Butyl glycollate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Remarks:

in vitro enzymatic hydrolysys to prove the read across assessment justification

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11-12-2018 to 12-12-2019

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:

To prove the read across approach on the basis of common breakdown products from the target substance and source substances and similar biological effects (NTP 2004, see assessment in Section 13.2)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Principles of Good Laboratory Practice

Version / remarks:

The method development part to determine the optimal experimental conditions for the
analysis of the test item and its possible metabolites was not performed under GLP. Final
experimental details were approved in the raw data and reported. This did not affect the
integrity of the study.

Deviations:

no

Remarks:

The report represents an accurate and complete record of the results obtained.

Principles of method if other than guideline:

The study consisted of three parts: a feasibility study, validation of the analytical methods developed for the test item and the reference items, and a metabolism study. The feasibility study was performed as a non-GLP part of the study during which the experimental details for the metabolism study were determined.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

Identification: Butyl glycollate, Polysolvan O ®
Batch (Lot) Number: 120001144747
Purity/Composition: 98.5%
CAS Number: 7397-62-8
EC Number: 230-991-7
Molecular weight: 132.16 g/mol

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Rat Liver S9 Homogenate, Rat Small Intestinal Mucosa Homogenate and Rat Caecum Content

Sex:

male/female

Details on test animals or test system and environmental conditions:

Not applicable, in vitro conditions

Administration / exposure

Route of administration:

other: in vitro

Vehicle:

water

Remarks:

Milli-Q water (MQ) Tap water purified by reversed osmosis and subsequently passed over activated carbon and ion exchange cartridges; Millipore, Bedford, MA, USA

Details on exposure:

not applicable

Duration and frequency of treatment / exposure:

see below details on study design

Doses / concentrations

Dose / conc.:

50 other: μM

Remarks:

incubations were performed at 37±1 °C in duplicate at a test item concentration of 50 μM using incubation times of 1±1, 30±1 (liver S9 only), 60±1 (small intestinal mucosa only), 120±1 and 180±1 minutes.

No. of animals per sex per dose / concentration:

Not applicable - in vitro

Positive control reference chemical:

Not applicable

Details on study design:

The study consisted of three parts: a feasibility study, validation of the analytical methods developed for the test item and the reference items and a metabolism study.
For validation of the analytical methods, the linearity of the analytical method developed for the test item and the stability of the test item under incubation conditions was determined. In addition, the analytical method and sample pre-treatment procedures developed for each of the reference items was validated.
In the metabolism study, incubations were performed with rat liver S9 homogenate, rat small intestinal mucosa homogenate and rat caecum content to determine the metabolic biotransformation of the test item in mixed gender Wistar rat. Additional incubations were performed with rat liver S9 homogenate (1, 30, 120 and 180 minute time points) and with rat small intestinal mucosa (1, 60, 120 and 180 minute time points) to determine if formation of the reference items occurred in the different matrices.
To determine the metabolic biotransformation time of the test item, incubations were performed at 37±1 °C in triplicate for all three matrices (rat liver S9 homogenate, rat small intestinal mucosa homogenate and rat caecum content) at a test item concentration of 50 μM using incubation times of 1±1, 5±1, 15±1, 30±1, 60±1 and120±1 minutes.
To determine the metabolite profile of the test item in rat liver S9 and small intestinal mucosa, incubations were performed at 37±1 °C in duplicate at a test item concentration of 50 μM and single incubation with vehicle (MQ) using incubation times of 1±1, 30±1 (liver S9 only), 60±1 (small intestinal mucosa only), 120±1 and 180±1 minutes.

Details on dosing and sampling:

See below in details of study design

Statistics:

not specified

Results and discussion

Preliminary studies:

During the feasibility study, analytical methods based on liquid chromatography (LC) and gas chromatography (GC) in combination with different sample pre-treatment procedures were evaluated for the test item, the reference items glycolic acid, glyoxylic acid, oxalic acid mono butyl ester, n-butyric acid, and the analyte n-butanol. It was concluded that a UPLC-MS method was suitable to detect the test item in incubation mixtures.

