2,2-bis(hydroxymethyl)butanoic acid

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2021 - January 2022

Reliability:

1 (reliable without restriction)

Objective of study:

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Remarks:

obtained from Charles River

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age on delivery: 8-12 weeks
- Weight at delivery: 255 g – 370 g
- Total number of animals: 48 (24 males + 24 females)
- Animal identification: Tail tattoo
- Housing: The animals were housed in Macrolon Tecniplast cages (2-4 animals of the same sex/cage) at a temperature of 19-25 °C, and the relative air humidity of 30 70 %, light: 12 hours per day
- Diet: ad libitum
- Water: e.g. ad libitum
- Acclimation period: 6-7 days
- Health status: Only animals in good health conditions were used for the study.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua pro injectione Ardeapharma

Duration and frequency of treatment / exposure:

28 consecutive days.

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose / concentration:

Number of groups: 2 treated groups
Number of animals per group: 24 (12 males + 12 females)

Control animals:

no

Details on study design:

ANIMAL SELECTION & GROUP ASSIGNMENT
All rats were randomized according to the body weight into groups. The body weights required for randomization were done at the delivery.
JUSTIFICATION OF DOSE LEVEL AND ROUTE
Oral administration as an intended route in animal is required by the corresponding international guideline. Doses were selected based on the data from previous carried studies provided by Sponsor.

Details on dosing and sampling:

DOSING PROCEDURE
Each animal from the dose groups D1 and D2 was administered by the determined dose of the Test Items as solution by oral gavage in volume of 5 mL/kg b.w. every day for 28 consecutive days. Individual doses were adjusted according to body weight
Daily Observations: All rats were observed for clinical signs, morbidity or mortality once a day during acclimatization and twice a day during the administration period
Clinical observations included: Signs of toxicity, changes in the skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling, the presence of clonic or tonic movements and stereotypes.
BODY WEIGHT:
All rats were individually weighed at delivery, before first administration on Day 1 and then weekly.
BLOOD SAMPLING FOR TOXICOKINETICS
Animals in each group were divided into 3 subgroups (A, B and C) of 4 males and 4 females. Blood samples were drawn under slight ether anesthesia from the retro-orbital venous plexus from each group for toxicokinetic analysis on Day 1 and Day 28 of administration period in specified time-points: Day 1 and Day 28: 0 (pre-dose), 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after the administration.

Statistics:

Statistical software GraphPad Prism (version 8.1.2., last internal validation August 2021) was used for statistical evaluation in this study.
Data for males and females were separately analyzed to determine whether analyzed parameters of group D1 are significantly different from group D2 at the 95.0 % confidence level.
Analyzed data were tested for normality (Kolmogorov-Smirnov test) and homogeneity of variance (Bartlett´s test or F test).
Data for males and females were analyzed using t-test. The t-test whether there are any significant differences amongst the means at the 95.0 % confidence level. With small sample sizes (less than 10), the normality of the data becomes increasingly uncertain and nonparametric methods such as Mann-Whitney test (which compares medians instead of means) are more appropriate, so we used it too.
Pharmacokinetic Data Evaluation: Phoenix WinNonlin™ software version 8.3 was used for evaluations.

Group	Day	Gender	AUC(0-t) (h*ug/mL)	Cmax (ug/mL)	Tmax (h)
D1	1	female	40.86	18.09	0.50
		male	30.22	11.64	0.50
D1	28	female	31.92	19.40	0.50
		male	35.43	20.39	0.50
D2	1	female	210.78	79.81	0.50
		male	171.59	45.38	1.00
D2	28	female	210.91	67.31	0.50
		male	211.32	54.59	0.50

Conclusions:

DMBA exposure, in term of its intensity, increases less than dose-proportionally in both male and female animals; the increase of exposure for both genders is more than dose-proportional.
Dose normalized TK parameters estimated in this study could suggest non linear toxicokinetics of DMBA at higher plasma levels.
Study population was too small, resulting in only eight complete concentration-time profiles, and precludes a definitive conclusion regarding linearity.

Executive summary:

The purpose of this study was to evaluate toxicokinetic profile of 2,2-bis(hydroxymethyl)butanoic acid as the Test Item after 28-day repeated oral administration in rats.

The study was performed in GLP condition according to OECD 417 and was carried out on 48 Wistar rats (24 males + 24 females) obtained from Charles River, Germany divided into two experimental groups D1 and D2, each contained 12 males and 12 females.

Each animal from the group D1 received oral administration of Test Item prepared as a solution of the dose 100 mg/kg and animals from the group D2 received the dose 500 mg/kg. Test Item solution was administered by oral gavage in volume of 5 mL/kg b.w. every day for 28 consecutive days. Individual doses were adjusted according to body weight.

Clinical signs and mortality were recorded in all the animals once a day during the acclimatization and twice a day during the study period for 28 consecutive days. Body weight was recorded at delivery, before the first administration on Day 1 and then weekly.

Blood samples for toxicokinetics were taken from all scheduled rats on Day 1 and Day 28 at following time points: 0 (pre-dose), 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after the administration. Plasma samples (aliquots A) were transported to the analyses on dry ice together to the Test Site (Quinta-Analytica).

Plasma samples obtained during in vivo phase were analyzed for content of 2,2‑bis(hydroxymethyl)butanoic acid (DMBA) using HPLC/MS/MS method over concentration range 1.00 – 1000.00 µg/mL for DMBA. K₃EDTA was used as an anticoagulant.

