reaction mass of 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,4,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

absorption

distribution

excretion

Principles of method if other than guideline:

Principle of test:
- Disposition of β-OTNE, was investigated following a single oral (20 mg/kg bw) dose of [14C]β-OTNE to male Fisher rats.
- Short description of test conditions:
Oral administration: Single radiolabeled oral doses were administered to male rats, by intra-gastric gavage, in a dose volume of 5 mL/kg bw via a syringe. Following administration, animals were returned to metabolism cages. Groups of 4 animals were sacrificed at 4, 8, 24 and 48 h following administration. A group of bile duct cannulated male rats were also given 20 mg/kg as above and sacrificed after 48 h following administration.
- Parameters analysed / observed:
Disposition of radioactivity at 4, 8, 24 or 48h following oral administration and 24, 48 or 96h after dermal application (tissues, organs, feces, urine, bile)
Blood or tissue concentration versus time profiles
Blood and tissue toxicokinetic parameters (T1/2, Mean Residence Time, AUC, Terminal elimination rate constant, Tmax, Cmax)
Excretion of radioactivity following administration

GLP compliance:

not specified

Radiolabelling:

yes

Remarks:

[14C]β-OTNE

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms (Germantown, NY), Bile duct cannulated male Fisher rats were obtained from Hilltop Labs (Scottsdale, PA)
- Age at initiation: 8-12 weeks
- Housing: individual all glass metabolism cages
- Diet (e.g. ad libitum): Purina rodent chow (5002) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.7–26.1
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.9%Saline:Alkamulus EL-620/L (9:1)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations contained [14C]β-OTNE (5 µCi/rat), an appropriate amount of non-radiolabeled OTNE in 0.9% saline:Alkamulus EL-620/L (9:1). Oral doses were administered by intra-gastric gavage in a dose volume of 5 mL/kg via a syringe equipped with a 16 gauge ball tipped gavage needle.

Duration and frequency of treatment / exposure:

Single doses were administered

Dose / conc.:

20 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

4 males per dose

Control animals:

no

Details on study design:

- Dose selection rationale: An oral dose of 20 mg/kg bw was selected based on the information available in the literature (ECHA registered substance database, 2013)

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, cage washes, bile, skin (ears), dose site skin (dermal groups only), spleen, liver, kidney, brain, heart, lung, pancreas, thymus, testes, bladder, muscle (abdominal and hind leg), adipose (perirenal and epididymal), and stomach, cecum, large intestine, and small intestine (all with contents)
- Time and frequency of sampling: urine and feces were collected separately into flasks cooled over dry ice at 0–4, 4–8, 8–24, 24–48, 48–72, 72–96 h intervals. In the bile collection group, bile, urine and feces were collected separately into flasks cooled over dry ice at 0–4, 4–8, 8–24, and 24–48 h intervals. Blood was sampled at the end of the study.

Statistics:

Non-compartmental analysis of blood or tissue concentration versus time data was conducted using Phoenix WinNonlin software, version 6.3 (Pharsight Corporation, Cary, NC). Individual animal data was modeled. Parameters estimated were: Lambda-z, rate constant of elimination; t1/2, half-life of elimination; Cmax, maximum concentration; Tmax, time at which the maximum concentration was achieved; AUC(0–t), area under the blood concentration versus time curve to last time point; AUC(0–1), area under the blood concentration versus time curve to infinity; AUC(0–1)/D, dose-adjusted area under the blood concentration versus time curve to infinity.

Type:

absorption

Results:

b-OTNE was well-absorbed after a single oral administration of 20 mg/kg bw [14C]β-OTNE in male rats. High fecal excretion was observed, due to excretion via bile. At least 86% of a 20 mg/kg bw oral dose was absorbed following administration.

Type:

distribution

Results:

Tissue/blood ratios (TBR) were highest in bladder (31±34), pancreas (11±4.6), liver (8.8±1.0), kidney (8.1±1.2), adipose (5.4±3.4), muscle (2±3.2) and spleen (2.6±1.2). AUC(0–48h) values were highest in the bladder, liver, kidney and pancreas.

Type:

excretion

Results:

28% and 39% of the dose was recovered in urine and feces, respectively, and 73% of a 20 mg/kg bw dose was excreted in bile within 48 h post-administration.

