CYROMAZINE
EXPLANATION
First draft prepared by Mrs E. Arnold,
Health and Welfare Canada, Ottawa, Canada
Cyromazine is an insecticide that interferes with the first
dipteran larval moult and to a certain extent with metamorphosis. It
was reviewed for the first time at the present meeting.
EVALUATION FOR ACCEPTABLE INTAKE
BIOLOGICAL DATA
Biochemical aspects
Absorption, distribution and excretion
Oral exposure
Rats
A single dose of 0.5 mg/kg bw of 14C-cyromazine (uniformly
triazine ring labelled) was given orally to two male and one female
Charles River white rats (not further identified). By 72 hours after
dosing, 95% of the administered dose had been excreted in urine,
essentially all within the first 24 hours.
About 3% was excreted in faeces, again predominantly in the first
24 hours. Negligible amounts were excreted as volatiles or CO2 in
another two males and one female given the same dose. Tissue residues
were below the level of detection except in liver; however, liver
levels were too low to permit accurate quantitation (about 0.007 ppm)
(Simoneaux and Cassidy, 1978).
Chickens
Two chickens (strain not indicated) were given daily oral doses
of 14C-cyromazine (uniformly triazine ring labelled) of 0.75
mg/hen/day by capsule for seven days. By 24 hours after the last dose
99.1% of the administered radioactivity had been recovered in the
excreta with essentially none in volatiles and CO2. Both egg whites
and egg yolks contained about 0.12-0.15 ppm consistently. Tissue
levels were: byproducts (i.e. head and feet) 0.047 ppm test material
equivalents; reproductive tract 0.047 ppm; liver 0.032 ppm; all other
tissues 0.008-0.019 ppm (Simoneaux and Cassidy, 1979).
Sheep
One sheep (strain not given) was given a daily dose of
14C-cyromazine (uniformly triazine ring labelled) of 0.15 mg/kg bw/day
by capsule for nine days. By 24 hours after the last dose about 90%
of the administered dose had been recovered in urine. A further 3.7%
was recovered in faeces. Only a trace was detected in CO2. Blood
levels appeared to plateau at about 0.1% of the administered dose
after five days. Tissue levels were: 0.174 ppm in liver, 0.164 ppm in
the GI tract, 0.048 ppm in kidney, 0.012-0.013 ppm in muscle, brain
and heart, and at or below the level of detection in fat (Simoneaux
and Cassidy, 1981).
Goats
One lactating goat/dose level was given a daily oral dose of
14C-cyromazine (uniformly triazine ring labelled) of 5 or 50
mg/goat/day by capsule for ten consecutive days. By 24 hours after
the last dose 90.4% of the low dose and 82.1% of the high dose had
been recovered in urine. Faecal excretion accounted for 7.5% of the
low dose and 5.7% of the high dose. There was some evidence of
plateauing of urinary and faecal excretion after 5 days. Blood levels
changed little on days 2-10 and were in the range of 0.05-0.09% of the
dose at both dose levels. Tissue levels were 0.01 ppm or less in the
low dose goat and 0.14 ppm or less in the high dose goat except in
liver (0.79 and 1.522 ppm, respectively), kidney (0.04 and 0.437 ppm)
and GI tract (contents) (1.27 and 1.11% of the administered dose).
Daily milk levels accounted for about 0.3% of the administered dose
(Simoneaux and Marco, 1984).
Monkeys
A single dose of 0.05 or 0.5 mg/kg bw of 14C-cyromazine
(uniformly triazine ring labelled) was given by capsule to groups of
two male and two female monkeys (Macaca fasicicula). Twenty four
hours after dosing 62.9-96.1% of the low dose and 47-82.2% of the high
dose had been recovered in urine. Only a small additional amount was
excreted in urine in the period 24-96 hours after dosing. Excretion
in faeces accounted for 1.14-1.31% of the low dose and 1.42-1.92% of
the high dose most of which was recovered in the first 24 hours after
dosing (Staley, 1986).
Because of the low total recovery of radioactivity in the above
study, a second study was undertaken using one male and one female of
the same strain of monkey and at the same dose levels given by
capsule. Greater recovery of radioactivity was not achieved. At the
high dose the results were similar to those in the above study. Most
of the recovered radioactivity was in urine excreted during the first
24 hours after dosing with only a small amount in faeces again mainly
in the first 24 hours. About 14% of the administered dose was
excreted in faeces mainly in the first 48 hours after dosing (Staley
and Simoneaux, 1986).
Dermal Exposure
In a preliminary study two male Harlan Sprague-Dawley albino rats
were exposed to 14C-cyromazine (uniformly triazine ring labelled) at
1.0 mg/rat applied dermally to a shaved area on the upper back. After
eight hours exposure 5.8-7.5% of the applied material had been
absorbed (recovered in urine, carcass, faeces and blood, listed in
order of decreasing amounts). About 16-22% of the applied dose was
detected in skin which had been washed to remove the test material.
About 60% of the administered dose was recovered in skin and cage
washes. Essentially no radioactivity was detected in volatiles.
Blood levels were at or below the level of detection (Murphy and
Simoneaux, 1985).
In the main part of this study, groups of three male Harlan
Sprague-Dawley albino rats/dose/time period were exposed to
14C-cyromazine (uniformly triazine ring labelled) at 0.1,1.0 or 100
mg/rat applied to a shaved area on the upper back. The rats were
sacrificed 1, 2, 4 and 10 hours after treatment. The percent of the
total dose applied which was absorbed was 4.5-11.0% at the low dose,
3.5-11.4% at the mid dose and 2.2-7.1% at the high dose with the
amount absorbed increasing with duration of exposure at all dose
levels. The amount of material bound in the skin also increased with
time. As in the preliminary study the highest recovery of absorbed
material was in the urine, followed by the carcass. Faecal and blood
levels were very low (Murphy and Simoneaux, 1985).
