757. Candelilla wax (WHO Food Additives Series 30)

CANDELILLA WAX

First draft prepared by
Dr D.L. Grant
Bureau of Chemical Safety
Health and Welfare
Ottawa, Ontario, Canada

1. EXPLANATION

This substance has not been previously evaluated by the Joint
FAO/WHO Expert Committee on Food Additives.

Candelilla wax is obtained from candelilla plants (Euphorbia
antisiphilitica, Euphorbia cerifers, Pedilanthus pavonis) which
are found in the dry regions of northern Mexico and to a lesser
extent in southern Texas, Arizona and California in the United
States. Candelilla wax is obtained through extraction from the
plants by immersion in a tank containing boiling water acidified
with sulfuric acid. Candelilla wax is a hard and brittle wax. It is
composed of about 20-29% wax esters, 12-14% alcohols and sterols,
49-50% hydrocarbons, 7-9% free acids, 2-3% moisture and 1% mineral
matter. The chemical and physical properties of the wax vary with
the age of the plant and the year in which it is collected. The wax
is insoluble in water but soluble in acetone, chloroform, benzene,
and other organic solvents (Rogers, 1978; FDA, 1982; ACT, 1984;
Ashraf-Khorassani & Taylor, 1990).

2. BIOLOGICAL DATA

2.1 Biochemical aspects

No information available

2.2 Toxicological studies

2.2.1 Acute toxicity studies

Species Sex Route LD₅₀ Reference
(mg/kg bw)

Rat Not defined oral > 5000 ACT, 1984

2.2.2 Short-term studies

2.2.2.1 Rats

Groups of 12 male and 12 female weanling Wistar rats, weighing
40-50 g, were fed a diet containing a mixture "(Product 12)" of gum
base and candelilla wax (composition not defined), at concentrations
of 0, 3 or 5%, equivalent of 0, 590 or 980 mg/kg bw/day for 8 weeks.
No significant differences were reported between groups, regarding
survival, body weight gains, food and water intake, urinalysis
(parameters measured not identified) haematology (parameters
measured not identified) nor gross pathology (Harrisson, 1946).

Groups of weanling Wistar rats (12 rats/sex/group) were fed
diets containing a 1:1 mixture of candelilla wax "(Product 16)" and
butadiene-styrene polymer "(Heveatex Polymer N-1017)", at
concentrations of 0, 1 or 5%, equivalent to 0, 750 or 3600 mg/kg
bw/day, for 8 weeks. The daily intake of candelilla wax was
approximately 0, 370 or 1800 mg/kg bw from the respective diets.
Food and drinking water were provided ad libitum. All rats were
observed for signs of toxicity and mortality. Food and water intakes
were measured twice weekly and body weights were recorded weekly. At
the 4th week of treatment and at termination, blood and urine
samples (procedure of collection not defined), from 4
rats/sex/group, were taken and examined for haematology
(haemoglobin, RBC, total and differential WBC) and urine parameters
(transparency, odour, colour, pH, albumin and microscopic elements).
At termination, all animals were sacrificed and autopsied. It was
reported that no treatment-related adverse effects were observed in
any of the investigated parameters (Harrisson, 1948).

Groups of weanling Wistar rats (17 rats/sex/group), weighing
50-60 g, were fed diets containing a mixture "(Product 17)" of
styrene-butadiene polymer (50%) and candelilla wax (50%) at
concentrations of 0, 1 or 5%, equal to 0, 680 or 3420 mg/kg bw/day
for 27 weeks. Rats were observed for signs of toxicity and
mortality. Food and drinking water intake and body weights were
recorded weekly. Blood samples were collected (procedure not
defined) from 5 rats/sex/group at the 13th and 25th week of
treatment and examined for haemoglobin, RBC, total and differential
WBC. On the same schedule, urine samples from 5 rats/sex/group were
collected and examined for transparency, odour, colour, pH, albumin
and microscopic elements. At termination, two-thirds of the animals
from treated groups were sacrificed and autopsied. Rats of the
control group were not sacrificed, but used as a control group in
another one-year study. The following tissues and organs from 3
males and 3 females, from each treated group, were microscopically
examined: heart, lung, spleen, kidney, pancreas, small and large
intestines, uterus, ovary, prostate, testicle and seminal vesicle.
The author indicated in the results that, with the exception of a
slight decrease of body weight gains in rats of both treated groups
(predominantly after 7th week of treatment), the other investigated
parameters did not indicate any toxic change attributable to the
treatment (Harrisson, 1949).