Details on absorption:

Following oral administration of 20 mg/kg bw in male Fisher rats, [14C]β-OTNE was excreted mainly via urine and feces. The total dose excreted in urine 48 h post administration was 28% with the majority excreted within 24 h (25%) post-administration suggesting rapid urinary excretion of [14C]β-OTNE following oral administration. However, fecal excretion continued through 48 h post administration with 39% excreted by 48 h while the levels of radioactivity in the GI tract contents decreased slowly, suggesting a long residence time of OTNE in the gut. In bile duct cannulated rats, about 73% of a 20 mg/kg bw was eliminated in bile within 48 h post administration while the excretion in urine and feces decreased to 12.8% and 2.8%, respectively, from 29% and 39% observed for intact animals. These data supports that the high fecal excretion observed following oral administration was not due to poor absorption but excretion via bile; at least 86% (73%+12.8%) of a 20 mg/kg bw oral dose of [14C]β-OTNE was absorbed following administration in male rats. Additionally, about 80% of the dose excreted in bile was excreted within 4 h suggesting rapid absorption.

Details on distribution in tissues:

In tissues: The concentration of radioactive equivalents in tissues 4, 8, 24, and 48 h following oral administration in (excluding digestive tract tissues) reached a maximum at 8 h post dosing; tissue/blood ratios (TBR) were highest in bladder (31±34), pancreas (11±4.6), liver (8.8±1.0), kidney (8.1±1.2), adipose (5.4±3.4), muscle (2±3.2) and spleen (2.6±1.2). The overall half-life was 34.3 hours in blood and most tissues. AUC(0–48h) values were highest in the bladder, liver, kidney and pancreas.

Details on excretion:

Following an oral administration of 20 mg/kg bw in bile duct cannulated male rats, 73% of the dose was recovered in bile 48 h post dosing. The dose recovered in urine and feces 48 h post dosing was 12.8% and 2.8%, respectively.

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: Blood: 34h, Adipose Tissue: 30h, Bladder: 8.7h, Brain: 25h, Heart: 24h, Kidney: 25h, Liver: 35h, Lung: 27h, Muscle: 27h, Pancreas: 19h, Skin: 22h, Spleen: 17h, Testes: 20h, Thymus: 24h

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: Blood: 20h, Adipose Tissue: 13h, Bladder: 14h, Brain: 19h, Heart: 17h, Kidney: 17h, Liver: 19h, Lung: 19h, Muscle: 15h, Pancreas: 16h, Skin: 16h, Spleen: 14h, Testes: 17h, Thymus: 17h

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: Blood: 26, Adipose Tissue: 71, Bladder: 521, Brain: 3.8, Heart: 19, Kidney: 182, Liver: 217, Lung: 25, Muscle: 34, Pancreas: 194, Skin: 17, Spleen: 45, Testes: 11, Thymus: 13

Remarks:

µg-eq.h/g

Metabolites identified:

yes

Details on metabolites:

It was deduced that the substance was conjugated based on the molecular weight.

Conclusions:

The data indicate that β-OTNE was well-absorbed following a single oral administration of 20 mg/kg bw [14C]β-OTNE in male rats. The reported oral absorption level of 86% is considered valid for use in further risk assessment for this substance and is based on radiolabel. the substance is fully metabolised and the key metabolite is the conjugated counterpart of OTNE. The DT50 is considered to be 34.3 hours also based on radiolabel.

Executive summary:

Disposition of β-OTNE, was investigated following a single oral (20 mg/kg bw) dose of [14C] β-OTNE to male Fisher rats. The study was rated Klimisch 2, since the study was not conducted according to a guideline (not available), but the study design is documented well and the results are presented clearly. Single radiolabeled oral doses were administered to male rats, by intra-gastric gavage, in a dose volume of 5 mL/kg bw via a syringe. Following administration, animals were returned to metabolism cages. Groups of 4 animals were sacrificed at 4, 8, 24 and 48 h following administration. A group of bile duct cannulated male rats were also given 20 mg/kg bw as above and sacrificed after 48 h following administration.

