Cyanoguanidine

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990 - 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

no estrous cycle & sperm parameters investigated

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

yes

Remarks:

no estrous cycle & sperm parameters investigated

GLP compliance:

yes

Remarks:

The study was conducted in compliance with the GLP Regulations as set forth in Title 40 of the U.S. Code of Federal Regulations Part 160 (effective May 1983).

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cyanoguanidine

EC Number:

207-312-8

EC Name:

Cyanoguanidine

Cas Number:

461-58-5

Molecular formula:

C2H4N4

IUPAC Name:

1-cyanoguanidine

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

other: Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina, USA
- Age at study initiation: (P) x 5 wks; (F1) x wks
- Weight at study initiation: (P) Males: 223-266 g; Females: 154-190 g;
- Fasting period before study: no
- Housing: individual housing in stainless-steel, hanging, wire-mesh cages
- Diet (ad libitum): Purina Certified Rodent Chow #5002
- Water (ad libitum): tap water, via an automatic watering system (or water bottles as appropriate)
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 65-80
- Humidity (%): 31-87
- Air changes (per hr): 21
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): fresh diets were prepared weekly and stored at room temperature
- Mixing appropriate amounts with (Type of food): Purina Certified Rodent Chow #5002
- Storage temperature of food: room temperture

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 21 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- On day 20 of presumed gestation dams were placed in polycarbonate nesting boxes
- in addition, nonconfirmed females were placed in polycarbonate nesting boxes at the end of the breeding phase

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability (14 days at room temperature) and homogeneity of dicyandiamide were established for the 5000 and 50000 ppm dose levels.
- For homogeneity analyses, duplicate samples were taken from the top, middle, and bottom of the mixes for the 5000 and 50000 ppm levels.
- Routine analyses for 100 % concentration were performed weekly for the first 4 weeks and monthly thereafter.

Duration of treatment / exposure:

F0 animals were treated (ad libitum) for 14 weeks prior to mating and throughout the mating, gestation, lactation, and postweaning periods, as appropriate. Treatment continued until termination.
F1 animals received the test diet (ad libitum) following weaning for at least 14 weeks prior to mating and throughout the mating, gestation and lactation periods. Animals selecetd as alternates or as part of the replacement pool also received the test diet beginning on Day 22 until their sacrifice within approx. 1 week of the start of the F1 maturation phase and 1 month after the last litter entered the maturation phase, respectively.

Frequency of treatment:

continuous tretament

Details on study schedule:

- Male and female F1 selected animals received the test diet ad libitum following weaning for at least 14 weeks prior to mating and throughout the mating, gestation, and lactation periods
- Animals selected as alternates or as part of the replacement pool also received the test diet beginning on Day 22 until their sacrifice within approx. 1 week of the start of the F1 maturation phase and 1 month after the last litter entered the maturation phase, respectively
Beginning at approx. Day 28, selected F1 breeder animals were housed individually in satinless-steel, hanging, wire-mesh cages and weekly body weights, food and compound consumption, and clinical observations were recorded thereafter


Mating procedures:
- Following a growth phase of at least 14 weeks, male and female F1 selected animals were mated placing one female with one male of the same dietary group until all the animals were paired.
- Each pair was given a maximum of 21 days to achieve mating.
- A daily examination was made for the presence of sperm or a retained copulatory plug.
- The day of observation of sperm or plug was designated as Day 0 of gestation.
- On day 20 of presumed gestation (or at the end of the breeding phase for unconfirmed females), dams were placed in nesting boxes.


- Treatment, clinical observations, and body weight, food consumption, and compound consumption measurements for all F1 generation were performed as described for the F0 generation. However, no pups from the F2 generation were retained to produce an additional generation. Rather, after weaning, all pups from the F2 generation were sacrificed via carbon dioxide inhalation and exsanguination and subjected to a gross examination of cervical, thoractic, and abdominal viscera for macroscopic abnormalities.


