Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 2-ethylhexyl ester

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Objective of study:

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Principles of method if other than guideline:

Data on the urinary deconjugation experiment with beta-glucuronidase/sulfatase at the high dose level were missing. This deviation did not affect the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Remarks:

14C- labeled phenolic ring

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

Strain as stated in the report: CD-l (albino) [Crl:CD1(ICR)]
Sex: Male
Bodyweight at dosing: 25 - 40 g
Age at dosing: 6-8 weeks
Supplier: Charles River UK Ltd., Margate, Kent, UK
Acclimatization: at least 5 days before the intended date of dosing.
Housing: During acclimatization animals were housed in groups of up to four mice in solid bottomed plastic cages.
After dosing, the animals were housed in sub-groups of 3 in glass metabolism cages (Metabowls®).
Diet ad libitum: VRFl diet manufactured by Special Diet Services
Water ad libitum: tap water

ENVIRONMENTAL CONDITIONS
Room temperature: 21 +/- 2°C.
Relative humidity: 55 - 15%.
Light/dark cycle: alternating 12 hour light/dark.
Air changes: approximately 15 per hour.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% carboxymethylcellulose in 0.1% Tween 80

Details on exposure:

14C-labelled test substance was stored in acetonitrile at a concentration of 495.91 µCi/ml (1.58 mg/ml) at or below -15°C. Dose formulations were freshly prepared on the day of administration. To confirm homogeneity and concentration of radioactivity in the dose formulations (Groups 1, 2 and 4) aliquots were taken during the dosing procedure and were diluted with acetonitrile. Subsequently aliquots were taken in triplicate for radioassay. Purity checks of the dose formulations were carried out at each dosing time and all were >95%.

Duration and frequency of treatment / exposure:

single gavage.

Dose / conc.:

0.5 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

No. of animals per sex per dose / concentration:

4 subgroups of 3 males (=12 males) received 50 mg/kg bw (designated as Group2)
8 subgroups of 3 males (=24 males) splitted into two treated groups received 0.5 mg/kg bw (designated as Group 1 and 4, respectively)

Control animals:

no

Details on study design:

Rationale for the selection of the administration route and dose levels
The oral route was chosen as these ia an expected route for human exposure to the test substance. Dose levels were selected upon consultation of the Regulatory Authority (BfR) at a level where no observable effects were expected (0.5 and 50 mg/kg bw in 10 mL/kg bw vehicle, respectively).

Dose preparation and administration
14C-labelled test substance was stored in acetonitrile at a concentration of 495.91 µCi/mL (1.58 mg/mL at or below -15°C. Dose formulations were freshly prepared on the day of administration and homogeneity and concentration of radioactivity in the dose formulations were confirmed. The dose solutions were administered orally using a graduated syringe with a rubber gavage tube. The exact amount of formulation administered was determined gravimetrically by weighing the dose syringe when loaded and again after dosing.
Administration of oral doses at 0.5 mg/kg bw (Groups 1 and 4):
A nominal portion of 141.33 - 141.48 µCi of the 14C- labeled test substance was dispensed into a scintillation vial. The solvent was evaporated under nitrogen gas at 37°C and re-suspended in vehicle (9 mL) by use of ultrasonication. Once suspended the formulation was stirred continuously.
Administration of oral dose at 50 mg/kg bw (Group 2):
Non-radiolabelled test substance (43.1 mg), was dispensed into a scintillation vial and 600 µCi 14C-labelled test substance stock solution (nominal) was added. The solution was stirred thoroughly and the solvent was evaporated under nitrogen gas at 37°C and re-suspended in vehicle by ultrasonication. Once suspended the formulation was stirred continuously. The radiochemical purity of the formulation was determined after dose administration.

Measurement of radioactivity
Radioactivity was measured by liquid scintillation counting (LSC) with automatic quench correction. Radioactivity below twice the background level was considered to be below the limit of detection.
Aliquots of liquid samples were mixed with Ultima Gold scintillator (Packard BioScience) or Monoflow 4 for measurement of radioactivity.
Solid samples were combusted in oxygen using a sample oxidizer. The combustion products were absorbed into CO2 sorbent and mixed with Permafluor E+ scintillator. The efficiency of the oxidizer was found to be above 95%. Measurements of radioactivity were corrected for oxidizer efficiency.

