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

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985/09/03 to 1992/05/18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

yes

Remarks:

See below

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Principles of method if other than guideline:

- The test substance was applied 5 days per week for 3 weeks instead of 7 days per week for 4 weeks.
- The spleen, testes, adrenals and heart were examined histopathologically only if macroscopic abnormalities had been detected post mortem.

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

Specific details on test material used for the study:

Storage: room temperature

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adults
- Source: Ray Nichols Rabbitry, Lumberton, Texas
- Weight at study initiation: Males (2.100 - 3.025 kg), Females (2.200 - 3.025 kg)
- Housing: One per cage
- Food: Purina Rabbit Chow; available ad libitum
- Water Type: Tap water; available ad Iibitum
- Acclimation period: 30 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 - 23.3 °C
- Humidity: 57 - 79 %
- Air changes: 13 changes per hour
- Photoperiod: 12 hours light, 12 hours dark; 6:30-6:30 PM

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

water

Remarks:

Deionised water

Details on exposure:

The animals were prepared prior to the initial treatment by clipping the back of the trunk of each animal free of hair to expose approximately 10% of the total body surface area. Clipping was repeated on Days 3, 7, 10, 14, and 17 within twenty-four hours prior to dosing to ensure intimate contact of the test material with the skin and to facilitate the scoring of dermal reactions.
Animals were treated five times weekly for three weeks. Treatment days were Days 1, 2, 3, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 20, and 21. The three additional animals were treated on Days 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 20, 21, 22, 23, and 24. On each treatment day, the back of each animal was moistened with 3.0 ml of deionized water. The appropriate amount of test material was applied evenly over the back of each animal in the test item groups. A 4 x 8 inch surgical gauze patch (two layers thick) was then placed over the exposure area of each animal and was secured in place with a strip of non-irritating adhesive tape. The entire trunk of each animal was then loosely wrapped with a semi-permeable dressing (orthopedic stockinette). To secure the wrappings in place, the edges of the dressing were wrapped with non-irritating adhesive tape. Six hours after each treatment, the wrapping gauze, and tape were removed from each animal and the skin was gently washed with room temperature tap water to remove remaining test material. The animals were dried with a clean cloth and returned to their cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability/Homogeneity were both confirmed.

Duration of treatment / exposure:

3 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5/sex/group
One additional male was added to the 50 mg/kg bw group and two males were added to the 500 mg/kg bw group due to early non-treatment related deaths.

Control animals:

yes

Details on study design:

The animals were assessed daily for any signs of systemic toxicity. Bodyweights and food consumption were recorded at intervals throughout the study. Twenty-four hours after the final application the animals were killed and blood samples taken for haematological and biochemical analysis. Each animal was subjected to a macroscopic examination post mortem, selected organs weighed and selected tissues examined microscopically.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Observations for skin irritation were made immediately prior to each application of Cyromazine. Observations for were made twice daily and observations for pharmacologic and/or toxicologic signs were made at least once daily. Individual body weights and food consumption were determined prior to the initial dosing, weekly throughout the study, and at termination of the study or at the time of discovery after death. Twenty-four hours after the final application the animals were killed and blood samples taken for haematological and biochemical analysis.

Sacrifice and pathology:

Twenty-four hours after the final application the animals were killed and blood samples taken for haematological and biochemical analysis. Each animal was subjected to a macroscopic examination post mortem, selected organs weighed and selected tissues examined microscopically.