- Following weaning of the F2 pups, and based on the decision not to rebreed following consultation with the Sponsor, all F1 males and females were sacrificed by exsanguination under sodium pentobarbital anestesia and subjected to a complete gross necropsy according to the same procedures as described for the F0 generation. Gross necropsies were performed on F1 males and females found dead or sacrificed via carbon dioxide inhalation and exsanguination in a moribund condition. The uteri and ovaries of females were examined for implantation sites and corpora lutea, respectively. An examination was performed to indicate apparently early or late resorptions or apparently normally developing fetuses

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

Nominal in diet, F0

Doses / concentrations 2

Dose / conc.:

5 000 ppm (nominal)

Remarks:

Nominal in diet, F0

Doses / concentrations 3

Dose / conc.:

15 000 ppm (nominal)

Remarks:

Nominal in diet, F0

Doses / concentrations 4

Dose / conc.:

50 000 ppm (nominal)

Remarks:

Nominal in diet, F0

No. of animals per sex per dose:

26 animals per sex per dose (F0 and F1 animals)

Control animals:

yes, concurrent no treatment

Details on study design:

No further details

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (at body weight intervals)


BODY WEIGHT: Yes
- Time schedule for examinations: weekly
- during gastation, F0 females were weighed on day 0, 7, 14, and 20 and aobserved closely for signs of abortion, premature delivery, or diffcult and prolonged parturition
- during lactation, F0 females were weighed on days 0, 7, 14, and 21


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- measured form day 0-7, 7-14, 14-20, and 0-20 for gestation and days 0-4, 4-7, 7-10, 10-14, and 0-14 of lactation
- after weaning, females resumed a weekly body weight schedule until sacrifice
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


HISTOPATHOLOGY:
- At the scheduled sacrifice, the vagina, uterus, ovaries, testes, epididymes, seminal vesicles, prostate, and unusual lesions were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and evaluated microscopically from F0 and F1 animals in Groups 1 and 4 selected for mating, as well as from all unmated animals in Groups 1 and 4 (excluding animals selected as alternates or part of the replacement pool). In addition, all tissues showing gross pathology and gross lesions were examined


OTHER:
- pregnant females were allowed to dliver and raise their young to day 21 postpartum
- all F0 males and females (including those that failed to deliver) were retained until the last litter was weaned (rest phase), when a decision was made not to rebreed based on reproductive performance of the first breeding; at that time, the F0 males and females were sacrificed via sodium pentobarbital injection and exsanguination and subjected to a complete gross necropsy, which included an examination of external surface, all orifices, cranial activity, cervical, thoractic, and abdominal viscera
- the following tissues, as appropriate, were saved from all F0 animals selected for mating and preserved in 10% neutral-buffered formalin: vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate, unusual lesions
- gross necropsies were performed on F0 males found dead or sacrificed (via carbon dioxide inhalation) in a moribund condition

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

- as soon as possible after birth, each pup (live or dead) was sexed
- Live pups were weighed and examined for external abnormalities
- On days 0, 4 (precull), 7, 14, and 21 of lactation, the number of live pups of each sex per litter, body weight of all live pups, and clinical observations, and clinical observations were recorded.
- On day 4 of lactation, all litters with more than eight pups were culled to that number by random card draw, to produce, as nearly as possible, litters containing four males and four females
- culled pups were sacrificed by intrapertoneal injection of sodium pentobarbital, examined for cervical, thoractic, or abdominal visceral abnormalities, and preserved in alcohol


PARAMETERS EXAMINED
- litters were observed daily for evidence for abnormal or ill health
- in addition, daily mortality records were maintained on each litter throughout lactation, and the number of pups of each sex which were missing, found dead, or sacrified in extremis, with or without evidence of cannibalization, was recorded
- pups which died during lactation were examined externally and internally in an attempt to determine the cause of death and preserved in alcohol


- at weaning, two male and two female pups from each litter, when possible were randomly selected as potential F1 parental animals
- pups selected for the F1 maturation phase and replacement pups were group housed by litter in clean nesting boxes with wire tops and water bottles
- the first pup/sex/litter was designated as the breeder and the second pup/sex/litter was designated as the alternate
- a replacement pool of five pups/sex/litter was selected from the available alternates and retained until the last litter entered the maturation phase
- sibling matings were avoided at the time of breeding
- pups not selected as breeder or alternates were sacrificed by carbon dioxide inhalation, subjected to a gross examination of the cervical, thoractic, and abdominal viscera, and discarded; these procedures were also followed for animals selected as alternates but not required as F1 parental animals

