Toxicological Evaluation and Limit Values for Nonylphenol, Nonylphenol Ethoxylates, Tricresyl, Phosphates and Benzoic Acid4. Toxicity, animal data4. Toxicity, animal data As for humans, the animal toxicity data include some information based sodium benzoate where relevant to describe the toxicological profile of benzoic acid. Conversion factor: 100 mg sodium benzoate is equivalent to 87.4 mg benzoic acid. A lot of animal exposure data exists on benzoic acid/sodium benzoate, however the majority of the studies mentioned below are of an earlier date and do not meet the requirement of today’s quality guidelines. For most of the studies the dose levels are given as percentage benzoic acid/sodium benzoate in the feed and therefore conversion from % in feed to mg/kg bw/day, which is needed for setting an ADI/TDI, has been carried out. In some cases this has been done according to a method used by IUCLID 1996, in other cases the following, widely used, calculation has been applied: Young rats consume 100 g feed/kg bw/day, old rats consume 100 g feed/kg bw/day and mice consume 150 g feed/kg bw/day. 4.1 Short term toxicity Inhalation An acute one hour inhalation toxicity study in rats using benzoic acid, stated to be in the vapour phase, has shown that LC50 is > 0.026 mg/l. The one hour exposure lead to generalised inactivity and lachrymation, but no mortality (Bio-Fax data sheet 1973 - quoted from IUCLID 1996). In a well performed four weeks study Sprague-Dawley rats (10 animals/ sex/group) were exposed by inhalation to benzoic acid (generated as a dust with an equivalent aerodynamic diameter of 4.7 µm). The animals were exposed to 0, 0.025, 0.25 or 1.2 mg benzoic acid/l for 6 hours/day, 5 days/week. All high- and mid-dose animals exhibited clinical signs of upper respiratory tract irritation (red material around the nares). Two animals (one/sex) in the high-dose group died. The cause of death was not determined. Statistically significant decrease in body weight gain, absolute and relative weights of liver, kidneys and trachea/lungs in high-dose animals were seen. Treatment related decrease in the number of blood platelets was seen in both sexes. The histopathological examination revealed compound-related lesions in the lungs: an increase in the intensity and extent of interstitial inflammatory cell infiltrate and an increase in the incidence and intensity of interstitial fibrosis. The lung lesions were seen in all dosed groups, and with respect to the cell infiltration the effect seems dose related. The author stated that the results of the study indicate that levels as low as 0.025 mg/l benzoic acid for four weeks produce toxic effects in the lungs (EPA/OTS 1992). Oral administration LD50-values Acute oral toxicity studies in mice, rats, rabbits and dogs have resulted in LD50 values within the range of 1700-2500 mg benzoic acid/kg bw. By i.p. and i.v. dosing slightly lower doses LD50 values were obtained. (Opdyke 1979, JECFA 1983, IUCLID 1996). Short-term studies The data are summarised in Table I. rats Groups of five male and five female Sherman rats were fed sodium benzoate for 30 days at levels ranging from 16-1090 mg/kg bw/day (corresponding to 14-923 mg benzoic acid/kg bw/day). A control group was included. No clinically adverse effects or histopathological changes in organs were detected. (Smyte & Carpenter 1948 - quoted from JECFA 1983). Twenty eight young rats (strain unknown) were given 0 or 5% sodium benzoate in the diet for three weeks. The animals consumed 39 g feed/ day which roughly corresponds to 3800 mg/kg bw/day expressed as benzoic acid. Nineteen animals died within two weeks and all others died within the following week. The food consumption was significantly reduced, the animals suffered from diarrhoea and crusted blood was seen around the nares. At autopsy intestinal bleeding was observed. (Kieckebusch & Lang 1960). Groups of three male and three female Sherman rats were fed 0, 2 and 5% sodium benzoate in the diet for 28 days (corresponding roughly to 1700 and 4250 mg/kg bw/day, expressed as benzoic acid). All animals on the 5% level died within the first two weeks showing hyperexcitability, urinary incontinence and convulsions. At the 2% level a decrease in body weight (only statistically significant for males) compared to that of controls was seen. No necropsy or histopathology data were reported. (Fanelli & Halliday 1963). Groups of 5-10 male Wistar rats were given 0 or 3% benzoic acid in the diet for five days (approx. 2250 mg/kg bw/day). After 4-5 days the animals showed disorders of the nervous system (excitation, ataxia and convulsions). After 3-5 days brain damage was demonstrated histologically. Another similar study with 15 male Wistar rats showed that the brain lesions were still present 19-30 days after cessation of dosing. (Kreis et al. 1967 - quoted from IUCLID 1996). Groups of 10 male and 10 female F344 rats were fed sodium benzoate in the diet at levels of 0, 0.5, 1, 2, 4 and 8% for six weeks (approx. 