N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985/06/25 to 1986/01/03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

EC Number:

266-257-8

EC Name:

N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

Cas Number:

66215-27-8

Molecular formula:

C6H10N6

IUPAC Name:

N2-cyclopropyl-1,3,5-triazine-2,4,6-triamine

Test material form:

solid: particulate/powder

Remarks:

Pale Yellow Crystalline

Test animals

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hra: (NZW)SPF

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 months old
- Source: Hazleton-Dutchland, Inc., Denver, Pennsylvania
- Weight at study initiation: 3.032 to 4.180 kg
- Housing: Upon arrival, all animals were individually housed in clean, stainless-steel, wire-bottom cages suspended above cage-board. Nesting material was not required for those females selected for the gestation day 29 Cesarean section as the sacrifice was scheduled prior to expected parturition. Those females selected to deliver were provided with nesting boxes and wood shavings beginning approximately on gestation day 25.
- Diet: Certified rabbit feed was provided ad Iibitum during the study.
- Water: Drinking water delivered by an automatic watering system was provided ad Iibitum during the study.
- Acclimation period: 35 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.4 ± 3°F
- Humidity (%): at least 40%
- Air changes (per hr): Minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark photoperiod

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

TEST SUSPENSIONS PREPARATION
- A total volume of 100 mL per test group was prepared fresh daily.
- Mixing test item with vehicle: The appropriate amount the test item, Cyromazine, (adjusted for purity) was weighed into a plastic weigh boat for each group, transferred to a mortar and ground with a pestle until uniform. The vehicle, 0.5% aqueous carboxymethylcellulose, was added to obtain a slurry. The resultant suspension was transferred to a volumetric ask via a series of vehicle rinses. Vehicle was then added in sufficient quantity to achieve the appropriate concentration for each group. A magnetic stir plate and bars were used following preparation and during administration to ensure adequate mixture. After mixing the test suspensions were transferred to storage containers and homogenized in a tissue homogenizer for 1 minute.
- Test suspensions were stored at room temperature, protected from light.

VEHICLE PREPARATION
The vehicle, 0.5% aqueous carboxymethylcellulose, was prepared by adding 5 g of the control material powder to 1000 ml of deionized water in an Erlenmeyer flask. The mixture was warmed gently and stirred using a magnetic bar and plate until clear. The vehicle was prepared fresh at least weekly and stored refrigerated between periods of use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of the study a representative batch of suspensions of each dose level was prepared according to the protocol and analyzed. Three aliquots of each dosage preparation were obtained from different strata (top, middle, bottom) within the test material/vehicle suspension and analyzed for concentration of the test material. Six hours later, an additional aliquot was taken from each dosage preparation and analyzed. On the first day of administration at approximately the midpoint of the dosing period (approximately gestation day 12) and on the last day of dosing, each dosing preparation was analyzed in duplicate for concentration of the test material. The test material/vehicle suspensions were judged homogeneous, stable and suitable for study initiation if the means of the homogeneity and stability assay values were within ±10% of the theoretical concentrations.

Details on mating procedure:

Not applicable, animals were inseminated artificially.

Duration of treatment / exposure:

13 consecutive days (GD 7-19)

Frequency of treatment:

Once daily

Duration of test:

Twenty-five rabbits were sacrificed on GD 29, 49 rabbits were sacrificed on lactation day 28.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Remarks:

High dose group

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group

Doses / concentrations 3

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

No. of animals per sex per dose:

74 female rabbits/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 74 artificially inseminated rabbits were dosed by gavage with 0 (control), 5, 10 or 30 mg/kg b.w./day on days 7-19 (inclusive) of gestation. On day 29 of gestation, twenty-five females with viable foetuses from each group were killed for teratology investigation. The uteri were examined for live foetuses and intra-uterine deaths. The foetuses were weighed, examined for external and visceral abnormalities, sexed, eviscerated and stained for skeletal examination. The remaining females were allowed to litter naturally. On the fourth day of lactation, six young per litter were randomly selected and retained for an assessment of post partum (p.p.) survival and growth up to lactation day 28. Remaining young were killed after weighing on day four p.p.. On lactation day 28, offspring were killed, sexed and necropsied. A gross post mortem examination was performed on all surviving F0 dams.

Examinations

Maternal examinations:

Detailed clinical observations were recorded individually from gestation days 0 through 29 for those females selected for Cesarean section and additionally from days 0-28 of lactation for those females designated for the postnatal phase of the study. All rabbits were examined daily for appearance and behavior prior to dosing, pharmacotoxic signs following dosing, as well as moribundity and mortality. A gross necropsy was performed on the females which died, sacrificed in extremis or aborted during the course of the study and a cause of death was determined, if possible. The number and location of implantation sites and corpora lutea were recorded.
Maternal body weights for all females were recorded individually on gestation days 0, 7, 10, 14, 20, 24 and 29 for those females selected for Cesarean section and additionally for lactation days 1, 7, 14, 21 and 28 for those females designated for the postnatal phase of the study. Individual food consumption was recorded daily from days 0 through 29 of gestation.

The females that were allowed to litter and rear their young until weaning were observed twice daily in the period of expected parturition. Parturition was observed for signs of dystocia. When parturition was judged complete, litters were examined for gross malformations and the numbers of stillbirths and live kits were recorded. Individual gestation length was calculated using the date on which delivery was initiated. The females which did not deliver were sacrificed on post-insemination day 35 or 36 to determine pregnancy status.

Ovaries and uterine content:

The trimmed uterus and contents were weighed and the number of corpora lutea on each ovary was recorded. The uterus was opened and the number and location of viable and nonviable fetuses, early and late resorptions, and the total number of implantation sites were recorded. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal uterine horn, noting the position of the cervix, and continuing from the proximal to distal right uterine born.
Uteri with no evidence of macroscopic implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of implantation loss.

Fetal examinations:

Teratological evaluation:
Each viable and nonviable fetus was weighed individually; crown-rump length was also determined for nonviable fetuses. A detailed examination of each fetus was conducted to include, but was not limited to, an examination of the eyes, palate and external orifices. The crown-rump length of late resorptions was measured.
Each viable and nonviable fetus was sexed internally and examined viscerally (including the heart and major vessels). The eviscerated fetuses were skinned; the brain was examined by a mid-coronal slice and the skeletons were fixed in 95% isopropyl alcohol. Following fixation in alcohol each eviscerated viable and nonviable fetus was macerated in potassium hydroxide and stained. The skeletal examination was conducted utilizing low power magnification provided by a stereomicroscope as necessary. External, visceral and skeletal findings were recorded as developmental variations or malformations. Body weights, visceral and skeletal findings
of nonviable fetuses were not tabulated. After the completion of the examination, the fetal skeletons were stored in thermoseal bags containing glycerin and phenol.

Postnatal evaluation:
Each litter was examined daily for survival. Stillborn kits and intact offspring dying from lactation days 0 to 4 were necropsied. A detailed gross necropsy was performed on any kit dying after lactation day 4 and prior to weaning.
All pups were individually identified by toe-marking on lactation day 0. To reduce variability among the litters, six kits from each litter were randomly selected on lactation day 4. Surplus offspring were weighed, sacrificed and necropsied on lactation day 4.
Litters were examined daily for any adverse physical signs. Each pup received a detailed physical examination on lactation days 1, 4, 7, 14, 21 and 28. Pups were weighed individually on lactation days 1, 4, 7, 14, 21 and 28. Pups were individually sexed at necropsy.
All surviving pups were sacrificed on lactation day 28 by carbon dioxide inhalation. A gross necropsy was performed on the major organ of the systems of the thoracic and abdominal cavities including the viscera. The brain was examined by a mid-coronal slice. The sex of each kit was determined internally.

Statistics:

All analyses were conducted using two-tailed tests for a minimum significance
level of 5% comparing each treated group to the vehicle control group; all means were presented with standard deviations. All statistical tests were performed by a Digital Computer with appropriate programming.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The incidence of clinical symptoms in this study was minimal. Defecation and urination were judged to be decreased during the treatment period in the 30 mg/kg bw/day group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Ten rabbits died during the experimental period; four in the control group, two in the 5 mg/kg bw/day group, three at the 10 mg/kg bw/day level and one in the 30 mg/kg bw/day group. Mortality incidence was not dose-related and not accompanied by relevant post mortem findings and was thus considered to be unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 30 mg/kg bw/day, there was a marked body weight loss in dams from days 7-20 of gestation. After the dosing period, body weight gain was statistically increased compared to the control group. Body weight gain during lactation in these females was generally comparable to controls.

Mean maternal body weights and body weight gains in the 5, 10 and 30 mg/kg bw/day groups were generally comparable to the control group throughout lactation. No statistically significant differences occurred.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption in the 30 mg/kg bw/day group was affected in a corresponding manner with the body weight changes. Conversely, food intake during the initial four days of the recovery period (gestation days 20-24) was increased with statistical significance in comparison with the control group.
There were no test article-induced alterations in food consumption during gestation in the 5 and 10 mg/kg bw/day groups nor were there any statistically significant differences.

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

A total of sixteen females aborted during the gestation period, four from the control group, five each from the low and high dose groups and two from the intermediate group. The abortions were not considered test item-induced as the incidence was similar between all study groups.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean numbers of viable and nonviable fetuses, early and late resorption sites, post-implantation loss, implantation sites and corpora lutea in all Cyromazine Technical treated groups were similar to the control group or the historical data.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean length of gestation in all treated groups was comparable to the control group.

Details on maternal toxic effects:

No signs of dystocia were observed in any study group.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights in all Cyromazine Technical treated groups were similar to the control group or the historical data.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Litter viability was not adversely affected on lactation day 0 in any dose group. Viability indices on lactation days 1, 4, 7, 14, 21 and 28 in all treated groups were either comparable to or exceeded the group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no remarkable differences in the pup sex ratios among treated dams receiving 5, 10 and 30 mg/kg bw/day Cyromazine in comparison with the control group.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There were no treatment-related alterations in gestation length, parturition, live birth and survival indices, sex ratio, live litter size, offspring body weight or condition throughout the lactation period.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no biologically meaningful or statistically significant differences in the number or percentage of foetuses with malformations or developmental variations in any of the cyromazine-
treated groups compared with controls.

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks:

Development

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Detailed results are provided in the overall remarks section below.

Applicant's summary and conclusion

Conclusions:

Based on the results of a prenatal developmental study with Cyromazine in rabbits, the NOAEL for maternal toxicity was concluded to be 10 mg/kg bw/day, based on decreased body weight, decreased food consumption and clinical signs of toxicity (decreased defecation and urination) at 30 mg/kg bw/day. The NOAEL for developmental toxicity was 30 mg/kg bw/day, based on the absence of developmental effects at all doses tested.

Executive summary:

Pottential maternal, embryotoxic and teratogenic effects of Cyromazine were evaluated in this teratology and postnatal study in rabbits. Groups of 74 artificially inseminated rabbits were dosed by gavage with 0 (control), 5, 10 or 30 mg/kg b.w./day on days 7-19 (inclusive) of gestation. On day 29 of gestation, twenty-five females with viable foetuses from each group were killed for teratology investigation. The uteri were examined for live foetuses and intra-uterine deaths. The foetuses were weighed, examined for external and visceral abnormalities, sexed, eviscerated and stained for skeletal examination. The remaining females were allowed to litter naturally. On the fourth day of lactation, six young per litter were randomly selected and retained for an assessment of post partum (p.p.) survival and growth up to lactation day 28. Remaining young were killed after weighing on day four p.p.. On lactation day 28, offspring were killed, sexed and necropsied. A gross post mortem examination was performed on all surviving F0 dams.

 

Based on the results of a prenatal developmental study with Cyromazine in rabbits, the NOAEL for maternal toxicity was concluded to be 10 mg/kg bw/day, based on decreased body weight, decreased food consumption and clinical signs of toxicity (decreased defecation and urination) at 30 mg/kg bw/day. The NOAEL for developmental toxicity was 30 mg/kg bw/day, based on the absence of developmental effects at all doses tested.