708. Erythorbic acid and its sodium salt (WHO Food Additives Series 28)

ERYTHORBIC ACID AND ITS SODIUM SALT

First draft prepared by
Dr R. Walker, Professor of Food Science,
Department of Biochemistry, University of Surrey, England.

1. EXPLANATION

Erythorbic acid (syn: isoascorbic acid, D-araboascorbic acid)
is a stereoisomer of ascorbic acid and has similar technological
applications as a water-soluble antioxidant. This compound was
previously evaluated under the name isoascorbic acid by the sixth
and seventeenth meetings of the Committee (Annex 1, references 6 and
32); at the last evaluation an ADI of 0-5 mg/kg b.w. was allocated,
based on a long-term study in rats, and a toxicological monograph
was prepared (Annex 1, reference 33). The name was changed to
erythorbic acid in accordance with the "Guidelines for designating
titles for specifications monographs" adopted at the thirty-third
meeting of the Committee (Annex 1, reference 83).

Since the previous evaluation further data have become
available and are included in the following monograph. The
previously published monograph has been expanded and is reproduced
in its entirety below.

2. BIOLOGICAL DATA

2.1 Biochemical aspects

2.1.1 Absorption, distribution and excretion

Erythorbic acid is readily absorbed and metabolized. Following
an oral dose of 500 mg of erythorbic acid to human subjects the
blood level curves for ascorbic acid and erythorbic acid showed a
similar rise. In five human subjects, an oral dose of 300 mg was
shown to have no effect on urinary excretion of ascorbic acid (Kadin
& Osadca, 1959). Erythorbic acid was found to have no antagonistic
effect on the action of ascorbic acid (Gould, 1948).

In hamster, rat and rabbit, for which ascorbate is not an
essential vitamin, intestinal absorption of L-ascorbic acid is low
and takes place by passive diffusion; conversely, in guinea pig and
human, ascorbate absorption is mediated by a saturable, sodium-
dependent, active transport mechanism. It follows that the former
species are not suitable models for human absorption. Since the
active transport system is saturable but since passive diffusion
might also be significant at high dose levels, the absorption of
ascorbic acid is dose dependent (Kallner et al., 1977; Kübler &
Gehler, 1970). Erythorbic acid appears to be another but much
poorer substrate for the same transport system and may thus act as a
weak competitive inhibitor of L-ascorbate uptake (Siliprandi et
al., 1979; Mellors et al., 1977). In studies using isolated
brush border vesicles from guinea pig ileum, the K₁ has variously
been estimated at about 11mM (Toggenburger et al., 1979) and
around 20mM (Siliprandi et al., 1979); this compares with an
apparent K_m for ascorbate uptake of about 0.3mM in the same
system.

In studies using gut sections of humans and guinea pigs, the
rate of ascorbate uptake was reduced by only 16% when erythorbate
was added at ten times higher concentrations. In other species
(hamster, rat, rabbit) in which absorption is by passive diffusion,
erythorbate is without effect on ascorbate absorption (Mellors et
al., 1977).

After i.p. or oral administration of [1-¹⁴C]-erythorbic acid
to guinea pigs, most of the labelled material was excreted within
24h after dosage, only minute amounts being retained in the tissues.
After i.p. administration, more than 75% of the dose was excreted in
urine, about 20% was exhaled as CO₂, and about 3% of the label
appeared in the faeces. The corresponding figures after oral
administration were 54% as CO₂ (after 12h), 30% in urine and 4% in
faeces. The highest activity in tissues was found in liver, lung
and kidneys but each organ accounted for less than 1% of the dose.

After simultaneous oral administration of equal amounts of [1-
¹⁴C]-erythorbic and [6-³H]-ascorbic acids there were no
differences in specific activities of the two in portal blood (ratio
0.8-1.15) during the first few hours but after 3.5h the ratio in
adrenals, liver, lung and spleen was approximately 4 in favour of
ascorbic acid, indicating a preferred uptake of ascorbic acid
compared to erythorbic acid. Lower ratios were found in kidney,
reflecting the large renal excretion rate of erythorbic acid
relative to ascorbic acid (Hornig, 1975).

The ascorbic acid transport system in to the brain and
cerebrospinal fluid is stereospecific with erythorbic acid being
transported in vivo less effectively than ascorbic acid (Spector &
Lorenzo, 1974).

When guinea pigs on a low ascorbate diet were given a
supplement of ascorbic acid or erythorbic acid at daily doses of 1.5
mg/kg b.w., ascorbic acid was deposited in the tissues while
erythorbic acid was not. Intramuscularly administered erythorbic
acid was not retained in the tissues to the same extent as a similar
dose of ascorbic acid. The authors concluded that ascorbic acid is
more readily absorbed from the gastro-intestinal tract and more
readily abstracted from the blood and/or retained by the tissues
than is erythorbic acid (Hughes & Hurley, 1969). When ascorbic acid
or erythorbic acid were administered in drinking water at doses of
180 mg/1, tissue levels of ascorbate were higher than erythorbate in
the tissues examined (spleen, adrenals, brain and eye lens). The
ratio of ascorbate: erythorbate concentrations achieved varied from
8:1 in the spleen to 2.8:1 in the brain. At higher concentrations
of 1% in drinking water, higher tissue levels of either ascorbate or
erythorbate were found and differences in tissue concentrations
between the two substances was reduced, the ratio varying from 1.7:1
in spleen to 1.1:1 in brain. It was concluded that the differences
arose from the lower absorption efficiency of erythorbic acid and
that this was partially overcome when higher concentrations were
given in drinking water rather than as a single supplement (Hughes &
Jones, 1970). It appears that in these high concentration
conditions, the active absorption mechanism for ascorbic acid might
have been approaching saturation with much of the dose of both
compounds being absorbed by passive diffusion.

The erythorbic acid content of the tissues (liver, adrenals,
kidneys and spleen) of guinea pigs was compared with that of
ascorbic acid after oral administration of the compounds at doses of
1, 5, 20 (erythorbic only) and 100 mg/day for 16 days. Only a small
amount of erythorbic acid was found in the four organs of animals
given 20 mg or more of erythorbic acid; conversely, ascorbic acid
was detected in the tissues of animals from all dose groups. Even
at the highest dose, much less erythorbic acid was retained in these
tissues than ascorbic acid (Suzuki et al., 1987).

Guinea-pigs were fed diets containing either 2% erythorbic acid
or 0.1% ascorbic acid for a period of 9 days followed by a depletion
period of 4 days during which they received an ascorbic acid-
deficient diet. At the end of the 9 day period, tissue levels of
erythorbic acid were about twice those of ascorbic acid, despite the
20-fold difference in dose. After the 4-day depletion period,
erythorbate levels were much lower than the ascorbate concentrations
in all the tissues examined except the adrenals. From the
calculated turnover rates, the t_1/2 was estimated to be 4-5 times
shorter for erythorbic acid than for ascorbic acid in all the organs
viz: brain, liver, heart, kidneys, adrenals and spleen.
Furthermore, erythorbic acid increased rather than decreased the
turnover of ascorbic acid (Pelletier, 1969b).