Main ADME results

Main ADME results 1

Type:

other: biotransformation rate

Results:

Test item was stable for at least 120 minutes under the incubation conditions used for caecum content incubations and for at least 180 minutes under the incubation conditions used for liver S9 and small intestinal mucosa incubations.

Main ADME results 2

Type:

other: metabolism profile

Results:

Test item was converted on average 97% in rat liver S9 and 22% in rat small intestinal mucosa > 120±1 min of incubation and highly stable in rat caecum content incubations. In vitro t1/2 values: 24 min liver and >120 intestinal mucosa and caecum content.

Main ADME results 3

Type:

other: metabolism profile

Results:

Test item was metabolized into the major metabolite glycolic acid in both liver S9 and small intestinal mucosa homogenate. In liver S9 incubations, minor amounts of the reference items glyoxylic acid and n-butyric acid were also detected.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Not applicable/in vitro

Details on distribution in tissues:

Not applicble/in vitro

Transfer into organs

Transfer into organs 1

Key result

Test no.:

#2

Transfer type:

other: metabilic biotransformation rate

Remarks:

Rapid biotransformed in in rat liver S9 on average 97% and 22% in rat small intestinal mucosa after 120±1 minutes of incubation.

Transfer into organs 2

Key result

Test no.:

#3

Transfer type:

other: test item is rapid converted into main metabolites glycolic acid and glyoxylic acid and butyric acid (generated from butanol)

Details on excretion:

not applicable/in vitro

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

glycolic acid, glyoxylic acid and oxalic acid mono butyl ester, butyric acid (generated from butanol).

Any other information on results incl. tables

Table 1. Converted Average Percentage of the Test Item after Various Incubation Periods in 3 Different Matrices

Test Item concentration	Matrix	Concentration Matrix Converted Percentage of Test Item
		1±1 min*	5±1 min	15±1 min	30±1 min	60±1 min	120±1min
50μM	Liver S9	0	11	31	55	82	97
	Mucosa	0	-5	-4	2	10	22
	Caecum	0	0	-1	2	2	2

*= Set at 0%.

Table 2. The Calculated Averaged in vitro t1/2 Values of the Test Item for 3 Different Matrices

Matrix	Averaged t1/2 (min)
Liver S9	24
Intestinal Mucosa	>120
Caecum Content	>120

Table 3. The Estimated Concentrations of the Test Item and Reference Items in Rat Liver S9 Incubation Samples

Sample	Concentration (μM)
	Repl.	Butyl glycolate	Glycolic acid	Glyoxylic acid 7.2 min	Glyoxylic acid 8.4min	Oxalic acid mono butyl ester	n-Butyric acid
Muc T=1	A	48.04	0.02	0.34	0.25	0.01	0.35
Muc T=1	B	50.25	0.14	-0.26	-0.11	0.00	0.16
Muc T=60	A	42.27	10.77	0.04	-0.06	0.01	0.31
Muc T= 60	B	39.69	9.85	-0.30	-0.04	0.01	0.32
Muc T=120	A	40.81	14.58	-0.49	-0.08	0.02	0.19
Muc T=120	B	42.52	14.80	-0.05	0.07	0.01	0.44
Muc T=180	A	39.32	21.36	-0.71	0.18	0.02	0.70
Muc T= 180	B	43.12	15.97	0.07	-0.07	0.03	0.25

* Glyoxylic acid displayed two peaks after derivatization, results for both peaks have been presented.

Repl.: Replicate.

Table 4. The Estimated Percentages of the Test Item and Selected Reference Items in Rat Liver S9 Incubation Samples

Sample	Percentage of Analyte Present
	Butyl glycolate	Glycolic acid	Glyoxylic acid*	Oxalic acid mono
S9 T=1	98.0	1.4	0.6	0
S9 T=30	35.5	62.1	2.4	0
S9 T=120	3.3	93.0	3.7	0
S9 T =180	0.8	97.6	1.5	0

* For glyoxylic acid, the averaged concentrations of the peaks at 7.2 and 8.4 minutes were used.