Details on dosing and sampling:

PHARMACOKINETIC STUDY
Excretion and tissue distribution after single oral doses (0.5 and 50 mg/kg bw; Group 1 and 2, respectively):
Following administration of radiolabeled test substance to four subgroups of three male mice, each subgroup was transferred into a glass metabolism cage.
Urine was collected into cooled receivers at time intervals of 0 - 6, 6 - 24 h, and subsequently at intervals of 24 h up to sacrifice after 168 h. Prior to dosing, pre-dose urine samples were collected for use as backgrounds.
Feces were collected separately into cooled receivers at 24 hr intervals up to 168 h. Cage washes were performed every 24 h throughout the sample collection period and retained for radioactivity measurement.
Blood samples were taken under isoflurane by cardiac puncture immediately prior to sacrifice at 168 h into heparinised tubes. Radioactivity of whole blood and plasma were measured.
At sacrifice the following tissues were removed for analysis of radioactivity:
Gastrointestinal tract with contents, Heart, Kidneys, Liver, Lungs, Muscles and Testes
Tissues were pooled for each subgroup of three mice and all samples were stored at or below -15°C prior to analysis. The residual carcass was retained. Radioactivity of tissues, carcass and feces was assessed after homogenization of the respective fraction.
Measurement of volatile radioactivity (0.5 mg/kg bw; Group 4):
Each subgroup of three mice were transferred to glass metabolism cages and 14CO2 of the expired air was trapped in a mixture of ethanolamine: 2-ethoxyethanol (1 : 3, v/v) during 24 hour intervals for up to 48 h. At 48 h the mice were sacrificed by cervical dislocation.

METABOLITE CHARACTERISATION STUDIES
Urine samples from the intervals 0 - 6 h, 6 - 24 h and 24 - 48 h from mice of group 1 and 2 (0.5 and 50 mg/kg bw, respectively) were pooled and duplicate aliquots were taken for radioassay. Urine pool concentrates were analyzed by reverse phase HPLC and normal phase TLC.
Fecal extracts from the 0 - 24 h interval were pooled (10% by weight), concentrated under nitrogen gas and duplicate aliquots were taken for radioassay. Fecal extracts were further analyzed by reverse phase HPLC and normal phase TLC.

PREPARATION OF URINE FOR MASS SPECTROMETRY ANALYSIS
To identify metabolites detected via HPLC/TLC a fresh 0 - 48 h urine pool was prepared from animals treated with 50 mg/kg bw (Group 2). Sub-samples of approximately 2 mL were freeze-dried and re-suspended in methanol. After centrifugation and further washing steps, the samples were analyzed by RP-HPLC. The major urinary metabolites were identified by mass spectrometry (ESI) coupled to a UPLC-UV-system at the Department of Mass Spectrometry for structural investigative work. Particular ions were selected for MS/MS analysis and the corresponding spectra were compared with that from the authentic test substance and the expected metabolite (Metilox acid) to confirm the structure of metabolites.

TREATMENT FOR CLEAVAGE OF CONJUGATES
Urine from sampling interval 0 - 48 h collected from groups 1 and 2, respectively was pooled and duplicate aliquots were taken for radioassay.
For the deconjugation experiments, portions of these pools were incubated with buffer and beta-glucuronidase/sulphatase at 37°C for 18h. Phenolphthalein glucuronic acid was added into a control sample to confirm beta-glucuronidase activity. Samples were analyzed by RP-HPLC and normal phase TLC.
Aliquots of the urine pools were further subjected to mild acid/base hydrolysis. Samples were acidified/basified and incubated at 37°C for 18 h. Upon neutralisation samples were analyzed by reverse phase HPLC and normal phase TLC.

Preliminary studies:

No preliminary study was conducted.

Type:

distribution

Results:

Upon sacrifice after 7 days, less than 0.3% radioactivity were detected in tissues at 0.5 and 50 mg/kg bw, respectively.

Type:

metabolism

Results:

A hydroxylated and a glucuronide conjugate of the hydroxylated metabolite, as well as the metabolite (14 - 18% and 0.4 - 1.1 % of dose, respectively) were detected at 0.5 and 50 mg/kg bw.

Type:

excretion

Results:

85% to 93% of applied dose were excreted within 48 hours.

