Cyanoguanidine

Endpoint summary

Description of key information

Groups of four SD rats per sex were fed with 0, 240, 2.400, 8.000 and 24.000 ppm Dicyandiamide (DCD) for 90 days according OECD 408 (1981). 
A repeated dose oral toxicity study according EPA OPP 82-1 was conducted in 2 dogs per sex and dose (initially 4 dogs). 
A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test [OECD 422] was conducted using SD rats at doses up to 1,000 mg/kg/day (NOAEL). 
In a 28-day oral toxicity dose range finding study similar to OECD 407 groups of ten Wistar rats per sex and dose were fed with up to 20.000 ppm DCD.  
In a 90-day dose range finding study similar to OECD 408 ten F344 rats per sex were fed diets containing 0, 1.25, 2.5, 5, and 10 % DCD.  The study language is Japanese. Only abstract and tables are available in English. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April - December 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

: no blood clotting time/potential; no Na, total cholesterol and urea; organ weights not determined for uterus, ovaries, thymus, spleen, brain heart; histopathology was not done on skin, salicary glands, mammary glands, and pro

GLP compliance:

yes

Remarks:

This study was conducted in compliance with GLP Regulations, Federal Register, December 22, 1978.

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York
- Age at study initiation: six weeks
- Weight at study initiation: males: 153.4-216.0 g; females: 119.3-161.8 g
- Housing: individuals in elevated stainless-steel wire-mesh cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002
- Water (e.g. ad libitum): tap water
- diet and water were analysed for contamination > there were no known contaminants in either the feed or the water which were expected to interfere with the objectives of the study
- Acclimation period: eleven days prior to sudy initiation


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.1 °C
- Humidity (%): 62.5 +/- 4.36 %
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): prior to study initiation
- Mixing appropriate amounts with (Type of food): feed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were conducted on freshly prepared control and test diets for homogeneity (preinitiation) and concentration (weeks 1, 4, 8, and 12) by the Analytical Chemistry Laboratory of Hazleton Biotechnologies Corporation, Vienna, Virginia
- Results of the analyses indicated that Dicyandiamide was mixed in a homogeneous manner and that diets presented to the animals contained Dicyandiamide within acceptable target levels

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

240 ppm

Remarks:

Nominal in diet

Dose / conc.:

2 400 ppm

Remarks:

Nominal in diet

Dose / conc.:

8 000 ppm

Remarks:

Nominal in diet

Dose / conc.:

24 000 ppm

Remarks:

Nominal in diet

No. of animals per sex per dose:

10 animals per sex per dose and satelitte group

Control animals:

yes, plain diet

Details on study design:

- Group assignment: computer-randomization (see also table 1)
- Post-exposure recovery period in satellite group: 4 weeks

Positive control:

No positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule dietary portion: once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Mortality, moribundity: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: at study initiation and once weekly for weeks 1-13 (through week 17 for satellite group)


FOOD CONSUMPTION (if feeding study):
- Time schedule for examinations: weekly, throughout the study (weeks 1-13; hrough week 17 for satellite group)


OPTHALMOLOGY:
- examinations were performed prior to initiation and at termination using an indirect opthalmoscope on all animals
- 1 % troicamide opthalmic solution (1 % Mydriacyl) was used as a mydriatic


CLINICAL PATHOLOGY:
- Thyroid function test were performed following ten weeks of treatment on ten animals/sex/group in the control and in the high dose groups, the satellite group was not evaluated
- Blood samples were collected via orbital sinus puncture following an overnight fast from food
- following parameters were evaluated: Triiodothyronine (T3), Thyroxine (T4), Thyroid-Stimulating Hormone (TSH)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: son termination on all animals in groups 1 through 5
- Animals fasted: Yes, overnight
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on termination on all animals in groups 1 through 5
- Animals fasted: Yes, overnight
- Parameters checked in table 2 were examined.


ORGAN WEIGHTS:
- liver, kidneys, adrenals, and testes with epididymides were examined


TISSUE PRESERVATION:
- the tissues (when present) in Table 4 from each animal were preserved in 10 % neutral buffered formalin

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3)
- Following 13 weeks of treatment (17 weeks for satellite group), all animals were weighed, anesthesized by an intraperitoneal injection of sodium pentobarbital and exsanguinated
- Necropsies were perfromed ba appropriately trained personnel using procedures approved by board-certified pathologists
- Gross observations were recorded
- Each necropsy included examinations as stated in table 3

HISTOPATHOLOGY: Yes (see table 4)
- tissues from all control and high-dose rats were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically
- Lesions, lungs, liver, and kidney from all low- and mid-dose animals were examined as well
- Due to the absence of distinct effects on the tissues of the high dose group, tissues from the satellite group were not examined microscopically

Other examinations:

No further examinations

Statistics:

- Total body weight change, absolute body weights, growth rates (Rao's growth parameters), total food consumption, clinical pathology data (except erythrocyte morphology), and organ weight data of the control group were compared statistically against the data of the compound-treated groups of the same sex.
- Absolute body weights were evaluated at week 13
- Total food consumption was evaluated from initiation through week 13
- Tests for homogeneity of variances and ANOVA were evaluated at the 5.0 % one-tailed probability level
- Control vs. compound-treated group mean comparisons of the above data were evaluated at the 5.0 % two-tailed probability level

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- No treatment-related clinical signs were observed
- Clinical signs which were noted were of the type and frequency commonly seen in rats at this laboratory


BODY WEIGHT, FOOD CONSUMPTION AND COMPOUND INTAKE
- Statistical analysis of mean total body weight change for groups 1 through 6 males and females (initiation through week 13) revealed no significant differences in the values between the control and compound treated groups
- In addition, the mean absolute body weights (week 13) and growth rates (initiation through week 13) were also comparable between the control and treated groups of both sexes
- Significant increases were noted in the mean total food consumption values of the Groups 3 and 6 females when compared to the control group. These findings, however, were not considered to be treatment related
All other food consumption values were comparable between control and compound-treated groups of either sex


OPHTHALMOSCOPIC EXAMINATION
- No treatment-related opthalmoscopic findings were noted during the study
- Sporadic incidences of commonly observed ocular findings were noted without relationship to treatment


CLINICAL CHEMISTRY
- Evaluation of clinical pathology data did not reveal any treatment-related effects
- Significant increases in total protein and albumin were noted in group 3 males. These findings, however, were considered incidental and not related to treatment


ORGAN WEIGHTS
- All organ weight data were comparable between control and treated groups of either sex


GROSS PATHOLOGY
- Observations noted at necropsy were similar between the control and treated groups and were considered to be incidental in nature and not treatment-related


HISTOPATHOLOGY: NON-NEOPLASTIC
- There were no histopathology changes which were considered to be associated with Dicyandiamide administration
- All lesions appeared to be incidental findings or part of spontaneous disease complexes of rats
- The lesions were typical of those which are commonly found in this strain of laboratory rat, and there was no discernable treatment-related alteration in the incidence or severity of the spontaneous lesions


In summary, the administration of Dicyandiamide, under the condition of this study was associated with no discernible gross or microscopic lesions in Sprague Dawley rats.

Key result

Dose descriptor:

NOAEL

Effect level:

> 24 000 ppm

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food consumption and compound intake; ophthalmoscopic examination; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Critical effects observed:

not specified

Conclusions:

Based upon the condition and findings of this study, toxicity of Dicyandiamide was not observed at any of the dose levels tested; therefore, a NOAEL was determined to be greater than 24000 ppm.

Executive summary:

This study investigated the toxicological effects elicited by the subchronic daily administration of Dicyandiamide to male and female Sprague Dawley rats via dietary admixture for 13 weeks.

Groups of 10 SD rats per sex and dose were dosed orally via diet with 0 (control), 240, 2400, 8000 and 24000 ppm. (24.000 ppm equals 2335 - 1291 mg/kg bw/day (male, week 1 & 13) and 2132 - 1664 mg/kg bw/day (female, week 1& 13)). An additional group of rats, the satellite group, received Dicyandiamide at a level of 24000 ppm for thirteen weeks followed by four weeks of treatment with only control diet. Criteria evaluated for signs of toxicity included survival, clinical signs, body weights, food consumption, ophthalmoscopic findings, clinical pathology, organ weight data, gross pathology, and histopathology.

No treatment-related effects were observed in any of the parameters evaluated. Based upon the condition and findings of this study, toxicity of Dicyandiamide was not observed at any of the dose levels tested; therefore, a "no effect" level was determined to be greater than 24000 ppm.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 1982- January 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

range-finding study

Deviations:

not applicable

Qualifier:

according to guideline

Guideline:

other: protocol approved by the sponsor, dated October 1982, and entitled "Protocol for a short-term (4-wk) oral toxicity study with dicyandiamide in rats"

Deviations:

not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

continous treatment

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

200 ppm

Remarks:

Nominal in diet

Dose / conc.:

2 000 ppm

Remarks:

Nominal in diet

Dose / conc.:

20 000 ppm

Remarks:

Nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects (slight changes of doubtful toxicological significance)

Critical effects observed:

not specified

- general condition, behaviour, survival and food intake were not adversely affected by the test substance in any of the dose groups

- there were no distinct or dose-related changes in growth among the various groups, body weights of males of the top-dose group were relatively low

- water intake was increased in the top-dose group in males

- total white blood cell count was slightly decreased in males of the top-dose group

- liver and kidney weights did not show any changes of toxicological significance

- gross and microscopic examinations of liver and kidneys did not reveal any abnormality that could be ascribed to the ingestion of dicyandiamid

Conclusions:

From the present study it appears that 2000 ppm Dicyandiamide did not induce any noticable toxic effects; at the next higher level of 20000 ppm only slight changes of doubtful toxicological significance were observed.

Executive summary:

This study investigated the toxicological effects elicited by the subacute administration of Dicyandiamide to male and female Wistar rate via dietary admixture for approximately 4 weeks.

Groups of ten male and female rats were dosed orally via diet with 0 (control), 200, 2000 or 20000 ppm.

A general battery of parameters was evaluated and included general condition, behaviour, survival, food intake, growth rates, body weight, water intake, blood cell count , liver and kidney weights, gross and microscopic examinations of liver and kidneys. General condition, behaviour, survival and food intake were not adversely affected by the test substance in any of the dose groups. There were no distinct or dose-related changes in growth among the various groups, body weights of males of the top-dose group were relatively low. Water intake was increased in the top-dose group in males. Total white blood cell count was slightly decreased in males of the top-dose group. Liver and kidney weights did not show any changes of toxicological significance. Gross and microscopic examinations of liver and kidneys did not reveal any abnormality that could be ascribed to the ingestion of dicyandiamid.

From the present study it appears that 2000 ppm Dicyandiamide did not induce any noticable toxic effects; at the next higher level of 20000 ppm only slight changes of doubtful toxicological significance were observed.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Not assignable: original study in foreign language (Japanese)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

No details reported (> only abstract in English)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

No details reported (> only abstract in English)

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No details reported (> only abstract in English)

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continous treatment

Dose / conc.:

0 other: %

Remarks:

Control

Dose / conc.:

1.25 other: %

Remarks:

Nominal in diet

Dose / conc.:

2.5 other: %

Remarks:

Nominal in diet

Dose / conc.:

5 other: %

Remarks:

Nominal in diet

Dose / conc.:

10 other: %

Remarks:

Nominal in diet

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

No details reported (> only abstract in English)

Positive control:

No

Observations and examinations performed and frequency:

No details reported (> only abstract in English)

Sacrifice and pathology:

No details reported (> only abstract in English)

Other examinations:

No details reported (> only abstract in English)

Statistics:

No details reported (> only abstract in English)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

- No animals died during the administration period.
- Inhibition of body weight gain was more marked in both sexes of the 10 % group and in females of the 5 % group as compared with the control group.
- Mean food intake in males of the groups treated with 5 % or 10 % and in females of the 10 % grop was significantly higher than that in the control group.
- Serum biochemical investigation revealed a higher level of serum BUN in both sexes of the 10 % group.
- On histopathological examination, toxic changes characterized by the occurrence of intranuclear eosinophilic inclusion bodies in the proximal tubular epithelium of the kidney were observed in both sexes of the 10 % group.
- Similar inclusion bodies were also seen in 2 out of 10 males of the 5 % group.

Key result

Dose descriptor:

NOAEL

Effect level:

2.5 other: % in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Dose descriptor:

LOAEL

Effect level:

5 other: % in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: decreased body weight gain

Critical effects observed:

not specified

Conclusions:

It can be concluded that a level of 10 % cyanoguanidine in the diet is unequivocally toxic. A dose level, 5 % cyanoguanidine, in the diet might be appropriate as a high dose for a carcinogenicity study.

Executive summary:

Male and female F344 rats were fed diets containing 0, 1.25, 2.5, 5, and 10 % cyanoguanidine for 13 weeks to determine appropriate dose levels for a subsequent 2-year carcinogenicity study. The rats were randomly allocated to 5 groups, each consisting of 10 males and females.

No animals died during the administration period. Inhibition of body weight gain was more marked in both sexes of the 10 % group and in females of the 5 % group as compared with the control group. Mean food intake in males of the groups treated with 5 % or 10 % and in females of the 10 % group was significantly higher than that in the control group. Serum biochemical investigation revealed a higher level of serum BUN in both sexes of the 10 % group. On histopathological examination, toxic changes characterized by the occurrence of intranuclear eosinophilic inclusion bodies in the proximal tubular epithelium of the kidney were observed in both sexes of the 10 % group. Similar inclusion bodies were also seen in 2 out of 10 males of the 5 % group.

From these results, it can be concluded that a level of 10 % cyanoguanidine in the diet is unequivocally toxic. A dose level, 5 % cyanoguanidine, in the diet might be appropriate as a high dose for a carcinogenicity study.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October 30, 1989 - April 6, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

yes

Remarks:

4 week study instead of 90 days; dose group should be 4 dogs/sex but palatability problem in highest dose group > half of the animals were sacrificed at the end of 4 weeks; reduced analytical program (see also "rational for realibility")

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products, Inc., Cumberland Virginia
- Vacciniations against: distemper, hepatitis, leptospirosis, parainfluenza, parvovirus, and rabies prior to receipt at Hazleton Laboratories America, Inc.
- Age at study initiation: approx. 5 months
- Weight at study initiation: males: 5.9 - 7.8 kg; females: 5.0 - 7.3 kg
- Housing: individually in a stainless-steel cage
- Diet (e.g. ad libitum): Purina Certified Canine Diet meal #5007
- Water (e.g. ad libitum): tap water (Fairfax County Virginia Water Authority)
- diet and water were analysed for contamination > there were no known contaminants in either the feed or the water which were expected to interfere with the objectives of the study
- Acclimation period: 2 weeks prior to inition of the treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 24.4 °C
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

other: oral: first 4 weeks in feed, then capsule

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly, throughout approx. 4 weeks of study
- Mixing appropriate amounts with (Type of food): feed


CAPSULE PREPARATION AND ADMINISTRATION:
- the appropriate amount of Dicyandiamde was weighed and transferred into a 1/2-oz gelatin capsule
- Storage: room temperature
- All doses were calculated based on most recently recorded body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- A 50-g sample was taken in duplicate from each level of the diet for each mixed batch (including control)
- One set of samples was forwarded to the Analytical Department of Hazleton Laboratories for subsequent analyses and the other set of samples was held in reserve (storage frozen)
- Diet samples were analyzed for homogeneity, stability, and concentration
- Homogeneity: analyzed from a pretreatment mix consisting of two samples from the top, middle, and bottom of the mixer for the 1.0 (low) and 5.0 % (high) levels. Analysis of a control sample was also conducted
- Stability: analyzed from a pretreatment mix for Day 0 and following 7 and 10 days of storage at room temperature (in the study room) for the 0 (control), 1.0 (low), and 5.0% (high) levels.
- Concentration: analyzed weekly (weeks 1-4) for all levels
- Analysis revealed homogeneous distribution of the test substance within the diet

Duration of treatment / exposure:

week 1-4: diet
week 5 until Sacrifice (week 8): capsule

Frequency of treatment:

- week 1-4: ad libitum
- week 5-8: once daily (capsula), generally between 9-11 a.m.