Comparative studies on the transport of ascorbic and erythorbic
acids across various tissue membranes have been carried out in vivo
using the guinea pig eye (Linner & Nordstrom, 1969) and in vitro
using guinea pig and rabbit ocular ciliary body-iris preparations
(Becker, 1967; Chu & Candia, 1988), cat retinal pigment cells
(Khatami et al., 1986), rabbit choroid plexus membranes (Spector &
Lorenzo, 1974) and human placental microvillus membranes (Iioka et
al., 1987). In all cases, a carrier-mediated ascorbate transport
system was identified which was inhibited competitively by
erythorbic acid. However, the affinity of erythorbate for the
carrier was weak and the K_m for ascorbate was in all cases several
times lower than the K₁ of erythorbate.

There are significant differences between ascorbic and
erythorbic acids in renal excretion in humans. Studies in vitamin
C-depleted humans indicated that the rate and extent of urinary
excretion of erythorbic acid are much greater than those of ascorbic
acid. At oral doses of 50-300 mg per person about 50-70% of the
dose of erythorbic acid was excreted in 24 hour urine (mainly in the
first 6 hours) but only 15% of a 100 mg dose of ascorbic acid
appeared in urine; the rate of urinary excretion of erythorbic acid
was 10-15 times that of ascorbic acid. (Ikeuchi 1955). Similar
results have been obtained in later studies in humans (Wang et al.,
1962; Rivers et al., 1963).

In guinea pigs receiving daily doses of ascorbic acid (2 mg/d)
or erythorbic acid (40 mg/d) the 12h urinary excretion of these two
compounds was found to be 0.13% and 1.9% of the daily dose
respectively at the end of the experiment. No further metabolites
of erythorbic acid were identified although the authors pointed out
that as so little was incorporated into organs it would be of
interest to determine how it is metabolized (Pelletier & Godin,
1969).

In trout and rats which received ¹⁴C-labelled ascorbic or
erythorbic acid (dose not specified) the rate of excretion of label

was about 10 times faster for erythorbic than ascorbic acid. The
primary urinary metabolite of ascorbate was ascorbate-2-sulfate but
the corresponding erythorbate-2-sulfate could not be detected in the
urine of rats receiving erythorbic acid (Baker et al., 1973).

At very high levels of intake (5% in the diet) in the rat, the
urinary concentration of erythorbate was about twice as high as that
of ascorbate (Fukushima et al., 1984).

In dogs, less marked differences in excretion between ascorbic
acid erythorbic acids have been reported. From equivalent doses on
a molar basis of 5 g of sodium erythorbate or 4.1 g of ascorbic
acid, about 19% and 12% of the dose respectively was excreted within
24 hours (Robinson & Umbreit, 1956). After a single i.v. dose of 1
g ascorbic acid or 1.23 g sodium erythorbate the plasma half-lives
were similar, indicating a similar rate of elimination (Robinson et
al., 1956).

Ascorbic acid is believed to be recovered from the glomerular
filtrate by active renal reabsorption involving a sterospecific,
energy-requiring transport process (Ahlborg, 1946). On the basis of
studies conducted in brush border membrane vesicles from kidney
cortex in vitro, erythorbate appears to be another, but poorer
substrate, of the same transport system. In view of the more than
fifty-fold difference between the K_m for ascorbate and the
competitive K₁ for erythorbate (approximately 16mM) (Toggenburger
et al., 1981) it appears unlikely that erythorbate would reduce
the reabsorption of ascorbate from the glomerular filtrate under
conditions likely to be encountered in vivo.

Since erythorbic acid is less readily resorbed than ascorbic
acid in the kidney, this affords a rational explanation for the
observation that whereas a daily intake of 600 mg erythorbic acid by
non-pregnant women resulted in a steady state plasma concentration
of about 16 µmol/1, a similar plasma level of ascorbic acid required
an intake only 60 mg/d (Sauberlich et al., 1989).

2.1.2 Biotransformation

The metabolism of erythorbate has not been examined in detail.
The occurrence of dehydro-erythorbate in the urine of erythorbate-
fed rats (Fukushima et al., 1984) indicates that ascorbate oxidase
may accept both stereoisomers as substrates. However, although
ascorbate-2-sulfate is quantitatively an important metabolite of
ascorbate in trout and rat, erythorbate-2-sulfate could not be
detected in these species (Baker et al., 1973).

There are indications that oxalate is a minor metabolite of
ascorbic acid and high doses are associated with an increase in
urinary oxalate. The urinary excretion of oxalate was examined in

women receiving increasing increments of 30, 60 or 90 mg ascorbic
acid per day for 10 days in the presence or absence of 600 mg/d of
erythorbic acid. Increasing the ascorbic acid intake from 30 mg/d
to 90 mg/d increased daily oxalate excretion by 67 µmol but an
intake of 600 mg/d of erythorbic acid increased daily oxalate
excretion by 67-133 µmol. This indicates that little if any of the
erythorbic acid was metabolized to oxalate (Sauberlich et al.,
1989).

2.2 Toxicological studies

2.2.1 Acute toxicity

Species Sex Route LD₅₀ Reference
(mg/kg b.w.)

Mouse Male oral 8,300 Orahovats, 1957

Rat Male oral 18,000 Orahovats, 1957

2.2.2 Short-term studies

2.2.2.1 Mice

Six groups of 10 male and 10 female eight week-old B6C3F₁ mice
were given sodium erythorbate in drinking water at concentrations of
0, 0.625, 1.25, 2.5, 5 or 10% for 10 weeks. At termination, all
survivors were sacrificed, autopsied and the major visceral organs
examined histologically.

The mean body-weight gain of male mice given 5% sodium
erythorbate was less than 90% of that of controls but the females in
this dose group had a higher weight gain than controls and it was
concluded that the MTD for males and females was 2.5% and 5%
respectively. Histological examination of organs from mice which
had received above the MTD of erythorbate showed marked atrophy of
liver cells, marked atrophy of splenic lymphoid follicles and
hydropic degeneration of renal tubular epithelium. No significant
changes were observed in the visceral organs of control mice nor of
mice which had received erythorbic acid at or below the MTD (Inai
et al., 1989). It is noted that this experiment was not
controlled for sodium ion concentration in the drinking water of
treated animals.

2.2.2.2 Rat

Groups of 10 male rats were fed for 36 weeks on diets
containing 0 or 1% of erythorbic acid. There was no difference
between treated rats and controls with respect to growth rate and
mortality. Gross post-mortem examination and microscopic studies of
various organs revealed no lesions attributable to erythorbic acid
(Fitzhugh & Nelson, 1946).

