Safety evaluation of certain food additives

T
a
bl
e 1.
 Results 
o
f stu
dies 
o
f 
gen
otoxicity i
n
 vi
tro w
ith cassia g
u
m
End-p
oint
Test
sy
stem
C
oncentration
Result
R
eferen
ce
Reverse mutation
S
a
lmon
ella
 typhimurium
 strain TA100,
E
scherichia coli 
WP
2uv
rA
R
ange-fin
ding stud
y: 3–50
00 μg
/p
la
te
, 
±S9
Positive/
n
egative
a
Ver
spe
ek-
Rip (1
998a)
S
. typhimurium 
stra
in
s T
A
98, TA153
5 and
TA1537
1
s
t experimen
t:
4–1000
μg/plate, ±S9
S
. typhimurium 
stra
in
s T
A
98, 
TA100,
TA1535 and
TA153
7, 
E. coli
WP2uvrA
2
nd experiment: 1
.6
–1000 
μg/plate, ±S9
Reverse mutation
S
. typhimurium 
stra
in
s T
A
98, 
TA100,
TA1535 and
TA153
7, 
E. coli
WP2uvrA
1
s
t experiment: 0.3–1
00 μg
/p
la
te
, 
±S
9
2
nd experiment: 0
.3
–100 
μg/plate, ±S9
Negative
b
Mee
rts (20
03)
S
. typhimurium 
stra
in
TA100, 
E. col
i
WP2uvrA
3
rd experimen
t:
10
0–5000
μg/plate, ±S9
Reverse mutation
S
. typhimurium 
stra
in
s T
A
97, TA98, 
TA10
0
and TA
102
1
s
t experiment: 0.05–
5 mg/plate, ±S9
2
nd experiment: 0
.0
5–5 mg
/pla
te
, 
±S9
Negative
c
Weidu (2
006)
Gene mutation
Mouse lymphoma
L517
8Y TK
+/
–
cells
1
s
t experimen
t:
0.003–10
μg/ml, ±S9
2
nd experiment: 0
.0
03–10 
μg/ml, ±S9
Negative
d
Ver
spe
ek-
Rip (1
998b)
Chromosomal
aberration
Human lymph
ocytes
1
s
t 
experimen
t:
1–10 μg/ml,
±S
9
2
nd experiment: 1–10
μg/ml, ±S9
Negative
e
Ber
tens (19
98)
S9, 9000 × 
g
supe
rnatant 
fr
om rat liver.
a
With semi-refined cassia gu
m, wit
h
and w
ithou
t metabolic activa
tion (S9), by the plate incorpo
ration method
, u
s
ing d
imethyl su
lfoxide (DMSO) as a vehicle.
S
light 
precipit
a
tion
occurre
d from 10
0 μg
/p
la
te
upw
ard
s 
in
t
he 
dose range
–find
in
g study an
d fro
m 62.5 
and 200
μg/plate upw
ards
in the first and second
expe
riments, 
resp
ectively. I
n
no
ne of 
the e
x
pe
riments was toxicity o
bserved. In two ind
epende
nt experiments, 
S. typhimurium
st
rain TA100 show
ed a
dose
-re
la
te
d 
increa
se in 
the number 
of revert
a
nts at pr
ecipitating concentrations in the pre
sence an
d absence of metabolic acti
vation,
whereas 
E. co
li 
WP2
uvr
A
sh
owed
ne
gative 
re
spo
nse
s. 
Salmonella typhimurium
stra
ins TA
9
8
, TA1535
a
nd 
TA1537 
show
ed 
negative responses in 
the
fi
rst
experime
nt,
but
in
the
seco
nd
exp
eriment
they
show
ed
dose-related
in
creases
in
the
number
o
f 
revertan
ts
at
precipitating
co
ncen
tr
ation
s
in
16
CASSIA GUM