In four separate studies, 12 male and 12 female weanling albino
rats (strain not identified) received a gum base containing 4.1 to
6.1% of candelilla wax in the diet, at concentrations ranging from
10 to 25%, for a period of 180 days (10% for the first 7 days, 20%
for the following 7 days, and 25% for the remaining treatment
period). During the entire treatment period, the daily consumption
of candelilla wax was estimated to be 2400 mg/kg bw. Rats of the
control group (12 males and 12 females) were fed a "colony" diet,
free of the test material. All rats survived the treatment. Food
intake and body weight gains (frequency of recording not defined)
were comparable between treated and control rats. Urinalysis (number
of animals used and frequency of collection not defined), gross and
histopathology (number of animals examined not defined) revealed no
changes that could be associated with the treatment (Hodge, 1973).

2.2.2.2 Dogs

Diets containing 0, 1 or 10% of a gum base containing 25%
candelilla wax were fed to groups of young dogs (strain not defined,
1-2 males and 2-3 females per group), for 6 months. Test dogs
consumed approximately 0, 60 or 600 mg/kg bw/day candelilla wax from
their diets. Dogs were housed individually and were fed twice daily
at regular intervals (amount of food per feeding was not defined).
They were observed daily and underwent careful physical examination
every second week. Each dog was weighed regularly (frequency not

defined). Blood and urine samples from an unstated number of dogs
were collected (frequency not defined) and analyzed for unidentified
parameters. At termination, 2-3 dogs from each treated group and 4
dogs (sex not defined) from the control group were necropsied and
the following tissues and organs were microscopically examined:
skin, stomach, small and large intestines, liver, pancreas, kidney,
urinary bladder, ovary, uterus, testicle, epididymis, prostate,
lung, mesenteric lymph node, spleen, tonsil, bone marrow, heart,
aorta, sciatic nerve, adrenal, thyroid and ribs (no individual
animal data for histopathology were provided). It was reported that
no treatment-related changes were observed (Harrisson, 1953).

2.2.3 Long-term/carcinogenicity studies

2.2.3.1 Mice

Diets containing 0, 0.8 or 5.0% (equivalent to 0, 1200 or
7500 mg/kg bw/day) of a mixture of gum base containing 25%
candelilla wax, were fed to groups of 15 black agouti (C57) mice of
each sex for 12 to 13 months. Mice were observed for signs of
toxicity and survival. Body weights were recorded (frequency not
defined). About 50% of the surviving mice/group were sacrificed at
the 12th month of treatment, and remaining mice at the 13th month.
It was not indicated which organs were examined or whether
histopathology was performed. It was reported that the number of
deaths in the 5% dose group exceeded those in the lower or control
groups. The causes of death were not defined. The authors concluded
that diets containing a gum base-25% candelilla wax mixture, at dose
levels as high as 7500 mg/kg bw/day, were not carcinogenic in mice,
under the conditions of this study (Hodge, 1973; FASEB, 1981).

2.2.3.2 Rats

Groups of Sprague-Dawley rats (30 rats/sex/group) were fed
diets containing 0, 0.8, 2.0 or 5.0% mixture of either gum base 11
or gum base 12 containing 25% candelilla wax for either 89 weeks
(gum base 12) or 2 years (gum base 11). It was estimated that the
actual intake of candelilla wax by rats was approximately 0, 125,
300 or 750 mg/kg bw/day. Rats were housed individually, provided
with food and drinking water ad libitum and observed for signs of
toxicity and mortality (frequency of observations not defined). Food
intake and body weights were recorded weekly. Blood and urine
samples from 2-6 rats/sex/group were collected at the 3rd, 6th, 12th
and 23rd month of treatment and analyzed for unidentified
parameters. One to seven rats/sex/group were sacrificed and
autopsied 6 months, 1 year or 1´ years after initiation of
treatment. At termination, the following tissues and organs from 3
males and 3 females (after 89 weeks) and 9 males and 9 females
(after 2 years), of the appropriate group, were microscopically

examined: lung, liver, spleen, kidney, adrenal, heart, pancreas,
thyroid, stomach, small and large intestine, sciatic nerve, ovary,
fallopian tube, uterus, testicle, seminal vesicle, prostate, bone
marrow, eye, bone, skin, and salivary gland (no individual animal
data for histopathology were provided). It was reported that no
significant differences were observed in any of the investigated
parameters, between animals of treated and control groups
(Harrisson, 1953).

2.2.4 Reproduction studies

No standard reproduction study with candelilla wax has been
conducted. The only available observations were incidental to the
short-term study reported above (Harrisson, 1949) in which 3 male
and 3 female rats were fed a diet containing a mixture of
styrene-butadine polymer "(Product 17)" (50%) and candelilla wax
(50%) at concentrations of 0, 1 or 5%, equivalent to 0, 680 or
3420 mg/kg bw/day for 5 months prior to mating. Two of the three
females from each dose level conceived on either the first or second
mating, and produced normal litters (FASEB, 1981).

