2-(2,6-diethyl-4-methyl-phenyl)propanediamide

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

absorption

distribution

excretion

metabolism

other: Prediction of in vivo exposure in the male and female rat

Qualifier:

no guideline available

Principles of method if other than guideline:

There are currently no specific regulatory guidelines that cover in vitro toxicokinetics. However, this type of work is exemplified in OECD 417 (22nd July 2010) Guideline for the testing of chemicals, Toxicokinetics, Supplemental approaches, use of in vitro information (paragraphs 59-61), and use of toxicokinetic modelling (paragraph 65). The work carried out also meets the spirit of the European Chemical Agency Guidance for the Implementation of REACH. i.e. Guidance on information requirements and chemical safety assessment, chapter R.7C: endpoint specific guidance (ECHA-12-G-23-EN), sections R.7.12.2.2 generating and integrating TK information, in silico methods – kinetic modelling, and Appendix R.7.12-2 prediction of toxicokinetics integrating information generated in silico and in vitro. All experimental work was carried out against appropriate well recognised and established control compounds detailed in the methods and the study data only reported when the control data fell within acceptable predefined limits.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Vehicle:

DMSO

MICROSOMAL INTRINSIC CLEARANCE

The in vitro CLint of the test substance in male Wistar rat microsomes was 1.45 ± 3.25 µL/min/mg protein. This was characterised by 9.6% depletion over 45 minutes. The in vitro CLint in female Wistar rat microsomes was 1.79 ± 3.85 µL/min/mg protein, characterised by 9.1% depletion over 45 minutes. In the absence of NADPH, there was approximately 4% depletion over 45 minutes in male microsomes and approximately 4.2% depletion over 45 minutes in female microsomes, indicating no non NADPH dependent metabolism. In the absence of microsomes, there was <1 % loss of the test substance over 45 minutes suggesting no potential chemical instability of the molecule in this matrix.

 

HEPATOCYTE INTRINSIC CLEARANCE

The in vitro CLint of the test substance in male Wistar rat hepatocytes was 1.54 ± 1.17 µL/min/10^-6 cells characterised by no depletion over 60 minutes. The in vitro CLint in female Wistar rat hepatocytes was 0.182 ± 0.651 µL/min/10^-6 cells characterised by 2.6% depletion over 60 minutes. In the absence of live hepatocytes, there was no loss of CA3250 at the 60 minute time point, suggesting no potential chemical instability of the molecule in this matrix. Due to the negligible intrinsic clearance in hepatocytes it was considered appropriate to use the microsomal data for modelling purposes.

 

PLASMA PROTEIN BINDING

The fraction of test substance unbound in plasma (fup) was 0.893 ± 0.142 in male plasma and in female plasma was 0.612 ± 0.0452. The recovery of test substance in the incubations was 82.6 and 93.4% respectively.

 

CACO-2 PERMEABILITY

The apparent permeability (Papp) of test substance across Caco-2 cell monolayers was 25.7 x 10^-6 cms-1 ± 1.86 x 10-6cms -1. The recovery of test substance from the incubation media was 92.9%.

 

CLOE PK™ - PK SIMULATION

The following parameters were used for Cloe PK™ modelling:

Molecular weight: 248.32

LogP: 1.1

pKa: Neutral

Aqueous solubility at pH 7.96 (g/L): 1.6

Fup: 0.893 for males 0.612 for females

Microsomal CLint (µL/min/mg): 1.45 for males 1.79 for females

Papp (cms-1 ): 25.7 x 10^-6

Certain physiological assumptions were made within the model such as body weight, hepatic blood flow, hepatic mass fraction and microsomal protein content.

 

TABLE 1: Median predicted pharmacokinetic parameters derived using Cloe PKTM simulating intravenous administration at doses of 15, 150 and 1000 mg/kg in male and female rats

 

Parameter	15 mg/kg		150 mg/kg		1000 mg/kg
	Male	Female	Male	Female	Male	Female
Cmax (µg/mL)	760	870	7600	8700	51000	58000
AUC (µg.h/mL)	63	81	630	810	4200	5400
T ½ (h)	33	28	33	28	33	28
Vd (L/kg)	11	6.8	11	6.8	11	6.8
Total CL (mL/min/mg)	4	3.1	4	3.1	4	3.1
MRTinf (h)	22	19	22	19	22	19
Vss (L/kg)	5.4	3.7	5.4	3.7	5.4	3.7
Elimination rate (h-1)	0.021	0.024	0.021	0.024	0.021	0.024