Details on distribution in tissues:

Following administration of 0.5 mg/kg bw radio-labeled test substance radioactivity in carcass, muscle, testes and plasma was below 0.001 µg equivalents/g wet weight. Radioactivity in the other tissues ranged from 0.01 (heart, kidneys, lungs) to a maximum of 0.02 % of administered dose in whole blood.
Following administration of 50 mg/kg bw radio-labeled test substance the tissue radioactivity was highest in whole blood and liver (0.293 and 0.128 µg equivalents/g wet weight, respectively). Radioactivity detected in kidney, lungs, heart and gastro intestine tract (including content) ranged from (0.083 to0.028 µg equivalents/g). All other tissue concentrations (carcass, muscle, plasma and testes) accounted for approximately 0.015 µg equivalents/g wet weight. The radioactivity detected in organs corresponded to 0.01 to 0.02% of the ingested dose.
Following administration of 0.5 mg/kg bw radio-labeled test substance to mice the mean excretion of radioactivity in the expired air during the 0 – 48 h intervall accounted for a total of 0.07% of dose.

Details on excretion:

The overall mean recovery following administration of 0.5 mg/kg bw radio-labeled test substance to mice accounted for 99.48% of the dose (Group 1). More than 85% of the given dose was excreted within 48 hours (44% via urine and 43% via feces). Urinary and fecal excretion during the 0 - 168 h interval accounted for 47.7 % and 43.8% of the dose and 7.94% of the dose were recovered in the cage-washes at the same time interval.
Radioactivity in tissues ranged from 0.01 to a maximum of 0.02 % of administered dose.

The overall mean recovery following administration of 50 mg/kg bw radio-labeled test substance accounted for 96.92% of the dose (Group 4).Approximately 93% of dose was excreted within 48 hours (38% via urine and 55%via feces). Urinary and fecal excretion during the 0 - 168 h interval accounted for 38.8% and 56.1% of the dose and 1.94% of the dose were recovered in the cage-washes at the same time interval
Radioactivity in tissues ranged from 0.01 to a maximum of 0.02 % of administered dose.

Following administration of 0.5 mg/kg bw radio-labeled test substance to mice the mean excretion of radioactivity in the expired air during the 0 – 48 h intervall accounted for a total of 0.07% of dose

Metabolites identified:

yes

Details on metabolites:

A total of 3 metabolites in the freshly prepared urine pools of animals treated with 50 mg/kg bw were identified via molecular weight and fragmentation pattern using a mass spectrometer operating in the negative electrospray mode (ESI LC/MS). Metabolites in the other urine and fecal fractions were correlated to these identified metabolites via their retention times.

Details on metabolites:

A total of 3 metabolites in the freshly prepared urine pools of animals treated with 50 mg/kg bw were identified via molecular weight and fragmentation pattern using a mass spectrometer operating in the negative electrospray mode (ESI LC/MS). Metabolites in the other urine and fecal fractions were correlated to these identified metabolites via their retention times.

In the feces collected 0-24 h after treatment the unchanged test substance was the major component (31.6% of applied dose). The main metabolite accounted for 7.4% while all other metabolites accounted for less than 5.6 % of dose.

Table 1: Radioactivity in selected tissues 7 days after single oral dose of radio-labeled test substance in µg equivalents/g.

Tissue	Dose (mg/kg bw)
	0.5 (group 1)	50 (Group 2)
Carcass	0.000 +/- 0.000	0.005 +/- 0.005
GIT	0.001 +/- 0.000	0.028 +/- 0.012
Heart	0.001 +/- 0.000	0.071 +/- 0.014
Kidney	0.001 +/- 0.000	0.083 +/- 0.015
Liver	0.002 +/- 0.001	0.128 +/- 0.025
Lungs	0.001 +/- 0.001	0.081 +/- 0.027
Muscle	0.000 +/- 0.000	0.013 +/- 0.015
Testes	0.000 +/- 0.000	0.008 +/- 0.001
Whole blood	0.003 +/- 0.001	0.293 +/- 0.093
Plasma	0.000 +/- 0.000	0.015 +/- 0.004
GIT: Gastro intestinal tract (with contents)

Deviations from the protocol:

No data on the urinary deconjugation experiment with beta-glucuronidase/sulfatase at 50 mg/kg bw were given.

Conclusions:

No bioaccumulation potential based on study results.

Reference 2

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

Strain as stated in the report: CD-l (albino) [Crl:CD1(ICR)]
Sex: Male
Bodyweight at dosing: 25 - 40 g
Age at dosing: 6-8 weeks
Supplier: Charles River UK Ltd., Margate, Kent, UK
Acclimatization: at least 5 days before the intended date of dosing.
Housing: During acclimatization animals were housed in groups of up to four mice in solid bottomed plastic cages.
After dosing, the animals were housed in sub-groups of 3 in glass metabolism cages (Metabowls®).
Diet ad libitum: VRFl diet manufactured by Special Diet Services
Water ad libitum: tap water

ENVIRONMENTAL CONDITIONS
Room temperature: 21 +/- 2°C.
Relative humidity: 55 - 15%.
Light/dark cycle: alternating 12 hour light/dark.
Air changes: approximately 15 per hour.

