6-nonyl-1,3,5-triazine-2,4-diamine

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Data are included to support toxicokinetik assessment

Objective of study:

excretion

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The metabolism of hexamethylmelamine (HMM), an antineoplastic drug, was studied in man and rats.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

14C

Species:

other: human / rat (Sprague-Dawley)

Sex:

male

Details on test animals or test system and environmental conditions:

PATIENTS
Patients selected for study were those accepted for Phase II or Phase III clinical cancer chemotherapy trials with HMM utilizing study protocols approved by the University of Wisconsin Center for Health Sciences Committee for Review of Clinical Research and Investigation Involving Human Subjects. Patient A. G. was a 62-year-old male with squamous cell tumor of the tonsil; Patient A. H. was a 51-year-old male with squamous cell lung carcinoma.

TEST ANIMALS
- Source: Sprague-Dawley, Inc., Madison, Wis.
- Weight at study initiation: 180 to 220 g
- Housing: in glass metabolism cages
- Individual metabolism cages: no data

Route of administration:

other: human: oral, added to orange juice / rat: i.p.

Vehicle:

other: HCl

Duration and frequency of treatment / exposure:

single dose

Remarks:

Doses / Concentrations:
human: 4 mg/kg HMM (clinical dose) (A. G., 200 mg; A. H., 350 mg) together with HMM-ring-14C, 100 µCi, at 7 a.m. on the day of the experiment, followed by another dose of unlabeled HMM, 4 mg/kg, at 7 p.m.
rat: 5 mg of HMM dissolved in a minimum of HCl containing 19.1 mCi of HMM-ring-14C

No. of animals per sex per dose / concentration:

2 male patients
4 male rats

Control animals:

no

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Metabolism)
HUMAN:
- Tissues and body fluids sampled: expired breath, urine, and feces, whole blood, hemolyzed erythrocytes, plasma proteins
- Time and frequency of sampling:
Plasma: 11 samples up to 24 hours, 1 sample after 48 hours p.a. (recognizably only from a diagram)
CO2, urine, feces: 24 hr, 48 hr, 72 hr p.a.

RAT:
- Tissues and body fluids sampled: CO2 , urine, feces
- Time and frequency of sampling: 24, 48, 72 h p.a.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Method type(s) for identification: gas chromatography and mass spectrometry, thin-layer chromatography, liquid scintillation counting

Type:

metabolism

Results:

The s-triazine ring is very stable and does not undergo cleavage. The rat urinary metabolites were the same as those observed in humans.

Type:

excretion

Results:

mainly in urine (human: 86 and 91%, respectively; rat: average 79%)

Details on distribution in tissues:

Plasma levels of radioactivity reached a maximum of 4380 and 3270 dpm/mL of plasma 1 hr after administration to Patients A. G. and A. H., respectively. The half-life of radioactivity in these patients was 13 hr. Hemolyzed erythrocytes that had been washed 3 times contained 6 to 9% of the radioactivity in 1 mL of whole blood. Proteins that were precipitated from plasma with sulfosalicylic acid and washed 3 times contained only 1 to 2% of the activity in 1 mL of plasma.

A tissue distribution study of radioactivity after administration of HMM-ring-14C in rats revealed no unusually high localization of radioactivity in any tissues.

Details on excretion:

Recovery of radioactivity in urine after administration of HMM-ring-14C to 2 cancer patients was as follows: in 24 hr each patient excreted 61 % of the total dose of radioactivity; A. G. excreted 25% and A. H. excreted 21 % from 24 to 48 hr; A. G. excreted 5% and A. H. excreted 4% from 48 to 72 hr. Total urinary excretion of radioactivity was 91 % for A. G. and 86% for A. H. (a portion of the urine sample from 0 to 24 hr was accidentally lost by A. H.). The amounts of radioactivity in the feces of Patients A. G. and A. H. within 48 hr after administration were 0.2 and 0.1%, respectively. No radioactivity could be found as expired 14CO2 in the breath of the patients during the 1st 6 hr after the administration of HMM-ring-14C.
The excretion of radioactivity in rats was similar to that of humans, except that rats appeared to excrete the radioactivity more rapidly; also the amount of radioactivity observed in the feces of rats (20%) was higher than that found in human feces (0.1%).

Toxicokinetic parameters:

half-life 1st: 13 hrs in plasma (human)

Toxicokinetic parameters:

C(time): 1 hr in plasma (human)

Metabolites identified:

yes

Details on metabolites:

The human urinary metabolites observed were pentamethylmelamine; N2, N2, N4, N6-tetramethylmelamine; N2, N2, N4-trimethylmelamine; N2, N2, N6-trimethylmelamine; N2, N2-dimethylmelamine; N2, N4-dimethylmelamine; monomethylmelamine; and melamine. The identities of the metabolites were confirmed by mass spectrometry. The rat urinary metabolites were the same as those observed in humans.