In a second dermal study, groups of male Harlan Sprague-Dawley
albino rats were exposed to 14C-cyromazine (uniformly triazine ring
labelled) at dose levels of 0.1, 1.0 or 10.0 mg/rat applied to shaved
areas on the upper back. The treated area was enclosed and covered by
a nonocclusive bandage. The hind legs of the rats were shackled to
prevent scratching of the treated area. Groups of four rats/dose were
sacrificed 2, 4, 10 and 24 hours after treatment. Additionally, two
groups of four rats/dose had the bandages removed and the treated area
washed 10 or 24 hours after treatment and then were returned to
metabolism cages for a 48 hour period prior to sacrifice. In all
groups, 62-86% of the applied dose was recovered in skin and cage
washings and bandages, thus was not absorbed. The amount bound in skin
tended to increase with duration of exposure but was reduced during a
post-exposure period. The amount absorbed (recovered in urine,
carcass, faeces and blood) appeared to be higher in the rats which
were maintained for 48 hours post-treatment. These data suggest that
the material apparently bound in the skin was absorbed into the body
(Murphy et al., 1987).
Biotransformation
Rats
Urine from a female rat given a single oral dose of
14C-cyromazine (uniformly triazine ring labelled) of 0.5 mg/kg bw was
analyzed using TLC and a cation exchange column chromatography system.
With both systems the majority of the urinary radioactivity was
determined to be in the form of unchanged parent compound. Unchanged
parent compound in urine accounted for about 80% of the administered
dose. Three metabolites were detected with each system and were
presumed to be the same compounds. These metabolites accounted for
2.2-3.2%, 3.0-5.5%, and 4.6-5.3% of the administered dose,
respectively, but no identification was made. Faecal material from a
male rat given the same dose as the above female was found to contain
little unchanged parent compound: <0.1% of the administered dose.
The same three metabolites as observed in urine were found and
represented 0.1, 0.1 and 4.1% of the administered dose, respectively
(Simoneaux and Cassidy, 1978).
One male and one female albino Sprague-Dawley rats were given
diet containing 3000 ppm of 14C-cyromazine for 10 days. In the male
the liver was found to contain 31.3 ppm cyromazine and 0.96 ppm
melamine and the kidney 62.4 ppm cyromazine and 1.3 ppm melamine. In
the female liver residues were 13.2 ppm and 0.51 ppm and kidney
residues 22.2 and 0.68 ppm of cyromazine and melamine, respectively.
This study indicated that there was some conversion of cyromazine to
melamine in vivo (Smith et al., 1983).
Chickens
Two chickens were each given one capsule containing 0.75 mg of
14C-cyromazine (uniformly triazine ring labelled) daily for seven
days. The chickens were sacrificed 24 hours after the last dose.
Parent compound accounted for about 75-78% of the radioactivity
recovered in excreta, 58% in egg white and 70 % in egg yolk. Other
metabolites in excreta were not resolved. In egg white and egg yolk
the major other peak accounted for 24.5% and 6.7% of the extractable
radioactivity, respectively. This metabolite was identified as
melamine. Minor peaks were present, accounting for 26% and 23.9% of
the radioactivity in whites and yolks, respectively, but none of the
metabolites was identified (Simoneaux and Cassidy, 1979).
Groups of two chickens were given diets containing 14C-cyromazine
(uniformly triazine ring labelled) at levels of 7.7, 32.9 or 84.3 ppm
daily for 7 days. All chickens were killed 24 hours after the last
dose. Egg fractions and livers from the high and low dose groups were
analyzed. At the low dose cyromazine accounted for 18.4%, 69.3% and
61.7% of the extractable radioactivity in egg white, egg yolk and
liver, respectively, while melamine accounted for 38.3%, 1.0% and
7.0%, respectively. At the high dose the extractable radioactivity
was 79.5%, 86.3% and 84.2% cyromazine and 3.8%, 2.0% and 5.6% melamine
in egg whites, egg yolks and liver, respectively. Another metabolite
designated "Metabolite A" was detected in egg white and accounted for
19.9% of the extractable radioactivity at the low dose and <1.0% at
the high dose. This metabolite was probably ammeline formed by
deamination of one of the amino groups of melamine (Simoneaux, 1981).
Groups of 30 hens were given diet containing 0 and 5 ppm
cyromazine (as 0.3% Larvadex Premix) for 56 days. The control group
received an equivalent amount of rice hulls in the diet. Following the
treatment period surviving birds in both groups were maintained on the
control diet for a 14 day depletion period. Three hens/group were
sacrificed on dose days 14, 28, 42 and 56 and on depletion days 1, 3,
7 and 14. Melamine was not detected in any of the tissue (breast,
thigh, liver, fat and skin) or egg samples. Maximum cyromazine
residues in eggs and tissues were reached in 3 and 14 days,
respectively, with no further increase with continued dosing.
Residues were no longer detectable in eggs and tissues 2 and 1 day
after removal of cyromazine from the diet (Boone and Cheung, 1985).
Goats
One lactating goat/dose level was given 14C-cyromazine (uniformly
triazine ring labelled) at nominal levels of 5 or 50 mg/goat/day for
10 consecutive days by capsule. The goats were sacrificed 24 hours
after the last dose. Unchanged cyromazine accounted for 43.7, 35.9,
0.2 and 32.5% of the extractable radioactivity in urine, faeces, liver
and milk, respectively, at the low dose and 78.8, 58.7, 1.9 and 41.0%
at the high dose, respectively. Melamine levels in urine, faeces,
liver and milk were 11.9, 14.3, 1.7 and 9.2% at the low dose and 7.8,
10.4, 5.6 and 4.5% at the high dose, respectively. An unidentified
metabolite designated "zone 4" accounted for 44.4, 17.4, 92.7 and 1.0%
of the extractable radioactivity at the low dose and 13.4, 1.8, 71.7
and 0.2% at the high dose in urine, faeces, liver and milk,
respectively (Simoneaux and Marco, 1984).
Sheep
One sheep was given a daily dose of 14C-cyromazine of 0.15 mg/kg
bw/day for nine days by capsule and then sacrificed 24 hours after the
last dose. Analysis by ion exchange column chromatography indicated
that parent compound accounted for 84% of the radioactivity in urine,
44% in faeces and 11.6% in liver. In all of these extracts four
metabolites were detected, accounting for: 0.1-6.8% of recovered
radioactivity in urine (metabolite b highest, metabolite d lowest);
0.3-6.4% in faeces (metabolite c highest, metabolite d lowest); and in
liver 1.3-2.9% as metabolites a, b and c but 43.0% as metabolite d.