The concentration-time profiles evince similar shape for both dose levels with single main peak. Plasma levels are fluctuating, and elimination followed multiexponential decline.

Toxicokinetic parameters AUC_(0-t), C_max, and t_max were used to assess toxicokinetics after peroral administration.

The maximum plasma concentrations (t_max) of DMBA were reached 0.50 hrs (the first post-dose sample) for both genders after administration of 100 mg and 500 mg.

The intensity of exposure achieved 11.64 – 18.09 and 45.38 – 79.81 µg/mL after administration of 100 and 500 mg dose, respectively, considering both genders on Day 1; and 19.40 – 20.39 and 54.59 – 67.31 µg/mL after administration of 100 and 500 mg dose, respectively, considering both male and female animals on Day 28.

The extent of exposure accounted for 30.22 – 40.86 and 171.59 – 210.78 h∙µg/mL after administration of 100 and 500 mg dose, respectively, considering both genders on Day 1; and 31.92 – 35.43 and 210.91 – 211.32 h∙µg/mL after administration of 100 and 500 mg dose, respectively, considering both male and female animals on Day 28.

DMBA exposure, in term of its intensity, increases less than dose-proportionally in both male and female animals; the increase of exposure for both genders is more than dose-proportional.

Dose normalized TK parameters estimated in this study could suggest non‑linear toxicokinetics of DMBA at higher plasma levels.

Study population was too small, resulting in only eight complete concentration-time profiles, and precludes a definitive conclusion regarding linearity.

Based on the results obtained during biological part of the study, no treatment-related clinical signs were observed in all animals after 28 consecutive days oral administration of Test Item - 2,2-bis(hydroxymethyl)butanoic acid at a dose 100 mg/kg (group D1) and dose 500 mg/kg (group D2).

All of these animals were in good health condition during the whole study period for 28 consecutive days. The body weight of animals from groups D1 and D2 was without major adverse fluctuations and gradually increased during the study period.

 

 

Description of key information

The purpose of this study was to evaluate toxicokinetic profile of 2,2-bis(hydroxymethyl)butanoic acid as the Test Item after 28-day repeated oral administration in rats.

The study was performed in GLP condition according to OECD 417 and was carried out on 48 Wistar rats (24 males + 24 females) obtained from Charles River, Germany divided into two experimental groups D1 and D2, each contained 12 males and 12 females.

Each animal from the group D1 received oral administration of Test Item prepared as a solution of the dose 100 mg/kg and animals from the group D2 received the dose 500 mg/kg. Test Item solution was administered by oral gavage in volume of 5 mL/kg b.w. every day for 28 consecutive days. Individual doses were adjusted according to body weight.

Clinical signs and mortality were recorded in all the animals once a day during the acclimatization and twice a day during the study period for 28 consecutive days. Body weight was recorded at delivery, before the first administration on Day 1 and then weekly.

Blood samples for toxicokinetics were taken from all scheduled rats on Day 1 and Day 28 at following time points: 0 (pre-dose), 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after the administration. Plasma samples (aliquots A) were transported to the analyses on dry ice together to the Test Site (Quinta-Analytica).

Plasma samples obtained during in vivo phase were analyzed for content of 2,2‑bis(hydroxymethyl)butanoic acid (DMBA) using HPLC/MS/MS method over concentration range 1.00 – 1000.00 µg/mL for DMBA. K₃EDTA was used as an anticoagulant.

The concentration-time profiles evince similar shape for both dose levels with single main peak. Plasma levels are fluctuating, and elimination followed multiexponential decline.

Toxicokinetic parameters AUC_(0-t), C_max, and t_max were used to assess toxicokinetics after peroral administration.

The maximum plasma concentrations (t_max) of DMBA were reached 0.50 hrs (the first post-dose sample) for both genders after administration of 100 mg and 500 mg.

The intensity of exposure achieved 11.64 – 18.09 and 45.38 – 79.81 µg/mL after administration of 100 and 500 mg dose, respectively, considering both genders on Day 1; and 19.40 – 20.39 and 54.59 – 67.31 µg/mL after administration of 100 and 500 mg dose, respectively, considering both male and female animals on Day 28.

The extent of exposure accounted for 30.22 – 40.86 and 171.59 – 210.78 h∙µg/mL after administration of 100 and 500 mg dose, respectively, considering both genders on Day 1; and 31.92 – 35.43 and 210.91 – 211.32 h∙µg/mL after administration of 100 and 500 mg dose, respectively, considering both male and female animals on Day 28.

DMBA exposure, in term of its intensity, increases less than dose-proportionally in both male and female animals; the increase of exposure for both genders is more than dose-proportional.

Dose normalized TK parameters estimated in this study could suggest non‑linear toxicokinetics of DMBA at higher plasma levels.

Study population was too small, resulting in only eight complete concentration-time profiles, and precludes a definitive conclusion regarding linearity.

Based on the results obtained during biological part of the study, no treatment-related clinical signs were observed in all animals after 28 consecutive days oral administration of Test Item - 2,2-bis(hydroxymethyl)butanoic acid at a dose 100 mg/kg (group D1) and dose 500 mg/kg (group D2).

All of these animals were in good health condition during the whole study period for 28 consecutive days. The body weight of animals from groups D1 and D2 was without major adverse fluctuations and gradually increased during the study period.