N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18 September 2016 to 26 June 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

New studies were conducted solely to comply with a non-EU national registration requirement, and have been included in the dossier in accordance with REACH, Article 22(1)e. The study was conducted under a National quality assurance scheme (HJ/T 155-2004) similar to OECD GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Test guideline

Qualifier:

according to guideline

Guideline:

other: The guidelines for the testing of chemicals (health effects, version 2) issued by the Ministry of Environmental Protection of the People's Republic of China in September 2013 (417, Toxicokinetics).

Deviations:

no

GLP compliance:

no

Remarks:

The study was conducted under a National quality assurance scheme (HJ/T 155-2004) similar to OECD GLP.

Test material

Test material information

Constituent 1

Reference substance name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

EC Number:

941-634-6

Cas Number:

1228284-78-3

Molecular formula:

C10H12Cl3NO

IUPAC Name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

Test material form:

other: liquid

Details on test material:

- Physical state: Brown liquid
- Purity test date: 28 May 2015
- Stability under test conditions: Recertification date 31 May 2017.
- Storage condition of test material: Room temperature.

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing WeiTongLiHua Test Animal Technology Co. Ltd.
- Age at study initiation: 6-8 weeks for males and females
- Weight at study initiation: 184.7 – 263.4 g (6 female rats) and 249.5 - 292 g (34 male rats)
- Housing: Transparent polycarbonate cages. Bedding was changed twice a week.
- Diet (e.g. ad libitum): Standard feed for test animal, ad libitum, Beijing Keao Xieli Feed Co. Ltd, SCXK (JING) 2014-0010.
- Water (e.g. ad libitum): RO water, ad libitum.
- Acclimation period: The animals were acclimatised and observed for at least 7 days prior to dosing. During this period, no abnormalities of the animals were observed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): Alternating 12-hour light and dark cycles

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Duration and frequency of treatment / exposure:

Different test goups were either given a single dose or repeat dosed for 7 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw (total dose)

Remarks:

single dose for toxicokinetics, tissue distribution and excretion groups

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

signle dose toxicokinetics

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day

Remarks:

7 day, repeat dose for toxicokinetics

No. of animals per sex per dose / concentration:

4 males in the TK studies, 2 males and 2 females in the tissue distribution study

Control animals:

no

Details on dosing and sampling:

DOSING: Single or repeat doses of test material were administered to each rat by oral gavage in a dose volume of 10 mL/kg. Feed was withheld from the animals for 12 hours prior to dosing.

SAMPLING:

TOXICOKINETIC STUDIES
- Tissues and body fluids sampled (delete / add / specify): Whole blood (ca 0.2 mL) was removed from each rat from the orbital venous plexus, the whole blood was collected into heparinised tubes and stored on ice. Plasma was separated from the red blood cells by centrifugation.
- Time and frequency of sampling:
50 mg/kg, single oral gavage: 5, 15 and 30 minutes, 1, 2, 4, 6, 8, 12 and 24 hours
500 mg/kg, single oral gavage: 5, 15 and 30 minutes, 1, 2, 4, 6, 8, 12, 24, 36, 48, 72 and 96 hours
50 mg/kg, repeat oral gavage: 5, 15 and 30 minutes and 1, 2, 4, 6, 8, 12 and 24 hours post administration days 1 and 7 and pre-dose, days 3-7

TISSUE DISTRIBUTION STUDY
- Tissues and/or body fluid, such as blood, heart, liver, spleen, lung, kidney and gastrointestinal tract and its contents, testes or ovaries, uterus or epididymis, muscle, eye, brain, thyroid gland, and fat
- Time and frequency of sampling:
0.5, 4 and 24 hours post administration

EXCRETION STUDY
- Urine and faeces samples were collected individually and separately from the rats in the excretion experiment at the following time points: 0-6, 6-12, 12-24, 24-48, 48-72, 72-96 and 96-120 hours post administration.
- Bile was collected individually from the cannulated rats in the biliary excretion experiment at the following time points: 0-1, 1-2, 2-4, 4-8, 8-12, 12-24, 24-36, 36-48 hours post administration.