The concentrated urine samples were also used for TLC. Assay of thin-layer chromatograms of urines on a radio-chromatogram scanner revealed the presence of 4 major radioactive metabolites in humans; the RF values of the radioactive metabolites were identical to samples of the authentic compounds indicated . Quantification of metabolites with a Disc integrator tracing of the radioactivity on the TLC plates gave the proportional amounts of the major radioactive metabolites in 24-hr urines. Multiplication of these proportions by the total percentage of radioactivity in the 24 hr urines gave estimates of the percentages of the various metabolites. The activity in the miscellaneous category includes a mixture of the other methylmelamines identified by gas chromatography and also of any possible unidentified metabolites. The percentage values of the metabolites correspond to the molar percentages of the metabolites since the specific activity per mole of melamine is identical to the specific activity of HMM-ring-14C.

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results
The experiments indicate that the s-triazine ring is very stable and that it does not undergo cleavage. This is suggested by the fact that there is no production of 14CO2 after administration of HMM-ring-14C to either man or rats. The identification of the major urinary metabolites as methylmelamines and melamine also confirms the stability of the s-triazine ring in mammalian systems.
Excretion is mainly via the urine. The rat urinary metabolites were the same as those observed in humans.

Executive summary:

The metabolism of hexamethylmelamine (HMM), an antineoplastic drug, was studied in man and rats. After administration of HMM-ring-14C to 2 patients, peak plasma levels of radioactivity were seen 1 hr after drug administration and the plasma half-life of radioactivity was 13 hr. Patients excreted 61% of the radioactivity in the urine within 24 hr and 89% within 72 hr. No expired 14CO2 was found in the breath of patients within 6 hr of administration, and less than 0.2% of radioactivity was found in the feces in 48 hr.

Rats that were given HMM-ring-14C i.p. excreted 74% radioactivity in urine, 19% in feces, and none as 14CO2 within 24 hr.

Urinary metabolites were isolated by ion exchange methods, identified by gas chromatography-mass spectrometry, and quantitated using thin-layer chromatography with a radiochromatogram scanner. The urinary metabolites of HMM in both rats and man were N-demethylated homologs of HMM. These experiments suggest that in man and rats there is no significant metabolomic cleavage of the s-triazine ring and that methyl-melamines appear to be the only major urinary metabolites of HMM.

Description of key information

Data from in vitro or in vivo studies, which were designed to identify the toxicokinetic properties of Caprinoguanamine, are not available.

Due to the lack of experimental data, 100% absorption by all routes is assumed as worst case default for chemical safety assessment.

Based on physicochemical properties, no potential for bioaccumulation is to be expected.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

Additional information

Data from in vitro or in vivo studies, which were designed to identify the toxicokinetic properties of Caprinoguanamine, are not available.Assessment is based on available toxicity data and physicochemical properties and is developed with the aid of ECHA guidance on information requirements and chemical safety assessment chapter R7c.

Furthermore, supporting information froma metabolism study withthestructurally similar substance Hexamethylmelamine is considered.

 

Summary of available physicochemical information for Caprinoguanamine:

Parameter	Value used for CSR
Molecular Weight	237.34 g/mol
Melting Point	110°C (melting range)
Boiling Point	> 400°C
Density	1.068 g/cm^3(estimated ACDlabs)
Vapour Pressure	1.62 x 10^-4Pa (at 25°C) estimated
Partition coefficient n-octanol/water (log Kow)	3.83 (measured)
Water solubility	7.317 mg/L (estimated)
pKa(base)	4.4 (estimated ACDlabs)
Particle size	D50: 25.42 µm; around 90% < 100 µm (measured)
Surface tension	57.7 mN/m (measured)

 

Oral absorption

Caprinoguanamine has a molecular weight < 500 g/mol and a log K_owbetween -1 and 4 which is favourable for absorption. The estimated pk_a(base) of 4.4 means that the substance will be present mainly ionised in the stomach (pH2) and not ionised in the small intestine (pH 8) which favours an intestinal absorption. But in general solids with a microscale particle diameter are too large to be directly taken up by pinocytosis and have to be dissolved before they can be absorbed. Caprinoguanamine is poorly water soluble (water solubility 7.317 mg/L) which will probably limit the absorption rate.

After oral exposure signs of systemic toxicity including death were observed in an acute toxicity study, thus absorption of Caprinoguanamine has obviously occurred.