320-5120 mg /kg bw/day expressed as benzoic acid). All rats on the 8% level and 19 rats on the 4% level died within four weeks. The author reported that animals treated with sodium benzoate showed "hypersensitivity" (probably hyper reactivity) as an acute effect. No other symptoms were seen. A statistically significant reduction in body weight was noted in the two highest dose groups compared to control group. By necropsy atrophy of the spleen and lymphnodes was observed in rats fed 4 and 8% sodium benzoate. (Sodemoto & Enomoto 1980). Six male and six female F344 rats were fed 0, 1.81, 2.09 or 2.40% sodium benzoate for 10 days (approx. 1150, 1330 or 1525 mg/kg bw/day, expressed as benzoic acid). One male in the 2.40% group, who showed hypersensitivity followed by convulsions, died. The mean body weight of both sexes in the highest dose group was statistically significantly reduced compared with the controls. The relative liver and kidney weights were statistically significantly increased at the 2.40% level in both sexes and in the liver in males at the 2.09% level. Histopathological examination were carried out on liver and kidneys from the 2.40% level. Only the liver from high dosed males showed signs of toxic effect. Eosinophilic foci in the periportal area and enlargement of the hepatocytes with glassy cytoplasm was seen. Statistically significant changes in some clinical chemistry parameters (e.g. albumin) compared to controls were seen at the 2.09 and 2.40% level. (Fujitani 1993). Benzoic acid was given orally to 4 groups of 25 CDBR rats at doses of 0, 30, 160 or 450 mg/kg bw/day on days 7 to 16 of gestation. Four deaths occurred in the high dose group. Further, at this dose level reduction in body weight , reduced food intake and increased liver weight were seen. Haemorrhages in the gastric mucosa was seen in the rats which died. (EPA/OTS 1992 - abstract quoted from Toxline 1990-1993). mice Albino Swiss mice, 4 mice per sex per group, were given 0, 0.5, 1, 2, 4, or 8% sodium benzoate in drinking water (approx. 0, 1000, 2000, 4000, 8000 or 16000 mg/kg bw/day, expressed as benzoic acid) for 35 days. Based upon survival rate, body weight and histological changes (not specified) the 2% dose level were found suitable for a life span study (but not stated as a NOAEL). All animals at the 8% level and 3/4 in both sexes died at the 4% level. (Toth 1984). Five male and four to five female B6C3F1 mice were fed 0, 2.08, 2.5 or 3% sodium benzoate for 10 days (approx. 2550, 3180 or 3810 mg/kg bw/day, expressed as benzoic acid). All of the animals in the 3% group showed "hypersensitivity" (probably hyper reactivity) as an acute effect. and 1/5 of males and 2/5 of females showed convulsions. Two females died. Absolute and relative liver weights (both sexes) and relative kidney weights (females) were dose-dependently increased (statistically significant from controls at 3% the level). Histopathological examination were carried out on liver and kidneys from the 3% level. Only the liver from high dosed males showed signs of toxic effect. Enlarged hepatocytes with eosinophilic cytoplasm, occasionally single cell necrosis and vacuolation of hepatocytes was seen. Statistically significant changes in some clinical chemistry parameters (e.g. cholinesterase) compared to controls were seen at the 2.5 and 3% level. (Fujitani 1993). cats Cats have a higher sensitivity to benzoic acid compared with other species (including humans) which may be due to its lack of ability to form benzoyl glucuronic acid (JECFA 1983). The application of 0 or 0.5% benzoic acid in the diet for 3-4 days to four male cats (approximately 300-420 mg/kg bw/day) resulted in convulsions, aggression, and hyperaesthesia. Two cats died. A histological examination revealed toxic effects in the liver (swollen hepatocytes with cell infiltration) and kidneys (swollen tubular cells). (Bedford & Clarke 1972 - quoted from IUCLID 1996). Four male cats were given 0, 100 or 200 mg benzoic acid/kg bw/day in the diet for 15 days. In another study four male cats were fed a diet containing 0 or 0.25% benzoic acid (approximately 130-160 mg/kg bw/day) for 23 days. No effects were observed. (Bedford & Clarke 1972 - quoted from IUCLID 1996). An outbreak of poisoning in 18 cats following ingestion of meat containing 2.39% benzoic acid have been reported. The effects were convulsions, nervousness, excitability, and loss of balance and vision. Seventeen cats died or were killed. Damage to the intestinal mucosa and liver was seen at autopsy. (Bedford & Clarke 1971 - quoted from JECFA 1983). Table I. Results of short-term oral toxicity studies with benzoic acid or sodium benzoate.