Metabolite d cochromatographed with melamine. The other three
metabolites were not identified but one was expected to be the
hydroxy-CGA-72662 compound (Simoneaux and Cassidy, 1981).
Monkeys
Urine from male and female monkeys (Macaca fasicicula) given
single oral doses of 14C-cyromazine (uniformly triazine ring labelled)
of 0.05 or 0.5 mg/kg bw by capsule was found to have the majority of
the radioactivity present in the form of unchanged parent compound.
Regardless of dose 93.6-96.1% of the urinary radioactivity was present
as unchanged cyromazine. Additionally, 2.9-6.4% of the radioactivity
as identified as melamine (Staley, 1986).
In a second study with the same strain of monkey given the same
dose levels, urine collected during the first 24 hours after dosing
had 95-100% of the recovered radioactivity in the form of unchanged
cyromazine. In one male dosed at 0.05 mg/kg bw, no melamine was
detected in the urine. In one female at 0.05 mg/kg bw and one monkey
of each sex given 0.5 mg/kg bw 3.0-3.9% of the urinary radioactivity
was in the form of melamine (Staley and Simoneaux, 1986).
Toxicological studies
Acute toxicity
The acute toxicity of cyromazine to rats, mice and rabbits is
given in Table 1.
Table 1. Results of acute toxicity assays with cyromazine.
Species Sex Route LD50 Reference
Mouse M, F oral 2029 Bathe and Sachsse, 1978a
Rat M oral 4050 Sabol, 1987
F oral 3530 ibid
M, F oral 3920 ibid
M, F oral 3387 Bathe and Sachsse, 1978b
Rabbit M, F oral 1467 Ullmann and Sachsse, 1978
Rat M, F dermal >1370 Bathe and Sachsse, 1978c
Rat M, F inhalation >2.7 Ulrich and Blair, 1979
Short-term studies
Oral studies
Rats
Groups of 20 weanling Charles River CD rats/sex/dose were given
diet containing 0, 30, 300, 1000 or 3000 ppm cyromazine (96.3% pure)
for 90 days. Additionally groups of 5 rats/sex/dose were given diet
containing 0 or 3000 ppm cyromazine for 90 days followed by control
diet for a 4 week recovery period. No treatment-related changes were
noted in behavior, appearance, haematological parameters, clinical
biochemistry or urinalysis. Mean body weight was about 5-10% lower
than controls in the 3000 ppm group and up to 4% lower in the 1000 ppm
group. In the recovery period males approached control weights but
the female weights were 5-6% lower than the controls. Food
consumption expressed as g/kg bw/day was reduced in both sexes at 3000
ppm during the first week of treatment. Absolute liver weights were
lower than controls in the 1000 and 3000 ppm groups in both sexes.
Liver/body weight ratios were lower than controls at 300 ppm and above
in males and at 30 or 1000 ppm in females. Liver/brain weight ratios
were lower than controls at 1000 and 3000 ppm in males and 30, 300 and
1000 ppm in females. The significance of these observations is not
clear. No treatment-related pathology was observed. Since there were
no pathological lesions to explain the liver weight effects the NOAEL
in this study was considered to be 300 ppm (equal to 23.5-26.9 mg/kg
bw/day) (Goldenthal and Hughes, 1979).
Dogs
Groups of 4 beagle (Ridglan) dogs/sex/dose were given diet
containing cyromazine (96.3%) at 0, 30, 300, 1000 or 3000 ppm for 90
days. An additional 2 dogs/sex/dose were given the 0 and 3000 ppm
diets for 90 days followed by control diet for a 4 week recovery
period. Slightly relaxed nictating membranes were observed quite
frequently in dogs at all levels of treatment. Slightly dry nose was
seen in dogs at 1000 and 3000 ppm. Body weight gain during the
treatment period was slightly reduced in males at 3000 ppm and females
at 1000 ppm or more. Some recovery of body weight was noted in the
recovery period, particularly in males. Food consumption was lower in
both sexes given 3000 ppm than in the controls during treatment and
higher during the recovery phase. RBC count, haemoglobin and
haematocrit were lower than controls in the males given 3000 ppm.
Haemoglobin was still depressed at the end of the recovery period. No
treatment- related effects were noted in clinical biochemistry
parameters, urinalyses or ophthalmoscopy. Absolute and relative liver
weights in 3000 ppm males were higher than controls. In females
relative liver weight was slightly but not statistically significantly
higher than controls. No treatment-related pathology was observed in
any of the groups. The NOAEL in this study was 300 ppm (equal to
11.4-12.0 mg/kg bw/day) since the incidence of relaxed nictating
membranes was apparently not dose-related and not clearly
treatment-related (Jessup and Hughes, 1979).
Groups of 6 young adult purebred beagle (Hazleton) dogs/sex were
given diet containing 0, 30, 300 or 3000 ppm of cyromazine (96.3%
pure) for 26 weeks. An additional 2 dogs/sex/group were given 0 or
3000 ppm for 26 weeks and then control diet for a 4 week recovery
period. There were no clearly treatment-related changes in behavior
or appearance. Body weight gains were lower in females at 30 and 300
ppm and both sexes at 3000 ppm. At 30 ppm body weight in females was
3.9% lower than controls. Some recovery of body weight was noted in
the recovery period. Food consumption was generally similar in all
groups although occasionally lower than controls in males at 3000 ppm.
Haematocrit, haemoglobin and RBC count were lower than controls in
both sexes at 3000 ppm. The differences were generally statistically
significant in males and occasionally in females. Serum cholesterol
levels were reduced at 3000 ppm especially in males, rising after
cessation of treatment in the recovery animals. SGOT was elevated in
males at 3000 ppm throughout the treatment period, returning to
control level in the recovery period. Serum glucose was slightly
increased in both sexes at 3000 ppm but the differences were
statistically significant only occasionally. There were no
treatment-related effects on urinalysis or ophthalmoscopy. In the
3000 ppm dogs organ weights tended to be higher than controls but this
was attributed to the reduced body weights. No treatment-related
pathology was observed. The body weight changes in females at 30 and
300 ppm were small and no other changes were noted in these groups.
Effects at 3000 ppm were more prominent in males than in females. It,
therefore, seems justified to consider 300 ppm (equal to 8.86-9.29
mg/kg bw/day) as an NOAEL for this study (Burdock et al,1980).
Inhalation study
Rats
Groups of 5 Tif:RAIf(SPF) albino rats (hybrids of
RII/1xRII/2)/sex were exposed to measured cyromazine aerosol
concentrations of 0, 57, 206 or 706 mg/m3 4 hours/day, 7 days/week
for 4 weeks. Satellite groups of 5 rats/sex/dose were exposed to 0 or
706 mg/m3 and then allowed a 3 week recovery period. Median
aerodynamic diameter of the particles was 1.6-6.0 µm with 52-57% of
the particles <3 µm. In all treated groups, piloerection, dyspnoea
and hunched position were observed in all animals with severity
increasing with dose. Reduced spontaneous activity was seen in the
two higher dose groups. In the recovery period activity returned to
normal and dyspnoea decreased but piloerection remained moderate. All
of the treated males had lower body weight gains than controls during
the treatment period but the differences were not dose-related.
Females had no body weight effects during treatment. Both sexes gained
more weight than controls in the recovery period. Food consumption
followed the same pattern as body weight. The high exposure males had
slightly higher RBC count, haemoglobin and haematocrit than controls
at the end of the treatment period which was not apparent after 2
weeks recovery. There were no treatment-related effects on clinical
biochemistry parameters. In the mid and high exposure females
absolute liver weight, liver/body weight ratio and liver/brain weight
ratio were higher than controls. In males, absolute pituitary weight
and pituitary/brain weight ratio were lower than controls. At the end
of the treatment period all the females in the mid and high exposure
groups had cytoplasmic vacuolation of liver hepatocytes which was not
seen in control or low exposure rats. However, after the recovery
phase 5/5 controls and 4/5 high exposure animals showed this change.
An NOAEL was not demonstrated in this study since clinical signs of
toxicity were apparent at all exposure levels (Hartmann et al.,
1988).
Long term/carcinogenicity studies
Mice
Groups of 68 Charles River CD-1 mice/sex/dose were given diet
containing 0, 50, 1000 or 3000 ppm cyromazine (95.3-95.5% pure). Of
these animals 8 mice/sex/dose were sacrificed after 12 months on test
and survivors to 24 months were all sacrificed. Homogeneity analyses
at the beginning of the study indicated a problem in obtaining uniform
dispersion of the test material in the diet especially at the low dose
level (50 ppm). The method of preparation was changed and better
homogeneity was achieved for the remainder of the study. For the
entire study weekly analyses indicated levels of 58-162% of nominal at
the low dose and 77-133% at the other two levels; mean concentrations
were 97-102% of nominal at all three dose levels. There were no
treatment-related clinical signs of toxicity. Mortality to 93 weeks
did not indicate a treatment-related effect. Survival at 104 weeks
was slightly lower in mid and high dose males and low and high dose
females. However, the mid dose females showed higher survival than
the controls suggesting that the differences observed were incidental.
Body weights of mid and high dose males were consistently about 5% and
7-9% lower than controls. Differences in low dose males and all
groups of females were not consistent. Although food consumption in
all treated groups was slightly lower than in controls there was no
dose-relationship to the differences. There were no treatment-related
effects on haematological parameters examined for 8 mice/sex/dose at
either 12 or 24 months. There were no treatment-related changes in
weights of liver, kidney, heart, testes or brain. A slight increase
in the liver/body weight ratio in high dose males was related to the
reduced body weight in these animals.
A slight increase in hepatocellular neoplasms (adenomas and
carcinomas) was noted in treated males but, since there was no
dose-relationship, no increase in nonneoplastic proliferative lesions
and no similar effect in females, it was not considered to be
treatment-related. There was a small increase in malignant lymphomas
(lymphocytic and histiocytic) in treated males that did increase with
dose. However, the incidence in the high dose group was only slightly
higher than in historical controls for the two types combined. There
was no similar trend in females. In high dose females the incidence
of mammary gland adenocarcinomas was higher than in controls or the
mid and low dose groups: 16% at 3000 ppm cf 4% in controls, 8.3% at 50
ppm and 5.7% at 1000 ppm. Historical control data indicated an
expected range of 0-5%. The lack of a dose-response relationship
suggests that these observations were not treatment-related. The
NOAEL in this study was 50 ppm (equal to 6.5 mg/kg bw/day) based on
the body weight effects in the male (Blair and Hardisty, 1982a).
Rats
Groups of 60 Charles River Sprague-Dawley CD rats/sex/dose level
were given diets containing 0, 30, 300 or 3000 ppm of cyromazine
(95.3-95.5% pure) for 104 weeks. Additionally, groups of 10 rats/sex
were given diets containing 0 or 3000 ppm of cyromazine for 52 weeks;
5/sex/group were sacrificed at 52 weeks and 5/sex/group were given a
4 week recovery period prior to sacrifice. Diets were prepared
weekly. During the study the method of preparation was altered to
improve the homogeneity of the diet particularly at the lowest dose
level. Overall mean concentrations were 96-101% of the nominal
concentrations. There were no treatment-related effects on general
clinical condition or on survival. At termination of the study
survival was 60-67% in males and 53-70% in females (lowest in
controls). Body weights were reduced in the high dose males and
females; weights were 11-13% lower than controls by Week 4 and about
30% lower by Week 79. Body weight was also reduced in the mid dose
females during much of the study (Weeks 12-79); the reduction in
weight was about 10% by Week 49 and remained in that range. During
the recovery period in rats treated at 3000 ppm for 52 weeks females
showed essentially complete recovery of body weight while males showed
some recovery but at the end of the four week period still had about
20% lower weights than the controls. Food intakes expressed as
g/rat/day were reduced in the high dose males and females but were
similar to the controls in the other two dose groups. Food intakes
expressed as g/kg bw/day were higher than controls in the high dose
males and females. Efficiency of food utilization was not
consistently affected during the first 25 weeks of the study. There
were no treatment-related effects on any of the haematology or
clinical biochemistry parameters examined. There was a tendency for
urine volumes to be greater in the high dose males and females but
unusually large volumes were seen only in occasional animals. The
observation was not consistent throughout the study and specific
gravity showed no consistent changes. Organ weight differences were
restricted to the high dose group and appeared to be related to the
body weight effects at this dose level rather than indicating a direct
effect on the organs.