Dose / conc.:

0 other: %

Remarks:

Nominal in diet

Dose / conc.:

1 other: %

Remarks:

Nominal in diet, weeks 1-4

Dose / conc.:

2.5 other: %

Remarks:

Nominal in diet, weeks 1-4

Dose / conc.:

5 other: %

Remarks:

Nominal in diet, weeks 1-4

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Capsular administration, weeks 5-8

Dose / conc.:

625 mg/kg bw/day (nominal)

Remarks:

Capsular administration, weeks 5-8

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

Capusular administration, weeks 5-8

No. of animals per sex per dose:

- week 1-4: 4 animals per sex per dose
- week 5 - sacrifice (week 8): 2 animals per sex per dose

Control animals:

other: week 1-4: plain diet, thereafter plain capsule

Details on study design:

- Group assignment: computer-randomization with Bartlett's test performed on pretreatment body weights to ensure homogeneity among groups (see also table 1)
- due to apparent palatability problem, the study was modified: half of the dogs were sacrificed following approx. 4 weeks (29 days) of dietary ingestion of Dicyandiamide and the remainig dogs received comparable doses via capsule for approx. 3 additional weeks (22 days). The modified design for capsule portion is presented in table 2

Positive control:

No positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule dietary portion: during mortality / moribundity check
- Time schedule capsule portion: approx. 1 hour postdose


DETAILED CLINICAL OBSERVATIONS: Yes
- Mortality, moribundity: twice daily
- Clinical signs: at a minimum once daily
- Detailed clinical observations were conducted once each week

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, throughout the study


FOOD CONSUMPTION (if feeding study):
- Time schedule for examinations: weekly, throughout the study

CLINICAL PATHOLOGY:
- clinical laboratory studies were performed on all surviving dogs during week -2 (for selection/assignment to study and as a baseline prior to the initiation of treatment), week 4, and week 7 of the study


CLINICAL PATHOLOGY:
- clinical laboratory studies were performed on all surviving dogs during week -2 (for selection/assignment to study and as a baseline prior to the initiation of treatment), week 4, and week 7 of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: see above (clinical pathology)
- Animals fasted: Yes, overnight
- How many animals: all dogs
- Parameters checked in table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above (clinical pathology)
- Animals fasted: Yes, overnight
- How many animals: all dogs
- Parameters checked in table 3 were examined.


ORGAN WEIGHTS:
- liver with drained gallbladder, kidneys, testes with epididymides, and thyroid with parathyroids were weighed following careful dissection and trimming to remove fat and other contiguous tissue in a uniform manner
- From these data, the organ/terminal body weight ratios were calculated


TISSUE PRESERVATION:
- the tissues (when present) in Table 5 from each dog were preserved in 10 % neutral buffered formalin
- additionally the tissues in table 6 were preserved for possible future examinations if indicated by signs of toxicity or target organ involvement


HISTOPATHOLOGY:
- the tissues in table 5 were enbedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically from all dogs in the control and high-dose groups

Sacrifice and pathology:

- Following approx. 4 weeks (last two dogs/sex/group) or 7 weeks (remainig animals) of treatment, surviving dogs were exsanguinated while under the influence of sodium thiamylal anesthesia
- Following an overnight fast from food, a complete gross necropsy was performed on each dog by trained personnel using procedures approved by board-certified pathologists
- parameters of necropsy see table 4

Other examinations:

No further examinations.

