FAO Nutrition Meetings
Report Series No. 48A
WHO/FOOD ADD/70.39
TOXICOLOGICAL EVALUATION OF SOME
EXTRACTION SOLVENTS AND CERTAIN
OTHER SUBSTANCES
The content of this document is the
result of the deliberations of the Joint
FAO/WHO Expert Committee on Food Additives
which met in Geneva, 24 June -2 July 19701
Food and Agriculture Organization of the United Nations
World Health Organization
1 Fourteenth report of the Joint FAO/WHO Expert Committee on Food
Additives, FAO Nutrition Meetings Report Series in press; Wld Hlth
Org. techn. Rep. Ser., in press.
CYCLOHEXYLAMINE
Biological data
Biochemical aspects
Following intragastric administration of 5 mg/kg body-weight of
14 C-CHA to a male rat, 95% was recovered in the first 24 hours, 1.3%
from cage washings and a trace of 14C-CHA in expired air (but not as
14CO2). Activity was evenly divided between urine and faeces.0.22%
were recovered between 24 and 48 hours. About 85% of the 14C-CHA in
the urine represented unchanged CHA. In 2 male dogs given oral doses
of 14C-CHA hydrochloride, more than 90% of the administered dose was
excreted in the urine within the first 24 hours. Chromatographic
determinations indicated that an average of 81% of the 14C was CHA
with an average of 18% representing an unknown metabolite. In a human
male receiving an oral dose of 14C-CHA about 82% of the dose was
recovered in the urine within 24 hours, less than 2% being found in
the faeces. Chromatography indicated that 98% of the 14C in the urine
was unchanged CHA, 1% was present as an unknown metabolite. 0.6% of
the 14C remained unidentified (Abbott Laboratories, 1967).
Orally administered CHA is excreted in the urine of rats to the
extent of over 99% with less than 1.5% in faeces. 80% of excreted CHA
is unchanged but 20% appears as unidentified metabolite. CHA is
rapidly absorbed from the gastrointestinal tract and appears in all
body tissues (Lethco et al., 1969). CHA-hydrochloride was fed to
groups of 16 rats at levels of 0, 0.01%, 0.05%, 0.1%, 0.25%, 0.5% and
1% for 3 months. Only cyclohexylamine was detected in the urine, while
faeces contained only traces. Blood levels were dose dependent but
very low, probably because of rapid excretion (Unilever Research
Laboratories, 1970).
Following the feeding of 0.17 g/kg of 1-14C-CHA to a rabbit 68%
of the radioactivity was recovered in the urine in the succeeding 60
hours. 45% of the administered dose was determined to be unconjugated
CHA, 0.2% was N-hydroxycyclo-hexylamine (Elliott et al., 1968).
Incorporation of inorganic S35 into protein polysaccharide was
used as a test system to show the slight inhibitory effect of CHA on
membrane processes (Wortman et al., 1968).
Acute toxicity
Animal Route LD50 mg/kg LD100 mg/kg References
body-weight body-weight
Mouse oral 500 Lomonova, 1963
" (single) oral 770 Lee & Dixon, 1969
" (groups
of 10) oral 520 Lee & Dixon, 1969
" s.c. 1150 Pliss, 1958
" i.p. 50 Buchel & Guyonneau, 1963
Rat oral 500 Lomonova, 1963
" i.p. 200 Mallette & Von Haam, 1952
Rabbit i.p. 500 Flinn, 1937
" dermal 632 Woodward Labs, 1964
CHA acts on the spinal motor centres and medulla producing delayed
convulsions several hours after administration. CHA is partly absorbed
through the skin but is also a caustic irritant (Carswell & Morrill,
1937; Lomonova, 1963). A rise in temperature and aggregation of
animals into groups raises the acute toxicity from 770 mg/kg for
single mouse at 20° to 445 g/kg for 10 mice at 28°C. This effect can
be prevented by chlorpromazine, and enhanced by SKF-S25A (Lee & Dixon,
1968; 1969). In man acute inhalation leads to lightheadedness,
faintness, tachycardia and vomiting (Watrous & Schulz, 1950).
One-tenth ml of CHA instilled into rabbits' eyes produced
permanent blindness (Woodward Labs, 1964). After exposure of
guinea-pigs to a theoretical concentration of 6.7 mg/L of CHA, they
exhibited salivation, dyspnea, rhinorrhea, ataxia, prostration. 8/10
guinea-pigs died within 80 minutes. The 2 survivors were observed for
7 days during which rhonchi were heard and slight rhinorrhea and
cloudy corneas were noted (Woodward Labs, 1963).