* The test compound is given as benzoic acid or sodium benzoate. See the text for further specification. The bracket gives the value converted into benzoic acid. bb: benzoic acid. n.a.d: no abnormalities detected. n.d.r: no data reported. Dermal contact Acute dermal toxicity studies in rabbits have shown that the LD50 value of benzoic acid exceeds both 5000 mg (Opdyke 1979) and 10000 mg/kg bw (Bio-Fax data sheet 1973 - quoted from IUCLID 1996). No information concerning mortality is available for the first mentioned study. No deaths were observed among rabbits given > 10000 mg/kg bw. skin irritation From a skin irritation study on rabbits, performed according to relevant guideline, it was concluded that benzoic acid is minimally irritating to the skin (RRC NOTOX B.V. 1988 - quoted from IUCLID 1996). In a similar study design sodium benzoate did not cause skin irritation (RRC NOTOX B.V. 1988 - quoted from IUCLID 1996). skin sensitisation Different types of sensitisation tests have been carried out on guinea pigs and mice. No sensitising effect was seen. (Gad et al. 1986 and Gerberick et al. 1992 - quoted from IUCLID 1996). Eye irritation Eye irritation studies in rabbits performed according to relevant guidelines revealed slightly (benzoic acid) to severely (sodium benzoate) irritating properties (Suberg 1986 and RRC NOTOX B.V. 1988 - quoted from IUCLID 1996). 4.2 Long term toxicity Inhalation No data have been found. Oral administration The data are summarised in Table II. rats A 90-days feeding study was carried out on groups of 8-10 Sherman rats. The animals were given sodium benzoate at levels of 0, 1, 2, 4 or 8% in the diet (approx. 540-5160 mg /kg bw/day expressed as benzoic acid). In the 8% group 4/8 died. The four survivors showed reduced weight gain compared to that of the controls. Increased kidney and liver weights together with pathological lesions (not specified) were seen in the 8% group. At the lower levels no adverse effects were observed. (Deuel et al. 1954 - quoted from JECFA 1983 and IUCLID 1996). In a chronic feeding experiment continuing over 4 generations, three groups of 20 male and 20 female rats (strain unknown) were fed 0, 0.5 and 1% benzoic acid (1% benzoic acid mentioned as equal to 150 mg/rat/ day, roughly corresponding to 600 mg/kg bw/day). The third generation was subjected to histopathological investigation after 16 weeks on test and organ weight was recorded for brain, liver, heart, spleen, kidney and testes. 1% benzoic acid was tolerated without adverse effect on growth, food utilisation and duration of life. No differences with respect to organ weights and histopathological findings (organ not mentioned, but probably the same as recorded for organ weights) were seen between dosed and control animals. The authors stated that 1% benzoic acid in the diet is near the upper limit of tolerability. (Kieckebusch & Lang 1960). Twenty male and 30 female rats were fed a diet containing 1.5% benzoic acid (approx. 1125 mg/kg bw/day) for 18 months. 13 males and 12 females served as controls. Reduced food intake, growth retardation and increased mortality (15/50 vs. 3/25 in the control) was seen in the dosed animals. No pathological data was recorded. (Marquardt 1960 - quoted from JECFA 1983 and IUCLID 1996). mice 50 mice per sex were given 0 or 80 mg benzoic acid /kg/day by gavage for 12 weeks. Reduced weight gain without reduced food intake was observed. No post mortem data recorded. (Shtenberg & Ignat’ev 1970 - quoted from JECFA 1983 and IUCLID 1996). dogs Seventeen fox terriers were fed 0.1 - < 7 g benzoic acid or sodium benzoate (not otherwise specified) once daily during 250 days. Seven g/day (approx. 1000 mg/kg bw/day) induced ataxia, convulsions and death. Below this level no adverse effects were seen. (Rost et al. 1933 - quoted from JECFA 1983 and IUCLID 1996). Table II. Results of long-term oral toxicity studies with benzoic acid or sodium benzoate.
