Reaction mass of 1-methyl-4-(1-methylethylidene)cyclohexyl acetate and p-menth-1-en-8-yl acetate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2019 - February 2020

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:

The information is used to support the read across of Terpinyl Acetate multi from Terpineol multi

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Objective of study:

metabolism

Principles of method if other than guideline:

Blank Wistar Han mix-gender rat plasma and whole blood with K2 EDTA as anticoagulant was obtained. Stability of Terpinyl acetate was evaluated in rat plasma and blood in two separate experiments. Both of the above mentioned experiments were performed in duplicate to prove the reproducibility. In both experiments rat K2EDTA matrix was spiked with analyte solution to achieve initial concentration at 50 μg/mL. The spiked matrix was kept at 37 °C. The spiked matrix samples were aliquoted at multiple time points between 0 and at least 4 hours and processed according to the established bioanalytical procedure (GC-MS MS).
In the same way Terpinyl Acetate multi was spiked to aqueous solution of pH2, 4 and 7 and the degradation was determined in the same way.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-methyl-4-(propan-2-ylidene)cyclohexyl acetate and 2-(4-methylcyclohex-3-en-1-yl)propan-2-yl acetate

EC Number:

904-693-9

Molecular formula:

C12H20O2

IUPAC Name:

Reaction mass of 1-methyl-4-(propan-2-ylidene)cyclohexyl acetate and 2-(4-methylcyclohex-3-en-1-yl)propan-2-yl acetate

Test material form:

liquid

Radiolabelling:

no

Test animals

Details on species / strain selection:

This is an in vitro study using blank Wistar Han mix-gender rat blood and plasma

Sex:

not specified

Administration / exposure

Doses / concentrations

Dose / conc.:

50 other: ug/ml (mg/l)

Results and discussion

Toxicokinetic / pharmacokinetic studies

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 50.7 minutes

Remarks:

plasma

Toxicokinetic parameters 2

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 8.75 minutes

Remarks:

Aqueous solutions pH2

Toxicokinetic parameters 3

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: 7.49 minutes

Remarks:

Aqueous solutions pH4

Toxicokinetic parameters 4

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 10.3 minutes

Remarks:

Aqueous solutions pH7

Toxicokinetic parameters 5

Key result

Toxicokinetic parameters:

half-life 1st: > 210 minutes

Remarks:

denaturated plasma

Metabolite characterisation studies

Metabolites identified:

not measured

Details on metabolites:

In the current test set up no degradation product(s) of Terpinyl acetate multi were found, while in denaturated plasma Terpinyl acetate multi (alpha and gamma) were recovered. Therefore the absence of Terpineol and other products are considered due to the limitation of the test method used. Further work will be ongoing.

Any other information on results incl. tables

The results from Terpinyl Acetate multi (gamma and alpha) are presented in the following table

Plasma 50 µg/mL
Name	Time (min)	Response TA gamma	Response TA alpha
Blank Plasma		35	54
QC-Plasma R(WH) t=240 min-1	240	14030	31692
QC-Plasma R(WH) t=240 min-2	240	25235	59035
QC-Plasma R(WH) t=120 min-1	120	43127	97085
QC-Plasma R(WH) t=120 min-2	120	41487	93463
QC-Plasma R(WH) t=60 min-1	60	89942	204366
QC-Plasma R(WH) t=60 min-2	60	90536	205301
QC-Plasma R(WH) t=30 min-1	30	131107	287963
QC-Plasma R(WH) t=30 min-2	30	105472	228612
QC-Plasma R(WH) t=15 min-1	15	118695	259819
QC-Plasma R(WH) t=15 min-2	15	100245	217221
QC-Plasma R(WH) t=9 min-1	9	121915	269120
QC-Plasma R(WH) t=9 min-2	9	124234	275049
QC-Plasma R(WH) t=6 min-1	6	141884	316293
QC-Plasma R(WH) t=6 min-2	6	166332	371893
QC-Plasma R(WH) t=3 min-1	3	141875	318123
QC-Plasma R(WH) t=3 min-2	3	116621	259510
QC-Plasma R(WH) t=1 min-1	1	152831	342982
QC-Plasma R(WH) t=1 min-2	1	178652	402596
QC-Plasma R(WH) t=0 min-1	0	171461	386289
QC-Plasma R(WH) t=0 min-2	0	185729	419585

Table2Peak area instrument response of Terpinyl acetate alpha and gamma isomers of samples aliquoted at different time points from spiked blood.

