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

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1987/09 to 1988/07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In compliance with EU B.8 testing method

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

Deviation from protocol:
- The exposure period was 4 hours per day. However, animals were exposed for 7 days per week for 28 days thus resulting in a similar total exposure as with 6 hours per day for 5 days per week.
- The stability of the test substance and the homogeneity of the atmosphere were not mentioned.
- The purity of the test substance is not reported.
The deviations are considered not to compromise the scientific validity of the 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

Specific details on test material used for the study:

Storage conditions: room temperature
Safety precautions: gloves and face masks

Test animals

Species:

rat

Strain:

other: Tif: RAIf (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Age at study initiation: approx. 8 weeks.
- Source: Animal Production CIBA-GEIGY Ltd., 4332 Stein / Switzerland.
- Weight at study initiation: 177 to 199 g in males and 168 to 202 g in females.
- Housing: Five animals per cage were housed in Macrolon cages type 4.
- Diet: Pelleted, certified standard diet (ad libitum).
- Water: Tap water was given ad libitum.
All batches of diet were assayed for composition and contaminant levels by the manufacturer. The drinking water quality fulfilled the specifications of the "Schweizerisches Lebensmittelbuch" (Ed. 1972). Routine chemical examinations of the water quality were performed by the water authority and in house.

ENVIRONMENTAL CONDITIONS:
- Temperature: 22 ± 3°C
- Humidity: 55 ± 15%
- Air changes: approximately 15 air changes/hour
- Photoperiod: 12 hours light per day

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.6 - <= 6 µm

Geometric standard deviation (GSD):

2.15

Remarks on MMAD:

Geometric standard deviation (GSD) of 1.5 to 2.8 at all exposure concentrations.

Details on inhalation exposure:

The stock aerosol was generated from the solid test material by means of a dual flexible-brush dust-feed mechanism, modified from a design by Milliman (Battelle Research Centres, Geneva/ Switzerland). In this instrument, powders are transferred from a hopper brush through a slit interface to the aspiration tube of a venturi by means of a spiral feed brush driven by a stepping motor. In the venturi, the dust particles are suspended in a jet of dry filtered carrier air, thus forming the aerosol.
The dust generator was operated at a total airflow (through the aspiration tube and the carrier jet nozzle) of 19 L/min (input pressure 350 kPa), and the aerosol was diluted with filtered humidified air to yield a total flow of 32 L/min to the distribution flask. After removal of secondary agglomerates by means of a cyclone, the aerosol was passed through a 3 liter round flask and distributed to four nozzles. Through three nozzles aerosol was drawn and diluted with fresh air to the appropriate end concentration of the indicated exposure group. Before entering the inhalation chambers the aerosol was passed through a second cyclone. The fourth nozzle was used to measure concentration and particle size of the stock aerosol.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The aerosol concentration in the chambers was determined gravimetrically twice during each exposure. Samples of the test atmosphere (2L) were passed through a GF 92 filter. The air flow rate for the sample collection was kept constant (2L/min) by means of a constant flow air sampler, regardless of filter loading. The mean and standard deviation of the aerosol
concentrations for the Whole exposure time was calculated.
Particle size analysis was conducted twice during each exposure with an APS—33 Aerodynamic Particle Sizer, equipped with appropriate dilution systems to avoid coincidence counts. The number distribution in the 48 size classes was converted to a mass distribution, based on the bulk density of the test substance, which was determinated separately.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

4 hours per day, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

58 mg/m³ air (analytical)

Doses / concentrations 2

Dose / conc.:

206 mg/m³ air (analytical)

Doses / concentrations 3

Dose / conc.:

706 mg/m³ air (analytical)

No. of animals per sex per dose:

5/sex/group

Control animals:

yes, sham-exposed

Details on study design:

Groups of 5 male and 5 female Tif:RAIf (SPF) rats were exposed nose-only to cyromazine in an aerosol at concentrations of 0, 58, 206 or 706 mg/m3 air for 4 hours daily during 28 consecutive days. The stock aerosol was generated from the solid test material by means of a dual flexible-brush dust-feed mechanism. Additional groups of 5 male and 5 females treated at the 0 and 706 mg/m3 air level were maintained for a 3-week recovery period. Control animals were exposed to filtered humidified air.
Mortality and clinical signs were checked at least daily, body weights and food consumption were recorded weekly. Samples for laboratory investigations (haematology and blood clinical chemistry) were taken from all rats at the end of the treatment period, and from recovery animals after a 2-week treatment free period. All animals were necropsied at terminal sacrifice, selected organs were weighed, and selected organs and tissues were examined histopathologically.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

* Signs of systemic toxicity: on exposure days: before and after exposure. on weekends: once daily.
* Mortality: daily, including weekends and holidays.
* Body weight: weekly (midweek).
* Food consumption: weekly.
* Laboratory investigations:
- Hematology.
- Coagulation.
- Blood chemistry.