 

TABLE 2: Median predicted pharmacokinetic parameters derived using Cloe PK™ simulating oral administration at doses of 15, 150 and 1000 mg/kg in male and female rats

 

Parameter	15 mg/kg		150 mg/kg		1000 mg/kg
	Male	Female	Male	Female	Male	Female
Cmax (µg/mL)	4.2	5.8	41	57	140	190
Tmax (h)	2.5	2.5	2.4	2.4	3.3	3.3
AUC (µg.h/mL)	60	78	590	780	2500	3200
T ½ (h)	34	29	33	29	36	31
Vd (L/kg)	11	6.9	11	6.9	12	7.5
Elimination rate (h-1)	0.021	0.024	0.021	0.024	0.019	0.023
Bioavailability (%)	0.93	0.94	0.93	0.93	0.57	0.57
Fraction absorbed	1	1	0.99	0.99	0.61	0.61

 

Conclusions:

Based upon an in vitro to in vivo extrapolation utilising recognised, established and validated in vitro systems carried out by Cyprotex Discovery Ltd. together with their commercially accepted physiologically based pharmacokinetic model (Cloe PK™), the test substance, when administered in vivo to male and female rats, is predicted to have a good fraction absorbed across the gastrointestinal tract following doses of 15, 150 or 1000 mg/kg (approx. 99% at lower doses reducing to 61% at 1000 mg/kg). This translates to a systemic oral bioavailability of 57-100% in male and female rats over this dose range. In general in vivo the test substance would be expected to be a low clearance compound that distributes widely into tissues with a mean residence time in the body of around 19-22 hours.

Executive summary:

This study was designed to assess the in vivo pharmacokinetic behaviour of the test substance via an in vitro to in vivo extrapolation (IVIVE) to help put into context the findings of toxicology studies carried out separately to this report. In vitro metabolic stability, plasma protein binding and Caco-2 permeability were determined by Cyprotex Discovery Ltd., Macclesfield, UK and the data used in the physiologically based pharmacokinetic (PBPK) model, Cloe PKTM, to simulate the predicted in vivo exposure in male and female rat at 100, 300 and 1000 mg/kg. Physical chemistry data including solubility, logP and pKa was provided by Syngenta for the purposes of modelling and simulation.

The in vitro CLint of the test substance in male Wistar rat microsomes was 1.45 ± 3.25 µL/min/mg protein. This was characterised by 9.6% depletion over 45 minutes. The in vitro CLint in female Wistar rat microsomes was 1.79 ± 3.85 µL/min/mg protein characterised by 9.1% depletion over 45 minutes. The in vitro CLint of the test substance in male Wistar rat hepatocytes was 1.54 ± 1.17 µL/min/10^-6 cells characterised by no depletion over 60 minutes. The in vitro CLint in female Wistar rat hepatocytes was 0.182 ± 0.651 µL/min/10-6 cells characterised by 2.6% depletion over 60 minutes. Due to the negligible intrinsic clearance in hepatocytes it was considered appropriate to use the microsomal data for modelling purposes. The fraction of test substance unbound in plasma (fup) was 0.893 ± 0.142 in male plasma and in female plasma was 0.612 ± 0.0452. The recovery of test substance in the incubations was 82.6 and 93.4%, respectively. The apparent permeability (Papp) of test substance across Caco-2 cell monolayers was 25.7 x 10^-6 cms^-1 ± 1.86 x 10-6cms^-1. The recovery of test substance from the incubation media was 92.9%. The predicted median pharmacokinetic parameters for the test substance using Cloe PKTM simulating oral administration at doses of 15, 150 and 1000 mg/kg in male and female rats is summarised below.

