Cyanoguanidine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In a two-generation feeding study according OECD 416 26 rats per sex and dose were exposed to 0, 5.000, 15.000 and 50.000 ppm Dicyandiamide (DCD). In addition, a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 was conducted was conducted using male and female SD rats (exposed to 0, 40, 200, 1,000 mg/kg/day).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

no estrous cycle & sperm parameters investigated

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

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

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

Dose / conc.:

0 ppm (nominal)

Remarks:

Nominal in diet, F0

Dose / conc.:

5 000 ppm (nominal)

Remarks:

Nominal in diet, F0

Dose / conc.:

15 000 ppm (nominal)

Remarks:

Nominal in diet, F0

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

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

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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

Key result

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight

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

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

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

15 000 ppm

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

15 000 ppm

Sex:

male/female

Basis for effect level:

body weight and weight gain

Reproductive effects observed:

not specified

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

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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

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 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

Parental animals: Observations and examinations:

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

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

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

The test substance had no effects on reproductive parameters such as the mating index, fertility index, numbers of corpora lutea or implantations, implantation index, delivery index, gestation index, gestation length, parturition or maternal behavior.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: This substance had no effects on reproductive parameters such as the mating index, fertility index, numbers of corpora lutea or implantations, implantation index, delivery index, gestation index, gestation length, parturition or maternal behavior.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

On examination of neonates there were no significant differences between the control and treated groups in the number of offspring or live offspring, sex ratio, live birth index, viability index or body weight. No abnormal findings ascribable to this substance were found for external examination or clinical signs or on necropsy of the offspring.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Reproductive effects observed:

not specified

Conclusions:

The NOAEL for reproductive and developmental toxicity is considered to be 1000 mg/kg/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test [OECD TG 422] (0, 40, 200, 1000 mg/kg/day) was conducted using SD rats. This substance had no effects on reproductive parameters such as the mating index, fertility index, numbers of corpora lutea or implantations, implantation index, delivery index, gestation index, gestation length, parturition or maternal behavior. On examination of neonates there were no significant differences between the control and treated groups in the number of offspring or live offspring, sex ratio, live birth index, viability index or body weight. No abnormal findings ascribable to this substance were found for external examination or clinical signs or on necropsy of the offspring. The NOAEL for reproductive and developmental toxicity is considered to be 1000 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

725 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Dietary exposure of rats to dicyandiamide in a two generations study according OECD 416 resulted in consistent and significant differences in body weights at the 50.000 ppm level as well as a slight reduction in fertility and pregnancy rates (50.000 ppm equals 2615 mg/kg bw/day (males) and 3551 mg/kg bw/day (females) as measured in week 13 -14). The study was designed to evaluate effects on gonadal function, mating behaviour, and fertility as well as growth and development of offspring and effects on gestation, parturition, and lactation. Gross pathology findings were observed and a histopathological examination of the reproductive organs and gross lesions of the parental animals was made. There was a minimal reduction of mean body weight at the 15.000 ppm level, but the overall weight change during the growth period was not statistically significant. There were no effects on body weight at the 5.000 ppm level and no adverse effects on reproductive performance at the 5.000 or 15.000 ppm levels. No effects on pup viability were noted at any dose level tested. The NOAEL is 15.000 ppm (equals 725 mg/kg bw/day (males) and 1002 mg/kg bw/day (females) as measured in week 13 -14).

In addition, a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test [OECD 422] (0, 40, 200, 1,000 mg/kg/day) was conducted using SD rats. This substance had no effects on reproductive parameters such as the mating index, fertility index, numbers of corpora lutea or implantations, implantation index, delivery index, gestation index, gestation length, parturition or maternal behaviour. On examination of neonates there were no significant differences between the control and treated groups in the number of offspring or live offspring, sex ratio, live birth index, viability index or body weight. No abnormal findings ascribable to this substance were found for external examination or clinical signs or on necropsy of the offspring. The NOAEL for reproductive and developmental toxicity in this study is considered to be 1,000 mg/kg/day.