Type of coverage:

occlusive

Vehicle:

polyethylene glycol

Duration of exposure:

Animals were treated for 6 h.

Doses:

0.5 mg/cm2, corresponding to 1mg/animal.

No. of animals per group:

12 males

Control animals:

no

Details on study design:

RATIONALE FOR THE SELECTION OF THE DOSE ROUTE AND LEVEL
The dermal route was chosen as this is a expected route for human exposure to the test substance. Dose levels were selected upon worst case calculation for dermal exposure of worker.

DOSE PREPARATION AND PRUITY CHECK
14C-labeled test substance was stored in acetonitrile at a concentration of 495.91 µCi/mL (1.58 mg/mL) at or below -15°C. Dose formulations were freshly prepared on the day of administration.
The solvent of 3.2 mg 14C-labeled test substance-Stock solution (1005 µCi) was evaporated and the residue was re-suspended in PEG. Homogeneity of the formulation was kept by stirring at 50°C. Aliquots of the formulation were taken throughout the dosing procedure and pipette tips used for application were washed to accurately determine the applied dose. Purity checks of the dosing formulation were carried out and were >95%.

DETAILS ON EXPOSURE
24 h prior dosing the dorsal skin was clipped free of hair and cleaned with solvent. The dose formulation (0.025 mL) was applied to 2 cm2 of the dorsal skin of lightly anesthetized animals. The treated area was covered by foil and animals were allowed to recover from the anesthesia. The treated area was washed with cotton wool swabs soaked in 1% Tween 80 in distilled water 6 h after treatment. The application site was re-covered with foil to avoid oral exposure and subgroups of three mice were transferred to glass metabolism cages.

EXAMINATIONS
Urine was collected at time intervals of 0 - 6, 6 - 24 h, and subsequently at intervals of 24 h up to sacrifice after 168 hours into cooled receivers. Prior to dosing, pre-dose urine samples were collected for use as backgrounds.
Feces were collected separately into cooled receivers at 24 hour intervals up to 168 hours. Cage washes were performed every 24 h throughout the sample collection period and retained for radioactivity measurement.
Upon sacrifice the gastrointestinal tract (plus contents) was removed. The radioactivity of the carcass, gastrointestinal tract and feces was assessed after homogenization of the respective fraction.
In addition, the treated skin area was dissected and tape stripped to remove the stratum corneum. The initial first two tape strips were collected separately and represented the non-absorbed dose. Subsequent tape strips (3-8) containing the stratum corneum were retained and analyzed in groups of three. After tape stripping, the residual skin membranes and the tape strips were seperately solubilised until digestion was complete (7 days). The weight of the digest was measured and duplicate weighed aliquots were taken for radioassay.
The first 3 swabs used to remove non absorbed test substance after 6 h were pooled for each group of 3 mice and were extracted with methanol by sonication followed by centrifugation. The supernatant was decanted, weighed and duplicate samples were taken for radioassay. The above procedure was repeated twice more.
The protective dressings and foils were pooled for each group of 3 mice and extracted and analyzed as described above.
All samples not analyzed immediately after collection were placed into a freezer at <-15°C until analyzed.

Measurement of radioactivity
Radioactivity was measured by liquid scintillation counting (LSC) with automatic quench correction. Radioactivity below twice the background level was considered to be below the limit of detection. Aliquots of liquid samples were mixed with Ultima Gold scintillator (Packard BioScience) or Monoflow 4 for measurement of radioactivity.
Solid samples were combusted in oxygen using a sample oxidizer. The combustion products were absorbed into CO2 sorbent and mixed with Permafluor E+ scintillator. The efficiency of the oxidizer was found to be above than 95%. Measurements of radioactivity were corrected for oxidizer efficiency.

Total recovery:

The overall mean recovery of radioactivity was 99.01% of the applied radioactivity. Mean tissue concentrations of radioactivity in carcass, gastrointestinal tract and treated skin corresponded to 2.26 %, 0.03 % and 0.07 % of applied dose.

Key result

Dose:

0.5 mg/cm2

Parameter:

percentage

Absorption:

4.42 %

Remarks on result:

other: 6 h

Remarks:

The absorbable fraction of test substance was calculated as the sum of excreta, tissues, treated skin after stripping and stratum corneum in percent of applied radioactivity.