Ta
b
le
 1
(cont
d)
the
presence
(TA1537 
only) 
and 
absence 
of 
metabolic activation. 
Howe
ver, the increa
ses observed
in st
rains TA98, TA15
35 and
TA1537
were within
the
historical con
tr
ol range.
b
With p
urified semi-re
fi
ne
d 
cassia gum (8
.6
mg total anthra
qui
nones/kg
), with 
a
nd wit
h
out metabolic activation (S9), by 
the
pla
te incorpo
ration method.
In the first 
an
d 
second experime
nts, ultrapur
e water 
w
a
s 
used as a 
vehicle
; 
slig
ht 
pr
ecip
itation occu
rre
d 
a
t 
33 and
100 μ
g/plat
e, but no 
to
xicity wa
s
o
bse
rved. In t
h
e t
h
ir
d experiment, DMSO wa
s 
used as a vehicle; sligh
t pre
cipitation o
ccurred at 
all 
con
cen
tr
ations,
but no
toxi
city w
a
s observed.
c
With 
cassia g
u
m, with 
and 
without 
metabolic 
act
ivation (S9),
by the plate incorp
oration meth
od, using
sterilized distilled wa
te
r as a vehicle. Th
e stud
y
w
a
s 
reported in a very limited manner, and no informa
tion
w
a
s provided
on 
th
e occurrence
of 
p
recip
itation 
or 
to
xicity.
d
With semi-refin
ed cassia gum, w
ith a
nd wi
thou
t me
tabolic activation (
S9), w
ith D
MSO as 
ve
hicle. 
In
both 
experimen
ts
, p
recipita
tion
occu
rre
d at 
10
μ
g/ml. In experimen
t 1, cells were exposed for 3 h and
h
arvest
e
d
3 days follo
wing exposure. No
to
xicity was obse
rved. In exp
eri
ment 2, 
in t
h
e 
absence
o
f 
metabolic activation, 
cells 
were e
x
po
sed
for 
24 h an
d harvested 
2 da
ys la
ter, whe
reas in the prese
nce
o
f 
me
tabolic activatio
n, cells we
re exposed
for 3 h 
a
nd harvested 3 da
ys later. Without meta
bolic activati
on, the
ce
ll coun
t 
and cloning e
ffi
cie
ncy w
ere reduced 
by 
42% and
8
1% 
at the high
est
concentratio
n t
e
sted, resp
ectively, bu
t 
with 
metabolic activa
ti
on, 
no 
toxicity was o
b
se
rved.
e
With semi-refin
ed cassia gum, w
ith a
nd wi
thou
t me
tabolic activation (
S9), w
ith D
MSO as 
ve
hicle. 
In
both 
experimen
ts
, p
recipita
tion
occu
rre
d at 
10
μ
g/ml. 
In the 
first 
exp
eriment, the
cells w
ere
exposed for
3 
h a
nd h
arvest
e
d
21 
h later. Th
e hi
ghe
st tested 
concentra
ti
on 
indu
c
ed
m
itotic inh
ibition
(22%) in 
th
e prese
nce, 
but not in 
th
e absence, of me
ta
bolic ac
tivation. In 
the second experiment, ce
lls we
re exposed 
fo
r 
24 
or 
48 h without S9 and
h
arvest
e
d
imm
ediately after 
e
x
po
sure. With S9, the ce
lls we
re 
tr
eated 
for 
3 h 
and h
arvest
e
d
an
other 
45 h
later. T
he h
ig
hest 
tes
ted
co
nce
ntrati
on
ind
uce
d mitotic 
inh
ib
ition (3
3%) in
the
presence, 
but no
t 
in the 
abse
nce
, 
of metab
olic activation.
CASSIA GUM
17

The results of two limitedly reported studies of genotoxicity in vivo (a sperm
abnormality test and a micronucleus test in mice) are summarized in Table 2. These
studies were performed with cassia gum. No statements regarding compliance with
GLP and QA were available (Weidu, 2006). 
Overall, the Committee concluded that cassia gum is not genotoxic.
2.2.5 Reproductive toxicity
In a two-generation study of reproductive toxicity, groups of 25 male and
25 female Ico:OFA.SD Sprague-Dawley rats were given diets containing 0, 5000,
20 000 or 50 000 mg semi-refined cassia gum/kg. These dietary concentrations
were equal to doses of 0, 510, 2060 and 5280 mg/kg bw per day for males and 0,
510, 2090 and 6120 mg/kg bw per day for females (calculated using the mean food
intake and mean body weights in weeks 1–10). An additional group received a diet
containing 50 000 mg of purified semi-refined cassia gum (resulting from an
additional isopropanol extraction step) per kilogram (equal to a dose of 5430 mg/kg
bw per day for males and 6230 mg/kg bw per day for females). All parental animals
(P) were treated for approximately 10 weeks before mating and during mating,
gestation and lactation. Pregnant females were allowed to rear their offspring (F
1a
)
to weaning. Rats in both 50 000 mg/kg diet groups exhibited low pregnancy rates,
and the non-pregnant rats were mated again with the same males. They were
allowed to litter, and the subsequent offspring (F
1b
) were terminated on days 5–7
postpartum. Selected F
1a
offspring were treated for a 10-week period of maturation
and during mating, gestation and lactation. Pregnant F
1a
females were allowed to
rear their offspring (F
2
) to weaning. The study was performed according to OECD
Test Guideline 416 (Two-Generation Reproduction Study) and was certified for
compliance with GLP and QA. The only significant findings were a slightly reduced
pregnancy rate in the 50 000 mg semi-refined cassia/kg diet group (pregnancy rate
Table 2. Results of studies of genotoxicity in vivo with cassia gum
End-point
Test system
Concentration
Result
Reference
Micronucleus test
Bone marrow
of KM mice
(males and
females)
625–2500 mg/kg
bw, by oral gavage
(divided over 2
doses in 30 h)
Negative
a
Weidu (2006)
Sperm abnormality
test
Male KM mice 625–2500 mg/kg bw
per day, by oral
gavage for 5 days
Negative
b
Weidu (2006)
a
Study was performed with cassia gum and was reported in a very limited manner. Bone
marrow was collected 6 h after second gavage, and micronuclei of 1000 polychromatic
erythrocytes (PCE) per animal were counted, followed by determination of the ratio of PCE
to normal chromatic erythrocytes (NCE).
b
Study was performed with cassia gum and was reported in a very limited manner. Sperm
was collected 30 days after last administration, and aberrations were counted in 1000 sperm
cells per animal.
18
CASSIA GUM

