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

Carcinogenicity

Administrative data

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1979/11 to 1982/06

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:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.32 (Carcinogenicity Test)

Deviations:

yes

Remarks:

See limitations below

Principles of method if other than guideline:

- Seminal vesicles were not listed in the report as being taken for histopathology.

GLP compliance:

yes

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:

coarse cream-coloured

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4 week old
- Source: Charles River Breeding Laboratories, Portage, Michigan
- Weight at study initiation: Male 14-31 gram, female 14-23 gram
- Housing: The mice were housed individually in hanging wire-mesh cages.
- Diet: Certified Rodent diet was available ad libitum
- Water: Water was available ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 21-25°C
- Humidity (%): 32-78%
- Photoperiod: 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: Diet was prepared every 4 weeks
- The appropriate amounts of the test item were each added to 500 grams of ground diet and mixed in a Hobart mixer for 5 minutes. These premixes were then added to sufficient ground diet to achieve concentrations of 50, 1000 and 3000 ppm and mixed in a twin-shell blender for 10 minutes.
Beginning week 35 and thereafter, the amount of ground diet used for the premix was increased to 2000 grams. Following the 5-minute Hobart mixing period, these premixes were then added to sufficient additional ground diet to achieve the desired concentrations and mixed in a twin-shell blender for 30 minutes.
- Storage temperature of food: Stored at room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each lot of test diet was analyzed by Raltech Scientific Services, St. Louis, Missouri.

Duration of treatment / exposure:

Following 1 year of test substance administration eight mice/sex/group were sacrificed and necropsied. The remaining animals were sacrificed and necropsied after 24 months of the study.

Frequency of treatment:

Continuous exposure through feed.

Post exposure period:

Not applicable.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 000 ppm

Remarks:

High dose group, equivalent to a mean daily intake of 384 mg/kg bw/d for male and 476 mg/kg bw/d for female.

Doses / concentrations 2

Dose / conc.:

1 000 ppm

Remarks:

Mid dose group, equivalent to a mean daily intake of 126 mg/kg bw/d for male and 164 mg/kg bw/d for female.

Doses / concentrations 3

Dose / conc.:

50 ppm

Remarks:

Low dose group, equivalent to a mean daily intake of 6.5 mg/kg bw/d for male and 8.24 mg/kg bw/d for female.

No. of animals per sex per dose:

68 animals/sex/dose

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

The mice were observed for appearance, behavior, overt signs of toxicity and moribundity three times daily on weekdays and twice daily on weekends and holidays. Individual detailed observations were recorded weekly. The mice were observed for mortality at least twice daily.
Individual body weights were recorded weekly the initial 13 weeks of study and bi-weekly thereafter.
Individual food (with test item) consumption was measured for 10 mice/sex/group (randomly selected) weekly the initial 13 weeks of the study and bi-weekly thereafter.
Hematological tests were performed on eight mice/sex/dose at 12 and 24 months of the study.
Hematological determinations included hemoglobin, hematocrit, erythrocyte count, total leucocyte count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, differential leucocyte count and reticulocyte count.

Sacrifice and pathology:

After 12 months of dietary administration of the test item, 8 mice/sex/dose were sacrificed by carbon dioxide asphyxiation and subjected to a complete postmortem examination. After 24 months, all survivors were similarly sacrificed and examined, and examinations were performed on all animals which died spontaneously or were sacrificed extremis.
Postmortem examinations were directly supervised by a pathologist, and included the external surfaces, all orifices, the cranial cavity, carcass, and the external and cut surfaces of the brain and spinal cord. The thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues were examined. Any morphologic changes observed were recorded.
Weights were obtained for the liver, kidney, heart, testis and brain from animals killed at the interim and terminal sacrifices. Before weighing, the organs were trimmed free of excess fat and connective tissue. Weights were obtained for those tissue masses which were subcutaneous, solid, and could be dissected out without damaging associated tissues.

All major tissues and organs were prepared and evaluated histopathologically.

Statistics:

Body weight gains, body weights, food consumption and hematological
parameters were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge significance of differences.
All statistical analyses compared the treatment groups with the control group, by sex.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment- related differences noted in clinical condition between control and treated mice.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no clear dose-related effect on survival. However, the lowest survival was in females fed 3000 ppm cyromazine (40% compared to 51% in controls at 104 weeks).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly lower body weight and body weight gains were noted for males fed 3000 ppm cyromazine throughout the study and 1000 ppm cyromazine from weeks 19 to 87. Females at these dose levels showed occasional statistically significant differences in body weight and body weight gain compared to controls. The differences at 1000 ppm were considered not to be biologically significant. Details are given below.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was statistically significantly decreased in males treated with 1000 and 3000 ppm cyromazine throughout the study and in females fed 3000 ppm cyromazine, particularly in the first year of the study.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the haematological parameters.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment-related effect on organ weights. Occasional statistically significant differences from control in absolute weights were considered to be incidental to treatment or, in the case of the liver weights in males in the 3000 ppm group, associated with the slight body weight reduction.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related macroscopic changes were noted at the 12 month interim examinations. At study termination, there was a slightly increased incidence of masses in the livers of treated male mice compared to controls.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination revealed a variety of non-neoplastic lesions. These lesions were of a similar incidence and distribution to spontaneous lesions in CD-1 mice of a similar age.
No treatment-related non-neoplastic proliferative hepatocellular changes were observed in mice of either sex. Due to the lack of a dose response, the absence of non-neoplastic proliferative changes and absence of similar findings in female mice, the slight increase in hepatocellular neoplasms in treated groups of male mice was considered to be unrelated to the administration of cyromazine in the diet.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination revealed a variety of neoplastic lesions. Except for hepatocellular neoplasms in male mice, these lesions were of a similar incidence and distribution to spontaneous lesions in CD-1 mice of a similar age.
Several liver neoplasms (hepatocellular adenomas and carcinomas) were observed in control and treated mice. The hepatocellular adenomas were nodular growths producing compression of the surrounding hepatic parenchyma. The larger adenomas produced elevation of the surface of the liver. They were composed of enlarged well-differentiated hepatocytes which formed nodules resembling normal liver. The hepatocellular carcinomas were generally larger than the hepatocellular adenomas and produced marked compression of the adjacent parenchyma. Most of the hepatocellular carcinomas had a distinct trabecular pattern with thickened hepatic plates. The carcinomas were composed of moderately well-differentiated hepatocytes. Nuclear and cytoplasmic pleomorphism was marked and the number of mitotic figures was increased in many of the hepatocellular carcinomas.
Pulmonary metastasis was observed in one male mouse at 1000 ppm that had hepatocellular carcinoma.
Although the incidence of total liver neoplasms was slightly greater in treated groups than in the control group, there was a distinct absence of a dose-related response in the incidence of these neoplasms. The incidence of liver neoplasms in the female mice was very low and with no treatment- related increase present in the female mice.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

164 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

2

Key result

Dose descriptor:

NOAEL

Effect level:

126 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Results are provided in the overall remarks section below.

Applicant's summary and conclusion

Conclusions:

Based on the results of a carcinogenicity study in mice, the NOAEL was set at 1000 ppm cyromazine (corresponding to 126 and 164 mg/kg b.w./day in males and females, respectively) based on reduced body weight gain at 3000 ppm. The study results did not indicate carcinogenic properties of Cyromazine.

Executive summary:

Cyromazine was dietary administered to male and female CD mice at doses of 0, 50, 1000 and 3000 ppm. Eight male and 8 female mice were assigned from each group for interim clinical pathology investigations after 52 weeks of treatment. The remaining mice were kept on the study for two years. Samples from all dietary levels (including controls) were taken at intervals throughout the study and analysed to confirm concentration, stability and homogeneity.

Clinical observations, bodyweights, food consumption and haematological parameters were measured throughout the study. At the end of the scheduled period the animals were killed and subjected to a full examination post mortem. Selected organs were weighed and specified tissues were taken for subsequent histopathology examination.

There was no evidence of inflammatory, degenerative or neoplastic lesions in mice, with the exception of a slightly higher number of hepatocellular neoplasms in male mice. However, the lack of non-neoplastic proliferative lesions in the liver, the absence of a dose-related response and absence of similar changes in female mice indicates that this observation was most likely not related to treatment with the test compound.

As a result, the NOAEL was set at 1000 ppm cyromazine (corresponding to 126 and 164 mg/kg bw/day in males and females, respectively), when administered in the diet to male and female mice for a period of two years, based on reduced body weight gain at 3000 ppm. The study results did not indicate carcinogenic properties of cyromazine.