Postmortem examinations (parental animals):

- all F0 males and females (including those that failed to deliver) were retained until the last litter was weaned (rest phase), when a decision was made not to rebreed based on reproductive performance of the first breeding; at that time, the F0 males and females were sacrificed via sodium pentobarbital injection and exsanguination and subjected to a complete gross necropsy, which included an examination of external surface, all orifices, cranial activity, cervical, thoractic, and abdominal viscera

- the following tissues, as appropriate, were saved from all F0 animals selected for mating and preserved in 10% neutral-buffered formalin: vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate, unusual lesions

- gross necropsies were performed on F0 males found dead or sacrificed (via carbon dioxide inhalation) in a moribund condition

Postmortem examinations (offspring):

Same procedures as for F0 animals

Statistics:

- performed on F0 and F1 animals on body weight, body weight change, and food consumption values during gestation and lactation
- performed on F0 and F1 maternal body weight, body weight change, and food consumption values during gestation and lactation
- performed on F0 and F1 duration of gestation and F1 and F2 litter data which included total number of pups delivered per litter, and the number of live pups (Days 0, 4 pre- and postcull, 7, 14, and 21)
- Mean data of the control group were compared statistically to corresponding data of the compoud-treated groups by ANOVA; when variances of untransformed data were heterogeneous, analyses were performed on rank-transformed data.
- Mean live male and female pup weight values per litter were analyzed by ANOVA for Days =, 4 (pre- and postcull), 7, 14, and 21 of lactation.
- The total of live and dead pups was used as the covariate for Day 0; the total number of pups in each litter was used as the covariate for the remianing intervals.
- On Day 4 postcull, the Day 4 precull litter size was used as the covariate.
- Group comparisons were performed routinely at the 5 and 1 % two-tailed probability levels.
- Statistical significance is designated throughout the text of this report by the term significant.

Reproductive indices:

not determined

Offspring viability indices:

not determined

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Males and nonconfirmed females: findings do not appear in a dose-related pattern and are not considered treatment related
- Gestation: observations are not considered treatment related
- Lactation: observations are not considered treatment related
- Rest phase: observations are not considered treatment related

BODY WEIGHT (PARENTAL ANIMALS)
- Males and nonconfirmed females:
consistently lower in Group 3 and 4 males throughout the F0 phase and in Group 4 females during the premating phase, weeks 1-15, when compared to the corresponding control values; significant differences for Group 3 males at weeks 1-8, 10-11, and 13-15, group 4 male values at weeks 1-25, and group 4 female values at week 1-14; slight differences were also noted between the control and group 3 females -> however, these differences were significant only at week 1;
significantly lower mean body weight change values, compared to the corresponding control values, were noted in the Group 4 males at weeks 0-1, 1-2, 2-3, 3-4, 5-6, 8-9, 0-14, and 23-24, Group 3males at weeks 0-1, Group 2, 3, and 4 females at weeks 0-1, and group 4 females at weeks 0-14;
significantly higher mean body weight change value, compared to the corresponding control value, was noted in the Group 4 males at weeks 7-8

- Gestation:
mean body weight values of the Group 4 females were consistently lower than the corresponding control values throughout gestation; these differences were significant in Group 4 at Day 7, 14, and 20 when compared to the corresponding control values; significantly lower mean body weight change values, compared to the corresponding control values, were noted in Group 4 at Days 0-7 and 0-20

- Lactation:
mean body weight values of the treated females were generally lower than the corresponding control values throughout lactation; a significantly lower mean body weight change value was noted in Group 5 at Day 4 when compared to the corresponding control value; significantly lower mean body weight change values, compared to the corresponding control values, were noted in Groups 2 and 4 at Days 0-4

- Rest phase:
the pattern of lower mean weights in Group 4 continued in the rest phase for the females; mean body weight values of the Group 4 females postweaning were generally lower than the control values, with significantly lower mean body weight values noted at weeks 0, 1, and 4-6; no significant mean body weight change values were noted during the rest phase among the control and treated groups