There was a slight increase in the incidence of bronchiectasis in
the high dose males and females; however, the incidence was considered
to be within the observed range in aging rats and not, therefore,
indicative of a treatment-related effect. Renal pelvic epithelial
hyperplasia was observed at a higher incidence in high dose females
than in other groups. The incidence at other dose levels was not
dose-related and the incidence of chronic nephropathy was lower in the
high dose group than in other groups. Since pelvic hyperplasia is
generally observed as part of the complex of changes comprising
chronic nephropathy the observed incidence probably relates to
variability in degree of change as a result of aging and was not
treatment-related. There was a slightly higher incidence of
interstitial cell tumours in high dose males (6/57 compared to 1/60 in
controls) and mammary gland adenocarcinomas in high dose females (9/59
compared to 3/53 in controls). Both of these incidences are within
the historical control range for the laboratory performing the in-life
portion. The NOAEL in this study was 30 ppm (equal to 1.8 mg/kg
bw/day) based on the body weight changes noted in females (Blair and
Hardisty, 1982b).
Reproduction study
Groups of 15 male and 30 female weanling Sprague-Dawley COBS CD
(Charles River) rats were given diets containing 0, 30, 1000 or 4000,
then 3000 ppm of cyromazine (95.3% pure). The high dose was reduced
from 4000 ppm to 3000 ppm at 4 weeks because of toxicity. The F0
animals were fed their respective diets for 100 days prior to mating.
Weekly diet analyses showed considerable variability in achieved
concentration, particularly in the 30 ppm diet. Mean concentrations
over the duration of the study, however, were close to the target
concentrations. There were no treatment-related changes in the
general condition of the animals during the study. Bodyweights were
lower than controls in male and female F0 and F1 rats given 1000 ppm
or 3000 ppm; the differences were statistically significant except in
the F1 males given 1000 ppm. Food consumption on a g/rat/day basis
was reduced in both sexes of both generations given 1000 ppm or 3000
ppm. There was no treatment-related effect on pregnancy in females.
In the F0 generation 6/15 males at 3000 ppm failed to sire a litter
compared to 2/15 males in each of the other groups suggesting an
effect on male fertility. However, this effect was not observed in
the F1 males. There was no effect on duration of gestation. Mean
litter size was slightly reduced in the 3000 ppm group in the F1
generation. In both generations there was an increase in perinatal
mortality at 3000 ppm (dead at birth or dying by Day 4). In both
generations pup weights in the 3000 ppm group were lower than controls
at birth and throughout the lactation period. In the 1000 ppm group
only the F1 male pups at 21 days of age were statistically
significantly lighter than the controls. There were no
treatment-related effects noted on pup behavior or appearance. Organ
weight differences appeared to be attributable to the body weight
changes observed and were not indicative of toxic effects per se.
No treatment-related gross or histopathology was noted in any tissue
in either adults or pups. The NOAEL in this study was 30 ppm (equal
to 2.0 mg/kg bw/day) based on body weight effects in adult animals
(Blair et al., 1981b).
Special studies on genotoxicity
Cyromazine was negative in 10 of 11 genotoxicity studies. In the
inconclusive study, a mammalian spot test in mice, interpretation of
the results was confounded by reduced reproductive performance of the
high dose group resulting in a much smaller number of observations.
Survival of offspring to 12 days of age was reduced at the top two
dose levels, the levels which gave equivocally positive results. This
study cannot be considered to be conclusive (Table 2).
Special studies on embryo/fetoxicity
Rats
Groups of 25 mated Charles River COBS CD female rats were given
daily oral (gavage) doses of cyromazine (96.3% pure, suspended in 0.1%
aqueous carboxymethylcellulose) at 0, 100, 300 and 600 mg/kg bw/day in
a volume of 10 mg/kg bw/day on Days 6 through 19 of gestation. The
day evidence of mating was observed was designated Day 0 of gestation.
A red nasal discharge was observed in all rats given 300 or 600
mg/kg bw/day approximately 1 1/2 hours after dosing on one occasion
during the first part of the dosing period. At 600 mg/kg bw/day this
was accompanied by increased activity. A clear oral discharge and
inactivity were observed in all animals at 600 mg/kg bw/day on several
days in the middle of the dosing period about 1-4 hours after dosing
and in 11 of these rats at subsequent dosing. An oral discharge was
observed in five rats at 300 mg/kg bw/day prior to dosing on several
days at the end of the dosing period and in two rats at 100 mg/kg
bw/day on two occasions. There were no deaths during the study. Dams
given 300 and 600 mg/kg bw/day lost weight during the first three days
of dosing and had reduced weight gains for the entire dosing period
compared to the controls. There were no effects on the number of dams
with litters, the numbers of viable fetuses/litter or the number of
resorptions/litter. No dead fetuses were observed in any group. Mean
body weight of fetuses from dams dosed at 600 mg/kg bw/day were
statistically significantly lower than that of controls. At 300 mg/kg
bw/day fetal body weight was slightly lower than controls but was not
statistically significant. Sex ratio was similar in all groups.
There was no evidence of a treatment-related teratogenic effect in the
fetuses. The incidence of unossified sternebrae was increased
significantly at 600 mg/kg bw/day and slightly at 100 and 300 mg/kg
bw/day. A NOAEL was not clearly demonstrated in this study but
effects at 100 mg/kg bw/day were very slight (Rodwell et al., 1979).