Six groups of 10 male and 10 female F344/DuCrj rats were given
sodium erythorbate in drinking water at concentrations of 0, 0.625,
1.25, 2.5, 5.0 or 10.0% for 13 weeks. All rats given the 10%
solution refused to drink and died within 2 to 5 weeks. Three males
and one female out of the group receiving 5% erythorbate died
during the first 4 days. All the rats receiving erythorbate at
concentrations of 2.5% or less survived to the end of the study.
The 2.5% solution depressed body weight gains by 12% in males and 6%
in females compared to untreated controls. This study was a pilot
for a long-term carcinogenicity study and no further details were
reported (Abe et al., 1984).

2.2.2.3 Guinea pig

In an experiment on the influence of protein-induced
differences in growth rate on tissue concentrations of erythorbic
acid, two groups of 10 male guinea pigs were fed an ascorbic acid-
free diet with either dried skimmed milk or gluten as protein
source. After a vitamin C depletion period of 10 days, the animals
were given daily doses of sodium erythorbate of 800 mg/kg b.w.
intraperitoneally. No adverse effects to the treatment with
erythorbate were noted and body weight gains were similar to those
of guinea pigs receiving ascorbic acid orally at a dose of 5 mg/kg
b.w. The highest tissue levels of ascorbic acid were found in the
adrenals and dietary protein source had no influence on organ
distribution (Williams & Hughes, 1972). Note that in this study,
erythorbic acid i.p. showed antiscorbutic activity (see also 2.2.12
Special studies of vitamin C activity of erythorbic acid).

2.2.2.4 Dog

Groups of 2 male and 2 female beagles received per os daily
doses of either 1 g erythorbic acid for 240 days or 5 g ascorbic
acid for 50 days then 7.5 g for a further 190 days; a third group
served as control. No signs of toxicity were observed.
Biochemical, haematological and urine analysis showed no treatment
related changes in haemoglobin, haematocrit, RBC and WBC counts,
differential counts, sedimentation rate, urea N, fibrinogen,
glucose, total and free cholesterol, total protein, albumin,
globulin, inorganic phosphorus, alkaline phosphatase nor in urinary
S.G., pH, urine blood, sugar or protein. At termination all the

animals were autopsied and no gross or histological changes were
found (Orahovats, 1957). Note that this report was available in
summary only.

2.2.3 Long-term/carcinogenicity studies

2.2.3.1 Mice

Sodium erythorbate was administered to groups of 50 male
B6C3F₁ mice in drinking water at concentrations of 0, 1.25 or 2.5%
(MTD); groups of 50 females received concentrations of 0, 2.5 or 5%
(MTD). Treatment commenced at eight weeks of age and continued for
96 weeks; the test substance was then withdrawn for a further 14
weeks when the study was terminated. The animals were weighed at
regular intervals throughout and any mouse found dead or moribund in
the course of the experiment was autopsied. At termination all
surviving animals were sacrificed and autopsied. All visceral
organs and any tumours were weighed and examined grossly and
histologically.

The mean body weights of the treated mice were generally
similar to those of controls throughout but, at the end of the
study, there was a dose-related increase in body weight and an
associated decrease in relative organ weights of heart, lungs,
kidney and brain. Survival rates were higher in treated mice.
Histological examination did not show any significant differences
between the treated and control groups.

Tumours were observed at various sites including liver,
haematopoietic system, lung and integumentary tissue but the tumour
incidence, time to death with tumours or histological distribution
of tumours differed significantly from untreated controls at none of
the sites. The authors concluded that the study did not demonstrate
any tumorigenic effect of sodium erythorbate on oral administration
to B6C3F₁ mice (Inai et al., 1989).

2.2.3.2 Rat

Groups of 10 rats (sex not specified) were fed diets containing
0 or 1% erythorbic acid for two years. The growth rate, mortality
and histopathology were not affected by the treatment (Lehman et
al., 1951).

Groups of 52 male and 50 female F344/DuCrj rats were given
sodium erythorbate in drinking water at concentrations of 0
(control), 1.25 or 2.5% from 8 weeks of age for 104 weeks. The
sodium erythorbate was then removed and the animals received plain
tap water for a further 8 weeks when the experiment was terminated.
Autopsies were performed on all rats; major organs and lesions
(details not given) were prepared for histological examination.

At the 2.5% level, sodium erythorbate significantly inhibited
weight gain in both sexes from weeks 40 to 90, the deficit being
maximally 8.5% in males at week 88 and 15.5% at week 85 in females,
relative to controls. No suppression of weight gain was observed at
the 1.25% dose level. The total intakes achieved in the treated
groups were estimated to be 217 g/rat and 430 g/rat for males in the
1.25% and 2.5% dose groups respectively; the corresponding intakes
for females were 206 and 583 g/rat. Between 60% and 82% of animals
in the various groups survived the treatment period and the mean
lifespan of tumour bearing rats was similar in the three groups,
viz: 117, 114 and 111 weeks for control, 1.25% and 2.5% males, and
114, 113 and 113 for the corresponding females.

All males except two in the higher dose group showed testicular
interstitial-cell tumours (typical of the strain of rat used). The
incidence of other tumours was 80%, 69% and 78% in control, 1.25%
and 2.5% males respectively, the more common tumours encountered
being leukaemia, phaeochromocytoma, mammary fibroadenoma and
mesothelioma with incidences of 6 - 18%. Aggregate tumour
incidences in females were 94%, 88% and 78% in control, 1.25% and
2.5% groups respectively, the latter group incidence being
significantly lower than controls, and the pattern of occurrence of
various tumour types was similar in the three groups. There was no
treatment-related acceleration in tumour development nor in
malignant transformation of benign tumours and the authors concluded
that sodium erythorbate was not carcinogenic to F344 rats (Abe et
al., 1984).

2.2.4 Special studies on bone mineralization

Male guinea pigs, 14 days old, were given 8.7% ascorbic acid in
the diet for up to 8 weeks. Two comparison groups were fed
corresponding amounts of ascorbate or erythorbate as a mixture of
the respective sodium, potassium and calcium salts and a control
group received 0.2% ascorbic acid. In a later 6-week experiment
using a similar protocol, guinea pigs were given 8.7% free
erythorbate acid in the diet. The results of bone and urinary
analysis demonstrated that the animals given 8.7% ascorbic acid had
decreased bone density and lower urinary hydroxyproline compared to
controls. No significant bone changes were observed in any of the
other groups, including those animals given free erythorbic acid.
It appears that the observed bone demineralization was a combination
of an effect of an acid diet together with a more specific effect of
ascorbate, not shared by erythorbate (Bray & Briggs, 1984).

