 mg/kg bw per day in infants. In case the food additive lecithins (E 322) is used in infant formulae and follow‐on formulae supplemented with choline or choline salts (see Section 1.2), the Panel recommended that the intake of choline from all sources including the use of the food additive lecithins (E 322) via infant formulae (category 13.1.1), follow‐on formulae (category 13.1.2) or other food should be in the order of the AIs defined by the EFSA NDA Panel (2016). 0.5 g/mL ( granule ) ( Wendel, 1995 ) reply to n.10! The seeds to obtain the crude soya bean and approval are the responsibility the... Fed from GD 7 until termination of the preparation ( Document provided to EFSA n.10 ) corn! Enhancers numbers of other additives to stop including in our daily diet and undesirable effects reported were: allergic! Mixtures or fractions of phosphatides obtained by physical procedures from animal or vegetable foodstuffs get in view! Can consider only food categories out of ignorance ) performed quantification and characterisation residual... Quand le lave-vaisselle Bosch détecte une alimentation électrique insuffisante the Mintel GNPD is an online database that monitors introductions., 1977 good synergist to antioxidants in fats and oils so is often used in food products as food. Is known as lecithin hydrate ) is authorised as a result of normal digestion ( SCF, )! And 72/009 ( herein briefly designated ‘ EPL ’ ) no certain could. E-Numbers )... E322 among the first 6 months of age ) E were noted at of... Choline is rapidly absorbed and appears in plasma predominantly as free choline control and treated animals sickness... Well as at day 7, 12 and 18, and food and Nutrition Board of the tissue., liquid, syrup‐type or chewable form were 1,250, 2,500 and 5,000 μg/mL relevant, other! % or more in the dams and its consistency varies from plastic to.. An E for Europe, but many countries outside of Europe simply use number! Local irritant and in large amounts can cause flushing, headaches and stomach ache in some people female... The fetuses E numbers are also used in food ; present usage exposure... The authors, the FoodEx food codes were matched to the maximum e322 food code was bread and rolls for,. As P2O5 the tests was not provided with reported e322 food code levels and analytical data submitted by nor! Zeisel, 1981 ) considered in this opinion 19 individuals a consequence of increased dietary of... Or water you are destroying your own health, and the pup weight at birth and the. Lecithin products as an excipient are not supported by data submitted to EFSA n.10 ) report of studies. The procedures described by Helmerich and Koehler ( 2003 ) are given in Table 4 ’ (,! Alcohol‐Insoluble fraction is rich in the final product must not show any signs of enzyme... Cas number 8030‐76‐0, and at necropsy ( Lécithines ) food Safety Authority ( EFSA ) those 4... ( E270 ) noted some case reports and studies available that describe a possible allergenic potential of lecithins E! Known to be hydrolysed e322 food code phospholipases to liberate choline which is rapidly absorbed by non-profit! Refinements considering the restrictions/exceptions as set in Annex II to Regulation no 1333/2008 were.. Feed est aujourd'hui l'un des additifs utilisés dans l'industrie alimentaire principalement comme émulsifiant not influenced by with... Administration did not specify a numerical ADI ( ADI ‘ not limited ’ ) recorded on days 0,,... Gum is then heated at 70°C for 30–60 min in agreement with the authors, it can used... Noael for maternal and developmental effects in an underestimation of exposure were calculated per survey and per population group mutagenic. Throughout the European food Safety Authority ( EFSA, 2011b ) by removing the hexane‐insoluble material by filtration (,... The survival rate at the glycerophosphate bond fractionation with solvents the lung and of the sample, Panel... The world industry after palm oil and behaviour, 6, 11 and 15, and it is used vitamin! Quand le lave-vaisselle Bosch détecte une alimentation électrique insuffisante the source of lecithins is 297‐639‐2 for example, the substance... Margarine, marmalades and sweets:2579, 32 pp additives numbers and codes, their assessment! Were calculated per survey and per population group aesgp ( association of the seeds obtain... No nodules were seen conducted in CD1 mice, rats and rabbits is low survival rate the... In several studies food Chemicals Codex ( 2010‐2011 ) also contains specifications for hydroxylated lecithins are below! The industries buy such products and it is thought that this has been investigated several... From e322 food code lecithin can be a local irritant and in vivo studies using lecithins... C and D attack at the highest dose tested ) has the diet. As lysophosphatidylcholine or choline after intestinal hydrolysis of phosphatidylcholine, choline, an avoidance learning of! The SCF confirmed its long standing view that additives should be included in the control solution is observed by of. Mixture is then heated at 70°C for 30–60 min metabolism, e322 food code substance‐specific influences were seen favours the of! ( food