In the absence of specific data, for the oral absorption was set to 100 % by default.

 

Respiratory absorption

Caprinoguanamine is solid at room temperature and has a very high boiling point (> 400°C) together with a low vapour pressure (estimated: 1.62 x 10^-4Pa (at 25°C)) therefore substance evaporation and uptake by inhalation as vapour is unlikely.

The uptake after direct inhalation of substance dust particles may be of relevance because 90% of particles have a diameter below 100 µm and are therefore potentially inhalable. As well particles with diameters below 15 µm, which may reach the alveolar region of the respiratory tract, may be present.

 

The poor water solubility of Caprinoguanamine will limit the amount that can be absorbed directly. In general poorly water-soluble dusts depositing in the nasopharyngeal region could be coughed or sneezed out of the body or swallowed. Such dusts depositing in the tracheo-bronchial region would mainly be cleared from the lungs by the mucocilliary mechanism and swallowed. A small amount may be taken up by phagocytosis and transported to the blood via the lymphatic system.

Poorly water-soluble dusts depositing in the alveolar region would mainly be engulfed by alveolar macrophages. The macrophages will then either translocate particles to the ciliated airways or carry particles into the pulmonary interstitium and lymphoid tissues.

Because around 90% of particles are potentially inhalable and most of the inhaled Caprinoguanamine particles will be swallowed and then absorbed via GI tract for exposure assessments via inhalation a worst case assumption of 100% is considered to be appropriate.

 

 

 

Dermal absorption

Based on above data the substance may be absorbed dermally in relevant amounts. The molecular weight < 500 g/mol, a -1 < log K_ow< 4 and a pK_a(base) of 4.4 meaning at skin pH of 5.5 below 10% of all Caprinoguanamine molecules will be ionised favour a dermal absorption. Taking in account the low water solubility a low to moderate dermal absorption is expected. As surface tension of Caprinoguanamine is above the threshold of

10 mN/m, the substance is not expected to be a skin penetration enhancer.

 

In the absence of specific data, the dermal absorption was set to 100 % by default.

 

Distribution

The substance is neither highly lipophilic nor highly hydrophilic this makes an estimation on which body compartment would be preferred for distribution in the human body practically impossible such that a more detailed description is futile.

 

Metabolism

Data from in vitro or in vivo metabolism studies are not available for Caprinoguanamine. For the assessment of the metabolism of Caprinoguanamine supporting information from a metabolism study with Hexa methylmelamine, an antineoplastic drug, is taken into account.

Caprinoguanamine, consists of ans-triazine ring with two amino and one alkyl (C9) substituent, while Hexamethylmelamine consists of ans-triazine ring with three methylated amino substituents (see Fig. below). Therefore both substances share the same heterocyclic core structure and have structurally similar substituents.

Structures were outlined in the attached document.

The results of the metabolism study with radiolabelled Hexamethylmelamine (HMM) in man and rats indicate that the s-triazine ring is very stable and that it does not undergo cleavage. This is suggested by the fact that there is no production of¹⁴CO2 after administration of HMM-ring-¹⁴C to either man or rats. The identification of the major urinary metabolites as methylmelamines and melamine also confirms the stability of the s-triazine ring in mammalian systems (Worzalla JF, Kaiman BD, Johnson BM, Ramirez G, Bryan GT. 1974. Metabolism of Hexamethylmelamine-ring-14C in rats and man. Cancer Res. 34(10):2669-74).

 

In addition the results of the hydrolysis tests performed with the structurally similar source substances Benzoguanamine and Acetoguanamine at pH 4.0, 7.0 and 9.0 at 50°C in which no relevant degradation was observed indicate that the test substance may not be hydrolytically cleaved in the GI tract.

These results show that a metabolic cleavage of the triazine ring is quite unlikely and may not be considered any further. The conversation of the alkyl chain adjacent to the triazine ring e.g. by oxidation by a cytochrome P450 is potentially possible but only a minor alternative route and is therefore not closer examined, too.

Based on its structure the expected main metabolic pathway of Caprinoguanamine is the conjugation of its two (nucleophilic) amino-groups by sulfotransferases and acetyltransferases. Caprinoguanamine may also be subject to glucuronidation and glutathione (GSH) conjugation.

 

 

Excretion

The conjugation reactions with charged groups will increase the low water solubility of the substance and improve urinary excretion, which may be the most relevant way of excretion for this substance. Another relevant pathway for excretion may be by faeces, especially for the fraction, which has not been absorbed in the gastrointestinal tract after oral uptake.

Excretion by exhalation does not seem to be relevant.