Statistics:

For all batches, the calibration standard and quality control sample data were considered satisfactory with reference to the assay acceptance criteria and pharmacokinetic analysis was based upon the reported data. Acceptance criteria for standard curve: at least six point accuracy between 85% and 115%, calculation with standard curve regression, the correlation coefficient for standard curve is >0.99. Acceptance criteria for QC sample: at least 2/3 QC sample (at three concentrations) accuracy between 85% and 115%.

Results and discussion

Preliminary studies:

In preliminary studies where the substance was administered as a single oral dose (20 and 1000 mg/kg), although the concentration-time profiles were variable amongst the animals following single oral administration (20 or 1000 mg/kg) of the substance, the mean AUC0-t for the male and female rats were similar and therefore, with the exception of the tissue distribution study, only male rats were used in the main tests. Since the concentration-times profiles for the male and female 1000 mg/kg oral dose groups were not well characterised, to the extent that the terminal phases for the individual animals were not made apparent, a 500 mg/kg oral dose group was used in the main study for the top dose. The low oral dose groups for the main studies were set at 50 mg/kg, 10-fold less than the 500 mg/kg dose.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Following single oral administrations of CA5204A to male rats (n = 4), a supraportional increase in CA5204A systemic exposure (AUC0-∞) was observed between 50 and 500 mg/kg, however, the CA5204A oral bioavailability for the 50 mg/kg (26.1±6.14%) and 500 mg/kg (150.6±18.3%) doses have been determined using the AUC0-t and should therefore be considered an underestimate of the absolute oral bioavailability.
Following 7 days repeat oral administration of 50 mg/kg/day CA5204A to male rats (n = 4/group) CA5204A predose concentrations in the plasma were approximately 2-fold greater than the LLOQ (50 ng/mL) and an increase (approximately 2-fold) in the mean CA5204A systemic exposure (AUC0-t) of the last exposure group was observed, compared to the mean CA5204A systemic exposure of the first group.

Details on distribution in tissues:

Following single oral administration of 50 mg/kg CA5204A to male and female rats, CA5204A was widely and rapidly distributed across all tissues. Concentrations of CA5204A were higher for all tissues, compared to plasma and the tissues having the highest concentrations were the excretion organs (kidney and liver) and the fat. Peak tissue concentrations were reached by 4 hours and for the male rats the concentrations then declined in line with plasma for most of the tissues. For the male fat and epididymis and the female fat, muscle, ovary, uterus and eye, unlike plasma, concentrations of CA5204A were maintained until 24 hours.

Details on excretion:

Following single oral administration of 50 mg/kg CA5204A to male rats, the pattern of excretion was similar between the animals. Although low, the major route of excretion of CA5204A was predominantly via the faeces, with 2.30-5.26% of the dose being recovered by 120 hours. Elimination via the urine was a minor route of excretion, with ≤0.01% being recovered by 120 hours.

Toxicokinetic parameters

Toxicokinetic parameters 1

Test no.:

#2

Toxicokinetic parameters:

AUC: 9212 ng/ml*h

Remarks:

mean

Toxicokinetic parameters 2

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 5.17 h

Remarks:

mean

Toxicokinetic parameters 3

Test no.:

#2

Toxicokinetic parameters:

Tmax: 6 h

Remarks:

mean

Toxicokinetic parameters 4

Test no.:

#2

Toxicokinetic parameters:

Cmax: 1454 ng/mL

Remarks:

mean

Toxicokinetic parameters 5

Test no.:

#3

Toxicokinetic parameters:

AUC: 547502 ng/L*h

Remarks:

mean

Toxicokinetic parameters 6

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: 10.1 h

Remarks:

mean

Toxicokinetic parameters 7

Test no.:

#3

Toxicokinetic parameters:

Tmax: 12 h

Remarks:

mean

Toxicokinetic parameters 8

Test no.:

#3

Toxicokinetic parameters:

Cmax: 23259 ng/L

Remarks:

mean

Toxicokinetic parameters 9

Test no.:

#4

Toxicokinetic parameters:

AUC: 9212 ng/mL*h

Remarks:

mean first group

Toxicokinetic parameters 10

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 5.62 h

Remarks:

mean first group

Toxicokinetic parameters 11

Test no.:

#4

Toxicokinetic parameters:

Tmax: 6 h

Remarks:

meran first group

Toxicokinetic parameters 12

Test no.:

#4

Toxicokinetic parameters:

Cmax: 1454 ng/mL

Remarks:

mean first group

Toxicokinetic parameters 13

Test no.:

#4

Toxicokinetic parameters:

AUC: 18840 ng/mL*h

Remarks:

mean 2nd group

Toxicokinetic parameters 14

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 4.21 h

Remarks:

mean 2nd group

Toxicokinetic parameters 15

Test no.:

#4

Toxicokinetic parameters:

Tmax: 4 h

Remarks:

mean 2nd group

Toxicokinetic parameters 16

Test no.:

#4

Toxicokinetic parameters:

Cmax: 2455 ng/mL

Remarks:

mean 2nd group

Metabolite characterisation studies

Metabolites identified:

not measured

Any other information on results incl. tables

Table 2. Concentration of test substance (ng/mL) in male rat plasma following 50 mg/kg oral gavage administration

Time (h)	R01	R02	R03	R04	Mean	SD
0.083	BLLOQ	BLLOQ	BLLOQ	BLLOQ	BLLOQ
0.25	68.7	63.4	100	62.0	73.6	18.0
0.5	104	160	207	137	152	43.2
1	137	253	568	197	289	192
2	397	668	693	439	549	153
4	291	1409	637	1132	867	499
6	493	1924	1327	697	1110	649
8	1047	806	1001	602	864	204
12	1435	715	747	202	775	506
24	BLLOQ	BLLOQ	BLLOQ	BLLOQ	BLLOQ

BLLOQ - Below the Lower Limit of Quantification, LOQ = 50 ng/mL

Table 3. Concentration of test substance (ng/mL) in male rat plasma following 500 mg/kg oral gavage administration

Time (h)	R01	R02	R03	R04	Mean	SD
0.083	56.0	259	77.2	169	140	93.1
0.25	797	1971	1301	1215	1321	486
0.5	1438	2566	2304	1834	2035	501
1	2802	3171	5584	3515	3768	1245
2	3967	5503	7028	5338	5459	1252
4	7158	5481	12078	5680	7757	3385
6	5989	3278	15009	4906	7296	5262
8	5392	2921	23086	11154	10638	8987
12	4348	25943	24744	29978	21003	11246
24	13371	10041	9389	9314	10529	1923
36	10406	5380	5944	5837	6892	2356
48	8449	3092	3653	3613	4702	2511
72	420	463	762	677	581	165
96	BLLOQ	BLLOQ	72.5	66.1	BLLOQ

BLLOQ - Below the Lower Limit of Quantification, LOQ = 50 ng/mL

Table 4. Concentration of test substance (ng/mL) in male rat plasma following 50 mg/kg oral gavage administration - first exposure group

Time (h)	R01	R02	R03	R04	Mean	SD
0.083	BLLOQ	BLLOQ	BLLOQ	BLLOQ	BLLOQ
0.25	68.7	63.4	100.3	62.0	73.6	18.0
0.5	104	160	207	137	152	43
1	137	253	568	197	289	192
2	397	668	693	439	549	153
4	291	1409	637	1132	867	499
6	493	1924	1327	697	1110	649
8	1047	806	1001	602	864	204
12	1435	715	747	202	775	506
24	BLLOQ	BLLOQ	BLLOQ	BLLOQ	BLLOQ

BLLOQ - Below the Lower Limit of Quantification, LOQ = 50 ng/mL

Table 5. Pre-dose concentration of test substance (ng/mL) in male rat plasma following oral gavage administration of 50 mg/kg/day for 7 days

Time (h)	L01	L02	L03	L04	Mean	SD
24	BLLOQ	BLLOQ	99.7	50.6	75.2	34.7
48	55.0	52.9	124	79.3	77.9	33.2
72	89.7	81.2	231	89.9	123	72.1
96	102	86.4	147	119	114	26.0
120	93.1	95.3	59.6	62.6	77.6	19.2
144	87.0	99.4	119	89.0	98.7	14.7

BLLOQ - Below the Lower Limit of Quantification, LOQ = 50 ng/mL

Table 6. Concentration of test substance (ng/mL) in male rat plasma following oral gavage administration of 50 mg/kg/day – last exposure group

