3.1.3.1 Glycerol produced from fats and oils

According to Christoph et al. (2006), glycerol is mainly produced from fats and oils by high pressure splitting. It is obtained by feeding water and fat in counter current into a splitting column at 2–6 MPa and 220–260°C. This glycerol is marketed as 88% saponification- or hydrolysis-crude glycerol (Christoph et al., 2006).

The production of glycerol after saponification (treatment with caustic alkali or alkali carbonates) of neutral fats and oils is only partially applied. The final product is obtained from crude glycerol by purification via distillation or ion exchange chromatography (Christoph et al., 2006).

According to CEFIC (2013a [Documentation provided to EFSA n. 3]), ‘vegetal’ glycerol can be produced from vegetable oils after a treatment at a temperature higher than 100°C at low pressure, followed by distillation and further purification. The Panel noted that refined oils and fats may be contaminated with glycidol (EFSA CONTAM Panel, 2016). Glycidol is characterised as probably carcinogenic to humans 2A (IARC, 2000; BfR, 2009) and as a carcinogenic and genotoxic compound by the EFSA CONTAM Panel (EFSA CONTAM Panel, 2016).

3.1.3.2 Glycerol produced by chemical synthesis

Glycerol can be produced from propene (Christoph et al., 2006). Three pathways are known: (i) via the conversion of propene to acrolein and allyl alcohol; (ii) via the conversion of propene to propylene oxide and allyl alcohol; and (iii) via the conversion of propene to allyl chloride and epichlorohydrin.

In the pathway via acrolein, propene is oxidised to acrolein, which is then reduced to allyl alcohol. Allyl alcohol is then epoxidised with hydrogen peroxide to glycidol, which is further hydrolysed to glycerol.

In the pathway via propylene oxide, propene is epoxidised to propylene oxide, which is then isomerised to allyl alcohol. In a second step, epoxidation is carried out to form glycidol, which is hydrolysed to glycerol.

In the pathway via allyl chloride, propene is oxidised with hypochlorite to dichlorohydrin, which, in the presence of calcium hydroxide or sodium hydroxide, is converted to epichlorohydrin. Epichlorohydrin is then hydrolysed to glycerol at 80–200°C with an aqueous solution of sodium hydroxide or sodium carbonate.

The Panel noted that in the synthesis pathway of glycerol via either acrolein or via propylene oxide, glycidol is produced as an intermediate, which may be present in the final product.

The Panel also noted that in the synthesis pathway of glycerol via allyl chloride, epichlorohydrin is formed as an intermediate, which may be present in the final product. Epichlorohydrin is classified as carcinogen 2A according to the International Agency for Research on Cancer (IARC, 1999).

The Panel further noted that there are no limits in the specifications for other starting or intermediate chemicals used in the synthesis of glycerol, including glycidol, propene, allyl alcohol, propylene oxide, allyl chloride and dichlorohydrin, and no numerical limit in the specifications for acrolein.

3.1.3.3 Glycerol produced by microbial fermentation

The Panel noted that processes are described for glycerol production via microbial fermentation using either yeasts (Saccharomyces cerevisiae or osmotolerant yeasts), bacteria (Lactobacillus lycopersici and Bacillus subtilis) or algae (Dunaliella tertiolecta and Dunaliella bardawil) (Wang et al., 2001). However, the Panel has no indication if these processes are used on an industrial level.

Schier et al. (2009) presented a literature survey of potential contaminants/intermediates resulting from the manufacturing or purification processes of glycerol. The authors indicated that when, e.g. glycerol is produced via hydrogenolysis of carbohydrates, ethylene glycol can be present as a contaminant. It is also indicated that when glycerol is produced via microbial fermentation, dioxane and aniline can be present as contaminants. All these contaminants are substances of toxicological concern.

Overall, the Panel noted that in addition to the information provided by CEFIC (2013), glycerol (E 422) can be produced by a variety of methods and that many of them lead to the presence or formation of contaminants, which are of toxicological concern. This should be considered in the EU specifications for glycerol (E 422). The Panel considered that the manufacturing process for glycerol (E 422) should not allow the production of a food additive which contains residuals of genotoxic or/and carcinogenic concern at a level which would result in a margin of exposure (MOE) below 10,000.

3.3.1.1 Summarised data on reported use levels in foods provided by industry

Industry provided EFSA with data on use levels (n = 91) of glycerol (E 422) in foods for 20 out of the 68 food categories in which glycerol (E 422) is authorised.

These data were provided by FoodDrinkEurope (FDE), the International Chewing Gum Association (ICGA), the Association of the European Self-Medication Industry (AESPG), Cutisin, Nutricia and Riemser Arzneimittel AG. Data were mainly reported for edible ices (FCS 03).

The Panel noted that the use levels of glycerol (E 422) reported by the food industry were high in some foods (> 50,000 mg/kg in dietary supplements, chewing gums and fine bakery wares) and varied largely between food categories: from < 0.01 mg/kg in breakfast cereals to 100,000 mg/kg in chewing gums.

Appendix A provides data on the use levels of glycerol (E 422) in foods as reported by industry.

3.3.1.2 Summarised data on concentration levels in food submitted by Member States

In total, 7,745 analytical results were reported to EFSA by one country, Germany. These data were mainly for wine and other products defined by the Council Regulation (EC) No 1234/20071717 Council Regulation (EC) No 1234/2007 of 22 October 2007 establishing a common organisation of agricultural markets and on specific provisions for certain agricultural products (Single CMO Regulation). OJ L 299, 16.11.2007, p. 1–149.
(FCS 14.2.2). Foods were sampled between 2000 and 2013, and all of them were analysed in the year of collection.

Almost 98% of the analytical results on glycerol (E 422) were quantified. In 72 samples, the results were not quantified (< limit of quantification (LOQ)) and in 89 samples, the results were reported as not detected (< LOD).

Complete information on the methods of analysis (e.g. validation) was not made available to EFSA, but all samples were derived from accredited laboratories.

In order to include only data over the last 10-year period, analytical results sampled before 2004 (n = 522) were excluded from further analyses.

