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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
in rabbits
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

open allclose all
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

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
Observed effects were either incidental or spontaneous in nature.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

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.