Special studies
CHA has been shown to have pressor activity. I.v. injection of doses
of 0.4 and 3.7 mg/kg produced a sympathomimetic pressor action in
cats, rats and guinea-pigs (Classen et al, 1968). I.v. injection of 1,
3 or 10 g/kg CHA into the anaesthetised cat produced a rise in blood
pressure and tachycardia. These effects were present after vagotomy,
adrenochetomy, nephrectomy or destruction of the spinal cord. A
positive inotropic effect was shown in the cat, in isolated perfused
rat and guinea-pig hearts and in rabbit atria but high doses caused a
negative inotropic effect. Direct stimulation of skeletal muscle and
cholinergic stimulation of smooth muscle was demonstrated. The
sympathomintic effect of CHA were probably mediated through the
release of endogenous catecholamines and could be absorbed by drugs
blocking alpha-adrenergic receptors (Rosenblum & Rosenblum, 1968). CHA
5 mg/kg had no effect on responses to catecholamines, cholinomimetic
or histamine. Administration of reserpine or phenoxybenzamine
inhibited the pressor response to CHA. The inhibitory effect of
reserpine on responses to CHA is reversed by adrenalin infusion.
Pretreatment with the MAO inhibitor, pargyline had no effect on the
pressor response to CHA (Hamamura et al., 1968). The effects of CHA on
the blood pressure are those of an indirectly acting sympathomimetic
but there is also some direct action on the heart and blood vessels.
CHA releases H3-labelled noradrenaline from the heart tissue. The cat
is the oat sensitive animal to the pressor effect of CHA and the
effects last longer than the rabbit, rat or guinea-pig. The pressor
response in all 4 species can be blocked by phentolamine (an alpha
receptor blocking drug). Intragastric or intraduodenal administration
has also a pressor effect but ten times the i.v. dose is needed.
Tachyphylaxis has been reported (Unilever Research Laboratories,
1970). I.v. CHA-sulfate given to dogs at the doses of 3 g/kg produces
pressor effects by release of noradrenaline from tissue stores
(Wechsler et al., 1969).
CHA produces allergic reactions of the delayed hypersensitivity
type in guinea-pigs (Chung, 1969).
Short-term studies
Rat. 12 rats were given 100 mg/kg body-weight/day CHA orally
for 82 days without obvious adverse effects (Carswell & Morrill,
1937).
Groups of 5 males and 5 females were fed diets containing 0, 300
and 3000 ppm of CHA for 38 weeks. No gross or histopathological
changes attributable to treatment were noted (Flinn, 1937).
Guinea-pig. 12 guinea-pigs were given 100 mg/kg body-weight of
CHA orally for 82 days without obvious adverse effects (Carswell &
Morrill, 1937).
Rabbit. 12 rabbits were given 100 mg/kg body-weight of CHA for
82 days without adverse gross effects (Carswell & Morrill, 1937).
Dog. Groups of 3 male and 3 female dogs were given by capsule
0, 0.15, 1.5 and 15 g/kg/day of CHA sulfate. The results based upon 2
years of feeding do not indicate any CHA effects in terms of
body-weight, food consumption, mortality, behaviour, haematology and
chemistry. Gross and microscopic pathology for 1 male and 1 female per
group sacrificed at 1 year did not indicate any adverse effects. The
study is still in progress (Industrial Bio-test Lab. Inc., 1969b).
Long-term studies
Rat. Groups of 22 males and 28 females were fed 0.5 ml/day of
5% oily solution of CHA for 12 months and were observed for another 6
months. No increase in mortality or tumour incidence was noted but
there were hepatic and renal degenerative changes (Pliss, 1958).
Four groups of 35 male and 35 female rats were given 0, 0.15
mg/kg, 1.5 mg/kg, and 15 mg/kg body-weight CHA-sulfate daily for 2
years. 5 males and 5 females were sacrificed from each group at 52
weeks. At the end of the study only 75% the animals died from disease
unrelated to the compound administered. No test-related histological
abnormalities were seen in the various groups except for one bladder
tumour in one male rat in the highest dose group, believed to be a
carcinoma. No other details are available (Oser et al., 1969).