In total, 1,133 analytical results reported for glycerol (E 422) in foods were taken into account by the Panel in the exposure assessment considering only direct addition of glycerol (E 422) to food according to Annex II of Regulation (EC) No 1333/2008. These data covered 14 out of 68 food categories in which glycerol (E 422) is authorised as a food additive according to Annex II to Regulation No 1333/2008.

The Panel noted that the remaining 6,090 analytical results were reported in food categories in which glycerol (E 422) is not authorised for direct addition according to Annex II of Regulation (EC) No 1333/2008, including: ‘Honey as defined in Directive 2001/110/EC1818 Council Directive 2001/110/EC of 20 December 2001 relating to honey. OJ L 10, 12.1.2002, p. 1–6.
’ (FCS 11.3) and ‘wine and other products defined by Regulation (EC) No 1234/2007 and alcohol-free counterparts’ (FCS 14.2.2). It should be noted that glycerol (E 422) was quantified in almost all samples of these two food categories. The authorised use of glycerol (E 422) according to Annex III to Regulation (EC) No 1333/2008 (Parts 1, 2, 3, 4 and 5, Section A) may have resulted in carry-over and its detection in the wine food category. Natural fermentation could also result in the presence of glycerol in wine and other alcoholic beverages (VCF, 1963-2016), as well as in honey (Huidobro et al., 1993). Therefore, the analytical results in these food categories (honey (FCS 11.3), wine (FCS 14.2.2), other alcoholic beverages (FCS 14.2.3, 14.2.4, 14.2.5, 14.2.7.1, 14.2.7.2, 14.2.7.3, 14.2.8)) were not considered in the exposure assessment of glycerol (E 422) as a food additive according to Annex II, but included in the overall estimation of the exposure to glycerol (E 422) according to Annex II, via carry-over (Annex III) and natural sources.

Appendix B shows the analytical results of glycerol (E 422) in foods as reported by Member States.

When both concentration levels, i.e. reported use and analytical levels, were available (only for a few food categories), they were comparable (Appendices A and B).

3.3.3.1 EFSA Comprehensive European Food Consumption Database

Since 2010, the EFSA Comprehensive European Food Consumption Database (Comprehensive Database) has been populated with national data on food consumption at a detailed level. Competent authorities in the European countries provide EFSA with data on the level of food consumption by the individual consumer from the most recent national dietary survey in their country (cf. Guidance of EFSA on the ‘Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment’ (EFSA, 2011a)). New consumption surveys recently2121 Available online: http://www.efsa.europa.eu/en/press/news/150428.htm
added in the Comprehensive database were also taken into account in this assessment.1010 Available online: http://www.efsa.europa.eu/en/datexfoodcdb/datexfooddb.htm

The food consumption data gathered by EFSA were collected by different methodologies and thus direct country-to-country comparisons should be interpreted with caution. Depending on the food category and the level of detail used for exposure calculations, uncertainties could be introduced owing to possible subjects’ underreporting and/or misreporting of the consumption amounts. Nevertheless, the EFSA Comprehensive Database represents the best available source of food consumption data across Europe at present.

Food consumption data from the following population groups: infants, toddlers, children, adolescents, adults and the elderly were used for the exposure assessment. For the present assessment, food consumption data were available from 33 different dietary surveys carried out in 19 European countries (Table 4).

Consumption records in the EFSA Comprehensive Database were codified according to the FoodEx classification system (EFSA, 2011b). Nomenclature from the FoodEx classification system has been linked to the food categorisation system (FCS) as presented in Annex II of Regulation (EC) No 1333/2008, part D, to perform exposure estimates. In practice, FoodEx food codes were matched to the FCS food categories.

3.3.3.2 Food categories considered for the exposure assessment of glycerol (E 422)

The food categories in which the use of glycerol (E 422) is authorised were selected from the nomenclature of the EFSA Comprehensive Database (FoodEx classification system), at the most detailed level possible (up to FoodEx Level 4) (EFSA, 2011b).

For the following food categories, the restrictions/exceptions which apply to the use of glycerol (E 422) could not be taken into account, and therefore, the whole food category was considered in the exposure assessment. This applies to 2 food categories and may have resulted in an overestimation of the exposure.

In the EFSA Comprehensive database, no information is provided on the type of food supplements consumed by infants and young children. In the exposure assessment, even if this food category refers in the EU regulation to foods supplements excluding infants and young children, it was in this opinion therefore assumed that the food supplements consumed in these population groups were the same as those consumed in the older population groups for which concentration data were supplied, resulting in an overestimation of the exposure to glycerol (E 422) in these two population groups.

Furthermore, 30 food categories were not taken into account because no concentration data (reported use levels or analytical data) were provided to EFSA. For the remaining food categories, the refinements considering the restrictions/exceptions as set in Annex II to Regulation No 1333/2008 were applied.

Overall, 28 food categories out of 68 were included in the present exposure assessment to glycerol (E 422) (Appendix D).

3.4.1.1 Maximum level exposure assessment scenario

The regulatory maximum level exposure assessment scenario is based on the MPLs as set in Annex II to Regulation (EC) No 1333/2008. As glycerol (E 422) is authorised according to QS in all food categories, a maximum level exposure assessment scenario was performed based on the maximum reported use levels provided by industry or high level of analytical data provided by MSs, as described in the EFSA Conceptual framework (EFSA ANS Panel, 2014), whichever was highest or available. The same food categories as included in data set 1 were considered for this scenario.

The Panel considers the exposure estimates derived following this scenario as the most conservative for the food categories taken into account, since it is assumed that the consumer will be continuously (over a long period of time) exposed to glycerol (E 422) in foods belonging to these food categories at maximum reported use levels/high level of analytical data.

3.4.1.2 Refined exposure assessment scenario

The refined exposure assessment scenario is based on use levels reported by industry and analytical results reported by Member States. This exposure scenario can consider only food categories for which these data were available to the Panel.

If both usage and analytical data were available for the same food category, the most reliable value under consideration was used. For glycerol (E 422), the Panel considered due to a low sample size of the analytical data, that the reported use levels were more reliable. However, the Panel also noted that the levels were comparable for both sources in the respective food categories.