Effects on developmental toxicity

Description of key information

DCD was administered by gavage to five groups of six mated female Crl:CDBR rats at doses of 0, 250, 500, 1000, or 2000 mg/kg bw/day during gestation Days 6-15. The study was designed as pilot for a teratogenicity study according EPA OPP 83-3. 
In addition, a GLP guideline study according to OECD 414 in rabbit was conducted. No test item related effects on development have been reported in either of these studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-20 to 2015-09-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

in rabbits

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Centre LAGO, 01540 Vonnas, France.
- Age at study initiation: 17 to 19 weeks old
- Weight at study initiation: 3.2 to 4.3 kg.
- Housing: individually housed in composite plastic and metal cages in compliance with European Regulations (Directive 2010/63/EU)
- Diet (e.g. ad libitum): The rabbits were given approximately 200 g of food per day during the study (Diet reference SAFE 110C-10)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days between animal arrival and start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C (target range)
- Humidity (%): 35 to 70 % (target range)
- Air changes (per hr): Minimum 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h / 12h

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % (w/v)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared as a suspension in the vehicle at concentrations of 40, 80 and 200 mg/mL according to Standard Operating Procedures of the Test Facility. The first preparations were mixed using sonication. However, since there were difficulties to handle these preparations and administer them to the animals, the suspensions used from 1 April 2015 were mixed using sonication followed by ultra-turrax at 13500 rpm (4000 rpm for suspensions used from 13 April 2015) for a few minutes.

volume applied: 5 mL/kg

VEHICLE
- Concentration in vehicle: 0.5 % (w/v) CMC 300-600 centipoises in water for injection
- Amount of vehicle (if gavage): 5ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations:
4 x 1 g samples were taken under magnetic stirring from the top, middle and bottom of each formulation (middle only for control), used on the first day of treatment and on a suitable day during the last week of treatment, and stored under the specified stability conditions until analysis.
Additional analyses were performed to complete stability data at room temperature for 10 days.
2 x 1 g samples were taken under magnetic stirring from the top, middle and bottom of formulation used on 03 April 2015. An additional sample (volume of 40mL) of this preparation was taken under magnetic stirring from the middle of formulation and was kept at room temperature (between +15 and +25°C) for 4 and 10 days. 2 x 1 g samples were taken under magnetic stirring from the top, middle and bottom of this additional sample on 07 and 13 April 2015 for analysis.
According to these results, the frequency of preparation was changed during the study (see section 3.3).
Duplicate samples from group 2 sampled on 20 April 2015 (last week of treatment) were analysed to confirm the initial analytical results.

Acceptance criteria:
Acceptable results were obtained if the difference between the actual mean value and the targeted concentration was within ± 15 %.

Details on mating procedure:

88 virgin mated females to obtain at least 16 full term gestating females per group.
The females were mated at the supplier and delivered to the Test Facility the same day (gestation day 0 (G 0))

Duration of treatment / exposure:

From Day 6 (G 6) to Day 28 (G 28) of gestation inclusive

Frequency of treatment:

Once daily, at approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose

Duration of test:

29 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
based on the results of the dose range-finding study (AB20818) where oral (gavage) administration of Dicyandiamide to New Zealand White rabbits from Days 6 to 28 of gestation at dose levels of 500, 750 and 1.000 mg/kg/day was associated with slight transient reduction in mean body weight gain and reduction in food consumption in the high dose group and slight reduction in mean body weight gain in the 750 mg/kg/day group. There was no evidence of embryo-foetal toxicity in any group. Therefore the dose levels of 200, 400 and 1000 mg/kg/day were selected by the Study Sponsor in agreement with the Study Director for this current study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
All animals were observed at least twice daily. Animals found dead were necropsied

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were observed daily for clinical signs. During the treatment period, the animals were observed once before and at least once after dosing to detect any clinical signs or reaction to treatment.

BODY WEIGHT: Yes
All animals were weighed on Days 0, 6, 9, 12, 15, 18, 21, 24, 27 and 29 of gestation

FOOD CONSUMPTION: Yes
Food consumption of each animal was measured daily from the day of arrival to Day 29 of gestation. The mean (g/animal/day) was calculated for the periods (Days) 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 27 and 27 to 29 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
Animals were dissected and examined for macroscopic pathological changes.
- Organs examined:
The ovaries and uterus of each female were removed and examined. The placentae were also examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter ]

Statistics:

Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
- The best transformation for the data (none, log or rank) was determined depending upon (a) the normality of the data distribution tested by the Shapiro-Wilk's test and (b) the homogeneity of the variances across groups tested by the Bartlett's test.
Non- or log-transformed data were analysed by parametric methods.
- Rank transformed data were analysed using non-parametric methods.
- Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
- Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
- If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
- The number of resorptions, number of dead foetuses and all litter-based percentages were analysed using non-parametric methods, i.e. Kruskal-Wallis test followed by non-parametric Dunnett’s test if the Kruskal-Wallis is significant.
- Selected incidence data were analysed using a chi2 test for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.