Statistics:

- Body weight changes at week 0-4, total food consumption at weeks 1-4, clinical pathology data at weeks -2 and 4 (with the exception of cell morphology gradings and tsh) and organ weight data of the control group were compared statistically to the data from the treated groups of the same sex.
- Tests for homogeneity of variances and ANOVA were evaluated at the 5.0 % one-tail probability level.
- Control versus compound-treated group mean comparisons were evaluated at the 5.0% two-tailed probability level.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- No dogs died during the study.
- Emesis was the most frequentzly noted finding but was not considered to be the result of treatment with Dicyandiamide due to its presence throughout the dose groups


BODY WEIGHT, FOOD CONSUMPTION AND COMPOUND CONSUMPTION
- Regardless of dietary or capsule administration of Dicyandiamide, most dogs gained weight throughout the study.
- No significant differences were observed in mean body weight change and total food consumption during the dietary portion of the study.
- However, mean body weight change values for group 4 males and females (weeks 0-4) were 33 and 64 % lower, respectively, when compared to the corresponding control value of the same sex.
- No distinct differences were observed between the dietary and capsule portions of the study in relation to body weight and food consumption data
- Based on mean body weight and food consumption data with respect to dietary levels, the average ingested intake for the dogs treated with Dicyandiamide throughout the dietary portion of the study was as shown in table 7.


HAEMATOLOGY
- There were no apparent treatment-related changes in associated hematology data which could be attributed to ingestion of Dicyandiamide

CLINICAL CHEMISTRY
- significant differences in clinical chemistry data of treated dogs when compared to the corresponding control value of the same sex included a decreased mean blood urea nitrogen value for the group 4 females at week -2, decreased mean inorganic phosphorous values for the group 4 dogs for both sexes at week 4, and a decreased mean chloride value for the group 3 females at week 4
- Although statistical analyses of week 7 clinical chemistry data was not performed due to the small sample size/group, inorganic phosphorus values for the remaining group 4 dogs of both sexes were slightly decreased at week 7
- Due to comparable differences in inorganic phosphorus at the pretreatment interval and the low magnitude of these differences, the biological importance of these variations is unclear
- All remaining significances were considered incidental to treatment with Dicyandiamide.


ORGAN WEIGHTS
- No statistically significant differences were noted in any of the associated organ weight data.
- The group 4 mean absolute testis/epididymis organ weight and organ-to-terminal-body-weight ratio values were slightly decreased when compared to the concurrent control value.


GROSS PATHOLOGY
- There were no gross necropsy findings which were considerd the direct result of administration with Dicyandiamide (Cyanoguanidine)
- The small testes noted in two Group 3 and two Group 4 dogs may have been related to the smaller body weight of the dogs.


HISTOPATHOLOGY: NON-NEOPLASTIC
- There were no compound-related histomorphologic lesions in any of the representative sections of tissues examined

Dose descriptor:

NOEL

Remarks:

phase 1: diet (week 0-4)

Effect level:

> 25 000 ppm

Sex:

male/female

Basis for effect level:

other: overall effects (25000 ppm: ~ 900 mg/kg/day)

Dose descriptor:

NOEL

Remarks:

Phase 2: capsule (week 5-7)

Effect level:

625 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

Table 7: Compound Consumption (weeks 1-4)

Group	Dietary Level %	Males mg/kg/day	Females mg/kg/day
2	1.0	333.32	351.13
3	2.5	904.41	895.16
4	5.0	1749.67	1601.40

 

Conclusions:

Based on the data generated from this study, the non-observable-effect level of Dicyandiamide (Cyanoguanidine), administered to male and female beagle dogs is greater than 2.5 % (approx. equivalent to 900 mg/kg/day) in the diet for 4 weeks followed by 625 mg/kg/day administered via capsule for an additional 3 weeks.

Executive summary:

This study investigated the toxicological effects elicited by the subchronic administration of Dicyandiamide (Cyanoguanidine) to male and female dogs via dietary admixture for approximately 4 weeks followed by capsular administration for an additional 3 weeks.

Groups of four male and female beagle dogs were dosed orally via diet with 0 (control), 1.0, 2.5, and 5.0 %. However, due to an apparent palatability problem, noted especially in the high-dose animals, two dogs/sex/group at the end of the 4 week dietary portion of the study were sacrificed. The remaining two dogs/sex/group were dosed with the test material via capsule administration at target concentrations of 250, 625, and 1250 mg/kg/day. 

A general battery of parameters was evaluated and included mortality and moribundity, clinical observations, body weight and food consumption measurements, clinical pathology, organ weight evaluations, and macroscopic and microscopic examinations of protocol-specified tissues. 