To consider left-censored analytical data (i.e. analytical results < LOD or < LOQ), the substitution method as recommended in the ‘Principles and Methods for the Risk Assessment of Chemicals in Food’ (WHO, 2009) and the EFSA scientific report ‘Management of left-censored data in dietary exposure assessment of chemical substances’ (EFSA, 2010) was used. In the present opinion, analytical data below LOD or LOQ were assigned half of LOD or LOQ, respectively (medium bound (MB)). Subsequently, per food category, the mean or median, whichever was highest, MB concentration was calculated.

3.4.1.3 Dietary exposure to glycerol (E 422)

Table 5 summarises the exposure to glycerol (E 422) from its use as a food additive according to Annex II to Regulation (EC) No 1333/2008 in six population groups (Table 4) according to the different exposure scenarios. Detailed results per population group and survey are presented in Appendix E.

In the maximum level exposure assessment scenario, mean exposure to glycerol (E 422) ranged from 10 mg/kg bw per day in infants to 583 mg/kg bw per day in toddlers. The 95th percentile of exposure ranged from 161 mg/kg bw per day in adults to 938 mg/kg bw per day in toddlers.

In the refined brand-loyal exposure scenario considering data set 1, the mean exposure to glycerol (E 422) ranged from 9 mg/kg bw per day in infants to 453 mg/kg bw per day in toddlers, and the 95th percentile from 136 mg/kg bw per day in adults and the elderly to 796 mg/kg bw per day in toddlers. In the non-brand-loyal scenario, the mean exposure to glycerol (E 422) ranged from 6 mg/kg bw per day in infants to 308 mg/kg bw per day in toddlers, and the 95th percentile from 98 mg/kg bw per day in the elderly to 460 mg/kg bw per day in children.

The Panel noted that the exposure to glycerol (E 422) using data set 2 (Appendix F), both brand-loyal and non-brand-loyal scenarios, did not significantly change the exposure to glycerol (E 422) using data set 1 (Table 5).

The Panel also noted that only one, rather high, use level (30,000 mg/kg) was reported by industry for food category 07.1 ‘bread and rolls’ (Appendix A) (no analytical data were reported for this food category). This level was for a niche product (‘chapattis’), i.e. a not commonly eaten food item. This use level was applied to the whole food category, which will result in a significant increase in the exposure estimates (between 1.7 and up to 19 times more depending on the population groups at the mean for the non-brand-loyal scenario), especially since the consumption of food category 07.1 ‘bread and rolls’ is fairly high in many surveys.

3.4.1.4 Main food categories contributing to exposure to glycerol (E 422)

3.4.1.5 Uncertainty analysis

Uncertainties in the exposure assessment of glycerol (E 422) have been discussed above. In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2007), the sources of uncertainties have been summarised and evaluated in Table 9.

Glycerol (E 422) is authorised as a Group I food additive in 66 food categories and has a specific authorised use in two food categories (Table 3). Since the majority of food categories correspond to the general Group I food additives authorisation, glycerol may not necessarily be used in some of these food categories. This may explain why reported use levels and analytical data of glycerol (E 422) were only available for 28 food categories, including one food category for which glycerol (E 422) has a specific use. The Panel calculated, based on the information in the Mintel GNPD, that out of the foods authorised to contain glycerol (E 422) according to Annex II to Regulation (EC) No 1333/2008, 18–50% of the amount of food consumed per population group was reported to potentially contain glycerol (E 422) as a food additive. Based on this, the Panel noted that the information from the Mintel GNPD supported the observation that due to its Group I authorisation, glycerol may be used in less food categories than it is authorised.

Furthermore, the Panel noted that information from the Mintel GNPD (Appendix C) indicated that 54 out of 76 food subcategories, categorised according to the Mintel GNPD nomenclature, in which glycerol (E 422) was labelled were included in the current exposure assessment. These 54 food subcategories represented approximately 94% of the food products labelled with glycerol (E 422) in the database. In the remaining 22 food subcategories, in which glycerol (E 422) was labelled but which were not included in the exposure assessment, glycerol (E 422) was authorised in 18 food subcategories.

Given these observations, the Panel considered overall that the uncertainties identified would, in general, result in an overestimation of the exposure to glycerol (E 422) from its use as a food additive according to Annex II in both the maximum level and refined exposure scenario.

The Panel noted that food categories which may contain glycerol (E 422) due to carry-over (Annex III, Part 1, 2, 3, 4 and 5) were only partly considered (via wine and other alcoholic beverages) in the current exposure assessment using data set 2, and that the exposure to glycerol via natural sources included only wine, other alcoholic beverages and honey. Taking these sources also into consideration did, however, not significantly change the exposure to glycerol (E 422) (Appendix F).

3.4.5.1 Glycerol as an ingredient in food for sports people

The use of glycerol in beverages for athletes to enhance and maintain hydration status and to improve endurance exercise performance is described (Van Rosendal et al., 2010; Savoie et al., 2016). Glycerol when used as a component of a hyperhydration strategy is prohibited by the World Anti-Doping Agency (WADA) (Martindale, online; Savoie et al., 2016; WADA, 2016).

3.4.5.2 Pharmaceutical use

Glycerol is used in pharmaceutical products as an active ingredient as well as an excipient, e.g. solvent, plasticiser or lubricant (EMA, 2003; Martindale, online). When glycerol is used as an excipient in medicinal products for oral administration and the glycerol intake is equal or above 10 g per single dose of the medicinal product, the information ‘May cause headache, stomach upset and diarrhea’ is required for the package leaflet (EMA, 2003). As an active ingredient, glycerol is used as an osmotic dehydrating agent. It is given orally, e.g. for the reduction in intra-ocular pressure before and after ophthalmic surgery and as an adjunct in the management of acute glaucoma or to reduce intracranial pressure (Martindale, online) (see Section 3.5.7).

3.5.1.1 Absorption

3.5.1.2 Distribution

In a study in rats, it was shown that there was a net synthesis of blood glucose and liver glycogen as a result of ¹⁴C-glycerol administration (88–89 mg/rat; intraperitoneal (i.p.)). Fifteen minutes after administration, 70% of newly formed blood glucose was derived directly from glycerol and 30 min after treatment this value increased to 100%. After 30 minutes, 39% of the newly formed liver glycogen was derived from the administered ¹⁴C-glycerol. This value decreased during the following 1–6 h after application and fell to 15% at 6 h (Gidez and Karnovsky, 1954).