Indices:

The data concerning the control pregnant females and the historical data were used to evaluate the effects of the test item.
For caesarean data, the group mean values are calculated on a litter basis. Foetal observation data are presented as the percentage of affected foetuses and percentage of affected litters.
Foetal abnormalities are categorised as follows:
- Malformations - structural defects which are rare in the control population and are thought to be life threatening or of major physiological consequence.
- Anomalies - minor abnormalities or defects which are relatively rare in the control population and/or are considered not to be of major physiological consequence.
- Variations - minor abnormalities, defects or alternative forms which are either common in the control population or are of no known physiological consequence.
For each group, the following parameters were calculated:
Pre-implantation loss (in %): ((Number of corpora lutea - Number of implantations)/ (Number of corpora lutea)) x 100
Post-implantation loss (in %): ((Number of implantations - Number of viable foetuses)/ (Number of implantations)) x 100

Historical control data:

yes

Details on maternal toxic effects:

Maternal toxic effects: no effects

Details on maternal toxic effects:
Mortality:
There were no test item-related deaths. One animal from the 200 mg/kg/day group was found dead on day 10 of gestation. The animal had no abnormal macroscopic findings at necrospy and death was attributed to misgavage and is therefore not related to the test item. Moreover, one female from the 1000 mg/kg/day group was found dead on day 25 of gestation. At necropsy, this animal had a mottled liver and dark areas on the lungs. In the absence of any comparable clinical or terminal observations amongst the other females in the group, this isolated finding was considered to be incidental.

Clinical Observations:
There were no treatment-related clinical signs.
One female in the 400 mg/kg/day group and one female in the 1000 mg/kg/day group had red traces in the cage on several occasions which was possibly associated with resorption of the implantations because these females had no viable foetuses at necropsy. These isolated cases were considered incidental.
Difficulties in test-item administration were observed for three females in the 200 mg/kg/day group which resulted on a sore on the tongue observed immediately after dosing on day 9 of gestation for one female and red traces on the cannula used for gavage on day 22 of gestation for two females.
Scab(s), lacrimation, nasal discharge, sneezing and reduced faeces occasionally observed in treated or control groups were considered incidental.

Body weight:
There was no adverse effect of treatment on mean body weight in any group.
Overall mean body weight gain in the 1000 mg/kg/day group was statistically significantly higher (+41 %) than the control during the dosing period (G 6 to G 29).

Food Consumption:
There was a transient slight reduction in mean food consumption (not statistically significant) during the first few days of the dosing period (gestation days 6 to 12) in the 1000 mg/kg/day group compared with the control.
There was no effect of treatment on mean food consumption in the lower dose groups.

Necrospy findings of adult females:
There were no treatment-related macroscopic findings at necropsy.
One control female had absence of the right adrenal gland, kidney and uterine horn.

Gravid Uterus Weight:
There was no effect of treatment on mean gravid uterus weight in any group.

Pregnancy Incidences:
There were 19, 17, 19 and 20 pregnant females at the terminal caesarean sections in the control, 200, 400 and 1000 mg/kg/day, respectively. One, 1 and 2 of these females in the control, 400 and 1000 mg/kg/day groups, respectively had no viable foetuses.

Pre-Implantation data:
Despite some incidental variation in the mean corpora lutea count and percentage pre-implantation loss, the mean number of implantation sites was comparable in all groups.

Post-Implantation data:
There was no treatment-related effect on embryo-foetal survival in any group.
One or 2 female(s) in each of the control, 400 and 1000 mg/kg/day group had 4 to 7 early or late resorptions and no viable foetuses. One female in the 1000 mg/kg/day group had a dead foetus. In the absence of any trend of increased embryo-foetal death amongst the other females in each of these groups, these isolated findings were considered to be incidental.

Mean live litter size was comparable in all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
Foetal Weight:
Mean foetal weight was comparable in all groups.

Foetal Sex:
The percentage of male foetuses was incidentally high in the control group (63 %) but was normal (close to 50 % per sex) in each of the treated groups.