There was no mortality nor were there any clinical observations indicative of toxicity noted throughout the study.

Statistical evaluations of body weight data failed to reveal any significant differences between the dose groups and their respective controls, although mean body weight gain data did suggest that those responses toward a depression in weight gain was apparent. Similarly, mean food consumption values were depressed, albeit not statistically significant in the high-dose males and females as compared to the respective control values.

Statistical evaluation of clinical pathology data revealed a number of differences which were only noted at the pretreatment period. Post-treatment findings were considered spurious in nature based on the low magnitude of differences or the failure to demonstrate a dose response.

There were no statistically significant differences in mean organ weight data when treatment group values were compared to respective control values, although gross necropsy and absolute/relative testes weight evaluations described somewhat smaller-sized testes in the group 3 and 4 males. Histomorphologic examination of protocol-specified tissue sections, however, failed to reveal any compound-related lesions involving the testes or any other tissues examined.

Based on the data generated from this study, the non-observable-effect level of Dicyandiamide (Cyanoguanidine), administered to male and female beagle dogs is greater than 2.5 % (approx. equivalent to 900 mg/kg/day) in the diet for 4 weeks followed by 625 mg/kg/day administered via capsule for an additional 3 weeks.

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 wks
- Weight at study initiation: male; 344 - 376 g, female; 204 - 245 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: From: To: no data

Route of administration:

oral: gavage

Vehicle:

other: 3% gum arabic solution

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0, 8, 40, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

male: 44 days,
female: from 14 days before mating to day 3 lactation

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

male: 12
female: 12

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: males and females; 0, 3, 7, 14 day from dose start, after then, one day per week. At mated female, 0, 7, 14, 20 day from pregnancy and 0, 4 day from lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: males and females; 0, 3, 7, 14 day from dose start, after then, one day per week. At mated female, 0, 7, 14, 20 day from pregnancy and 0, 4 day from lactation.

OTHER: Mortality: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: males and females; 0, 3, 7, 14 day from dose start, after then, one day per week. At mated female, 0, 7, 14, 20 day from pregnancy and 0, 4 day from lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: males and females; 0, 3, 7, 14 day from dose start, after then, one day per week. At mated female, 0, 7, 14, 20 day from pregnancy and 0, 4 day from lactation.

OTHER: Mortality: Yes
- Time schedule: daily

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Details on results:

In addition no effects on necropsy findings. No histopathological changes in organs were found in either sex.

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

A preliminary test to decide the highest level at 100, 300, 1000 mg/kg/day for 14 day was conducted. At each dose, no change was observed. Then the highest dose level for the test was set at 1000 mg/kg/day.

Conclusions:

The NOAEL for the repeat dose toxicity is considered to be 1000 mg/kg/day for both sexes.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test [OECD TG 422] was conducted using SD rats at doses of 0, (vehicle; 3% gum arabic solution), 40, 200, and 1000 mg/kg/day. The dosing period for males was 44 days, and females were dosed from 14 days before mating to day 3 of lactation. This substance had no effect on clinical signs, body weights, food consumption or necropsy findings. The organ weights were similar among all groups. No histopathological changes ascribable to this substance in these organs were found in either sex. The NOAEL for the repeat dose toxicity is considered to be 1000 mg/kg/day for both sexes.

Reference 6

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Not assignable: Abstract; original study in foreign language (Japanese)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

No details reported (> only abstract in English)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

No details reported (> only abstract in English)

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No details reported (> only abstract in English)

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continous treatment (gavage with feed)

Dose / conc.:

0 other: %

Remarks:

Control

Dose / conc.:

1.25 other: %

Remarks:

Nominal in diet

Dose / conc.:

2.5 other: %

Remarks:

Nominal in diet

Dose / conc.:

5 other: %

Remarks:

Nominal in diet

Dose / conc.:

10 other: %

Remarks:

Nominal in diet

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

No details reported (> only abstract in English)

Positive control:

No

Observations and examinations performed and frequency:

No details reported (> only abstract in English)

Sacrifice and pathology:

No details reported (> only abstract in English)

Other examinations:

No details reported (> only abstract in English)

Statistics:

No details reported (> only abstract in English)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

- No animals died during the administration period.
- Inhibition of body weight gain was more marked in both sexes of the 10 % group and in females of the 5 % group as compared with the control group.
- Mean food intake in males of the treated with 5 % or 10 % and in females of the 10 % group was significantly higher than that in the control group.
- Serum biochemical investigations revealed a higher level of serum BUN in both sexes of the 10 % group.
- On histopathological examination, toxic changes characterized by the occurrence of intranuclear eosinophilic inclusion bodies in the proximal tubular epithelium of the kidney were observed in both sexes of the 10 % group. Similar inclusion bodies were also seen in 2 out of 10 males in the 5 % group.

Dose descriptor:

NOAEL

Effect level:

2.5 other: % in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Dose descriptor:

LOAEL

Effect level:

5 other: % in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: decreased Body weight gain

Critical effects observed:

not specified

Conclusions:

It can be concluded that a level of 10 % of cyanoguanidine in the diet is unequivocally toxic. A dose level of 5 % cyanoguanidine in the diet might be appropriate as a high dose for a carcinogenicity study.

Executive summary:

A 13-week subchronic oral toxicity study of cyanoguanidine was performed in male and female F344 rats by feeding of CRF1 powder diets containing 0, 1.25, 2.5, 5 and 10 % cyanoguanidine to determine appropriate dose levels for a subsequent 2 -year carcinogenicity study. The rats were randomly allocated to 5 groups, each consisting of 10 males and 10 females.

No animals died during the administration period. Inhibition of body weight gain was more marked in both sexes of the 10 % group and in females of the 5 % group as compared with the control group. Mean food intake in males of the treated with 5 % or 10 % and in females of the 10 % group was significantly higher than that in the control group.

Serum biochemical investigations revealed a higher level of serum BUN in both sexes of the 10 % group.

On histopathological examination, toxic changes characterized by the occurrence of intranuclear eosinophilic inclusion bodies in the proximal tubular epithelium of the kidney were observed in both sexes of the 10 % group. Similar inclusion bodies were also seen in 2 out of 10 males in the 5 % group.

From these results, it was concluded that a level of 10 % of cyanoguanidine in the diet is unequivocally toxic. A dose level of 5 % cyanoguanidine in the diet might be appropriate as a high dose for a carcinogenicity study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 291 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three out of four feeding studies in rats and dogs conclude a NOAEL of 24.000 - 25.000 ppm DCD. In addition, the NOAEL is reported to be 1000 mg/kg bw for male and female rats in study conducted according to OECD 422. In one 28-day study in Wistar rats slight changes of doubtful toxicological significance were observed resulting in a NOAEL of 2.000 ppm. A general battery of parameters was evaluated and included general condition, behaviour, survival, food intake, growth rates, body weight, water intake, blood cell count, liver and kidney weights, gross and microscopic examinations of liver and kidneys. General condition, behaviour, survival and food intake were not adversely affected by the test substance in any of the dose groups. There were no distinct or dose-related changes in growth among the various groups, body weights of males of the top-dose group were relatively low. Water intake was increased in the top-dose group in males. Total white blood cell count was slightly decreased in males of the top-dose group. Liver and kidney weights did not show any changes of toxicological significance. Gross and microscopic examinations of liver and kidneys did not reveal any abnormality that could be ascribed to the ingestion of DCD. The slight changes in male body weight, white blood cell count, and water intake are of doubtful toxicological significance and are not strong enough to challenge the remaining results.

Thus, DCD can be considered as non-toxic in repeated dose feeding studies up to a concentration of 24.000 ppm derived from the most reliable 90-day study in SD rats. 24.000 ppm equals 1291 mg/kg bw/day (males, week 13) and 1664 mg/kg bw/day (female, week 13).

Justification for classification or non-classification

The highest compound consumption in a 90 day study was 1291 mg/kg bw/day reported for male SD rats in the 24.000 ppm group (week 13) and 1664 mg/kg bw in females, respectively. These values are above the limit value of 1000 mg/kg bw/day given in OECD 408. DCD is considered to be non-toxic in rodents in repeated dose feeding studies.