In further experiments, the incorporation of ¹⁴C-glycerol into liver lipids was also reported (Gidez and Karnovsky, 1954). At a dose of 1 mM ¹⁴C-labelled glycerol (92 mg/rats; i.p.), most radioactivity was found in liver lipids (5.9% of applied dose) 6 h after application with only minor amounts found in other organs (e.g. 0.26% in the brain and 0.16% in the kidney).

3.5.1.3 Metabolism

Glycerol occurs naturally in fats (e.g. triglycerides, phospholipids) and is also an endogenous metabolite (Tao et al., 1983). After ingestion of triglyceride, hydrolysis in the gastrointestinal tract yields glycerol and fatty acids which are then absorbed by the mucosa. Glycerol is also released by adipose tissue lipolysis; the catabolic process leading to the breakdown of triglycerides stored in fat cells. Glycerol may then be metabolised through a number of pathways (Lin, 1977; Brisson et al., 2001).

Glycerol may be phosphorylated by glycerol kinase to glycerol phosphate followed by oxidation to dihydroxyacetone phosphate via the enzyme glycerol phosphate dehydrogenase (Tao et al., 1983; Brisson et al., 2001). Dihydroxyacetone phosphate can participate in glycolysis and be further oxidised to carbon dioxide (oxidised as an energy substrate) or gluconeogenesis after formation of the intermediate glyceraldehyde 3-phosphate (Tao et al., 1983; Brisson et al., 2001). Exogenous glycerol also participates in lipogenesis (e.g. formation of triglycerides). When rats were i.p. injected with radiolabelled glycerol, radioactivity was found in lipids (Gidez and Karnovsky, 1954). With respect to glycerol utilisation, the liver accounts for up to at least 75% of the total body glycerol-utilising capacity and the kidney accounts for up to 25% (Lin, 1977; Tao et al., 1983). At physiological serum concentrations up to 1 mM glycerol (92 mg/L), uptake into hepatocytes follows first order kinetics (Tao et al., 1983).

Gidez and Karnovsky (1954) examined the relationship between the amount of glycerol oxidised compared to the amount administered. At dose levels above 30 mg/rat (about 150 mg/kg bw), an approximately constant proportion of the administered glycerol appeared to be oxidised and exhaled as ¹⁴CO₂. At lower concentrations, the proportion converted to CO₂ was higher.

3.5.1.4 Excretion

3.5.3.1 Rats

In a study by Staples et al. (1967), female rats (10 animals/group; strain not reported) were treated three times daily for 3 days with 0, 0.75, 1.5 or 3 mL/kg bw undiluted glycerol by gavage (equivalent to 0, 2,800, 5,600 and 11,200 mg/kg bw per day), control animals received 3 mL water by gavage. The study was restricted to examining irritant effects in the stomach and duodenum. At 0.75 mL/kg bw (2,800 mg/kg bw per day) and higher, the authors reported dose-dependent local irritant effects including slight to severe hyperaemia, petechial haemorrhages and erosions but no systemic toxicity. Administration of 3,800 mg glycerol/kg bw (thrice daily) as either a 20, 40, 60, 80 or 100% aqueous solution (dose volume varied between 3 (for 100%) and 15 mL (for 20%)) resulted in local irritant effects at all dilutions but with severity reduced by dilution such that 1 animal in 10 showed slight effects in the 20% group.

Thirty adult male Wistar rats (6 animals/group) were treated by gavage with 0 (control/saline), 200, 400, 800 and 1,600 mg glycerol/kg bw per day (bidistilled glycerine, 99.85% glycerol) for 28 days (Lisenko et al., 2015). Glycerol did not induce any statistically significant change in average daily weight gain or water consumption or excretion levels. Glycerol supplementation did induce a significant decrease in the average daily feed intake levels. Supplementation with glycerol did not affect plasma triglyceride, glucose, total cholesterol, lipoprotein concentrations, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Plasma glucose and total cholesterol levels demonstrated a significant increase in values up to a dose of 800 mg/kg, followed by a decrease at 1,600 mg/kg bw per day. In terms of high-density lipoprotein levels, there was an increase up to a dose of 800 mg/kg bw per day, followed by a decrease at 1,600 mg/kg bw per day, but levels were still within the normal range for the species. For very low-density lipoprotein and low-density lipoprotein levels, there was a decrease with increasing glycerol doses, which remained within the standards. The liver enzymes AST and ALT showed a decrease trend up to the dose of 800 mg/kg bw per day, with an increase at 1,600 mg/kg bw per day. Glycerol supplementation did not result in any change in relative organ weights. Leucocyte levels differed between the groups, although all were within the normal range. The differential count for lymphocytes and neutrophils did not change between the experimental groups. No lesions were observed in any tissue (pancreas, kidney, liver) from animals receiving different levels of glycerol.

In a limited study by Fischer et al. (1949), male rats (20 animals/group; strain not reported) were treated for 44 days with 0, 100, 500, 1,000 or 2,000 mg glycerol/kg bw per day by gavage. The authors reported no clinically adverse signs at any dose; no effects on mortality or body weight gain. For the haematological parameters examined, there were no effects reported on haemoglobin, erythrocyte, leucocyte and lymphocyte counts and differential blood counts. Histopathological examination was restricted to liver, kidney and urinary bladder, and no effects were observed.

Adult male Wistar rats were subjected to 6 weeks of aerobic training (after an acclimatisation to swimming, the animals swam for 60 min daily, at a frequency of 5 times per week, with relative overload of 5% of their body mass) (Andrade et al., 2014). In the last 4 weeks, the animals were treated with saline, glucose or glycerol (800 mg/kg bw per day) by gavage (it is not stated how frequently animals were dosed). Treatment with glycerol did not induce statistically significant differences in body mass, water consumption, urinary production or relative weight of liver or kidney compared to negative control. Glycerol supplementation did induce a significant reduction in food consumption compared to negative control. In terms of blood biochemical parameters, no difference was observed in glycaemic level or lipoprotein concentrations in plasma compared to negative control. However, a statistically significant increase in both plasma triacylglyceride and total cholesterol levels was observed after supplementation with glycerol. Statistically significant increases in adipocyte diameter and area, as well as villous:crypt ratios of the duodenum and ileum were observed following glycerol supplementation. No differences were observed in adipocyte density or the jejunum villous:crypt ratio in glycerol-supplemented animals compared to negative control.