Rabbits
Groups of 16 virgin female Dutch Belted rabbits were artificially
inseminated with sperm from one of six males of the same strain and
then given an injection with chorionic gonadotropin to induce
ovulation. The day of insemination was considered to be Day 0 of
gestation. Each female was given daily oral (gavage) doses of
cyromazine (96.3% pure suspended in 1% aqueous carboxymethylcellulose)
at 0, 25, 50 or 75 mg/kg bw/day on Days 6 through 27 of gestation in
a volume of 1 mg/kg bw/day.
Table 2. Results of mutagenicity assays on cyromazine
Test system Test organism Concentration* Results Reference
Ames test Salmonella typhimurium 20-5000 µg/0.1 ml negative Deparade and Arni, 1988
TA98, TA100,
TA1535, and
TA1537
Chromosome assay Human Lymphocytes 0-1000 µg/ml negative Strasser and Arni, 1985
DNA repair assay Rat hepatocytes 0.0001-1 mg/ml negative Tong, 1982
DNA repair assay Mouse Hepatocytes 0.0005-1 mg/ml negative Tong, 1983
Dominant lethal test Mouse 0, 226, 678 negative Hool and Muller, 1981
mg/kg bw
Mammalian Mouse 0-600 mg/kg bw inconclusive Strasser and Arni, 1986
Micronucleus test Mouse 0, 360, 1080 negative Strasser et al., 1987
(bone marrow) mg/kg bw
Mouse lymphoma assay Mouse L5178Y TK +/- 0-500 µg/ml negative Beilstein and Muller, 1985
Nucleus anomaly test Hamster 0-8000 mg/kg bw negative Hool, et al., 1980
Point mutation test V79 Chinese hamster 0-4000 µg/ml negative Dollenmeier and Muller, 1986
cells
Yeast assay Saccharomyces cerevisiae D7 375-3000 µg/ml negative Hool and Arni, 1984
* Purity range 96.2 - 98.9%
There were 0, 1, 2 and 4 deaths at 0, 25, 50 and 75 mg/kg bw/day,
respectively. Two of these females (one each at 25 and 75 mg/kg
bw/day) died of heart failure. Another 75 mg/kg bw/day animal died
with pneumonia-pleuritis. Cause of death of the remaining animals was
not established. No treatment-related changes in appearance or
behavior of the animals was noted. Two of the animals that died
aborted prior to death: one each at 50 and 75 mg/kg bw/day. Three
other females aborted: one at 50 mg/kg bw/day and two at 75 mg/kg
bw/day. All three treated groups had a mean body weight loss over the
treatment period. Pregnancy rates were low in all groups apparently
partially due to technical error but were slightly lower in all
treated groups than in the controls. Pre-implantation loss was higher
in all treated groups than in the controls but a dose-relationship was
not apparent. Post-implantation loss was similar in controls and the
two lower dose groups but was slightly higher in the high dose group
(2.4 cf 0.6 in controls). Post-implantation loss in the 75 mg/kg
bw/day group was nearly equally attributable to early and late
resorptions while early resorptions accounted for nearly all the
post-implantation loss in the other groups. The mean number of viable
fetuses/dam was reduced in the 75 mg/kg bw/day group. Fetal weights
did not appear to be affected but there were fewer male fetuses in the
litters of dams given 75 mg/kg bw/day. Only three fetuses had
malformations: one fetus at 25 mg/kg bw/day had fused sternebrae and
two fetuses (from one litter) at 75 mg/kg bw/day showed fetal
anasarca. The incidence of fetal variations was not treatment-related
(Blair et al., 1981a).
The above study was repeated in the same strain of rabbit using
dose levels of 0, 10, 30 and 60 mg/kg bw/day. In this study there
were two deaths, both at 60 mg/kg bw/day, with severe lung congestion
and edema. A reduction in faecal excretion which was dose-related in
duration was reported in the treated groups. Two dams each at both 30
and 60 mg/kg bw/day aborted. In this study weight loss during
treatment was noted only at 60 mg/kg bw/day. Weight gain was lower
than in controls in the other two groups but the difference at 10
mg/kg bw/day was small and not considered to be biologically
significant. Pregnancy rates in this study were in the expected range
in all groups. No treatment-related effect was observed on numbers of
implantations/dam or numbers of viable fetuses/dam. Post-implantation
loss was slightly higher in the 60 mg/kg bw/day group than in the
controls (1.1 cf 0.6 in controls). In this study no malformations were
observed in control fetuses but there were a few in each of the
treated groups. Fetal anasarca was seen in one fetus at 30 mg/kg
bw/day. Fused sternebrae were observed in 1, 1 and 3 fetuses at 10,
30 and 60 mg/kg bw/day, respectively; the fetus at 30 mg/kg bw/day was
the same one that showed anasarca. Hydrocephalus was observed in two
fetuses: one each at 10 and 60 mg/kg bw/day; the latter also had skull
anomalies (nasals, premaxillae and jugals malformed and small
bilaterally). Two fetuses from the same litter at 60 mg/kg bw/day had
abdominal closure defects (omphalocele). The total numbers of
malformed fetuses were 5, 6 and 7 from 5, 4 and 4 litters at 10, 30
and 60 mg/kg bw/day, respectively. All treated groups had increased
incidences of 27 presacral vertebrae and a 13th full rib but there was
no dose-relationship to this observation. No treatment-related gross
pathology was observed in the dams (Blair et al., 1981a).
A second study conducted in the same strain of rabbit and at the
same laboratory could not be evaluated due to an infection of the dams
(Schardein, et al., 1985).
In a study in New Zealand White (BUK(CRL)NZW fBR), Charles River
derived, closed colony, outbred, SPF rabbits, groups of 18 sexually
mature virgin females were artificially inseminated with sperm from
one of four males of the same strain and source. Immediately after
insemination the females were injected with chorionic gonadotropin to
ensure ovulation. On gestation days 7 through 19 the groups were
given daily oral (gavage) doses of cyromazine (95.2% pure, suspended
in 0.5% aqueous carboxymethylcellulose) at 0, 5, 10, 30 or 60 mg/kg
bw/day. An additional group was untreated as an environmental
control.