17/25 compared with 24/25 in the control group) and the 50 000 mg purified semi-
refined cassia/kg diet group (pregnancy rate 18/25 compared with 24/25 in the
control group) of the P generation and a slightly, but not statistically significantly,
reduced mean pup weight of the F
1a
(–11%) and the F2 (–14%) generations in the
50 000 mg purified semi-refined cassia/kg diet group on day 21 postpartum. When
both 50 000 mg/kg diet treatment groups of the P generation were mated again
(resulting in the F
1b
generation), however, a reduced pregnancy rate was no longer
observed. Therefore, 50 000 mg/kg diet (equal to a dose of 5280 mg/kg bw per day)
was taken to be the NOEL (McIntyre, 1990).
There was also a one-generation study of reproductive toxicity with cats
available, in combination with a short-term study of toxicity. In this study, groups of
10 male and 20 female cats were given semi-refined cassia gum as part of a canned
food diet at a dietary concentration of 0, 7500 or 25 000 mg/kg for at least 83 weeks.
These dietary concentrations were equal to 0, 690 and 2470 mg/kg bw per day for
males and 0, 860 and 2950 mg/kg bw per day for females (calculated using the
mean food intake and mean body weights in weeks 1–83). The F
1
generation was
sacrificed after at least 13 weeks. The study was certified for compliance with GLP
and QA and was performed according to OECD Test Guidelines 415 (One-
Generation Reproduction Toxicity Study) and 409 (Repeated Dose 90-Day Oral
Toxicity Study in Non-Rodents). An unusually high mortality in the control group
resulted in a high litter loss, impairing appropriate comparison between control and
treatment groups. In general, no obvious effects on short-term toxicity parameters
were observed in both parental generation and offspring. Reproductive
performance was not affected, nor were growth and development of the offspring.
Only at the highest dose were some effects observed that were possibly related to
treatment: a slight decrease in food consumption during late gestation and a slight
increase in absolute and relative ovarian weights in parental females, together with
a significantly higher combined incidence of stillborns and neonatal deaths (Virat,
1989). As a result of the unusually high mortality in the control group, the Committee
considered this study not suitable for use for the evaluation.
In a developmental toxicity study that was reported in a limited manner,
groups of 12 pregnant female SD rats were administered 0, 250, 500 or 1000 mg
cassia gum/kg bw per day by oral gavage from day 7 until day 16 of gestation. No
treatment-related effects on maternal body weight, number of resorptions or dead
embryos, or weight and length of the fetuses were observed. No abnormalities were
detected upon observation of the skeleton and visceral examination of the fetuses.
It seems, therefore, that no adverse effects were observed in dams or offspring
at doses up to and including 1000 mg/kg bw per day, the highest dose tested
(Weidu, 2006).
In a developmental toxicity study, groups of 28 pregnant Crl:CD (SD)BR
Sprague-Dawley rats were administered semi-refined cassia gum in distilled water
by gavage twice daily at a total dose of 0, 350 or 1000 mg/kg bw per day from days
6 to 19 post-coitum. A fourth group of 29 pregnant rats was administered 1000 mg
purified semi-refined cassia gum/kg bw per day following the same dosing regimen.
All animals were sacrificed and examined on day 20 post-coitum. The study was
certified for compliance with GLP and QA and was conducted in accordance with
OECD Test Guideline 414 (Prenatal Development Toxicity Study). Two animals in

tải về 1.44 Mb.

Chia sẻ với bạn bè của bạn: