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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1979/06/26 to 1980/10/22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.27 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

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:

white

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adults (twenty-eight to thirty-five weeks old)
- Weight at study initiation: Males 7.9-14.6 kg, females 6.1-11.1 kg
- Housing: Individually housed in stainless steel cages
- Diet: Dog feed was available ad libitum
- Water: Water via an automated watering system was available ad libitum
- Acclimation period: Minimum of eighteen days

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was chosen because potential human exposure would be by the oral route.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- The test material and the ground dog feed were mixed at a rate of one minute/kg in a Patterson-Kelly twin-shell blender fitted with a pin-intensifier bar. Fresh diets were prepared and presented weekly.
- The required amount of test material for each compound-treated group was weighed on an Arbor balance, added to a small amount of ground dog feed, and mixed in a Waring blender.
- Fresh diets were prepared weekly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the control and mixed diets for each week were retained and shipped for diet analysis.

Duration of treatment / exposure:

Twenty-six weeks

Frequency of treatment:

Continuous exposure via feed

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 000 ppm

Remarks:

High-dose group (Group 4), equivalent to a mean daily intake of 750 mg/kg bw/d.

Doses / concentrations 2

Dose / conc.:

300 ppm

Remarks:

Mid-dose group (Group 3), equivalent to a mean daily intake of 75 mg/kg bw/d.

Doses / concentrations 3

Dose / conc.:

30 ppm

Remarks:

Low dose group (Group 2), equivalent to a mean daily intake of 7.5 mg/kg bw/d.

No. of animals per sex per dose:

Low- and mid-dose groups: 6 animals/sex/dose
Control and high-dose groups: 8 animals/sex/dose
Recovery groups (control and high dose only): 4 animals/ group

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

All of the dogs were observed twice daily for mortality and moribundity and once daily for appearance, behavior, appetite, elimination, and signs of toxic and pharmacologic effects. These observations were recorded daily. Body weights were recorded weekly beginning one week prior to the initiation of treatment. Food consumptions were recorded twice weekly (presented as weekly food consumptions).

Hematology, clinical chemistry and urinalysis examinations were performed on all dogs initially (Week 0), at Weeks 4, 8, 13, 17, 21, and 26, and at Week 30 for the recovery animals.
Ophthalmologic examinations were performed on all dogs prior to treatment and during Weeks 12 and 26.

Sacrifice and pathology:

Gross Pathology: Two males and two females from the control and high-dose groups were randomly selected by card draw to be maintained on study (without compound treatment for the high-dose dogs) for an additional four weeks (until completion of Week 30) as recovery animals. The remaining surviving dogs from the control and each test group were sacrificed after twenty-six weeks on study by exsanguination while under the influence of anesthesia and necropsied. The four males and four females designated as recovery animals were sacrificed and necropsied in a like manner four weeks later. Complete necropsies were also performed on all dogs which died or were sacrificed moribund.

Organ Weights: The organs from each dog were weighed and the organ/body ratios were determined.

Statistics:

Mean control body weight changes, weekly mean control food consumption data and total mean control weekly food consumption were compared statistically to the data of the treated groups of the same sex and at the same intervals by Bartlett's test for homogeneity of variance and the one-way classification analysis of variance ANOVA. If significant results were obtained from both Bartlett's test and ANOVA, a multiple pairwise comparison procedure (Games and Howell,1976) was used to compare the group mean values. If a significant result was not obtained from Bartlett's test, but was obtained from ANOVA, Scheffe's multiple pairwise comparison procedure (Scheffe, 1953) was used to compare the group mean values.
The clinical laboratory, terminal body weight, and absolute organ weight data for the control group were compared statistically to the data of the treated groups of the same sex by Bartlett's test for homogeneity of variance which was followed by a one-way classification analysis of variance ANOVA if the variances proved to be homogeneous. If the variances proved to be heterogeneous, a log10 transformation was performed which was followed by Bartlett's test. If the log10 was ineffective in removing variance heterogeneity, a loge transformation of the original data was performed which was followed by Bartlett's test. If homogeneity could not be achieved by transformation, ANOVA of the non-transformed data was completed. If ANOVA of homogeneous was significant, Scheffe's (1953) multiple pairwise comparison procedure was used to compare the group mean values. If ANOVA of heterogeneous data was significant, Games and Howell's (1976) multiple pairwise comparison procedure was used to compare the group mean values.
The relative organ weights for the control group were compared statistically to the data of the treated groups of the same sex by the Kurskal-Wallis non-parametric analysis of variance and the Wilcoxon's non-parametric comparison of group means.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No distinct treatment-related clinical signs were observed in any of the compound-treated animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two dogs (one from the low-dose group and one from the high-dose group) either died or were sacrificed moribund during the study. The low-dose male was found dead near the end of week 16. No premortem clinical signs were observed except for the presence of a large amount of stool in its cage. A high-dose male appeared depressed and had labored respiration on the last days of week 13. The dog was diagnosed with severe eye problems (including conjunctivitis, iridocyclitis, and corneal epithelial hyperplasia with bilateral iridal aneurysms of the nasal primary vessels and the superior vessels). On the following day the dog was sacrificed fir humane reasons.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Evaluation of the body weight data revealed weight losses or decreased rates of body weight gain for the high-dose males and females at all compound-treatment and recovery intervals. These findings are considered a result of treatment with cyromazine.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean weekly food consumption values for the high-dose males were less than the respective male control values.
These changes were not as evident in the female dogs, and in most instances, the female low- and mid-dose values were lower than those of the high-dose dogs. Total mean weekly food consumption of the high-dose males and females at both the treatment and recovery intervals were lower than the respective control values; however, as for the mean weekly food consumption values, the changes were not as evident in the female dogs.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related ophthalmologic findings were noted in any of the cyromazine-treated dogs.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Evaluation of the hematology data revealed a possible treatment-related decrease in the red cell mass of the high-dose males, but a similar trend was not quite as evident in the high-dose females.
The mean serum glutamic oxaloacetic transaminase values of the high-dose males exhibited significant elevations over respective control values in five of the six sampling periods during the compound-treatment phase, followed by a return to control values during the recovery phase. This finding is also suggestive of a treatment-related effect.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Evaluation of the urinalysis data revealed no trends or alterations which could be considered a result of treatment with cyromazine.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute organ weights and the remaining relative organ weights of the cyromazine-treated dogs were comparable to the respective control values.
Slight but consistent increases were noted for the relative brain, heart, and liver weights of the high dose males and females sacrificed at weeks 26 and 30 and the ovary weights of the high dose females sacrificed at weeks 26 and 30 when compared to respective control values. These findings were attributed to the decreased body weight gains noted for the high dose group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No distinct treatment-related findings were noted in any of the dogs sacrificed after twenty-six weeks of treatment or in any of the recovery dogs sacrificed four weeks later.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No compound-related histopsthological alterations were observed in treated animals.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

7.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (nominal)

System:

haematopoietic

Treatment related:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Results are provided in the overall remarks section below.

Applicant's summary and conclusion

Conclusions:

The no-effect level (NOAEL) of cyromazine, when administered in the diet to male and female beagle dogs for six months, was considered to be 7.5 mg/kg bw/d.

Executive summary:

This study was designed to characterize and evaluate the subchronic toxicity of cyromazine when administered in the diet for twenty-six weeks to two groups of six male and six female beagle dogs at levels of 30 (Group 2, low-dose) and 300 (Group 3, mid-dose) ppm, and one group of eight male and eight female beagle dogs at a level of 3000 (Group 4, high-dose) ppm. An additional group of eight male and eight female dogs served as a control (Group 1) and received the basal diet only. Two male and two female dogs each from the control and high-dose groups were maintained as recovery animals for an additional four weeks (receiving no treatment) after the initial twenty-six weeks of treatment. Criteria evaluated for compound effect included survival, appearance and behavior, appetite and elimination, body weights, clinical laboratory data, ophthalmologic examinations, terminal body weight and organ weight data, and gross and microscopic pathology. One low-dose male was found dead during Week 16 and one high-dose male was sacrificed moribund during Week 14. No premortem clinical signs were observed in the low-dose dog. Clinical signs observed in the high-dose dog were indicative of respiratory distress.

 

Losses in body weight or decreased rates of body weight gain were noted for the high-dose males and females at all treatment and recovery intervals evaluated. These changes were considered compound-related. The mean weekly food consumption values for the high-dose males were less than the respective male control values. These changes were not as evident in the female dogs, and in most instances, the female low- and mid-dose values were lower than those of the high-dose dogs. Total mean weekly food consumption of the high-dose males and females at both the treatment and recovery intervals were lower than the respective control values; however, as for the mean weekly food consumption values, the changes were not as evident in the female dogs.

 

A treatment-related decrease occured in the red cell mass of the high-dose males, with a similar but less pronounced decrease in the high-dose females. The changes were characterized by significantly decreased hematocrit and hemoglobin values for the males at all compound-treatment intervals when compared to the respective pretreatment or the control values. A stepwise progressive decrease in the hematocrit and hemoglobin values of the compound-treated males is positively correlated to increase in dose levels. Post-recovery hematocrit and hemoglobin values of the high-dose males approached the male control values obtained at the same interval. Decreased mean total cholesterol values at all of the compound-treatment intervals for the high-dose males followed by a near return to control values at the recovery interval suggests a treatment-related phenomenon. The mean serum glutamic oxaloacetic transaminase values of the high-dose males were significantly increased at five of the six compound-treatment intervals followed by a return to control values during the recovery period. This finding is also suggestive of a treatment-related effect. No other changes were noted in the clinical laboratory studies which were indicative of a cyromazine treatment-related phenomenon.

 

Slight but consistent increases were noted for the relative brain, and liver weights of the high-dose males and females sacrificed at Weeks 26 and 30 and the ovary weights of the high-dose females sacrificed at Weeks 26 and 30 when compared to respective control values. These findings were attributed to the decreased body weight gains noted for the high-dose dogs. No distinct treatment-related findings were observed with respect to clinical signs, ophthalmologic examinations, or gross and microscopic pathology. The no-effect level of cyromazine, when administered in the diet to beagle dogs for six months, was considered to be 30 ppm. The slightly lowered hematocrit and hemoglobin levels for the low-dose (30 ppm) males were considered within acceptable laboratory limits. The only findings noted in the mid-dose (300 ppm) dogs were similar, however slightly more pronounced effects in the hematocrit and hemoglobin levels.