2.2.5 Special study on mutagenicity

Four Ames tests with Saccharomyces cerevisiae, Escherichia
coli and Salmonella typhimurium showed that candelilla wax was not
mutagenic, either with without the activation system (Table 1).

2.3 Observations in humans

No information available.

Table 1. Results of mutagenicity assays on candelilla wax

Test System Test object Concentration Results Reference
of candelilla
wax

Ames test¹ Saccharomyces 1, 25, 2.50 Negative Brusick, 1976
cerevisiae D4 and 5.00%

Ames test¹ S. typhimurium 1, 25, 2.50 Negative Brusick, 1976
TA1535, and 5.00%
TA1537,
TA1538

Ames test² S. typhimurium 0.3-10 000 Negative Mortelmans
TA1535, µg/plate & Eckford,
TA1537, 1979
TA1538, TA98,
TA100

Ames test² Echerichia coli 10-10 000 Negative Mortelmans
WP2 µg/plate & Eckford,
1979

¹ Both with and without S-9 fraction of rat, mouse and monkey liver
² Both with and without S-9 fraction of rat liver

3. COMMENTS AND EVALUATION

A number of older studies in mice, rats, and dogs were
reviewed. A 6-month study in dogs and a 2-year study in rats, with
the highest dietary levels equivalent to 600 and 750 mg/kg bw/day,
respectively, showed no compound-related toxicity. These studies
were considered to be of fundamental importance in the safety
assessment. In addition, microbial tests for mutagenicity were
negative.

The Committee felt that the deficiencies noted in the
individual studies, particularly in the light of current criteria,
were counterbalanced to some extent by the consistent absence of
adverse effects, and concluded that the present functional uses
(glazing agent, component of chewing-gum base, surface-finishing
agent, and carrier for flavour) did not raise any toxicological
concerns.

4. REFERENCES

ACT (1984). Final report on the safety assessment of candelilla wax,
carnauba wax, Japan wax and beeswax. J. Amer. Coll. Toxicol., 3:
1-41.

ASHRAF-KHORASSANI, M., & TAYLOR, L.T. (1990). Analysis of crude,
purified, and synthetic candelilla wax using supercritical fluids.
Liquid and Gas Chromatogr., 8: 314, 316, 318, 320.

BRUSICK, D. (1976). Mutagenic evaluation of compound FDA 75-43,
MX8006-44-8 candelilla wax, L.F. refined. Project No. 2468, Litton
Bionetics, Inc.

FASEB (1981). Evaluation of the health aspects of candelilla wax as
a food ingredient. Federation of American Societies for Experimental
Biology. Prepared for FDA, Contract No. FDA 223-78-210. Unpublished
Report.

FDA (1982). Candelilla wax, proposed affirmation of GRAS status as a
direct human food ingredient. Fed. Reg., 47: 35776-35777.

HARRISSON, J.W.E. (1946). Product No. 12, type III GRS. Rubber and
candelilla wax. Report No. 36731. LaWall and Harrisson, Consultants,
Unpublished Report.

HARRISSON, J.W.E. (1948). Product No. 16, 50/50 mixture candelilla
wax and heveatex (Styrene and Butadiene) Polymer N-1017. Report
38222. LaWall and Harrisson, Consultants. Unpublished Report.

HARRISSON, J.W.E. (1949). Heveatex 50%, candelilla wax, hydrogenated
fat. Report 39348. LaWall and Harrisson, Consultants. Unpublished
Report.

HARRISSON, J.W.E. (1953). Long-term safety studies. Resume of
studies on young dogs and albino rats. LaWall and Harrisson,
Consultants. Unpublished Report.

HODGE, H.C. (1973). A critical review at the 1) Feeding studies of
candelilla wax incorporated in gum base, 2) together with studies of
the possible carcinogenic potential by other routes of
administration. University of California, San Francisco.

MORTELMANS, K.E. & ECKFORD, S.L. (1979). Microbial mutagenesis
testing of substances compound report: F76-048, candelilla wax.
Final Report, Project No. LSU-6909. SRI International.

ROGERS, R. (1978). Monograph on candelilla wax. Submitted under:
Contract No. FDA 223-76-2001. Informatic Inc., Maryland, USA.
Unpublished report.

    See Also:
       Toxicological Abbreviations
       CANDELILLA WAX (JECFA Evaluation)