2.2.5 Special studies on collagen and elastin synthesis in vivo

Based on observations in cultured smooth muscle and fibroblast
cells, it was hypothesized that elevated ascorbic acid levels should
increase collagen and decrease elastin deposition in neonatal rat

lung and a comparative study was conducted with erythorbic acid.
Rat pups, 2 days post partum, were given ascorbic or erythorbic
acids by daily oral gavage at a level of 2% of the total consumed
milk solids for 19 or 23 days; controls received saline. No
treatment-related differences were observed in lung, liver or body
weights, or collagen accumulation in the lung. The relative rates
of lung protein and elastin synthesis were lowered by both ascorbic
or erythorbic acid but mechanical lung function (pressure-volume
curves) was not influenced by treatment (Critchfield et al.,
1985).

2.2.6 Special studies on effects on bioavailability and
toxicity of metals

The concentration of metallothioneins, a group of low molecular
weight proteins which form complexes with various heavy metals, in
mouse liver was increased from 26 to 341 µg/g tissue after i.p.
administration of ascorbic acid at a dose of 1 g/kg b.w. The same
dose of erythorbic acid caused a similar increase in metallothionein
levels to 378 µg/g liver (Onasaka et al., 1987). The elevated
metallothionein level caused by ascorbic acid was associated with a
reduced mortality after administration of a lethal dose of cadmium
but erythorbic acid was not included in this test.

Ascorbic acid is known to increase the bioavailability of iron
and the activity of erythorbic acid in this respect was examined in
a haem-repletion assay in iron deficient male weanling rats. The
bioavailability of iron from bologna-type sausages cured with 550
mg/kg erythorbate and 156 mg/kg nitrite was assayed. Groups of 6
rats were limit-fed for 2 weeks diets that contained as their sole
protein sources uncured meat, meat cured with nitrite alone or with
erythorbic acid. Curing with nitrite and/or erythorbate had no
significant effect on iron absorption or iron incorporation into
tissues (Lee et al., 1984; Lee & Greger, 1985). In a critique of
the above study, similar results were confirmed in an analogous
experiment (Mahoney & Hendricks, 1985).

Adult male volunteers received a constant mixed diet which
contained 200g processed meat for 51 days. The processed meats
used were uncured sausage, sausage cured with nitrite (156 mg/kg
meat) and sausage cured with a mixture of nitrite (156 mg/kg meat)
and erythorbic acid (550 mg/kg meat). The dietary treatments had no
significant effects on apparent absorption of iron, zinc or copper,
nor on serum zinc or copper levels, plasma ferritin, transferrin or
ceruloplasmin levels. The authors concluded that commercial curing
processes do not adversely affect the bioavailability of zinc or
copper in meat (Greger et al., 1984; 1985).

2.2.7 Special studies on embryotoxicity and teratogenicity

2.2.7.1 Mouse

Groups of pregnant CD-1 mice were given erythorbic acid at
doses of 0, 10.3, 47.6, 221.9 or 1030 mg/kg by gavage on days 6-15
of gestation. On day 17 the pups were removed by Caesarian section
and the number of implantation sites and urogenital abnormalities
determined in the dams. The number of live foetuses and body
weights were determined and the foetuses were given a gross
examination for abnormalities. Foetuses were then prepared and
examined for skeletal or soft tissue anomalies. None of the
treatment groups showed any significant differences from controls in
these parameters (Food and Drug Research Laboratories, 1974).

2.2.7.2 Rat

Pregnant Wistar rats were fed a diet containing 0 (control),
0.05, 0.5 or 5% sodium erythorbate from day 7 through day 14 of
pregnancy. On day 20 of pregnancy, 5-7 dams from each group were
killed and the foetuses removed for teratological examination. All
gross anomalies were recorded and half of the foetuses from each
litter were examined for skeletal anomalies (Alizarin red). The
remaining foetuses were fixed in Bouin's solution and examined for
soft tissue defects using Wilson's technique.

Another 5 dams from each group were allowed to proceed to
parturition and the number of live and dead offspring delivered was
recorded. The litter size was standardized to 4 males and 4 females
per litter and development of the offspring monitored to weaning for
a further 10 weeks. The dams were killed at weaning and the number
of implantation remnants recorded.

No adverse effect on body weight gain nor any clinical sign of
toxicity was observed in any of the dams during pregnancy. There
were no significant differences between treated and control groups
in the incidence of intrauterine fetal death, number of live
foetuses per dam, sex ratio of fetuses, fetal body weight or
placental weight. External, skeletal and soft tissue examinations
did not reveal any evidence of teratogenicity and the post-natal
development of the offspring of treated dams was uneventful (Ema et
al., 1985).

A teratogenicity study was conducted in pregnant Wistar rats
given sodium erythorbate from day 6 of gestation for 10 consecutive
days at doses of 0, 9.0, 41.8, 194 or 900 mg/kg b.w. per os. No
differences were observed in implantation rates, live births or
gross, skeletal or soft tissue morphological abnormalities (Food and
Drug Research Laboratories, 1974).

2.2.7.3 Chick

Erythorbic acid was tested for embryotoxic and teratogenic
effects to the developing chick embryo under four sets of
conditions. The test compound was administered in aqueous solution
via the air cell or via the yolk at 0 or 96 hours of incubation and
at doses of 0, 0.5, 1, 5, 10 or 20 mg/egg. All the eggs were
candled daily and non-viable embryos removed; survivors were allowed
to hatch. Non-viable embryos and hatched chicks were examined for
gross anomalies (externally and by dissection) and for toxic
responses such as oedema and haemorrhage. Histological examination
was carried out on liver, heart, kidney, lung, brain, intestine,
gonads and some endocrine organs from a representative number of
animals from each group.

Erythorbic acid was quite embryotoxic under all test
conditions, except at the lowest dose level via the air sac at 0
hours. The LD₅₀ was estimated as 3.7 mg/egg and 4.5 mg/egg via the
air cell at 0 and 96 hours respectively. Yolk treatment at 0 and 96
hours resulted in LC₅₀s of 4.6 and 5.4 respectively. The incidence
of structural abnormalities of head, limbs, skeleton or viscera was
not significantly different from sham-treated controls (Hwang &
Conors, 1974). The data do not provide evidence of any teratogenic
effect but the significance of the embryotoxicity is difficult to
ascertain since the experiment was not controlled for sodium ion.

In a comparative study on embryotoxicity in the chick in which
both sodium ascorbate and sodium erythorbate were tested via
administration into the air sac at 96 hours incubation, the LC₅₀
was found to be 100 and 84 mg/kg egg respectively (approximately 6
mg and 5 mg/egg respectively) (Naber, 1975).