Sacrifice and pathology:

At the end of the test period, all animals were bled under ether anesthesia and subjected to detailed autopsy as scheduled.

Statistics:

For each time point and parameter a univariate statistical analysis was conducted. Due to the routine manner of the analysis system parameter free methods were applied. Each treated group was compared to the control group in of dispersion and displacement. In addition a trend test was applied considering all groups.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment with cyromazine triggered clinical signs that included piloerection, dyspnoea, hunched posture, and reduced spontaneous activity in all treated groups. The time of onset, the duration and severity was concentration-dependent and subsided during the recovery period.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in any of the dose groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant, dose related effects on body weights in either male or female animals, however the males of all exposure groups showed a slightly depressed body weight compared to control at the end of the exposure period. The depression was independent of the aerosol concentration and showed recovery by the end of the recovery period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Minor effects noted in haematology were slightly higher values for red blood cell parameters, specifically erythrocyte count, haemoglobin and packed cell volume, in the high dose males. At the end of the treatment-free recovery period the values for treated and control groups were comparable.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence that treatment with cyromazine had any influence on blood clinical chemistry parameters. Incidences of statistical significant differences between treated groups and control were sporadic and not dose related and were considered to represent the normal physiological variation of the respective parameters.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight analysis showed decreased pituitary weights in all treated males and increased liver weights at dose levels of 210 and 710 mg/m3 in treated females. However, pituitary weight in treated females was higher but did not reach statistical significance. In view of the small size of the pituitary and therefore the high variability in the weight, and in the absence of histopathological findings in this organ, the biological significance of this result is questionable.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross anatomical findings, both in the control and in the treated animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopical examination revealed 3 females from the 710 mg/m3 group and one female from the 55 mg/m3 group with small foci of lymphocytic infiltration in the adrenal cortex. This change was not seen in control female rats. All other changes were incidental in nature and not related to the test compound.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

55 mg/m³ air (analytical)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEC

Effect level:

210 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

206 mg/m³ air (analytical)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Any other information on results incl. tables

Table 1: Test atmosphere characteristics

Parameter	Intended aerosol concentration (mg/m^3)
	Control	55	210	710	Stock Aerosol
Analytical concentration (mg/m^3)	0	58 + 10	206 + 13	706 + 19	2850 + 349
Nominal concentration (mg/m^3)	0	55	210	710	3079 + 434
Mean particle size MMAD (µm)	-	1.9 - 5.3	1.6 - 5.0	2.0 - 6.0	-
Mean particle size GSD (µm)	-	1.6 - 2.8	1.5 - 2.7	1.8 - 2.7	-
Particles 7 µm (% w/w)	-	89 + 9	89 + 8	86 + 10	-
Particles 3 µm (% w/w)	-	56 + 14	57 + 13	52 + 11	-
Flow rate (L/min)
through generator	-	-	-	-	19
to distrib. flask	-	-	-	-	32
through chamber	48	32	64	64	-
to animal tube	2	2	2	2	-
Temperature (°C)	23.4 + 0.2	23.6 + 0.2	23.6 + 0.2	23.8 + 0.2	-
Humidity (%)	47 + 3	71 + 3	39 + 2	34 + 1	-
Oxygen content (%)	21	21	21	21	-

Table 2: Group Mean Bodyweights (g) (Selected Timepoints)

	Dose of cyromazine (mg/m^3)
	Males				Females
Week	0	55	210	710	0	55	210	710
-1	187	190	186	186	179	187	182*	185
1	235	234	235	228	203	205	200	208
3	307	288	291	285	225	228	226	230
4	334	307	312	307	238	236	229	241
7	369	-	-	361	249	-	-	268

* Statistically significant different from control, p<0.05

 