Statistics:

All statistics and calculations were performed by computer with appropriate statistical
software.
Hematology values (except for differential leukocyte counts), all clinical chemistry values, and
Day 21 body weights were analyzed by Analysis of Variance (ANOVA). Statistical differences
found by ANOVA were further analyzed by Dunnett’s test and/or Student’s t-test. If any parameter
for a treatment group was found significantly different from the control for the terminal values, then the corresponding baseline values were also analyzed by the Analysis of Variance. Organ to body weight ratios and differential leukocyte counts were analyzed by the Kruskal-Wallis test. Ryan’s procedure for the Mann-Whitney U test was used to elicit differences indicated by the Kruskal-Wallis test. All statistical analysis were tested for significance at 0.05 and 0.01 alpha levels. If p > 0.05, the difference was judged non-significant.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations were generally minor. Changes in bowel and bladder function were observed in all groups and occurred more frequently during the first part of the study; these changes could have been in response to wrapping and handling of the animals. Other clinical observations included very slight to moderate diarrhea, decreased activity, emaciation, lacrimation, yellow nasal discharge and ataxia, however, these effects were not dose-related and were considered not to be related to treatment.

Dermal irritation:

no effects observed

Description (incidence and severity):

The 21-day dermal exposure to cyromazine produced no observable skin irritation.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Six animals died during the study but without dose-response correlation to treatment i.e. there were no deaths at the highest dose, while deaths occurred in control (one male), at 50 mg/kg (one female) and at 500 mg/kg (two males and two females). Mortality was thus concluded not to be related to exposure to the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no dose related effects on body weight or food consumption.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no dose-related differences in the clinical chemistry or haematological parameters measured in treated animals compared to control.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum creatinines increased over the duration of the study in all four groups, both male and female. There was a decline of serum chloride and potassium values between baseline and terminal determinations in all groups. A significant increase in serum glucose between baseline and terminal measurements was observed in exposed males. However, in these cases mean values were within normal ranges, and there were no statistically significant differences among the groups.

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no dose-related differences in organ weights, organ/body weight ratios, organ/brain weight ratios or histopathology in treated animals compared to control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test material-related effects were observed in any of the animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A number of histopathologic changes indicative of low grade infection or parasite migration were observed in liver, kidneys and lungs taken at sacrifice from all four groups. These findings were considered not to be related to treatment.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1:

21 Day Dermal Study in Rabbits (mortality data)

Dose level	Time of death	Number of deaths
(mg/kg b.w.)	(Day)	Male	Female
0	20	1/5	0/5
	Total at day 21	1/5	0/5
50	21	0/6	1/5
	Total at day 21	0/6	1/5
500	4	1/7	0/5
	7	1/7	1/5
	11	0/7	1/5
	Total at day 21	2/7	2/5
2000	Total at day 14	0/5	0/5

See "Overall remarkes, attachement" for the rest of tables 

Applicant's summary and conclusion

Conclusions:

Based on the results of a dermal 28-day repeated dose toxicity study, the NOAEL was considered to be 2000 mg/kg bw/day.

Executive summary:

A 21-day dermal toxicity study was conducted on male and female albeno rabbits using Cyromazine. Twenty-three male and twenty female albino rabbits were selected for testing. Five males and five females were randomly selected for each of the four treatments groups. One additional male was added to the low dose group and two males were added to the mid dose group because of early non-treatment related deaths. The exposure areas of all animals were moistened with 3.0 ml of deionized water. The animals in the test item groups were treated with 50, 500, 2000 mg/kg bw of the test material, respectively. The Vehicle Control animals were treated only with 3.0 ml of deionized water. The exposure period lasted for 6 hours per day. The treatment regimen was repeated five times weekly for 3 weeks.

 

Several deaths occurred with no dose-response correlation to treatment, i.e., there were no deaths at the highest dose, while deaths did occur in Control (one male), low dose (one female), and high dose group (two males and two females). The mortality was concluded not to be related to the test item exposure. No dose-related effects on in-life observations, mortality, body weights, food consumption, skin reactions, hematology, clinical chemistry, organ weights, organ/body weight ratios, organ/brain weight ratios, or histopathology were produced by the repeated dermal administration of Cyromazine at up to 2000 mg/kg bw/day, two times the limit dose. Based on the absence of adverse effects, the NOAEL was concluded to be 2000 mg/kg bw/day.