2.2.8 Special studies on genotoxicity

Test system Test object Substance and Results Reference
Concentration

Ames test¹ S. typhimurium erythorbic weakly Ishidate et al.,
TA100, acid 5-50 positive 1984
TA92, TA1535, mg/plate negative
TA1537, TA94,
TA98

TA100, TA92 sodium negative Ishidate et al.,
erythorbate 1984
5-50 mg/plate

Ames test¹ S. typhimurium erythorbic negative Litton Bionetics,
acid 0.25-0.5% 1976

Mitotic rec. Saccharomyces erythorbic negative Litton Bionetics,
assay cerevisiae D3 acid 2.0-4.0% 1976

Ames test¹ S. typhimurium sodium negative Newell et al.,
TA1530, TA1535 erythorbate 1974
TA1536, TA1537 100 mg/plate
TA1538

Mitotic rec. S. cerevisiae D3 sodium negative Newell et al.,
assay erythorbate 5% 1974

Ames test S. typhimurium sodium equivocal Kawachi et al.,
TA100 erythorbate negative 1980
TA98 ?concn. not
given
?concn. not
given

(contd)

Test system Test object Substance and Results Reference
Concentration

Rec assay ¹ B. subtilis ? concn. not negative Kawachi et al.,
given 1980

Chromosome Chinese hamster erythorbic negative Ishidate et al.,
aberration fibroblast cell acid, sodium negative 1984
line (CHL) erythorbate
0-0.25 mg/ml

Chromosome CHL sodium negative Matsuoka et al.,
aberration¹ erythorbate 1979
2.0 mg/ml

Chromosome Human fibroblast sodium negative Sasaki et al.,
aberration cell line erythorbate 1980
(HE2144) 0.0099-0.0198
mg/ml

Rat bone marrow sodium positive² Kawachi et al.,
micronucleus erythorbate 1980

Rat bone marrow erythorbic Hayashi et al.,
micronucleus acid negative 1988
187.5-1500
mg/kg b.w.
i.p.; 4x705 negative
mg/kg b.w.
i.p.

(contd)

Test system Test object Substance and Results Reference
Concentration

Rat dominant rat sperm/off- sodium negative Newell et al.,
lethal assay spring erythorbate 1974
1x0.2-5.0 g/kg
b.w. orally;
5x0.2-5.0 g/kg negative
b.w. orally

Mouse in vivo mouse sperm/off- sodium negative Newell et al.,
heritable spring erythorbate 1974
trans-location 1% & 5% in
diet for 7
weeks

1. With and without rat liver S9 fraction
2. Insufficient detail to evaluate this study

2.2.9 Special studies on interactions between erythorbic acid and
ascorbic acid (see also Biochemical Aspects)

In view of the observations that ascorbic acid is absorbed from
the gut and selectively concentrated in various tissues by active
transport systems, and that their affinity for erythorbate is only
about one-fifth of that for ascorbic acid (see Biochemical Aspects
above), a number of studies have investigated the possibility that
erythorbic acid might antagonize the absorption/tissue uptake of
ascorbic acid and exert an anti-vitamin effect.

2.2.9.1 Mouse

In a study of the scorbutigenic activity of glucoascorbic acid
due to antagonism of ascorbic acid, an additional group of mice was
given 5% erythorbic acid in the diet for two weeks. Glucoascorbic
acid caused severe symptoms of scurvy in this species, which is not
dependent on dietary vitamin C (i.e. antagonized the synthesis or
effects of ascorbic acid in the tissues). Conversely, erythorbic
acid was without effect and the general health and weight gain of
treated mice was normal (Woolley & Krampitz, 1943).

Erythorbic acid or ascorbic acid was fed to female Swiss
Webster mice in the diet at a concentration of 5% for 2 months and
then at 10% of the diet for a further 5 months; a control group
received an ascorbic acid-free diet throughout. Urinary ascorbic
and erythorbic acids were determined two weeks prior to termination.
At the end of the study tissue levels were measured in plasma, liver
and brain. In the ascorbic acid treated animals there was a marked
elevation of urinary and plasma ascorbate and a 38% increase in the
ascorbate level in the liver but there was no substantial increase
in the four brain regions studied viz: cerebrum, cerebellum, medulla
and brain stem. In the erythorbic acid-treated animals, the
erythorbate was well absorbed and rapidly excreted in the urine. It
was found that these extremely high dietary levels of erythorbic
acid caused a reduction of tissue ascorbic acid of 45% in the liver
and 28-39% in the brain, interpreted by the authors as "replacing"
ascorbic acid in these organs. Although erythorbic acid was found
in high levels in the plasma, it did not cause a reduction in plasma
ascorbic acid concentration. The body weight gain in both the
erythorbic acid and ascorbic acid groups was reduced by 40% relative
to mice receiving control diet (Tsao & Salimi, 1983).

2.2.9.2 Guinea pig

The effect of co-administration of ascorbic and erythorbic
acids compared with ascorbic acid alone was investigated in two
groups of guinea pigs receiving a semi-purified diet containing 0.1%
ascorbic acid with or without 2% erythorbic acid. It was found that
the organs of the guinea pigs retained a significant quantity of

erythorbic acid which replaced a corresponding quantity (about half)
of the ascorbic acid. The erythorbic acid incorporated was lost
rapidly and replaced by ascorbic acid when treatment with erythorbic
acid was discontinued and ascorbic acid only was given (Pelletier,
1969a).

Erythorbic acid was fed to groups of 7 male guinea pigs, body
weight 220-250g, at daily dose levels of 0, 20, 50, 100 or 400 mg
together with ascorbic acid (20 mg/d). After three days on these
regimes, a single oral dose of ¹⁴C-ascorbic acid was given orally.
There was a dose related reduction in the amount of ¹⁴C taken up by
the tissues which was significant at the 50 mg erythorbic acid dose
level when there was a 17-26% reduction in activity in the lungs,
kidneys, testes, eyes and pancreas and a 55% reduction in the
adrenals. Higher dose levels of erythorbic acid did not further
decrease the retention of ¹⁴C-label in the adrenals and the
reduction in other organs never exceeded 50% (Hornig et al.,
1974).

In a study in guinea pigs of the absorption, transport through
cell membranes at the tissue level, and catabolism of ascorbic acid,
and of the effects of erythorbic acid, it was found that after oral
administration there was no difference in the absorption of these
compounds, whereas uptake by the tissues was approx. 4 to 1 in
favour of ascorbate. Feeding studies with daily co-administration
of erythorbic and ascorbic acids indicated that the availability of
ascorbic acid was diminished by 40-60% (Hornig, 1977).