One female died or was sacrificed in each of the 0, 30 and 60
mg/kg bw/day groups. Two treated animals reportedly died from
intubation errors. No treatment-related changes in appearance or
behavior were noted. Four females aborted: 1, 1 and 2 at
0(untreated), 30 and 60 mg/kg bw/day. During the early part of the
treatment period the 60 mg/kg bw/day group lost weight and the 30
mg/kg bw/day group gained less weight than the other groups.
Following treatment the 60 mg/kg bw/day group showed increased weight
gain but failed to completely recover to control weight. In this
group food consumption was reduced during treatment and increased
following treatment compared to controls. The number of females
pregnant was similar in all groups except the 10 mg/kg bw/day group in
which 11/18 (61.1%) were not pregnant. This was considered to be a
random occurrence. The numbers of early resorptions/dam were somewhat
higher than controls at 30 and 60 mg/kg bw/day as was the number of
late resorptions at 60 mg/kg bw/day but the numbers of live
fetuses/dam was similar in all groups. Mean fetal weight was not
affected by treatment. There were statistically significant
differences in sex ratio at 5, 10 and 30 mg/kg bw/day but not at 60
mg/kg bw/day. No dose-relationship was observed and the differences
were considered to be incidental. Malformations were observed in
7(6), 2(2), 4(3), 3(2), 10(6) and 15(6) fetuses (litters) at 0
(vehicle control), 0 (untreated control), 5, 10, 30 and 60 mg/kg
bw/day, respectively. At 60 mg/kg bw/day 4 fetuses from one litter
had open eyelids and 3 fetuses from another litter had short tails.
These malformations were not seen in any other group but since all
affected fetuses in each case were from a single litter it is doubtful
the observations were treatment-related. A number of unusual
malformations were observed in one or more groups. Cyclopia with
multiple head anomalies was seen in 2 fetuses: one each at 10 and 30
mg/kg bw/day. One fetus at 60 mg/kg bw/day had externally apparent
skull anomalies with cleft palate. All three of these fetuses were
sired by the same male. Hydrocephalus was observed in 1(1), 2(2) and
2(2) fetuses (litters) at 10, 30 and 60 mg/kg bw/day. Spina bifida
was observed in one fetus at 60 mg/kg bw/day, diaphragmatic hernia in
1(1) and 3(2) fetuses (litters) at 10 and 30 mg/kg bw/day and
umbilical hernia in one fetus at each 30 and 60 mg/kg bw/day. Some
fetuses in each group had vertebral anomalies and/or rib anomalies but
the numbers of litters involved did not suggest a treatment-related
effect. The incidence of accessory skull bones and rudimentary 13th
ribs was increased in the 60 mg/kg bw/day group (Nemec and Rodwell,
1985).
A study was conducted in order to determine the possible genetic
origin of the observed malformations. Three groups of females were
utilized. Two of the groups of 56 females were inseminated with sperm
from the male which sired the two cyclopic fetuses; one group was sham
gavaged, the other untreated. The third group of 59 females was
inseminated with sperm from two alternate males. No cyclopic fetuses
were observed in these groups; however, various head anomalies were
observed including cleft palate, acrania and anophthalmia.
Hydrocephalus, spina bifida, diaphragmatic hernia, gastroschisis,
short tail and vertebral anomalies with or without rib anomalies were
observed in some fetuses in one or more of these control groups. Some
additional malformations were also seen: conjoined twins,
carpal/tarsal flexure and bradydactyly. These results indicate that
the malformations observed in the treated groups of the cyromazine
study (Nemec and Rodwell, 1985) are consistent with those seen in this
colony of rabbits (BUK:(CRL)NZWfBR). In these control groups the
highest incidence of hydrocephalus was 1.1% of the fetuses (Nemec and
Rodwell, 1986a).
Another teratology study was carried out in New Zealand White,
Hra;(NZW)SPF (Hazleton-Dutchland), rabbits. Groups of 74 sexually
mature virgin females were artificially inseminated with semen from
seven bucks of the same strain and source. Immediately after
insemination the females were injected with chorionic gonadotropin to
induce ovulation. The day of insemination was designated Day 0 of
gestation. On gestation days 7 through 19 the females were given
daily oral (gavage) doses of cyromazine (95.2% pure, suspended in 0.5%
aqueous carboxymethylcellulose) at 0, 5, 10 or 30 mg/kg bw/day in a
volume of 1 ml/kg bw.
There were 4, 3, 3 and 1 deaths and 4, 5, 2 and 5 abortions at 0,
5, 10 and 30 mg/kg bw/day, respectively. The incidences of decreased
defecation and urination was highest at 30 mg/kg bw/day and somewhat
higher than controls at 10 mg/kg bw/day. The group given 30 mg/kg
bw/day lost weight during treatment and had a higher weight gain
following treatment than controls. The other groups were similar to
controls. Food consumption was reduced in the 30 mg/kg bw/day group
during treatment and increased following treatment. There were
similar numbers of females pregnant in all groups. For the teratology
phase of the study a minimum of 25 females with viable fetuses were
examined from each group. There was no apparent treatment-related
effect on numbers of dead fetuses or early or late resorptions but the
numbers of viable fetuses and total implantations was slightly lower
in the 30 mg/kg bw/day group than in the other groups. The difference
was not statistically significant and was probably not biologically
significant. Fetal body weights and sex ratios were similar in all
groups. There were a total of 8(7), 11(8), 12(8) and 6(5) fetuses
(litters) at 0, 5, 10 and 30 mg/kg bw/day which had malformations.
Soft tissue malformations were observed only in the treated groups but
were not dose-related. The only soft tissue anomaly seen in more than
one fetus was diaphragmatic hernia which was seen in 1(1), 3(2) and
1(1) fetuses (litters) at 5, 10 and 30 mg/kg bw/day, respectively.
External malformations seen only in treated groups included
omphalocele (1 at 5 mg/kg bw/day), umbilical hernia (1 at 5 mg/kg
bw/day), gastroschisis(1 at 30 mg/kg bw/day), spina bifida (1 at 30
mg/kg bw/day), microphthalmia(1 at 5 mg/kg bw/day), macroglossia (1 at
10 mg/kg bw/day) and agnathia (1 at 10 mg/kg bw/day). Skeletal
malformations and visceral and skeletal variations were similar in all
groups.