Time (h)	L05	L06	L07	L08	Mean	SD
144	76.8	188	54.6	163	121	64.9
144.083	67.4	195	56.4	136	114	64.8
144.25	59.6	235	55.1	133	121	84.0
144.5	65.1	338	60.2	158	155	130
145	184	735	163	273	339	268
146	565	1917	650	1216	1087	624
148	1837	3747	417	2341	2085	1375
150	1644	3247	1321	1170	1845	955
152	983	2081	1896	783	1436	648
156	341	791	337	1144	654	391
168	BLLOQ	196	BLLOQ	119	79	96.2

BLLOQ - Below the Lower Limit of Quantification, LOQ = 50 ng/mL

Applicant's summary and conclusion

Conclusions:

Following oral (50 and 500 mg/kg) administration of CA5204 to male rats, the data indicate that CA5204A is a low clearance (CLz = 1.25±0.130 L/h/kg) compound that non-specifically distributes into tissues and is eliminated with a half-life of ca. 3.25 – 7.49 hours for the 50 mg/kg dose group and 10.1±2.30 hours for the 500 mg/kg dose group. A supraportional increase in systemic exposure (AUC0-∞) of CA5204A in male rat plasma was observed between 50 and 500 mg/kg. The oral bioavailability of CA5204A in male rat plasma for the 50 mg/kg (19.1-23.6%) and 500 mg/kg (132-159%) dose groups has been determined using the AUC0-t and should therefore be considered an underestimate of the absolute oral bioavailability. Following repeat oral administration to male rats, CA5204A predose concentrations in the plasma were approximately 2-fold greater than the LLOQ (50 ng/mL) and an increase (approximately 2-fold) in the mean CA5204A systemic exposure (AUC0-t) of the last exposure group was observed, compared to the mean CA5204A systemic exposure of the first group. CA5204A was found to be a substrate for P450 mediated metabolism across species when incubated with liver microsomes or recombinant human P450 enzymes and was also found to have a high affinity for binding to rat dog and human plasma proteins.

Executive summary:

The purpose of the in vivo studies was to reveal the toxicokinetic characteristics of the test substance in the rat, to inform on the absorption, distribution, metabolism and excretion processes and characteristics. The study was also not conducted to Good Laboratory Practice Standards as defined by the OECD but according to Ministry of Environmental Protection of the Peoples Republic of CHINA: HJ/T 155-2004; the guidelines for chemical testing good laboratory practices, implemented in June 2004.

Following single oral administrations of CA5204A to male rats (n = 4), a supraportional increase in CA5204A systemic exposure (AUC0-∞) was observed between 50 and 500 mg/kg, however, the CA5204A oral bioavailability for the 50 mg/kg (26.1±6.14%) and 500 mg/kg (150.6±18.3%) doses have been determined using the AUC0-t and should therefore be considered an underestimate of the absolute oral bioavailability.

Following 7 days repeat oral administration of 50 mg/kg/day CA5204A to male rats (n = 4/group) CA5204A predose concentrations in the plasma were approximately 2-fold greater than the LLOQ (50 ng/mL) and an increase (approximately 2-fold) in the mean CA5204A systemic exposure (AUC0-t) of the last exposure group was observed, compared to the mean CA5204A systemic exposure of the first group.   Following single oral administration of 50 mg/kg CA5204A to male and female rats, CA5204A was widely and rapidly distributed across all tissues which is reflective of the high volume of distribution that was determined following single IV administration of 5 mg/kg to male and female rats.  Concentrations of CA5204A were higher for all tissues, compared to plasma and the tissues having the highest concentrations were the excretion organs (kidney and liver) and the fat.  Peak tissue concentrations were reached by 4 hours and for the male rats, concentrations then declined in line with plasma for the majority of the tissues.  For the male fat and epididymis and the female fat, muscle, ovary, uterus and eye, unlike plasma, concentrations of CA5204A were maintained until 24 hours.  

Following single oral administration of 50 mg/kg CA5204A to male rats (n = 4), the pattern of excretion was similar between the animals.  Although low, the major route of excretion of CA5204A was predominantly via the faeces, with 2.30-5.26% of the dose being recovered by 120 hours.   Elimination via the urine was a minor route of excretion, with ≤0.01% being recovered by 120 hours.  Following IV bolus administration of 5 mg/kg CA5204A to male rats, excretion via the bile was very low, with ca. 0.03% of the administered dose being recovered by 48 hours.