After a vitamin C depletion period of 12 days, groups of 7-9
male guinea pigs were given daily supplements of ascorbic acid,
erythorbic acid or a mixture of both isomers at dose levels of 5, 50
or 5+50 mg/kg b.w. for 16 days. The animals were then given an oral
dose of 1-¹⁴C-ascorbic acid and respiratory CO₂, urine and faeces
were collected for 96 hours. In comparison with animals treated
with 5 mg/kg ascorbic acid alone, body weight gains were depressed
by 49g and 22g in animals given erythorbic acid alone or with
ascorbic acid, respectively. No differences were observed in faecal
or urinary excretion of radioactivity between the three groups but
the exhalation of ¹⁴CO₂ was increased in both groups receiving
erythorbic acid. Kinetic analysis of the data showed that the
disappearance of ascorbic acid from the organism was accelerated
during the initial phase by erythorbic acid at a dose of 50 mg/kg
b.w./day and the half-life was shortened from 97h in animals
receiving ascorbic acid alone to 50h or 59h in the groups given
erythorbate alone or with ascorbic acid, respectively. However,
during the later, linear phase of disappearance the half-lives were
not significantly different between the groups receiving ascorbic
acid with or without erythorbic acid (Hornig & Weiser, 1976; Hornig,
1977). The increased catabolism of ascorbic acid was accompanied by

a lower ascorbic acid body pool, which was reduced by 30% in animals
receiving ascorbic acid plus erythorbic acid compared with animals
receiving ascorbic acid alone.

In a more recent study, groups of male guinea pigs, initial
b.w. 220 g were given a scorbutigenic diet together with 5 mg
ascorbic acid/day, 100 mg erythorbic acid/day, a combination of
both, or no supplement. On days 1, 4, 10, 16 and 30 of the
treatment period, tissue concentrations of ascorbic and erythorbic
acids in the liver, adrenals, spleen and kidneys were determined
following a 24 hour fasting period by a HPLC method. The ascorbic
acid content in the tissues of animals given ascorbic plus
erythorbic acids was lower than that of animals given only ascorbic
acid. However, the rate of disappearance of ascorbic plus
erythorbic acids was lower than that of animals given only ascorbic
acid. However, the rate of disappearance of ascorbic acid from the
tissues of ascorbic acid-deficient animals was similar to that of
animals given erythorbic acid alone. The authors concluded that
erythorbic acid does not accelerate the catabolism of ascorbic acid
but interferes with its uptake into or its storage in the tissues
when given at twenty-fold higher amounts (Arakawa et al., 1986).
The reviewer concluded that the results are also consistent with
accelerated catabolism limited to freshly absorbed ascorbic acid
before it has entered the tissues.

Groups of male guinea pigs, initial body weight about 220 g,
were given daily doses of 5 mg ascorbic acid and 1, 5, 20 or 100 mg
erythorbic acid; or 1 mg ascorbic acid and 1 or 10 mg erythorbic
acid; or 20 mg ascorbic acid and 20 mg erythorbic acid for 16 days.
The animals were then sacrificed and the ascorbic and erythorbic
acid content of the liver, adrenals, spleen and kidneys determined
by HPLC. The tissue content of ascorbic acid of animals given less
than 5 mg erythorbic acid with 5 mg ascorbic acid was not
significantly different from that of animals given 5 mg ascorbic
acid alone. The co-administration of 100 mg erythorbic acid caused
a decrease in the amount of ascorbic acid in the tissues of animals
given 5 mg ascorbic acid. The tissue content of animals given
erythorbic acid together with 1 mg ascorbic acid was not
significantly different from that of animals given 1 mg ascorbic
acid alone. In the case of animals given equal amounts of ascorbic
and erythorbic acids, the tissue levels of the former were
consistently much higher than the latter. The results were taken to
indicate that relatively small amounts of erythorbic acid do not
appear to reduce the availability of ascorbic acid (Suzuki et al.,
1986).

The activities of some ascorbic acid-dependent enzymes, liver
aniline hydroxylase and acid phosphatase, and serum alkaline
phosphatase, as well as liver cytochrome P450 content, were assayed
to investigate the effect of erythorbic acid administration on

ascorbic acid availability in male guinea pigs. The animals were
given 5 mg ascorbic acid and 1, 5, 20 or 100 mg erythorbic acid; or
1 mg ascorbic acid and 1 or 20 mg erythorbic acid daily for 16 days.
The body weight gains were similar in all groups. The liver
ascorbic acid content of animals receiving 5 mg ascorbic acid and
100 mg erythorbic acid was about 50% lower than that of animals
receiving 5 mg ascorbic acid alone, however neither liver cytochrome
P450 levels nor any of the enzyme activities of animals receiving 5
mg ascorbic acid were affected regardless of erythorbic acid
supplement. In animals given 1 mg ascorbic acid, liver aniline
hydroxylase and acid phosphatase activities were significantly
different from those in animals receiving 5 mg ascorbic acid;
however, the enzyme activities in animals given 20 mg erythorbic
acid together with 1 mg ascorbic acid were similar to those of
animals given 5 mg ascorbic acid alone. These results were taken to
indicate that erythorbic acid had no effect on these parameters in
animals receiving adequate (5 mg daily) amounts of ascorbic acid but
that administration of 20 mg erythorbic acid was effective in
maintaining normal levels of hepatic aniline hydroxylase and acid
phosphatase in animals receiving marginal amounts (1 mg daily) of
ascorbic acid (Suzuki et al., 1988).

In follow-up experiments, four groups of male guinea pigs, body
weight 220 g, received ascorbic acid (5 mg/d), erythorbic acid (100
mg/d) a combination of both isomers (5 mg + 100 mg/d) or no
supplement for a period of up to 30 days (16 days in the
unsupplemented group). Liver aniline hydroxylase, cytochrome P450
and acid phosphatase, and serum alkaline phosphatase showed
significant differences between the ascorbic acid deficient
(unsupplemented) group and the group receiving 5 mg/d. No
significant differences were seen between the other three groups.
In a further experiment, guinea pigs depleted of ascorbic acid for
16 days were divided into three groups which subsequently received
ascorbic acid (5 mg/d), erythorbic acid (100 mg/kd) or a combination
of the two (5 mg + 100 mg/d) for up to 20 days. During the
repletion period a similar pattern of recovery was observed and
there were no significant differences in enzyme activities or
cytochrome P450 content among the animals given ascorbic acid and/or
erythorbic acid. The results demonstrate that, using these
criteria, erythorbic acid in adequate amounts has a vitamin C-like
activity. The authors suggested that erythorbic acid administration
may not affect ascorbic acid availability in guinea pigs but, as the
ascorbic acid was given at above minimal requirement levels and the
high dose of erythorbic acid has a significant antiscorbutic effect,
the position with respect to effects on ascorbic acid availability
was not clearly demonstrated (Suzuki et al., 1989).