Blood 50 µg/mL
Name	Time (min)	Response TA gamma	Response TA alpha
Blank Plasma		22	179
QC-Blood R(WH) t=240 min-1	240	43684	94716
QC-Blood R(WH) t=240 min-2	240	30814	64668
QC-Blood R(WH) t=120 min-1	120	70343	151922
QC-Blood R(WH) t=120 min-2	120	61753	132424
QC-Blood R(WH) t=60 min-1	60	75564	162611
QC-Blood R(WH) t=60 min-2	60	72902	155776
QC-Blood R(WH) t=30 min-1	30	89811	195186
QC-Blood R(WH) t=30 min-2	30	96779	210157
QC-Blood R(WH) t=15 min-1	15	111385	238551
QC-Blood R(WH) t=15 min-2	15	90042	190433
QC-Blood R(WH) t=9 min-1	9	112311	244318
QC-Blood R(WH) t=9 min-2	9	104542	225536
QC-Blood R(WH) t=6 min-1	6	122965	270586
QC-Blood R(WH) t=6 min-2	6	128977	285491
QC-Blood R(WH) t=3 min-1	3	105291	232263
QC-Blood R(WH) t=3 min-2	3	99631	219965
QC-Blood R(WH) t=1 min-1	1	108633	241289
QC-Blood R(WH) t=1 min-2	1	121681	271784
QC-Blood R(WH) t=0 min-1	0	129673	289621
QC-Blood R(WH) t=0 min-2	0	119319	266485

The modelled kinetic parameters are presented below. The figure showing the decrease in time in plasma is in the illustration section. The aqueous results from pH2, 4 and 7 are presented in the study report in table 4,5 and 6 and figures 6.7 and 8.

y = A1*exp(-x/t1) + y0	y0	A1	t1	k	t½ in minutes	R-Square
TA at pH 2	34270	377072	12.6	7.92E-02	8.75	0.983
TA at pH 4	30744	433347	10.8	9.25E-02	7.49	0.969
TA at pH 7	32834	320195	14.8	6.75E-02	10.3	0.967
TA in Plasma*	38592	309706	73.1	1.37E-02	50.7	0.866

*Terpinyl Acetate multi in blood were very similar as in plasma as can be seen from the table above but these data were not modelled yet.

Applicant's summary and conclusion

Conclusions:

Terpinyl acetate alpha and gamma are decreased by half within 60 minutes in plasma at a concentration of 50 ug/ml (mg/l). In aqueous solutions the substance has a half-life of ca 10 minutes. This means that Terpinyl Acetate multi will not be in the systemic circulation.

Executive summary:

The stability of Terpinyl Acetate multi was evaluated in rat plasma and blood in two separate experiments. Both these experiments were performed in duplicate. In both experiments rat k2 -EDTA matrix was spiked with analyte solution to achieve an initial concentration at 50 ug.ml. The spiked matrix was kept at 37oC. The spiked matrix samples were aliquoted at different time points: 0, 1,3, 6, 9, 15, 30, 60, 120 and 240 minutes. In addition, the stability of Terpinyl Acetate multi was evaluated in aqueous solutions of different pH: 2,4 and 7. Results: Two peaks were seen in the chromatogram: Terpinyl Acetate alpha and gamma presumably being the two key constituents. The substance decreased steadily. DT50 in plasma was calculated to be ca 50 minutes for both constituents. The DT50 regression line for plasma is modelled from the equation: y=A1 (response value)*(exp(-x/t1)+y: A=309706 (response value)*exp(-x/t1=73.1)+y=0 = 38592, k=0.0137 and r2 0.866. In the control where proteins including esterases were denaturated no decrease of Terpinyl Acetate alpha or gamma was seen. The degradation product(s) could not yet be captured in the chromatograms and was considered to be due to the limitation of the method. Further work is ongoing to detect the degradation products. In aqueous solutions the decrease of Terpinyl Acetate multi was <=10 minutes. Conclusion: Terpinyl Acetate is not stable in plasma and aqueous solutions and therefore no Terpinyl Acetate multi will be in the systemic circulation. Due to the absence of any metabolite/degradation product further work is needed to know what these are. Further work is currently initiated.