FOOD CONSUMPTION (PARENTAL ANIMALS)
- Premating:
food consumption values were generally similar in all groups during the growth phase; occasional weeks showed significant differences among groups; however, there were no consistent or apparently tretament-related patterns; significantly lower mean food consumption values, compared to the corresponding control values, were noted in teh Group 3 and 4 males at weeks 5-6 and 9-10 and Group 2 nd 4 females at weeks 4-5

- Gestation:
mean food consumption values were similar between control and treated groups throughout gestation; mean compound consumption values decreased at each interval for all treated groups

- Lactation:
mean food consumption values were similar between control and treated groups throughout lactation; mean compound consumption values increased at each interval for all treated groups -> this is consistent with the increased nutritional requirements of lactating females and the beginning of solid food consumption by pups in the second week of the lactation period

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- the female fertility indices (percent pregnant) were 83, 88, 88, and 72 % for the Groups 1-4, respectively
- the male fertility indices were 83, 88, 88, and 69 % for the Groups 1-4, respectively
- the gestation index and mean duration of gestation were 100 % and 22 Days, respectively, for all groups; there were no abortions, early deliveries, abnormal gestations, or abnormal deliveries noted.

GROSS PATHOLOGY AND HISTOPATHOLOGY (PARENTAL ANIMALS)
- findings did not suggest an adverse effect of exposure to the test substance

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING) - F1
- no adverse effects on offspring viability
- the livebirth and Day 4 viability indices were 97, 99, 100, 98 % and 99, 95, 97, 93 % for the groups 1-4, respectively
- the weaning indices were 98, 99, 100 an 98 % for the groups 1-4, respectively
- the mean number of live pups per liter was slightly but not significantly lower in the high-dose group when compared to the control groups
- there were 9, 5, 1, and 5 stillborn pups in groups 1-4, respectively; there were no total litter deaths.
- the percentages males per litter were similar in each group


CLINICAL SIGNS (OFFSPRING) - F1
- pup clinical observations noted were few and did not appear in a dose-related pattern


CLINICAL OBSERVATION - F1
- Males and nonconfirmed females:
clinical observations noted during the growth phase included isolated incidences of anorexia, chromodacryorrhea, sores, alopecia, and urine stains; a large moveable mass was noted in one Group 3 female during weeks 11-16; cageside observations noted for the males included hunched posture and thin appearance, tremors, and ataxia; there were no remarkable cageside observations noted for females

- Gestation:
observations noted during gestation were not considered to be treatment related

- Lactation:
observations were few and not considered treatment related

- Rest phase:
observations were few and not considered treatment related


BODY WEIGHT (OFFSPRING) - F1, pups
- the mean pup weights of group 4 were consistently lower than the control weights at the same intervals during lactation
- male and female mean pup weight values were significantly lower compared to the corresponding control values in group 4 at days 0, 4 (pre- and postcull), 14, and 21
- Group 2 female pup weights and group 3 male and female pup weights were also significantly lower than the control weights at day 21


BODY WEIGHT (OFFSPRING) - F1
- Males and nonconfirmed females:
mean body weight values were consistently lower in the group 4 males and females during the growth phase when compared to the corresponding control values; these differences were significant in group 4 for males at weeks 0-27 and for the females at week 0-16 when compared to the corresponding control values; slight differences were noted between the control and group 3 values of both sex, with significances noted in group 3 male4s at week 0 and group 3 females at weeks 0 and 1;
mean weight change values for the growth period, weeks 0-14, were similar between groups 2 and 3 and the control group; significantly lower mean body weight change values, compared to the corresponding control values, were noted in group 4 males at weeks 0-1, 1-2, 2-3, and 0-14, and group 4 females at weeks 0-14; a significant difference was also determiend for the group 2 males at weeks 15-16relative to the control value; significantly higher mean body weight change values, compared to the corresponding control values, were noted in the group 2 and 4 females at weeks 16-17

- Gestation:
mean body weight values of the group 4 females were consistently lower than the corresponding control values throughout gestation, these differences were significant at days 0,7,14, and 20 when compared to the corresponding control values; mean body weight change values were lower in group 4 when compared to the corresponding control values, although the differences were not significant