2.2.9.3 Monkey

Two groups of 4 male Cynomolgus monkeys were depleted of
ascorbic acid by feeding an ascorbic acid-free total liquid diet for
eight weeks; by this time plasma ascorbic acid levels had fallen
from 1.1 mg/dl to 0.04 mg/dl but the animals showed no signs of
scurvy. The animals were given a daily oral dose of ascorbic acid
of 10 mg/kg b.w. with or without 200 mg/kg b.w. erythorbic acid. In
all animals repletion was accomplished in two to three weeks using
return to initial plasma levels as the criterion. After treatment
for 4 weeks, the total amount of "apparent ascorbic acid" (ascorbic
plus erythorbic acid) was determined in whole blood 21 hours after
the last administration of the supplements. No difference was found
between the two treatment groups. Based on the assumption that most
of the erythorbic acid would have been excreted in the 21 hours
before blood samples were taken and therefore did not significantly
inflate the apparent blood ascorbic acid levels, the authors
concluded that erythorbic acid at the dose used did not antagonize
ascorbic acid (Turnbull et al., 1979).

2.2.10 Special studies on nitrosation in vivo

Co-administration of aminopyrine (0.4 mmol/kg b.w.) and sodium
nitrite (1.0 mmol/kg b.w.) caused alterations in serum GOT and GPT
activities and in hepatic G-6-PDH, microsomal drug metabolizing
enzymes, and lysosomal enzymes attributed to the formation of N-
nitrosodimethylamine in vivo. Sodium erythorbate (1.0 mmol/kg)
had no effect on these parameters per se but repressed the changes
induced by aminopyrine + nitrite (Kawanashi et al., 1981).

2.2.11 Special studies on tumour promotion

In a series of studies on the promoting effect of a series of
antioxidants in rats, mice, or hamsters, sodium erythorbate was
found to have no effect on the induction of tumours by methyl-N-
nitroso guanidine (MNNG)in the stomach, by DMH in the colon, by N-
ethyl-N-hydroxyethyl nitrosamine in the liver or by DMBA in the ear
duct. However, sodium erythorbate (and to a greater extent sodium
ascorbate) enhanced the induction of bladder tumours by N-butyl-N-
(4-hydroxybutyl) nitrosamine (BBN) when administered at 5% of the
diet for 32 weeks after treatment with the carcinogen (Ito et al.,
1986a,b; 1987).

In similar studies, the lack of effect of sodium erythorbate on
MNNG-induced stomach tumorigenesis was confirmed when the compound
was administered in drinking water at a concentration of 2.5% (Abe
et al., 1983) while a level of 5% in the diet was reported to
cause a decreased incidence of dysplasia of the pylorus and, more
marginally, papilloma of the forestomach (Shirai et al., 1985).
In the latter study, sodium ascorbate (1% or 5%) or ascorbic acid
(5%) in the diet had no effect.

With regard to the reported potentiation of BBN-induced bladder
carcinogenesis by sodium erythorbate, this was supported by the
observation that this compound at a dietary level of 5% caused a
significant increase in bladder tumours (Fukushima et al., 1984)
or premalignant papillary or nodular hyperplasias (Miyata et al.,
1985) in BBN-pretreated rats. However, using a similar protocol,
free erythorbate acid had no such promoting effect but actually
reduced the incidence of preneoplastic changes and tumours
(Fukushima et al., 1987) while high dietary levels (0.375 - 3.0%)
of sodium bicarbonate increased the incidence of urinary bladder
carcinomas in the BBN-treated rat (Fukushima et al., 1988).
Further, while free ascorbic acid was without effect, high dietary
concentrations of the sodium salt (5% but not 1%) had a promoting
effect on the BBN-treated rat (Fukushima et al., 1983).

2.2.12 Special studies on vitamin C activity of erythorbic acid

(a) in vivo studies

In studies on the anti-scorbutic effect of erythorbic acid in
guinea pigs, as much as 250 mg per day did not support animals fed a
vitamin C-deficient diet although administration of erythorbic acid
tended to slow down the development of acute vitamin C deficiency.
In vitamin C-depleted guinea pigs, erythorbic acid had no
therapeutic effect whereas the animals responded to vitamin C;
however, animals maintained on a suboptimal intake of ascorbic acid
showed some response to erythorbic acid. The authors concluded that
erythorbic acid has a protective effect on ascorbic acid in the body
but no significant antiscorbutic activity per se (Reiff & Free,
1959).

Groups of seven young adult male guinea pigs were fed a
scorbutigenic diet and supplemented with daily oral doses of 1, 2,
10, 50, 100 or 200 mg erythorbic acid for 38 days (3 animals from
the 10 mg/d group were treated for 115 days). Comparison groups
which received 10 mg erythorbic acid/d survived, including those
maintained for 115 days, although they had a slightly reduced weight
gain. For ascorbic acid, a dose of 1-2 mg/d was sufficient to
sustain appropriate growth. In contrast to the preceding study, the
authors concluded that daily oral doses of 10 to 200 mg erythorbic
acid replaced the anti-scorbutic activity of L-ascorbic acid and
this prevented them from developing any sign of scurvy discernible
at autopsy (Fabianek & Herp, 1967).

After 6 days on a scorbutigenic diet, female guinea pigs were
given daily supplements of 10 mg ascorbic acid, 100 mg erythorbic
acid or combinations of 100 mg erythorbic acid with 0.5 or 5 mg
ascorbic acid for 7 weeks. Their responses were judged by body
weight gain, serum alkaline phosphatase (SAP) levels, wound healing
and tooth structure. The supplement of 100 mg erythorbic acid

resulted in normal growth, SAP levels, tooth structure development
and collagen formation after wounding and the addition of 0.5 or 5
mg ascorbic acid did not further improve growth nor collagen
deposition after wounding. It was concluded that erythorbic acid
has about 1/20th the antiscorbutic potency of ascorbic acid and its
additive effect to sub-minimal levels of ascorbic acid implied that
there was no competitive inhibition in the utilization of the two
compounds. The authors further concluded that the weakly
antiscorbutic effect of erythorbic acid relative to ascorbic acid is
due to its poor absorption and tissue retention, and that to the
degree that it is taken up and retained by the tissues, it may be
equal in potency to ascorbic acid (Goldman et al., 1981).

Groups of seven male guinea pigs were depleted of vitamin C for
17 days then the scorbutic animals were treated daily with 40 mg
erythorbic acid or 2 mg ascorbic acid for 2 months. Both isomers
restored the growth of the animals and caused the disappearance of
scorbutic symptoms. Animals given erythorbic acid ate less and had
lower weight gains than those given ascorbic acid but this was
overcome by pair feeding. At autopsy, none of the animals had the
enlarged kidneys or adrenals characteristic of chronic
hypovitaminosis C and scurvy. Only a small proportion of the
erythorbic acid administered was recovered in organs or urine. The
total "ascorbic acid" (erythorbate plus ascorbate) content of the
erythorbic acid treated animals was less than that of the ascorbic
acid treated and the low content of ascorbic acid in the organs of
erythorbic acid-treated animals indicated that erythorbic acid had
"no significant sparing action on ascorbic acid". From the relative
tissue concentrations it may be concluded that the activity of
erythorbic acid in the organs is similar to that of ascorbic acid
but, as a result of less efficient uptake after dietary exposure and
more rapid excretion the apparent physiological activity is about
1/20th of that of ascorbic acid (Pelletier & Godin, 1969).