 

Parameter	15 mg/kg		150 mg/kg		1000 mg/kg
	Male	Female	Male	Female	Male	Female
Cmax (µg/mL)	4.2	5.8	41	57	140	190
Tmax (h)	2.5	2.5	2.4	2.4	3.3	3.3
AUC (µg.h/mL)	60	78	590	780	2500	3200
T ½ (h)	33	28	33	28	33	28
Vd (L/kg)	11	6.8	11	6.8	11	6.8
CL (mL/min/kg)	4	3.1	4	3.1	4	3.1
MRTinf (h)	22	19	22	19	22	19
Vss (L/kg)	5.4	3.7	5.4	3.7	5.4	3.7
Bioavailability (%)	0.93	0.94	0.93	0.93	0.57	0.57
Fraction absorbed	1	1	0.99	0.99	0.61	0.61

 

Based upon an in vitro to in vivo extrapolation utilising recognised, established and validated in vitro systems carried out by Cyprotex Discovery Ltd. together with their commercially accepted physiologically based pharmacokinetic model (Cloe PK™), the test substance, when administered in vivo to male and female rats, is predicted to have a good fraction absorbed across the gastrointestinal tract following doses of 15, 150 or 1000 mg/kg (approx. 99% at lower doses reducing to 61% at 1000 mg/kg). This translates to a systemic oral bioavailability of 57-100% in male and female rats over this dose range. In general in vivo the test substance would be expected to be a low clearance compound that distributes widely into tissues with a mean residence time in the body of around 19-22 hours.

Additional information

Information based on physico-chemical properties

The substance is a yellowish powder with a molecular weight of 248.3. Its solubility in water is 1.6 g/litre at 25 °C and it possesses a Log Pow value of 1.1 at 25 °C. Based upon its structure, molecular weight and log P value, some absorption of the orally administered substance across the gastrointestinal mucosa would be expected. Hydrolysis studies have shown that the substance is resistant to hydrolysis across a range of pH values from 3.8 to 9.1 and at 50 °C. The half life was estimated to exceed one year at each these extreme pH values and also at pH 7.1. Consequently, the substance would not be expected to be hydrolysed in the gastrointestinal tract before absorption. The molecular weight of the absorbed substance is below the biliary exclusion limit in the rat, consequently, it would not be excreted directly in bile. Its aqueous solubility would allow some direct excretion in urine; however, the absorbed substance would be expected to be subject to some biotransformation to promote its excretion. The alkyl groups on the phenyl ring are all subject to progressive oxidation, initially to an alcohol, but then possibly to a carboxylic acid. Direct hydroxylation of the phenyl ring is also possible. All such oxidised metabolites would be more polar, favouring urinary excretion. Such metabolites would also be subject to conjugation including sulphates and glucuronides, the increased molecular weights of which would support biliary elimination for subsequent faecal excretion. The amide moieties are likely to be acetylated to promote urinary excretion. Deamination is also possible, leading to the formation of a carboxylic acid group that would also favour elimination directly in urine. The absorbed substance is therefore likely to be excreted intact or to be readily metabolised to promote fairly rapid excretion.

 

Information based on toxicity studies

There were signs of severe toxicity following a single oral dose of an aqueous preparation of 2000 mg/kg to female rats with 4 of the 5 animals killed in extremis on day 1. However, the surviving rat had recovered by the second day. At examination post mortem, the only observation was red staining around the nose and mouth of one rat. Following a single oral dose of 300 mg/kg to females, there were signs of slight systemic toxicity with complete recovery by day 2 and with all animals showing an overall weight gain during the study. At examination post mortem, there were no signs of any treatment-related abnormalities.

Following a single 24 hour occluded application of 2000 mg/kg to male and female rat skin, applied as a paste moistened with water, there were no signs of systemic toxicity. Signs of slight skin irritation were seen in all animals, but was completely resolved by day 14 and all animals showed an overall weight gain during the study. There were no treatment-related abnormalities at examination post mortem.

Following a 4 hour occluded application of 500 mg of the substance to rabbit skin, applied as a paste in a small volume (0.5 mL) of water, very slight erythema was seen in all animals at 1 hour after application and very slight oedema in one animal at the same time. Accordingly, the substance did not fulfil the criteria for classification of irritating to the skin under the conditions of this test. In a rabbit eye experiment, the substance produced mild irritation, but not sufficient for classification to the unrinsed eye, and no toxicokinetic information could be concluded from this finding.

In the 28 day oral dose study, the dose-related effects on the liver, notably on liver weight, together with perturbations in clinical chemistry and haematology parameters are indicative both of absorption of an oral dose and of a dose-related increase in absorption. There was no evidence of any dermal penetration of the test material in the rat or rabbit studies, despite occlusion of the application sites that would have promoted any absorption. This was not inconsistent with results of a local lymph node assay, the conclusion from which was that the substance was unlikely to be a skin sensitiser under the conditions of the test.