NON-ABSORBED RADIOACTIVITY

Following the topical application of 0.5 mg/cm2 14C-labeled test substance in PEG to mice under occlusive conditions, approximately 29% of the radioactivity applied was detected in the swabs used to clean the treated skin after 6 h. Another 65.4% of the applied radioactivity was extracted from the protective dressing.

The dose recovered from the treated skin (0.07%) and in the first two tape strips (0.27%) accounted for 0.34% of applied radioactivity. The mean total amount of non-absorbed (sum of the dose removed during the washing procedure and the dose remaining on the skin surface) accounted for 94.32% of applied radioactivity.

ABSORBED RADIOACTIVITY

Radioactivity detected in the stratum corneum corresponded to 0.26 % of the applied radioactivity. Radioactivity in urine, feces and cage washes accounted for 0.90%, 0.71% and 0.20% of applied radioactivity, respectively.

Radioactivity recovered in the gastrointestinal tract (including contents) accounted for 0.03% while 2.26% of applied radioactivity was detected in the carcass. The mean concentrations of radioactivity in carcass, treated skin, and gastrointestinal tract were 0.085, 0.021 and 0.006 µg equivalents/g wet weight, respectively.

The overall mean absorbable proportion of 14C-labelled test substance (sum of direct absorption [excreta, tissues], treated skin and stratum corneum) was 4.42% of applied radioactivity.

Table 1: Distribution of radioactivity detected after topical application of 14C-labelled test substance to male mice in percent of applied radioactive dose.

Sample	Time point/interval (hours after dosing)
	6 h	168 h	0 - 168 h
NON-ABSORBD DOSE
Swabs	28.95 +/- 5.62
Dressing extracts	59.62 +/- 5.60	5.75 +/- 2.17
Dose on surface (Strips1,2)		0.27 +/-.0.09
TOTAL		94.59 +/- 2.50
DIRECT ABSORPTION (Tissues & Excreta)
Urine total			0.90 +/- 0.21
Feces total			0.71 +/- 0.22
Carcass		2.26 +/- 2.06
Gastrointestinal tract		0.03 +/- 0.02
Cage wash		0.20 +/- 0.03
TOTAL			4.09 +/- 2.33
ABSORBABLE
Skin (after stripping)		0.07 +/- 0.02
Stratum corneum		0.26 +/- 0.16
TOTAL ABSORBABLE
Direct Absorption + treated skin + Stratum corneum			4.42 +/- 2.40
OVERALL RECOVERY
Total			99.01 +/- 1.95

Conclusions:

After topical application of 14C-labeled test substance to male CD-1 mice according to OECD guideline 427, the mean total absorbed dose was 4.42 % of the applied radioactivity. Mean tissue concentrations of radioactivity in carcass, gastrointestinal tract and treated skin corresponded to 2.26%, 0.03% and 0.07% of applied dose.
The mean total non-absorbed dose was 94.59 % of the applied radioactivity. The mean overall recovery of radioactivity was 99.01% applied radioactivity.

Description of key information

Toxicokinetic assessment of a strucutral analogue substance is available.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - dermal (%):

4.42

Additional information

Assessment of read-across substance (EC 406-040-9):

Information of uptake and metabolism after oral application was obtained from a study in rats (Huntington, 2006), wheras information on absorption through the skin was obtained from a study in mice (Huntington, 2006). Both studies were performed under GLP and according to OECD testing guidelines 417 and 427, respectively. 14C-labelled substance was used for tracing.

Upon single oral dosing, a total of 3 metabolites in the freshly prepared urine pools of animals treated with 50 mg/kg bw were identified via molecular weight and fragmentation pattern using a mass spectrometer operating in the negative electrospray mode (ESI LC/MS). Metabolites in the other urine and fecal fractions were correlated to these identified metabolites via their retention times. Upon sacrifice after 7 days, less than 0.3% radioactivity were detected in tissues at 0.5 and 50 mg/kg bw, respectively.

Upon single dermal exposure, the absorption rate was calculated to be 4.42 % at 6 h (The absorbable fraction of test substance was calculated as the sum of excreta, tissues, treated skin after stripping and stratum corneum in percent of applied radioactivity.) Mean tissue concentrations of radioactivity in carcass, gastrointestinal tract and treated skin corresponded to 2.26%, 0.03% and 0.07% of applied dose.

Based on the experimental data, the substance is considered to be non bioaccumulating.