Guinea pigs maintained on a scorbutigenic diet could be
maintained in good health if erythorbic acid was included in
drinking water at a concentration of 0.1%. However, animals
pretreated with erythorbic acid were depleted of vitamin C twice as
fast as those which received ascorbic acid prior to the depletion
phase (Hughes et al., 1971).

Scorbutic (low collagen) granulomas were induced by s.c.
injection of carrageenan to vitamin C-deficient male guinea pigs.
Isoascorbic acid and ascorbic acid (6 doses of 100 mg i.p. at 12 h
intervals) were similarly effective in restoring collagen synthesis
in the granuloma although the concentration of erythorbic acid 12 h
after injection was lower than that of ascorbic acid 24 h after
injection (Robertson, 1963).

(b) in vitro studies

Ascorbic acid and erythorbic acid demonstrated similar activity
in promoting the hydroxylation of peptidyl proline in a cell-free
system (Hutton et al., 1967; Kutnink et al., 1969) and these
observations have been confirmed using a purified prolyl 4-
dihydroxylase preparation (Kurata et al., 1987).

Erythorbic acid had a similar effect to ascorbic acid in
protecting hepatic microsomal UDP-glucuronyltransferase activity
towards p-aminophenol against excess substrate but no protection was
afforded by ascorbate-2-sulfate or alpha-tocopherol (Neumann &
Zannoni, 1988).

The effects of ascorbate and erythorbate on collagen synthesis
were studied in cultured human skin fibroblasts. At concentrations
of 0.25 mM in the culture medium both ascorbate and erythorbate
increased collagen synthesis about eightfold with no significant
change in synthesis of non-collagen protein. Lysyl hydroxylase
activity increased 3-fold in response to ascorbate or erythorbate
administration. After prolonged exposure of cells to ascorbate or
erythorbate, prolyl hydroxylase activity was decreased to a similar
extent. The results were taken to indicate that collagen
polypeptide synthesis, posttranslational hydroxylations and
activities of the two hydroxylases are independently regulated by
ascorbate, with erythorbate having similar effects at the high
concentrations used (Murad et al., 1981).

In further studies using human skin fibroblasts, ascorbate
stimulated the rate of incorporation of labelled proline into total
collagenase-sensitive protein without changing the specific activity
of intracellular free proline. The effect of ascorbate was maximal
at a concentration of 30 µM and resulted in a four-fold increase of
incorporation. Erythorbate also stimulated collagen synthesis but
at considerably higher concentrations of 250-300 µM. The
stimulation of collagen synthesis by ascorbate and erythorbate was
accompanied by a decline in prolyl hydroxylase activity and a rise
in lysyl hydroxylase activity; again ascorbate was the more
effective (Murad et al., 1983).

The protective effects of ascorbic and erythorbic acid against
carbon tetrachloride-induced lipid peroxidation were investigated in
guinea pigs using exhalation of pentane and ethane as an index of
in vivo lipid peroxidation. It was observed that equal doses (750
mg/kg b.w., i.p.) of ascorbic acid or isoascorbic acid provided the
same degree of protection for a period of at least 4 hours (Kunert &
Tappel, 1983). The authors concluded that the antioxidant function
of ascorbic acid is relatively non-specific and that the two
stereoisomers do not differ with regard to their antioxidant
properties in vivo.

2.3 Observations in humans

In order to determine whether erythorbic acid could displace
ascorbic acid from the tissue, urinary levels of ascorbic acid were
measured after ingestion of 300 mg erythorbic acid by 5 healthy
human volunteers who had been previously repleted by administration
of 500 mg ascorbic acid for 7 days. Urinary analyses indicated that
ascorbic acid excretion was not affected by treatment with
erythorbic acid and that there was no significant displacement of
ascorbic acid from tissues (Kadin & Osadca, 1959).

The influence of erythorbic acid on ascorbic acid metabolism
and status was investigated in 11 healthy, non-pregnant women
volunteers. The volunteers were maintained in a metabolic unit and
fed a formula diet devoid of vitamin C for 54 days. After depletion
of 24 days, the subjects received increasing supplements of ascorbic
acid (30 mg/d, 60 mg/d and 90 mg/d for successive periods of 10
days) in the presence or absence of 600 mg/d of erythorbic acid.
The depletion resulted in a marked decrease in ascorbic acid in all
blood indices and during the study some subjects developed signs of
scurvy. Ascorbic acid supplements of 30 mg/d for 10 days failed to
increase plasma ascorbate concentrations; 60 mg for 10 days caused a
small increase and 90 mg/d resulted in a mean ascorbic acid
concentration of 29 mmol/l. Erythorbic acid did not cause any
adverse effects but rather had a small ascorbic acid-sparing effect
(Sauberlich et al., 1989).

3. COMMENTS

At the last evaluation an ADI of 0-5 mg/kg b.w. was allocated
based on a long-term study in the rat. The present Committee
reviewed new toxicological studies on isoascorbic acid and its
sodium salt, and metabolic and nutritional studies of the
interactions with ascorbic acid.

In rodent tests for embryotoxicity and teratogenicity,
erythorbic acid was without effect at dose levels up to 1 g/kg b.w.
and the Committee did not consider that chick embryo tests were
indicative of potential teratogenicity or fetotoxicity for man.

New long-term toxicity and carcinogenicity studies in rats and
mice did not show any specific toxic or carcinogenic effects up to
the maximum tolerated dose and most genotoxicity studies were
negative. Studies on tumour promotion were also negative with
exception of those on bladder tumours initiated by N-butyl-N-(4-
hydroxybutyl) nitrosamine in which high doses of sodium erythorbate
(but not free erythorbic acid) showed effects. Similar effects were
seen with sodium ascorbate (but not ascorbic acid) and various
sodium salts and the Committee concluded that this was not a
specific effect of erythorbate.

Erythorbic acid is much more poorly absorbed and retained in
the tissues than ascorbic acid, is poorly reabsorbed in the kidney
and rapidly excreted. As a result it has low anti-scorbutic
activity and only interferes significantly with ascorbic acid uptake
and retention in the tissues when concentrations are at least an
order of magnitude higher than ascorbic acid. Human studies showed
that daily doses of 600 mg per capita had no adverse effects on
ascorbic acid repletion in depleted volunteers.

4. EVALUATION

A new ADI "not specified" was allocated to erythorbic acid and
its sodium salt.

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    See Also:
       Toxicological Abbreviations