Less severe skeletal anomalies and variations:
Evidence of potential embryo-foetal effects of Dicyandiamide was restricted to slightly higher incidences of foetuses with an absent interparietal bone, unossified phalanges of the forepaw and 5th sternebrae, and variations in the number of full ribs (n=12 unilaterally or bilaterally) in the 1000 mg/kg/day group compared with the concurrent control and historical control data. A similar trend was noted for the interparietal bone only in the 400 mg/kg/day group. Although an association with treatment cannot be completely excluded, these minor findings in isolation are of no toxicological significance.
Summary of malformations_individual descriptions:

Dose level Female Foetus Malformation(s) (including external, visceral and skeletal examinations)
(mg/kg/day) number number

0 10 3 Multiple abnormalities of the great vessels (descending aorta arising from pulmonary trunk; aortic arch termina ting with right subclavian, and right and left carotid arteries; innominate artery absent; left subclavian artery ari sing from pulmonary trunk)
200 35 2 Fused sternebrae (3rd to 5th)
40 1 Malrotated left forepaw with hyperextended 1st digit, multiple abnormalities of sternebrae and ribs, multiple ab normalities of cervical and thoracic vertebrae (scoliosis)
41 5 Multiple abnormalities of thoracic vertebrae (scoliosis)
9 Multiple abnormalities of sternebrae (2nd bipartite, asymmetric 2nd to 4th, unossified 5th and 3rd and 4th fused)
44 1 Hyperflexed left forepaw, sternoschisis
400 46 6 Fused sternebrae (4th and 5th)
49 3 Multiple abnormalities of sternebrae (2nd to 5th fused, 2nd bipartite and 2nd to 5th asymmetric)
60 6 Multiple abnormalities of heart (fifth chamber communicating with right ventricular and atrial chambers)
1000 72 4 Dilated aortic arch and pulmonary artery, malpositioned testis
88 11 Omphalocele


In total, there were 1 (1), 5 (4), 3 (3) and 2 (2) foetuses (litters) with malformations in the control, 200, 400 and 1000 mg/kg/day groups, respectively, none of which were attributed to Dicyandiamide. The principal changes included sternebral defects for 4 foetuses in the 200 mg/kg/day group and 2 in the 400 mg/kg/day group.
However, since there was no similar finding in the 1000 mg/kg/day group and these changes are part of the historical background for the strain of rabbit, there was considered to be no association with treatment. The other malformations noted (hyperflexion or malrotation of a forepaw, great blood vessel changes, vertebral defects leading to scoliosis, a heart defect and omphalocele) were considered to be incidental since they are part of the background of changes noted in the strain of rabbit, the incidence did not increase in a dose-dependent manner, were isolated findings and there was a lack of any associated increases in embryo-foetal death and in less severe structural changes.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse signs of toxicity observed

Key result

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

Observed effects were either incidental or spontaneous in nature.

Key result

Developmental effects observed:

no

Conclusions:

Oral (gavage) administration of Dicyandiamide (> 99.7% a.i.) to New Zealand White rabbits from days 6 to 28 of gestation at doses of 200, 400 and 1000 mg/kg/day was well tolerated. No treatment related effects were observed in dams. There was no adverse effect of treatment on embryo-foetal survival or morphological development at any dose.

Executive summary:

In a developmental toxicity study (OECD guideline 414) Dicyandiamide (>99.7% a.i.) was administered to 22 female New Zealand White rabbits/dose by gavage at dose levels of 0, 200, 400 and 1000 mg/kg bw/day from days 6 through 28 of gestation.

There were no treatment-related effects in mortality, clinical signs, body weight, food consumption, or caesarean parameters. The maternal NOAEL exceeds 1000 mg/kg bw/day. 

The principal changes in developmental parameters included sternebral defects for 4 foetuses in the 200 mg/kg/day group and 2 in the 400 mg/kg/day group. However, since there was no similar finding in the 1000 mg/kg/day group and these changes are within the historical background for the strain of rabbit, there was considered to be no association with treatment. Thus, the developmental NOAEL exceeds 1000 mg/kg bw/day.  

The developmental toxicity study in the rabbit is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rabbit.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - October 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

modified for a Pilot Study

Deviations:

yes

Remarks:

only 6 females per group; food consumption not recorded; b.w. only every 4 days not 3 days recorded; only external alterations, no skeletal or soft tissue alterations investigated. This is range-finding study only.

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 Ferderal Regulations Part 160, issued November 29, 1983.