In a study by Anderson et al. (1950), female rats (5 animals/group; strain not reported) were treated for 26 weeks with 0 or 5% glycerol by volume in the drinking water (either derived from natural sources or synthetically synthesised), equivalent to 0 and 4,500 mg glycerol/kg bw per day). Haematology was performed monthly and was composed of erythrocyte and leucocyte counts, haemoglobin and differential blood counts. At 26 weeks, necropsy and histopathology of heart, lungs, liver, spleen, stomach, intestines, kidneys, thymus, thyroid and adrenals was performed, and no effects were observed by the authors except mineralisation in renal tubules. Glycerol treatment had no effects on haematological parameters. The Panel considered mineralisation in renal tubules to be due to the high dose of glycerol used in the study.

In a 3-month study by Löser et al. (1954), male and female rats (strain unspecified, 6 animals/group) were treated with glycerol as a 0, 1%, 2%, 5%, 10% or 20% solution in drinking water (equivalent to 0, 800, 1,600, 4,000, 8,100 and 16,200 mg/kg bw per day and 0, 900, 1,800, 4,500, 9,100 and 18,200 mg/kg bw per day for males and female, respectively). No effects were noted in the animals receiving 1%, 2%, 55 or 10% glycerol. In the group receiving 20% glycerol, 2 animals out of 12 animals died. The remaining 18 exhibited some retardation in weight gain and development. However, they recovered while under treatment and had no abnormalities at the end of the study. The authors reported that some organs were investigated (liver, spleen, kidney, endocrine organs) and that no effects of glycerol were found. Identical results for glycerol were reported in a shorter version in a publication, by Bornmann (1954). The Panel considered it probable that this paper reported the same study.

3.5.3.2 Dogs

In a study by Staples et al. (1967), mongrel dogs (1–2 animals/group) were treated three times daily for 3 days with 0, 0.75, 1.5 or 3 mL/kg bw undiluted glycerol (equivalent to 0, 2,800, 5,600 and 11,200 mg/kg bw per day) by gavage, control animals received 3 mL water by gavage. The study was restricted to examining irritant effects in the stomach and duodenum. At 5,600 mg/kg bw per day and higher, the authors reported dose-dependent local irritating effects including hyperaemia, petichial haemorrhages and erosions.

Overall, the Panel noted that no studies were performed according to current test guidelines. The Panel also noted that in a subchronic toxicity study (in drinking water) in rats (Löser et al., 1954) the effects reported were observed with doses in the range of the LD₅₀ for glycerol. The Panel considered that the local irritating effects of glycerol in the gastrointestinal tract reported in some gavage studies in rat (100% glycerol at 2,800 mg/kg bw per day, the lowest dose tested (Staples et al., 1967)), and dogs (100% glycerol at 5,600 mg/kg bw per day (Staples et al., 1967)) was likely due to the hygroscopic and osmotic effects of large doses of glycerol administered by gavage.

3.5.4.1 In vitro

Numerous studies have been published for glycerol on reverse gene mutation in bacteria, chromosome aberrations and gene mutations in mammalian cells. A summary of available data is presented in Table 12.

The most reliable data for the bacterial reverse mutation assay in Salmonella Typhimurium strains TA100, TA1535, TA1537, TA98, TA1538 and Escherichia coli WP2uvrA were presented by Shimizu et al. (1985). The study is comparable to current standards. Glycerol concentrations up to 5 mg/plate were tested, the maximal dose level recommended in OECD Guideline 471. Negative results were obtained with and without addition of S9 mix (prepared from livers of male rats pre-treated with polychlorinated biphenyl). Vehicle and positive controls were performed and showed the expected results. No mutagenic activity was detected in other studies (Clark et al., 1979; Stolzenberg and Hine, 1979; Yamaguchi, 1982; Haworth et al., 1983; Ishidate et al., 1984; Doolittle et al., 1988; Higashimoto et al., 1991; Fujita et al., 1994).

Ishidate et al. (1984) published a chromosome aberration test in Chinese hamster lung fibroblast cells (see Table 12). The cells were exposed to three different doses for 24 and 48 h without addition of a metabolic activation system. The maximum dose tested was 1 mg/mL. This concentration represented the highest non-cytotoxic dose level tested in preliminary tests (50% cell-growth inhibition). One hundred metaphases per concentration were analysed for polyploid cells and structural chromosomal aberrations (no further details). Chromosome and chromatid gaps were included in the evaluation of clastogenic effects. No data were presented about concurrent positive controls but clastogenic effects were reported in parallel experiments with other test substances. The exposure to glycerol resulted in no increase in polyploidy or chromosome aberration. The authors considered glycerol to be non-clastogenic and non-mutagenic in this test system, but concentrations up to the cytotoxic threshold were not tested. However, no cytotoxic effects, nor clastogenic activity, was reported by Galloway et al. (1987) at higher concentrations (92 mg/mL), a dose level 18 times greater than the maximum dose level recommended in vitro by OECD Guideline no. 473. In this study, no concurrent positive control was tested for verification of the results. The concentrations used by Doolittle et al. (1988, see Table 12) also gave negative results but did not induce cytotoxic effects nor reach the recommended maximum concentration. Overall, however, the available studies are sufficient for evaluation of this endpoint and were considered by the Panel to be sufficiently robust to support the results reported, indicating no clastogenic activity of glycerol in in vitro studies.

Equivocal results were reported in a mammalian cell gene mutation assay in trials without addition of a metabolic activation system (Doolittle et al., 1988). An increase in the mutation frequency compared to negative (4.8-fold increase) and solvent control (12-fold increase) was found at a concentration of 0.8 mg/mL but no clear increase at the highest dose level of 1 mg/mL. The effect was not dose-dependent and therefore considered by the authors to be without biological relevance. The Panel noted that mutation frequencies in the concurrent untreated and solvent treated controls were unusually low. No independent second trial was performed. Negative results were obtained with addition of a metabolic activation system. Other studies on this endpoint were not available.