The females not sacrificed for the teratology portion of the
study were allowed to deliver their litters and rear their offspring
to weaning. Mean gestation length was 31.7, 31.8, 32.3 and 32.4 days
at 0, 5, 10 and 30 mg/kg bw/day, respectively. No treatment-related
effects were noted in the number of dams with live litters, the number
of kits/litter, the total number of dead kits/group, kit survival to
day 4, the number of dams which failed to raise their litter to
weaning, sex ratio of kits or body weights of kits. Following culling
day 4 there was a slightly higher number of kit deaths in the 30 mg/kg
bw/day group during days 4-28 (37 cf 12 in controls). Among the kits
culled at day 4 only one (at 5 mg/kg bw/day) had a cataract; no other
malformations were observed. Among kits examined at weaning one
control kit had carpal flexure and one kit at each of 5 and 30 mg/kg
bw/day had a cataract; no other malformations were reported. Among
the kits that died during days 0-4, 1, 0, 0 and 4(2) fetuses (litters)
had malformations. The control had omphalocele. One kit at 30 mg/kg
bw/day had cyclopia, omphalocele and cleft palate. Other
malformations in this group were tarsal flexure, absent kidney and
ureter (each in one kit) and hydrocephalus (in 2 kits). No
malformations were observed in kits dying at days 4-28. No
treatment-related gross pathology was observed in dams at any dose
level. The NOAEL in this study was considered to be 5 mg/kg bw/day
(Nemec and Rodwell, 1986b).
Special studies on irritation and sensitization
Guinea pigs
A series of 10 sensitizing injections of cyromazine was given to
10 male and 10 female Pirbright white guinea pigs every other day
intracutaneously in the back. Fourteen days later a challenge
injection was given in the flank. Following the third and fourth
sensitizing injections each of the cyromazine-treated animals showed
some reaction. After the challenge injection only two of the animals
showed any reaction and in both cases the reaction was less than that
following the sensitizing injection. None of the animals was scored
as positive. There was no evidence of skin sensitizing (contact
allergic) potential of cyromazine in guinea pig (Ullman and Sachsse,
1978c).
Rabbits
Following treatment of the left eyes of 3 male and 3 female
Himalayan rabbits with 0.1g cyromazine, the eyes of the females were
flushed about 30 seconds after treatment. No irritation was observed
in cornea, iris or conjunctiva in any rabbit with or without rinsing
after treatment (Ullman and Sachsse, 1978a).
In Himalayan rabbits treated dermally with technical cyromazine
very slight to well-defined erythema and very slight to moderate
oedema was observed on scarified skin and no reaction to very slight
erythema and oedema on intact skin at 24 hours after treatment. By 72
hours after the start of treatment (48 hours after removal of
dressings) no reactions were observed on either intact or scarified
skin. The mean score was 1.1 of a possible 8. The test material
caused mild irritation to rabbits (Ullman and Sachsse, 1978b).
Observations in humans
No information available.
Oncogenicity studies of melamine, a metabolite
Several toxicological studies of melamine have been conducted in
rats and mice by the U.S. National Toxicology Program (NTP/NIH, 1983).
COMMENTS
Cyromazine administered orally to rats, monkeys, sheep or goats
was rapidly absorbed and excreted, predominantly in urine. Absorption
and excretion were also rapid in the hen. Tissue levels were highest
in the livers of rats, sheep and goats. Other tissue levels were low.
Cyromazine was excreted in small amounts in goat milk and in eggs.
The majority of the material excreted in the urine of rats,
monkeys, sheep and goats and in the excreta of hens was unchanged
cyromazine. Melamine was determined to be the major metabolite of
cyromazine in all these species. Other metabolites were not
identified.
WHO has classified cyromazine as slightly hazardous on the basis
of its acute toxicity and has concluded that it is "unlikely to
present acute hazard in normal use" (WHO, 1990).
In short-term rat and dog studies and in long-term studies in
mice and rats, the most consistent effect was a reduction in body
weight gain at high dose levels during the treatment period. The
effect appeared to the largely reversible upon cessation of treatment.
Red blood cell counts and haemoglobin levels were reduced in male dogs
at 3000 ppm. There was a slight increase in the number of females
with mammary gland adenocarcinomas in the high dose group (3000 ppm)
in both mice and rats. However, in rats the incidences at other dose
levels did not suggest a dose-response relationship and in the mouse
the incidence was within the historical control range. Therefore,
this effect was not considered to be treatment-related. No other
tumour incidence appeared to be affected by treatment.
There were ten negative genotoxicity studies and one inconclusive
study (mouse spot test).
In a multigeneration study in rats, cyromazine did not affect
fertility, but at maternally toxic doses there was increased perinatal
pup mortality and reduced pup weight. Studies in rats did not
demonstrate a teratogenic effect. Data from several rabbit
teratogenicity studies showed inconsistent results but no dose-related
effects. The Meeting felt that there was no evidence of
teratogenicity in rabbits.
The Meeting was aware of toxicological data on melamine, notably
the National Toxicology Program (USA) carcinogenesis bioassays on mice
and rats. The observation of bladder tumours in the rats in one of
these studies was related to the formation of bladder stones when the
rats were given high doses of melamine. Since this is an indirect
mechanism of carcinogenesis limited to this species, concern was
alleviated with respect to melamine residues in food.
The ADI was estimated utilizing a hundred-fold safety factor and
the NOAELs in the rat long-term (1.8 mg/kg bw/day) and reproduction (2
mg/kg bw/day) studies.
TOXICOLOGICAL EVALUATION
Level causing no toxicological effect
Mouse: 50 ppm in the diet, equal to 6.5 mg/kg bw/day
Rat: 30 ppm in the diet, equal to 1.8 mg/kg bw/day
Rabbit: 5 mg/kg bw/day
Dog: 300 ppm in the diet, equal to 9.1 mg/kg bw/day
Estimate of acceptable daily intake for humans
0-0.02 mg/kg bw
Studies which will provide information valuable in the
continued evaluation of the compound
- Observations in humans
- Identification of unknown metabolites
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See Also:
Toxicological Abbreviations