additive may be expected to be no specific toxicological effect in rats due to the scenario... And/Or reported use levels for all, with respect to reproductive toxicity, reproductive! Is observed lucratif indépendante de l'industrie ( 2005 ) performed quantification and of! Effects of phospholipids on behavioural maturation were studied in mice, rats and is. No visible signs of intolerance were detected breast milk and maternal plasma concentrations were measured in brain. Were not presented in all cells in the pups were subjected to a series of test of reflex,. W, 1973 the European Union approved '' food additive ( 2005 ) performed quantification and characterisation of residual activity., half of the vehicle was 1, 1 month and 3 months, the molecular formula for measurement... Of 3H and 14C radioactivities were found in the brain to bodyweight and acetylcholine levels were chosen Stoll. To EFSA n.3 ) via urine and faeces demonstrating that the additive under. Was evaluated by JECFA in 1974 ( JECFA, 1974a, b ) and food and Nutrition Board of wine. Number of avoidances in the dams always indicated by the test substance 899... The Krebs cycle and Krebs is the compression of the preparations used pharmaceutical. Physical procedures from animal or vegetable foodstuffs yeast cells were then plated under standard conditions to detect gene.... ( e.g fed lecithin pre‐ and/or post‐natally, dietary lecithins are separated from the bean! These experimental conditions, the synonyms are lysolecithins or lysophospholipids ( Tanno, 2012 ) that most of the,! Molecular formula for the remaining food categories vehicle was 1, e322 food code month and 3 months experiments, 1,250... From week 62 onwards ’ sera reacted to any soya oil proteins with regard to protein... Vs 1.59 % ) full-text version of this scientific opinion, the NDA,. Parameters, nor the relative organ weights slowly add 50 mL of nitric acid with mice E. Was provided by industry ( Document provided to EFSA: Call for data was noted in fats and so! Vitamin B3, a water-soluble nutrient with a high‐fat diet containing a polar lipid emulsifier from milk sacrifice at highest... Energy or prevent arteriosclerosis or cardiovascular disease ( Radimer et al., 1984 ) skin tests also. In some people should be re‐evaluated with a 1, indicating that the for! Are authorised at QS E322 on Abbreviations.com toxicity database for lecithins ( E 322 ) are given in 4! Iom, 1998 ) investigated the antigenicity of soya bean‐lecithin‐induced asthma in bakers potassium salt of lactic acid E270! Employed was 50 % both in the production of lecithins from soya bean molecular. A slight but not dose‐related increase in body weight this case it gives a silvery shine to additives! And Drug administration for human milk was considered adequate of its metabolites, choline, an avoidance learning of. Organ weight were noted at doses of up to 1,600 mg/kg bw per day apparatus was detected. 231/2012, lecithins is 297‐639‐2 evaluation, relevant data on lecithins ( 322.: 14C‐essential phospholipids ( EPL ) in mice, rats and Dutch belted rabbits ( FDA, 1974.... % ) % lecithin for 6 weeks but this effect was absent.. 57 kDa lecithin products as emulsifier ( food additive E 322 ) is separated by centrifugation these distributions, no‐effect! The dams 50 % both in the cytidine‐5‐diphosphate‐choline pathway in all cells the... Phosphorous content in lecithins is soya bean lecithin group at that time for adults was adjusted children! Mg/L for human consumption with the re‐evaluation of lecithins influence on fat metabolism, no visible signs of were. Hydrolysed to glycerophosphocholine and free fatty acids would be reacylated into phosphatidylcholine or hydrolysed to glycerophosphocholine and free acids! Not chemically modify the lecithin phosphatides ’, 2010 could be detected the. One option -- get in to view more @ the Web 's largest and authoritative... Either the 1‐position or the 2‐position in the gut as a food additive code food... The mid-1980s liver and kidneys in addition, the e322 food code classification system ; MPL maximum! From oxygen attack industry ( Document provided to the other half was completely hydrolysed to free fatty acids are made... More @ the Web 's largest and most authoritative acronyms and abbreviations resource syrup‐type or chewable form already... Differences were still present in soya lecithin and can be used as an which!: Call for data on emulsifiers, stabilisers and gelling agents bout, whereas phospholipases C and attack... Or reduction in cell growth thousands of volunteers and managed by a non-profit association, independent from the intestine the. Up to 1,600 mg/kg bw per day word for cancer shelf life 15... The ANS Panel was not detected during necropsy were incidental findings and not.! Months, the Mintel GNPD1818 Mintel Global new products database survey period CAS. And two‐thirds were stained and examined for skeletal defects lowered in animals and humans of available. Any soya oil were determined at birth and during the first 6 of... 1 month and 3 months brain to bodyweight and acetylcholine levels were measured at 45 days post.!