- Lactation:
No significant mean body weight change values were noted between the control and treated groups

- Rest phase:
mean body weight values of the group 4 females postweaning were consistently lower than the control values, with significantly lower mean body weight values noted in group 4 at week 2-6; a significantly higher mean body weight change value was noted in group 3 at weeks 1-2 when compared to the respective control value


FOOD CONSUMPTION - F1
- Premating:
A significantly higher mean food consumption value, compared to the correspomding control value, was noted in group 4 females at weeks 14-15; other mean food consumption values were similar among the control and treated groups

- Gestation:
mean food consumption values were similar among the control and treated groups

- Lactation:
mean food consumption values were similar among the control and treated groups


REPRODUCTIVE PERFORMANCE - F1
- the female fertility inidces (percent pregnant) were 86, 79, 78, and 67 % for the groups 1-4, respectively
- the male fertility indices were 86, 83, 78, and 67% for the groups 1-4, respectively
- the gestation index and mean duration of gestation were 100 % and 22 days, respectively, for all groups
- there were no abortions, early deliveries, abnormal gestations, or abnormal deliveries noted


GROSS PATHOLOGY (OFFSPRING) - F1 (pups and parental animals)
- neither the type nor frequency of the observations indicated a treatment-related pattern


HISTOPATHOLOGY (OFFSPRING) - F1 (parental animals)
- neither the type nor frequency of the observations indicated a treatment-related pattern



RESULTS - F2 LITTER DATA (pup survival, body weights, and clinical observations)
- livebirth and viability indices were 96, 95, 98, and 96 % and 91, 89, 96, and 92 % for groups 1-4, respectively
- the weaning indices were 89, 92, 97, and 97 % for the groups 1-4, respectively
- the mean number of live pups per litter was slightly but not significantly lower in the high-dose group when compared to the control group; there were 6, 14, 5, and 3 stillborn pups in groups 1-4, respectively;
- there was 1 total litter death each in groups 1, 2, and 4
- the percent of males per litter was similar in all groups at Day 0, Day 4 precull , and at weaning
- male and female mean pup weight values were significantly lower compared to the corresponding control values in Group 4 at Days 0, 14, and 21
- pup obseravtions noted included pale and weak appearance, cold-to-touch, and dehydration; these observations did not appear in a dose-related pattern


RESULTS - F2 LITTER DATA (pup gross pathology)
- neither the type nor frequency of the observations indicated a treatment related pattern

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

15 000 ppm

Sex:

male/female

Basis for effect level:

body weight and weight gain

Results: F2 generation

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

15 000 ppm

Sex:

male/female

Basis for effect level:

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Compound consumption (week 13 -14) in mg/kg bw/day according table 11b of the study report

	5.000 ppm	15.000 ppm	50.000 ppm
male	241	725	2615
female	322	1002	3551

Applicant's summary and conclusion

Conclusions:

Dietary exposure of rats to dicyandiamide in a 2-generation study resulted in consistent and significant differences in body weights at the 50000 ppm as well as a slight reduction in fertility and preganancy rates. The no adverse effect level in this study is considered to be 15000 ppm (ca. 725 mg/kg bw/day in male rats (week 13-14)).

Executive summary:

In a 2-generation reproduction study Dicyandiamid (99.5%) was administered to 26 Crl:CD BR rats/sex/dose in diet at dose levels of 0, 5000, 15000, 50000 ppm. 

Significant differences in body weights were reported at the highest dose level as well as a slight reduction in fertility and pregnancy rates. These effects can be attributed to overt toxicity. There was a minimal reduction of mean body weight at the 15000 ppm level, but the overall weight change during the growth period was not statistically significant. There were no effects on body weight at the 5000 ppm level and no adverse effects on reproductive performance at the 5000 or 15000 ppm levels. No effects on pup viability were noted at any dose level tested. The LOAEL is 50000 ppm (2615 mg/kg bw/day in males, 3551 mg/kg bw/day in females (week 13)), based on body weight changes. The NOAEL is 15000 ppm (725 mg/kg bw/day in males, 1002 mg/kg bw/day in females), respectively. 

This study is acceptable and satisfies the guideline requirement for a 2-generation reproductive study according OECD 416 in rats.