Table 3: Intergroup Comparison of Selected Haematology Parameters

		Dose of cyromazine (mg/m^3)
		Males				Females
Parameter/week		0	55	210	710	0	55	210	710
Erythrocytes [x10^12/L]	Week 5	8.5	8.9	8.5	9.0*	8.6	8.7	8.8	9.0
Hb [mmol/L]	Week 5	10.6	10.9	10.6	11.1*	10.4	10.4	10.6	10.9
Hct [1]	Week 5	0.49	0.51	0.48	0.51*	0.47	0.48	0.48	0.49

* Statistically significant different from control, p<0.05

 

Table 4: Intergroup comparison of selected organ weights (absolute, relative to body weight, relative to brain weight)

	Dose of cyromazine (mg/m^3)
	Males				Females
Organ	0	55	210	710	0	55	210	710
Liver (g)	11.9	11.2	10.9	10.7	8.2	7.9	11.0*	11.8*
rel. to b.w.	36.7	39.0	37.9	37.5*	37.24	36.08	48.52*	49.71*
rel. to br.w.	547	510	523	484	390	376	522*	553*
Pituitary (mg)	17.0	13.1*	12.8	12.5*	15.5	22.5	17.8	19.8
rel. to b.w.	0.053	0.045	0.045	0.044	0.071	0.102	0.077	0.084
rel. to br.w.	0.786	0.596	0.615	0.566*	0.741	1.068	0.838	0.926

* Statistically significant different from control, p<0.05

Applicant's summary and conclusion

Conclusions:

Based on the results of a repeated dose toxicity study with exposure via inhalation, the NOAEC was concluded to be 55 mg/m3 based on adverse effects in females at higher concentrations.

Executive summary:

In a repeated dose toxicity study, sixty albino rats were used to examine the 28-day inhalation toxicity of Cyromazine by a nose-only exposure system. Three groups of five males and five females each were exposed to a respirable dust during 4 hours a day on 7 days per week for 4 weeks, followed by laboratory investigations on hematology, blood chemistry and pathology. Additionally one group exposed to the highest concentration of Cyromazine served as a satellite group surviving a recovery period of 3 weeks after the last exposure. One control group and one satellite control group of five males and five females each were treated the same way in the inhalation chamber without test article exposure.

Exposure concentrations
The intended exposure levels were 55, 210, and 710 mg/m3 (including satellite group). The effectively measured exposure concentrations were 58 ± 10, 206 ± 13, and 706 ± 19 mg/m3. The control animals were exposed to filtered humidified air (including satellite group).

No mortality occurred in any of the groups. No clinical symptoms and no signs of systemic toxicity were observed in the control animals.
The test article exposed animals of all exposure groups showed an impairment of the general condition with the symptoms piloerection, dyspnea, hunched posture, and reduced spontaneous activity. The time of first occurrence, the duration, and the degree of clinical signs was concentration dependent.
The males of all exposure groups showed a slightly but not significantly depressed body weight compared with the controls at the end of the exposure period. The depression was independent of the aerosol concentration. No significant or exposure-correlated influence on the food consumption and the food consumption rate could be determined. The hematological investigation revealed higher values of red blood cell parameters (erythrocyte hemoglobin, packed cell volume) in the males of the high group (710 mg/m3) at the end of the treatment period. At the end of the treatment-free recovery period the values of treated and control groups were comparable. The analysis of the blood chemistry data revealed no evidence that the treatment with Cyromazine had any influence on clinical chemistry parameters. The weight of the pituitary glands of the test article exposed males was dose-dependently lower compared to controls. Because of high variance this effect was not significant in the mid dose group. The liver of the females of the mid and the high dose group showed a significantly higher weight than that of the controls. Macroscopical examination revealed no changes which could be attributed to effects of Cyromazine. Microscopical examination revealed 3 females from group and one female from group 2 with small foci of lymphocytic infiltration in the adrenal cortex. This change was not seen in control female rats.
All other changes seen in some control and treated animals are incidental in nature and not related to the test compound.

Overall it is concluded that an aerosol concentration of 55 mg/m3 of the test article resulted only in marginal effects on the animals general condition. In the absence of a substantial impairment of body weight development, hematological and pathological parameters, and clinical signs of only slight degree it can be assumed to be a marginal ”no observable effect level".
At exposure concentration of 210 mg/m3 signs of toxicity occurred to a moderate degree.
The exposure to 710 mg/m3 air produced moderate to severe clinical signs already after one week of exposure.