Based upon particle size distribution in the air elutriation study, 13.12% of the substance is capable of becoming airborne, being equal to or less than 115 µm aerodynamic equivalent diameter (AED). However, none of the substance possessed an aerodynamic particle diameter of ≤ 15 µm, that would be capable of penetrating lower into the respiratory tract than the nasopharyngeal region, and is therefore not classified as the respirable fraction by the HSE. Based upon the current EU and ISO definitions adopted by the UK (BS EN481) this substance is considered to be of low inhalability with none of the sample being respirable. Any particulate substance deposited in the nasopharyngeal region would pass rapidly to the gastrointestinal tract.

 

Absorption, distribution & excretion

In the 28 day oral dose study, the dose-related effects on the liver, notably on liver weight, together with perturbations in clinical chemistry and haematology parameters are indicative both of absorption of an oral dose and of a dose-related increase in absorption. There was no evidence of any dermal penetration of the test material in the rat or rabbit studies, despite occlusion of the application sites that would have promoted any absorption. This was consistent with results of a local lymph node assay, the conclusion from which was that the substance was unlikely to be a skin sensitiser under the conditions of the test.

Based upon particle size distribution in the air elutriation study, 13.12% of the substance is capable of becoming airborne, being equal to or less than 115 µm aerodynamic equivalent diameter (AED). However, none of the substance possessed an aerodynamic particle diameter of ≤ 15 µm, that would be capable of penetrating lower into the respiratory tract than the nasopharyngeal region, and is therefore not classified as the respirable fraction by the HSE. Based upon current UK and ISO definitions, this substance is considered to be of low inhalability with none of the sample being respirable. Any particulate substance deposited in the nasopharyngeal region would pass rapidly to the gastrointestinal tract.

 

Metabolism

In the bacterial mutagenicity assays in S. typhimurium and E. coli the substance induced significant, reproducible and dose-related increases in the numbers of revertant colonies in strains TA 1537 and TA98 in the presence of S9-mix. No mutagenic effects were observed in these strains in the absence of S9-mix, nor in any of the other strains tested, either in the presence or absence of S9-mix. These results indicate that a metabolic product of the substance, mediated by the presence of S9-mix, possesses the potential to induce reversions in these two strains of S. typhimurium.

In the mouse lymphoma mutation assay, no significant increases in mutation frequency were observed in cultures treated in vitro with the substance either in the presence or absence of S9-mix. Hence, the substance is not mutagenic under the conditions of this assay. Accordingly, no conclusions can be drawn on its metabolism from this study.

 

Conclusion

Toxicity studies have shown clear evidence for the absorption of the substance following oral administration, with a progressive increase in clinical signs as the dose level increased.

There was no evidence of any dermal penetration of the substance in the rat and rabbit, despite occlusion of the application sites. The results from the local lymph node assay indicated that this substance showed no capacity to cause skin sensitisation and therefore showed no evidence of any dermal absorption.

An assessment of the potential absorption of the substance is also based upon its physico-chemical properties; these suggest that the intact molecule would be subject to some absorption across the gastrointestinal mucosa following oral administration. The modest aqueous solubility of the substance would allow some direct urinary excretion of the absorbed molecule. However, based upon its structure, some biotransformation of the substance would be expected to promote its excretion. The alkyl groups on the phenyl ring are each subject to progressive oxidation, initially to an alcohol and possibly to a carboxylic acid. Direct hydroxylation of the phenyl ring is also possible. All such oxidised metabolites would be more polar, favouring urinary excretion, but each would also be subject to conjugation including sulphates and glucuronides, the increased molecular weights of which would favour biliary elimination for subsequent faecal excretion. The amide moieties are also likely to be acetylated to promote urinary excretion. Deamination is also possible, leading to the formation of a carboxylic acid group that would also favour elimination directly in urine. Absorbed test material is therefore likely to be excreted intact or to be readily metabolised to promote fairly rapid excretion. The results of the mutagenicity assays in S. typhimurium confirmed that substance was subject to metabolism, based upon its potential to induce reversions, but only following metabolic activation mediated by the presence of S9-mix.