Table 5. The Estimated Percentages of the Test Item and Selected Reference Items in Rat Small Intestinal Mucosa

Homogenate Incubation Samples

Sample	Percentage of Analyte Present
	Butyl glycolate	Glycolic acid	Glyoxylic acid*	Oxalic acid mono
S9 T=1	99.7	0.2	0.1	0
S9 T=30	79.8	20.3	0	0
S9 T=120	74.1	26.1	0	0
S9 T =180	68.6	31.0	0.4	0

* For glyoxylic acid, the averaged concentrations of the peaks at 7.2 and 8.4 minutes were used.

Applicant's summary and conclusion

Conclusions:

In conclusion, In liver S9 homogenate incubations, the butyl glycolate was completely metabolized after 180 minutes of incubation whereas in small intestinal mucosa homogenate incubations the conversion was approximately 20% after 180 minutes of incubation. Butyl glycollate was metabolized in the incubation with rat liver S9 and small intestinal mucosa whereas the test item showed high metabolic stability in rat caecum content incubations. The extent of conversion of the butyl glycolate was on average 97% in rat liver S9 and 22% in rat small intestinal mucosa after 120±1 minutes of incubation. The calculated averaged in vitro t1/2 values for butyl glycolate acid were on average 24 minutes in liver S9 and >120 minutes in small intestinal mucosa and caecum content incubations. In total, three of the proposed metabolites were detected in liver S9 incubations (the major metabolite glycolic acid and the minor metabolites glyoxylic acid and n-butyric acid) whereas in small intestinal mucosa incubations only one reference item (glycolic acid) was detected. Based on these findings and the proposed metabolic pathway presented in Figure 1, it is most likely that the test item is first metabolized enzymatically by esterases into glycolic acid and n-butanol. Glycolic acid can be further metabolized by glycolate oxidase into glyoxylic acid, however, this is only a minor pathway as mainly the primary metabolite glycolic acid was detected in combination with only small amounts of the secondary metabolite glyoxylic acid. It was shown that n-butanol can be further metabolized by alcohol dehydrogenase into nbutanal which can subsequently be further metabolized by aldehyde oxidase into n-butyric acid.

Based on the metabolic profile, we can conclude that that butyl glycolate is not metabolized via alcohol dehydrogenase/aldehyde oxidase pathway.

Executive summary:

An in vitro enzymatic hydrolysis with rat liver S9 homogenate, rat small intestinal mucosa homogenate and rat caecum content were performed to determine the biotransformation of butyl glycolate in mixed gender Wistar rat. Additional incubations were performed with rat liver S9 homogenate (1, 30, 120 and 180 minute time points) and with rat small intestinal mucosa (1, 60, 120 and 180 minute time points) to determine if formation of the predicted metabolites occurred in the different matrices. Several metabolites were identified glycollic acid, glyoxylic acid, andn-butyric acid (as the metabolite of n-butanol). In liver S9 homogenate incubation, butyl glycolate were biotransformed on average 97% and 22% in rat small intestinal mucosa after 120±1 minutes of incubation. The calculated averaged in vitro t1/2 values for the test item were on average 24 minutes in liver S9 and >120 minutes in small intestinal mucosa and caecum content incubations.

 

Based on the results, butyl glycolate is rapidly metabolized into by esterases into glycolic acid and n-butanol. Glycolic acid can be further metabolized by glycolate oxidase into glyoxylic acid, however, the primary metabolite is glycolic acid in combination with only small amounts of the secondary metabolite glyoxylic acid. Also, n-butanol can be further metabolized by alcohol dehydrogenase into n-butanal

which can subsequently be further metabolized by aldehyde oxidase into n-butyric acid.