Limit test:

no

Species:

rat

Strain:

other: Crl:CD BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: approx. 11 weeks
- Weight at Day 0 of gestation: 244- 302 g
- Housing: individually in stainless-steel wire cages
- Diet: ad libitum, Purina Certified Rodent Chow #5002
- Water: ad libitum, tap water
- Food and water were analyzed for minerals and contaminants on a regular basis; there were no known contaminants determined to be in either the feed or water at levels sufficient to interfere with the study
- Acclimation period: acclimated to laboratory conditions for approximately 2 weeks prior to study initiation


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 23.3
- Humidity (%): 31 - 69
- Air changes (per hr): 17
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

VEHICLE PREPARATION
- Rate of preparation of diet (frequency): daily
- Mixing appropriate amounts with water: to yield a 0.5% solution
- Storage temperature of food: room temperature

VEHICLE
- Concentration in vehicle: for each dose level, the test material was ground into a powder with a mortar and pestel and the required amount weighed on a balance (mg)
- Amount of vehicle (if gavage): 5 ml/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HOMOGENEITY:
- determined prior to initiation of dosing
- duplicate samples were taken from the top, middle, and bottom of the mixes for 250, 500, 1000, and 2000 mg/kg dose levels, as well as a single control solution sample

ROUTINE ANALYSES:
- concentration analyses were performed on duplicate samples of each dose level taken at Day 1 and 12 and on the Group 5 sample at Day 4, as well as a single control sample

ANALYTICAL METHODS:
- the samples were analyzed by reverse phase high-performance liquid chromatography techniques for concentration of Dicyandiamide

RESULTS:
- homogeneity: mean % sample variability within each mix, i.e. top, middle, and bottom, was within +/- 6 % of the target levels
- concentration: concentration of the test material in the dosing suspensions presented to the animals were within +/- 14 % of the target levels

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
* M/F ratio per cage: 1/1
* Length of cohabitation: until confirmation of mating (presence of sperm or vaginal plug)
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation

Duration of treatment / exposure:

during gestation days 6-15

Frequency of treatment:

daily treatment

Duration of test:

March 12, 1990 (females were placed into breeding) - April 1, 1990

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

six mated female rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

no further details reported

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily throughout gestation (approx. 1 hour postdose on gestation days 6-15)
- all animals were observed twice daily for mortality and moribundity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to dosing at each weighing interval
- animals wre observed for signs of inappetence


BODY WEIGHT: Yes
- Time schedule for examinations: on gestation Days 0, 6, 8, 12, 16, and 20


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20; all surviving females were weighed, sacrificed by carbon dioxide inhalation and exsanguination
- animals were examined grossly for abnormalities of the thoractic, abdominla, and pelvic viscera

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: any abnormalities of the uterus or embryonic sac

Fetal examinations:

External examinations: Yes:
- each fetus was sexed, weighed, and examined externally for abnormalities
- fetuses were sacrificed via intraperitoneal injection of sodium pentobarbital and discarded
- gross external findings were judged to be either variations or malformations

Statistics:

Statistical analyses of the data were not required

Indices:

No data

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects: yes

Details on maternal toxic effects:
MORTALITY AND CLINICAL OBSERVATIONS:
- All females survived until the scheduled sacrifice
- two, one and one female in Groups 1, 2, and 4, respectively, were not pregnant
- during the study, there were no remarkable cageside or clinical observations noted in any of the females

BODY WEIGHTS
- all of te mean body weights and body weight change values were similar between the control and groups 2, 3, and 4
- the group 5 mean body weights from initiation of dosing and mean body weight changes for the study period were generally slightly lower than the control

GRAVID UTERINE WEIGHTS AND NET BODY WEIGHT CHANGES
- similar between the control and treated groups
- the net body weight change from day 0 value was slightly lower in group 5 when compared to the corresponding control value

GROSS PATHOLOGY
- findings noted were hydrometa in two nonpregnant females (one each in group 1 and 2) and pale areas on the liver in one group 2 female
- these findings were considered to be incidental

CESAREAN SECTION
- the pregnancy rate was 67, 83, 100, 83, and 100 % in Groups 1-5, respectively
- the mean preimplantation loss (which indicates implantation efficiency) was 4.2, 5.0, 22.0, 18.3, and 9.4 % for Groups 1-5, respectively
- the mean number of implantations and corpora lutea did not indicate a treatment-related effect on implantation efficiency

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
FETAL VIABILITY, SEX, AND BODY WEIGHTS
- the mean percent of total resorptions (early plus late) was 4.3, 5.6, 3.8, and 6.6, and of live fetuses was 95.7, 94.4, 96.2, 95.8, and 93.4 in Groups 1-5, respectively
- the mean fetal body weights were similar in Groups 1-4
- fetal body weights were slightly lower in Group 5 than in the control group
- one dead fetus was noted in Group 4
- the sex ratio was similar between the control and treated groups

FETAL EXTERNAL EXAMINATION
- the only external variation noted was anasarca in one Group 4 fetus
- there were no fetal external malformations noted in this study

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Oral administration of Dicyandiamide during gestation days 6-15 did not result in either maternal or embryo/fetal toxicity at doses up to 1000 mg/kg in Crl:CD BR rats. No indications of an adverse effect on fetal development were seen at any dose tested. Maternal and embryo/fetal toxicity were indicated by slightly lower body weights in the highest dose group receiving 2000 mg/kg bw/day. No indications of an adverse effect on fetal development were seen at any of the doses tested.