No genotoxic activity was reported in limited studies on DNA damage in mammalian cells (Doolittle et al., 1988). There were only limited details reported for the rec assay in B. subtilis (Nonaka, 1982).

3.5.4.2 In vivo

Barilyak and Kozachuk (1985) reported a bone marrow chromosome aberration assay and a dominant lethal assay, both performed in male rats. No other in vivo genotoxicity data were available for glycerol.

A single gavage dose of 1 g/kg bw glycerol (n = 10, controls n = 11) induced an increase in chromosomal aberrations (2.2 ± 0.6% vs 0% in control; without gaps) and polyploidy cells (3.2 ± 0.8% vs 0.5 ± 0.2% in control) (Barilyak and Kozachuk, 1985). The bone marrow was processed for cytogenetic analysis 50 h after treatment and at least 50 metaphases per animal were analysed. However, the Panel noted that incidence of chromosomal aberrations observed was low and fell within the range of values often observed in untreated or solvent control animals. Moreover, the Panel also noted methodological shortcomings (e.g. collection of bone marrow outside the recommended range of 12–36 h, no positive control; no aberrations in 1,000 control metaphases; no statistical analysis and one dose level only) and limited information (e.g. no data on toxic effects). On this basis, the Panel considered this study not reliable. In the same publication, a rat dominant lethal assay was documented. Male rats were exposed to 0, 10, 100 or 1,000 mg/kg bw glycerol ‘at the stage of late spermatids’ (2 weeks before fertilisation) and mated with untreated females (n = 11–12 per dose). A dose-dependent and significant increase in post-implantation loss was reported with a statistically significant increase at ≥ 100 mg/kg bw. The Panel noted that the reported positive results were based on insufficient number of female animals to assess dominant lethal mutations and the number of treated male animals was not indicated. On this basis, the Panel considered this study not reliable.

Overall, glycerol did not show any genotoxic activity in different in vitro assays, which include negative results in the bacterial reverse mutation assay (Ames test), in chromosome aberration assays and in studies on DNA damage in mammalian cells. Questionable results obtained in a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene mutation assay did not show a dose–response effect and were therefore judged of no biological significance. A lack of valid in vivo genotoxicity data is not of concern since clear negative findings were observed in in vitro assays. On this basis, the Panel considered that glycerol as a food additive did not raise concern with respect to genotoxicity.

3.5.5.1 Mice

Witschi et al. (1989) investigated the effects on lung and liver tumour incidences in male and female CH3 mice (between 31 and 51 mice/group) after administration for 1 year of drinking water containing 0% or 5% glycerol (equivalent to 0 and 6,500 mg glycerol/kg bw per day for males and 0 and 8,300 mg/kg bw per day for female). Although the reporting of this study was limited, the Panel noted that lung and liver tumour incidences were not significantly elevated in male or female mice (no further parameters examined).

3.5.5.2 Rats

As reported by JECFA (1976), male and female Sprague–Dawley rats (9 animals/group) were fed a diet supplemented with 0%, 5%, 10% or 20% natural or synthetic glycerol (equivalent to 0, 2,200, 4,500 and 9,000 mg/kg bw per day for male and 0, 2,900, 5,800 and 11,600 mg/kg bw per day for female) for 50 weeks (Atlas Chemical Industries Co. Ltd., 1969, as referred to by JECFA, 1976). At necropsy, growths in the pituitary and histopathological effects were observed in the kidney in females treated with either test compounds. The Panel noted limited methodological detail, effects only in females and data lacking any dose–response effects.

Hine et al. (1953) examined the effects of supplementing the diet with glycerol (natural and synthetic, no further details on test substances) for up to 2 years on Long-Evans male and female rats (between 10 and 14 animals per group) at 0, 2,500, 5,000 or 10,000 mg/kg bw per day. The Panel noted that the study has a number of limitations – all rats in the high-dose group were culled after 1 year, with no concurrent control group; no clinical chemistry; limited parameters in haematology and urinalysis and limited histopathology. Body weight and food consumption were measured weekly and body weight gain calculated at 4 week intervals. Clinical signs were recorded daily and careful inspections for health and activity were made once a week. Urinalysis and blood sampling for haematology were performed at 3, 6, 12, 18 and 24 month (n = 5 per dose, no data about sex). Urinalysis was limited to microscopical examination and determination of glucose and albumin concentrations. Haematology was limited to erythrocyte counts, leucocyte counts and haemoglobin levels. High-dose rats were sacrificed after 1 year and liver glycogen and lipids determined (no control values were available). For the other groups, including the control group, the study was terminated after 105 weeks. In all groups, complete necropsy was performed and weights for the liver, lung, kidney, heart and spleen determined. Histopathology was limited to the liver, spleen, adrenals, kidney, small intestine, reproductive organs and urinary bladder. Lipid stains in the liver were prepared for all groups sacrificed after 105 weeks. The results were similar for the groups treated with natural or synthetic glycerol. No data were presented on food consumption, clinical signs or mortality. No significant effects were found on the body weight gain (statistical analysis restricted to mean values at week 13, 52 and 105); data on absolute body weight were not given. No consistent treatment-related effects were found in haematology, urinalysis, evaluation of organ weights and pathology. Liver glycogen and lipids did not differ between the two high-dose groups, a comparison with the control group was not possible. Similarly, data on organ weights and pathological effects in the high-dose group (10,000 mg/kg bw per day) were compared with a control group sacrificed at 105 weeks, limiting the validity of the results in the high-dose group. No no observed adverse effect level (NOAEL) was identified by the authors of the study. Although this study had minor limitations (e.g. lacking concurrent control in the 1-year study group), the Panel noted that no adverse effects were reported in animals receiving doses up to 10,000 mg/kg bw per day for 1 year, the highest dose tested. The Panel also noted that there was no increase in the tumours incidences up to doses of 5,000 mg/kg bw per day for 2 years, the highest dose tested.