In a 2 year study in rats, groups of 25 male and 25 female rats
received CHA sulfate in their diets adjusted so as to provide daily 0,
0.15, 1-5 and 15 mg/kg. No compound related effects were noted with
respect to food consumption, mortality, behaviour, haematology,
chemistry, or organ weights. Males of the 15 mg level showed a reduced
weight gain during the first 13 months. Microscopic pathology revealed
urinary bladder changes in 2 rats of the 15 mg/kg group. One female
sacrificed at 1 year showed epidermoid metaplasia and one male
sacrificed at 2 years had a transitional cell carcinoma. Bladder
lesions in these rats are extremely rare (Industrial Bio-test Lab.
Inc., 1969a).
In another study, 5 groups of 30 male and 30 female rats were
given 0, 15, 50, 100 and 150 mg/kg of cyclohexylamine base in their
diet and will be observed for 2 years. So far at 12 weeks, growth
depression had been observed in males at 100 and 150 mg/kg levels and
in females at 5% 100 and 150 mg/kg body-weight levels. These studies
are being continued (Food and Drug Research Lab., 1970)
Reproduction teratogenicity studies
Rat
In phase 1 of a 3-phase study, groups of 10 male and 20 female
rats received intragastrically, 0, 1.5 and 15 mg/kg/day of CHA
sulfate, the males being placed on this regimen beginning at age 30
days, the females at age 86 days. These animals were mated within
their groups at 100 days. No effects were noted upon growth, libido,
or fertility. For females sacrificed at day 14 of pregnancy; control
and test animals did not differ as to corpora lutea, implantation and
resorption sites or numbers of viable fetuses. For females allowed to
go to term, no effects were noted upon gestation time, maternal weight
or progeny number. Population data, however, showed increased pup
mortality in the 15 mg/group during the lactation period. The survival
index at day 4 post-partum for the high dose group and the survival
indices for both test groups at day 21 were reduced compared to the
control group. In the teratological phase of this study, groups of 20
female rats received intragastrically 0, 1.5 and 15 mg/kg/day of CHA
sulfate on days 6-15. Dams were sacrificed on day 20 and litters
delivered by caesarean section. Findings revealed no abnormalities
with respect to growth or mortality of the dams; corpora lutea,
implantation and resorption sites and fetus viability were normal. No
abnormalities were seen in the foetuses based upon gross observation,
free-hand sectioning or alizarin staining. In the third phase, groups
of 20 pregnant rats received 0, 1.5 and 15 mg/kg/day of CHA sulfate
beginning on day 15 of pregnancy and continuing through weaning of the
litters, all of which were cast naturally. While no effects were seen
with respect to maternal body weight and litter size, the 21-day
survival and lactation indices for both test groups were lower than
control indices (Industrial Bio-test Lab. Inc., 1967).
In two further reproduction and teratology studies essentially
identical to those already described (Industrial Bio-test Lab. Inc.,
1967), no effects were seen in one study except for decreased survival
indices in the high dose level rats during phase 1 (Industrial
Bio-test Lab. Inc., 1968) and in the second study a questionable
dose-related decrease in growth during lactation in phase 3 of the
study (Foster D. Snell, Inc., 1968).
Chicken
Fresh fertile eggs were injected directly into yolk or through
the air cell with 0.2 ppm CHA at 0 and 96 hours of incubation.
Viability of embryos was reduced and teratogenic effects were noted
when compared with controls (Verrett, 1969).
Mutagenicity tests
CHA was shown to produce chromosomal alterations at certain
concentrations in microbial cell cultures. The host mediated assay
using the mouse and S.typhimurium was negative for CHA. In vivo
cytogenetic analysis of somatic and reproductive cells showed that
CHA, at 1 mg/kg injected i.p. into the rat daily for 5 days, increased
chromasom breaks in spermatogonenal cells and 10 mg/kg injected daily
for 5 days had the same effect on bone marrow cells. The dominant
lethal test in rats for CHA at 200 mg/kg showed preim-plantation loss
hence some effect on sperms was possible (US Food and Drug Admin.,
1969).
Rats given CHA i.p. showed a dose related incident of chromosomal
breaks in leucocytes of the bone marrow (Legator et al., I969).
In groups of 12 rats given 50 mg/kg/day of CHA orally or i.p. for
5 days no effects on chromosomes of bone marrow cells could be
demonstrated (Dick & Biava, 1970).