Executive summary:

In a developmental toxicity study Dicyandiamide (99.5%) was administered to 6 female Crl:CD BR rats/dose by gavage at dose levels of 0, 250, 500, 1000, or 2000 mg/kg bw/day from days 6 through 15 of gestation.

Maternal toxicity occurred at the highest dose level. There were no treatment-related effects in mortality, clinical signs, or cesarean parameters. The maternal LOAEL is 2000 mg/kg bw/day, based on body weight changes. The maternal NOAEL is 1000 mg/kg bw/day.

No indications of an adverse effect on fetal development were seen at any of the doses tested. The developmental NOAEL is 2000 mg/kg bw/day.

The developmental toxicity study in rat is classified acceptable (range-finding study). It satisfies the requirements for a developmental toxicity study (modified OPP 83-3 in rats) but deviates from OECD 414. Major deficiencies are the relative small group size of 6 females and the lack of an investigation of skeletal or soft tissue alterations.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

DCD was administered by gavage to five groups of six mated female Crl:CDBR rats at doses of 0, 250, 500, 1000, or 2000 mg/kg bw/day during gestation Days 6-15. The study was designed as pilot for a teratogenicity study according EPA OPP 83-3. No indications of adverse effects on fetal development were seen at any of the doses tested. Maternal toxicity and embryo/fetal toxicity were indicated by slightly lower body weights in the highest dose group receiving 2000 mg/kg. All dams were necropsied. The uterus of each dam was excised and the uterine contents examined. The number of corpora lutea, implantations, resorptions, and viable fetuses was determined. All fetuses were examined for external abnormalities, weighed and sexed. There were no mortalities or clinical signs of toxicity throughout the study. Similarly, maternal body weights and body weight changes in 250 – 1000 mg/kg bw/day groups did not indicate any adverse effects on maternal toxicity. There were no treatment-related gross tissue abnormalities noted at the necropsy of the dams. Based on the maternal effect a NOAEL of 1000 mg/kg bw/day was derived. Major deviations in this study were a low number of animals and a restricted set of examinations. Current guidelines on prenatal development toxicity recommend using 20 female animals with implantation sites at necropsy per dose. In contrast only 6 females per group were used in this study. In addition food consumption was not recorded and only external but no skeletal or soft tissue alterations were investigated.

However, the negative results were confirmed in rabbit as second species. In a developmental toxicity study according to OECD 414, Dicyandiamide (>99.7% a.i.) was administered to 22 female New Zealand White rabbits/dose by gavage at dose levels of 0, 200, 400 and 1000 mg/kg bw/day from days 6 through 28 of gestation. There were no treatment-related effects in mortality, clinical signs, body weight, food consumption, or caesarean parameters. The maternal NOAEL exceeds 1000 mg/kg bw/day. Changes in developmental parameters included sternebral defects for 4 foetuses in the 200 mg/kg/day group and 2 in the 400 mg/kg/day group. However, since there was no similar finding in the 1000 mg/kg/day group and these changes are within the historical background for the strain of rabbit, there was considered to be no association with treatment. Thus, the developmental NOAEL exceeds 1000 mg/kg bw/day. The developmental toxicity study satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rabbit.

Justification for classification or non-classification

A well conducted two-generation study and two prenatal developmental toxicity studies in rat and rabbit do not indicate any adverse effect on reproduction in rodents. A NOEAL of 15.000 ppm DCD was derived from the 2-Generation study based on body weight changes as well as a slight reduction in fertility and pregnancy rates at the highest dose. The NOAEL value equals 725 mg DCD/kg bw/day in male rats at the end of the study (week 13-14) and 1000 mg DCD/kg bw/day in female rats, respectively. No test item related signs of toxicity to prenatal development were reported in rats and rabbits at the limit dose of 1000 mg/kg bw/day. Therefore, Dicyandiamid is not classified for reproductive/developmental toxicity.