JECFA reported a study in rat (Atlas Chemical Industries Co. Ltd., 1969, as referred to by JECFA, 1976) in which male and female Sprague–Dawley rats (24 animals/group) were fed a diet supplemented with 0%, 5%, 10% or 20% glycerol (equivalent to 0, 2,200, 4,500 and 9,000 mg/kg bw per day for male and 0, 2,900, 5,800 and 11,600 mg/kg bw per day for female) for 2 years. Animals were treated with natural glycerol (99.8% glycerol, similar caloric density for test and control diets) or synthetic glycerol (99.76% glycerol and 1,2,3-butanetriol and 1,2,4-butanetriol, 0.09%, similar caloric density for test and control diets). For both natural and synthetic glycerol, at all dose levels, glycerol treatment resulted in an increase in body weight gain (no further details). At the 10% dose level, there was an increase in relative heart weights (no data regarding other dose levels). At necropsy, the 20% glycerol group was examined for mortality, body weight, efficiency of caloric utilisation, water consumption, urinalysis (volume, specific gravity, sediment, acetone, albumin, sugar and oxalate), haematology (white blood cells and differential count, red blood cells, haematocrit, and haemoglobin) and clinical chemistry (blood glucose, urea, nitrogen, plasma cholesterol, serum alkaline phosphatase and transaminases, and bromosulfophthalein retention). In addition, organ weights were determined and complete histopathology performed (no further details). The only reported change noted was an increase in relative kidney weight at 20% glycerol dose level with both natural and synthetic glycerol. The Panel considered that the increase in relative heart weights did not show a dose–response relationship and that the increase in relative kidney weight at 20% glycerol dose level in both studies as adaptive since no histopathological changes were reported.

Overall, the Panel considered glycerol to be of no concern with regard to carcinogenicity.

3.5.5.3 Promoting and/or co-carcinogenic effects of glycerol

The Panel noted a series of studies, which had investigated potential modulating effects of glycerol administration on the development of cancer in animal models treated with an initiating agent (Inayama, 1986; Inayama et al., 1986; Nagahara, 1987; Witschi et al., 1989; Nagahara et al., 1990; Yano et al., 1993, 1994; Itoh et al., 2002; Kanojia and Vaidya, 2006). The Panel considered these studies not relevant for the risk assessment of glycerol as a food additive.

3.5.6.1 Reproductive toxicity

In a study reported by Johnson et al. (1933), rats were kept on diets containing 61% starch and no glycerol (control), or 20% starch plus 41% glycerol (equivalent to 20,500 mg/kg bw per day), or no starch plus 61% glycerol (equivalent to 30,500 mg/kg bw per day), limited information, e.g. no data about number of animals per group. Reproduction was impaired by diets containing 41% glycerol and no pregnancies occurred in rats that were on diets containing 61% glycerol (undernourished conditions). However, this was an old study and the documentation of the results is not sufficient for evaluation of this endpoint.

In a study performed by Guerrant et al. (1947), groups of male and female rats (3 males and 6 females per group) received a synthetic diet containing 0 or 30% glycerol (equivalent to 15,000 mg/kg bw per day) and were carried through seven successive generations. Young rats from treated females weighed on average 20% less than controls. The Panel noted this study was limited: the number of animals per group was low and the diet changed during the generations.

Ten male and 10 female rats were orally gavaged daily either with glycerol (20% solution in water) at a dose level of 2,000 mg/kg bw per day or with distilled water (control) for 8 weeks before mating (Wegener, 1953). After mating, one subgroup of females (n = 5) received glycerol throughout pregnancy and up to parturition. The other subgroup (n = 5) was treated until weaning of the F₁ generation. Body weights of F₁ rats were measured every second day during the post-natal period from day 15–60. At post-natal day 100, F₁ rats were sacrificed except for 10/sex which were used to produce the F₂-generation. The authors reported that there were no effects of glycerol on fertility of parental (P) rats, F₁ litter size or F₁ weight gain (however, data were not presented). In total, 12 F₁ rats died during post-natal day 1–50 in the treatment group and three rats in the control; this mortality was not further discussed and could not be judged by the Panel as the total number of pups/dam was not presented. The onset of oestrus cycle in F₁ females was comparable in both groups and histopathology of F₁ rats revealed no effects on testes, ovaries, thyroid glands, pituitary and adrenal glands (no other organs were examined). F₂ litter size was comparable to control. No further parameters were investigated. The Panel noted that the study has limitations due to some methodological weaknesses (e.g. low number of pregnant P rats, no F₁ pup weights, no data about external malformations or toxic effects in P generation) but there is no indication for adverse effects on reproductive performance of P rats at a dose of 2,000 mg/kg bw per day, the highest dose level tested. The Panel, therefore, considered that this study was limited as the number of animals per (sub) group was very low and the reporting was limited.

3.5.6.2 Developmental toxicity

Several developmental toxicity studies of glycerol were conducted in CD-1 mice, Wistar rats golden hamsters and Dutch-belted rabbits (FDRL, 1974a,b,c). Animals were administered different doses of glycerol (not specified) in water by gavage; the control groups were vehicle-treated (dose volume is not reported). Body weights were recorded at regular intervals during gestation and all animals were observed daily for appearance and behaviour. All dams were subjected to caesarean section, and the numbers of implantation sites, resorption sites, live and dead fetuses, and body weight of live fetuses were recorded. All fetuses were examined grossly for external abnormalities, one-third underwent detailed visceral examinations and two-thirds were stained and examined for skeletal defects.

Groups of 25 adult female albino CD-l outbred mice were dosed by gavage from gestation day (GD) 6 to 15 with 0, 12.8, 59.4, 276 or 1,280 mg glycerol/kg bw per day (dose volume 10 mL/kg bw per day) (FDRL 1974a). At necropsy on GD 17, the surviving dams appeared to be completely normal and the number of implantations, and live fetuses was comparable to the control group. Doses up to 1,280 mg glycerol/kg bw per day had no effects on implantation nor on maternal and fetal survival. The numbers of live or dead fetuses, resorptions and average implant sites and also fetal weights did not differ among the groups. The sex distribution of fetuses was not affected by the treatment. The number of abnormalities seen in either soft tissues or skeletons at fetal pathological examination of the glycerol-treated groups, did not differ from the number in vehicle-treated dams of the control group.