* The test compound is given as benzoic acid or sodium benzoate. See the text for further specification. The bracket gives the values converted into benzoic acid. bb: benzoic acid. n.a.d: no abnormalities detected. n.d.r: no data reported. Dermal contact No data have been found. 4.3 Reproductive / developmental effects In a four generation feeding experiment three groups of 20 male and 20 female rats (strain unknown) were fed 0, 0.5 and 1% benzoic acid (1% benzoic acid mentioned as equal to 150 mg/rat/day, roughly corresponding to 600 mg/kg bw/day). No effects on fertility and litter size neither at birth or weaning, were observed. (Kieckebusch & Lang 1960). Rats were given 0, 1, 2, 4, or 8% sodium benzoate (approx. 590-4720 mg/kg bw/day expressed as benzoic acid) in the diet during the whole gestation period (20 days). 4 and 8% sodium benzoate caused maternal toxicity, 100% perinatal death and many abnormalities of organs (eye, brain and kidneys) and skeletal system were found in foetuses from these groups. No statistically significant differences in organs and skeletal abnormalities were detected between the 1 and 2% levels and the controls. Maternal as well as teratogenic NOAEL was 2% corresponding to 1180 mg/kg bw/day. (Onodera et al. 1978 - quoted from IUCLID 1996). Benzoic acid was given orally to 4 groups of 25 CDBR rats at doses of 0, 30, 160 or 450 mg/kg bw/day on days 7 to 16 of gestation. Four deaths occurred in the high dose group. At this dose level reduction in body weight , reduced food intake and increased liver weight were seen. Reduction in body weight was also observed at 160 mg/kg/day. No compound-related effects on reproductive parameters (not specified) were observed. Foetal weight gain was significantly decreased at the two highest dose levels. Other effects reported in the high dose group were significant increases in malformations (not specified), foetal development variations (not specified), and variations due to retarded development (not specified). The maternal and foetal NOAEL reported in this study was 30 mg/kg/day. (EPA/OTS 1992 - abstract quoted from Toxline 1990-1993). In rats (up to 150 mg/kg bw/day on days 6 to 15 of gestation), mice (up to 150 mg/kg bw/day on days 6 to 15 of gestation), rabbits (up to 210 mg/kg bw/day on days 6 to 18 of gestation) and hamsters (up to 250 mg/kg bw/day on days 6 to 10 of gestation) sodium benzoate (value expressed as benzoic acid) was given orally. No effect on nidation or foetal and maternal survival was seen and the number of abnormalities of soft and skeletal tissues did not differ from controls. No maternal toxicity was reported. (Food and Drug Research Laboratories, Inc. - quoted from IUCLID 1996). It has been shown that benzoic acid penetrate the placental barrier readily after s.c. administration the maternal rats (Maickel & Snodgrass 1973 - quoted from IUCLID 1996). In an uterotrophic assay performed on mice and rats it was found that benzoic acid does not possess oestrogenic properties. This finding was supported by an in vitro test. (Ashby 1997). 4.4 Genotoxic effects In vitro tests mutagenicity Benzoic acid and sodium benzoate was not found mutagenic when tested in Ames test and E. coli reversion assay with and without metabolic activation (Ishidate et al. 1984, Zieger et al. 1988 and Prival et al. 1991 - all three quoted from IUCLID 1996). Ambiguous results was observed when sodium benzoate was tested in a B. subtilis recombination assay with and without metabolic activation (Ishizaki & Ueno 1989 - quoted from IUCLID 1996). chromosomal aberration Benzoic acid was tested in two different Chinese hamster cell lines without metabolic activation; negative or ambiguous results were obtained (Ishidate et al. 1984 - quoted from IUCLID 1996). Sodium benzoate was tested in two different Chinese hamster cell lines without metabolic activation; positive results were seen in both cell lines (Ishidate et al. 1977,1984 and Abe & Sasaki 1977 - quoted from IUCLID 1996). effects on DNA Sodium benzoate was tested for Sister Chromatide Exchange in Vicia faba root tip cells, hamster cell line and human lymphocytes without metabolic activation; a positive result in Vicia faba root tip cells and human lymphocytes was observed (but only one dose level tested). Ambiguous result was seen in the hamster study. (Abe & Sasaki 1977 and Xing & Zhang 1990 - quoted from IUCLID 1996). Benzoic acid was tested in human lymphocytes without metabolic activation; a negative result was obtained. (Tohda et al. 1980 and Jansson 1988 - quoted from IUCLID 1996). In vivo tests chromosomal aberration Rats were given 50-5000 mg sodium benzoate/kg orally for one to five days. No detectable significant aberrations of the bone marrow chromosomes. Negative results were also obtained in a dominant lethal assay using the same study design (Litton Bionetics Inc. 1974 - quoted from IUCLID 1996). 4.5 Carcinogenic effects rats Rats were fed 0, 1 or 2% sodium benzoate in the diet (approx. 500-1000 mg/kg/day) for 18-24 months. No differences in mortality were seen. No significant differences between groups with respect to number of tumour-bearing animals and time to occurrence of tumours. (Sodemoto & Enomoto 1980). mice Sodium benzoate was administered as a 2% solution in drinking water (approx. 4000 mg/kg/day) for the life span to 50 mice per sex. The control group consisted of 100 animals per sex. The author reported that no carcinogenic effect observed and the treatment has no effect on survival (Toth 1984). However, a higher incidence and earlier onset of mammary gland tumours was seen in dosed females (8%) compared to controls (2%). No statistical analysis were performed. The study is only reported as a short communication.
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