Groups of 25–29 virgin adult female Wistar rats were dosed by gavage from GD 6 and to 15 with 0, 13.1, 60.8, 282 or 1,310 mg glycerol/kg bw per day (dose volume 1, 1, 1, 2, or 5 mL/kg bw per day) (FDRL, 1974b). At necropsy on GD 20, doses up to 1,600 mg glycerol/kg bw per day appeared to be completely normal and had no effects on implantation or on maternal and fetal survival. The numbers of live or dead fetuses, resorptions, average implantations and fetal weights did not differ among the groups. The sex distribution of fetuses was not affected by the treatment. The number of abnormalities seen in either soft tissues or skeletons at fetal pathological examination of the glycerol-treated groups, did not differ from the number in vehicle-treated dams of the control group.

Groups of 15–20 virgin adult female Dutch-belted rabbits were treated by gavage once daily from GD 6 to 18 with doses of 0, 11.8, 54.8, 254.5 or 1,180 mg glycerol/kg bw per day (dose volume 1, 1, 1, 2 or 5 mL/kg bw per day) (FDRL, 1974c). Only 10–14 rabbits became pregnant and were evaluated at termination. At necropsy on GD 29, the surviving dams appeared normal throughout the observation period and had normal fetuses. No effect was observed on the number of implantations. The numbers of live or dead fetuses, resorptions, average implant sites or fetal weights did not differ among the groups. The sex distribution of fetuses was not affected by the treatment. The number of abnormalities seen in either soft tissues or skeletons at fetal pathological examination of the glycerol-treated groups, did not differ from the number in vehicle-treated dams of the control group.

The Panel noted that the identically performed tests with glycerol in mice, rats and rabbits by the FDRL (1974a,b,c) were reported in a limited way but cover the organogenesis period of the reproductive cycle. These prenatal developmental toxicity studies showed no dose-related developmental and maternal effects up to the highest dose tested 1,280, 1,600 or 1,180 mg glycerol/kg bw per day for mice, rats and rabbits.

3.5.7.1 Observations in humans

Glycerol has been used therapeutically by intravenously or oral administration to mobilise oedema and to induce osmotic diuresis. Clinical experience and data from clinical studies are found in the literature indicating that doses up to 60,000 mg intravenously, infused over 1–4 h, for a treatment duration of 1 week and in some studies, several weeks (Frank et al., 1981), and bolus oral doses (initial dose 1,500 mg/kg bw, followed by 500–700 mg/kg bw every 3 h) (Cantore et al., 1964) or 500–1,000 mg/kg bw (Wald and McLaurin, 1982).

The Panel considered that the therapeutic administration of glycerol by oral administration to patients reported to be suffering disease that could impact significantly on their physiological functionality was not relevant in the safety assessment of glycerol (E 422) as a food additive. However, glycerol has been prescribed for reduction in intraocular pressure in patients with glaucoma and may be prescribed prior to intraocular surgery (Bartlett, 1991). For this indication, glycerol was reported to be administered orally as a bolus dose of 1,000–1,500 mg/kg bw (as a 50% solution) with a daily total oral dose not higher than 120,000 mg (about 1,700 mg/kg bw) (Gilman, 1990). The Panel considered that the therapeutic administration of glycerol by oral administration to patients reported to be suffering ocular disease – such as glaucoma and administration of glycerol to control participants in clinical studies – were relevant for the safety assessment of glycerol (E 422) as a food additive.

The Panel noted that for oral therapies to patients with ocular disease or healthy controls, glycerol was administered as a bolus at 1,350 mg/kg bw (Buckell and Walsh, 1964); 1,500 mg/kg bw (Drance, 1964); 1,500 mg/kg bw (Consul and Kulsretha, 1965); 1,000 mg/kg bw (Kornblueth et al., 1967); 1,000–1,270 mg/kg bw (McCurdy et al., 1966); 1,350 mg/kg bw (Charan and Sarda, 1967); 1,200 mg/kg bw (Krupin et al., 1970) and 1,260 mg/kg bw (Friedman et al., 1980). The only side effects observed in these oral therapies to patients with ocular disease or healthy controls were either none or one or more of nausea, headache and/or vomiting.

Guidelines for glycerol use in hyperhydration and rehydration associated with exercise report 28 studies in which oral doses between 500 mg/kg bw and 1,500 mg/kg bw were given in the total of 238 subjects (Van Rosendal et al., 2010). Three studies reported side effects after rapidly administering the glycerol as a concentrated bolus followed by fluid ingestion. Four subjects in two of the trials were nauseous after glycerol ingestion. In another study, two subjects developed diarrhoea in the 24 h after the trial. In a further three studies, a low incidence of gastrointestinal distress (bloatedness) or light-headedness were reported.

Using the studies of Wald and McLaurin (1982), the Panel noted that patients were administered glycerol orally as bolus doses of 500–1,000 mg/kg bw every 3–4 h, dependent on the patients’ intracranial pressures (ICPs). Specific individual dosages ranged from 4,000 mg to 70,000 mg (average 54,000 mg) and it was administered via a nasogastric tube as a 50% solution (by mixing a 100% glycerol solution with an equal volume of either 5% dextrose in water or 5% dextrose in 0.4% normal saline, depending upon the systemic electrolyte status). If no change in ICP was achieved or a significant volume of solution was aspirated from the stomach, intravenous mannitol was administered and another trial of the drug was initiated 4–24 h later. For the six patients in which data were reported in this study, maximum ICP reduction and maximum serum glycerol concentration occurred around 60–90 min after oral bolus ingestion. In most cases, the serum glycerol concentration returned to pre-treatment levels around 3 h after oral administration.

Given the dose- and time period-range reported in this study, the Panel calculated that the dose of glycerol required to induce a therapeutic reduction in ICP was within the range of 125–333 mg/kg bw per hour.

The Panel therefore considered that a conservative estimate of the lowest oral bolus dose of glycerol required for therapeutic effect was 125 mg/kg bw per hour. The Panel considered this dose would also be responsible for the side effects (nausea, headache and/or vomiting) observed in some patients.