Disposition of radioactivity was measured at 4, 8, 24 or 48h following oral administration (tissues, organs, feces, urine, bile). Blood and tissue toxicokinetic parameters (T1/2, Mean Residence Time, AUC, Terminal elimination rate constant, Tmax, Cmax) were determined, as well as excretion of radioactivity following administration. The data indicate that the substance was well-absorbed following a single oral administration of 20 mg/kg bw [14C] β-OTNE in male rats. At least 86% of a 20 mg/kg bw oral dose was absorbed. b-OTNE was distributed to tissues, with bladder, pancreas, liver, adipose, and kidney showing the highest exposure to the test substance. Excretion of the test substance was mainly via urine and feces following both routes of administration. Biliary excretion and enterohepatic recirculation contributed to the high and prolonged excretion in feces. The reported oral absorption level of 86%, is considered valid for use in further risk assessment for this substance. The substance is extensively metabolised because no beta-OTNE could be detected, the glucuronic conjugated is one of the key metabolites. The DT50 for OTNE based on radiolable is 34.3 hours: 1.43 days.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Principles of method if other than guideline:

Principle of test method:
- Disposition of radioactivity was determined following a single application of 55 or 550 mg/kg bw [14C]β-OTNE to covered or uncovered dose sites in male Fisher rats.
- Short description of test conditions:
Dermal administration: Male rats were surgically fitted with indwelling jugular vein cannulae at the study laboratory. Single dermal doses of 55 or 550 mg/kg radiolabeled OTNE were applied to a 3x4 cm clipped area on the back of the animal in a dose volume of 1 mL/kg bw. Exposure was performed both with and without occlusion to protect from grooming. All animals were returned to metabolism cages following dosing. Groups of 4 animals were sacrificed at 24, 48, and 96 h following application.
- Parameters analysed / observed:
Disposition of radioactivity at 24, 48 or 96h after dermal application (tissues, organs, feces, urine, bile)
Blood or tissue concentration versus time profiles
Blood and tissue toxicokinetic parameters (T1/2, Mean Residence Time, AUC, Terminal elimination rate constant, Tmax, Cmax)
Excretion of radioactivity following administration

GLP compliance:

not specified

Test material

Test material information

1

Reference substance name:

Reaction Mass of 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,4,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

EC Number:

915-730-3

Molecular formula:

C16H26O

IUPAC Name:

Reaction Mass of 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,4,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

Constituent 1

Reference substance name:

1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

EC Number:

259-174-3

EC Name:

1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

Cas Number:

54464-57-2

Molecular formula:

C16H26O

IUPAC Name:

1-(2,3,8,8-tetramethyl-1,2,3,4,5,6,7,8-octahydronaphthalen-2-yl)ethanone

Constituent 2

Reference substance name:

1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

EC Number:

268-978-3

EC Name:

1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

Cas Number:

68155-66-8

Molecular formula:

C16H26O

IUPAC Name:

1-(2,3,8,8-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-2-yl)ethanone

Constituent 3

Reference substance name:

1-(1,2,3,4,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

EC Number:

268-979-9

EC Name:

1-(1,2,3,4,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

Cas Number:

68155-67-9

Molecular formula:

C16H26O

IUPAC Name:

1-(2,3,8,8-tetramethyl-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl)ethanone

Test material form:

liquid

Radiolabelling:

yes

Remarks:

[14C]β-OTNE

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms (Germantown, NY), Bile duct cannulated male Fisher rats were obtained
from Hilltop Labs (Scottsdale, PA)
- Age at initiation: 8-12 weeks
- Housing: individual all glass metabolism cages
- Diet (e.g. ad libitum): Purina rodent chow (5002) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 3 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.7–26.1
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:

other: open or site protected from ingestion (semi-occlusive)

Vehicle:

ethanol

Duration of exposure:

PREPARATION OF DOSING SOLUTIONS:
dose formulations contained [14C]β-OTNE (10 µCi/rat) and an appropriate amount of non-radiolabeled OTNE in ethanol in a dose volume of 1 mL/kg bw.

Doses:

- Nominal doses: 55 mg/kg bw and 550 mg/kg/bw
- Dose volume: 1 mL/kg bw
- Rationale for dose selection: dermal doses selected were approximately 0.01 and 0.1% of LD50 values reported in rodents

No. of animals per group:

4 animals per dose

Control animals:

no

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: dose formulations were prepared to contain [14C]β-OTNE (10 µCi/rat) and an appropriate amount of nonradiolabeled OTNE in ethanol in a dose volume of 1 mL/kg, method was not further specified.

TEST SITE
- Preparation of test site: on the day of dosing, the fur was clipped from the animal’s back and the clipped area on each animal was examined for nicks.
- Area of exposure: the outline of the dosing area of a 3 x 4 cm was inscribed on the back of the animal with a marker and defined as the dose area.
- Type of cover / wrap if used: a protective foam appliance with a non-occlusive cotton cloth cover and an aluminum grate
- Other: single dermal doses of 55 or 550 mg/kg bw were applied to the marked area using a syringe fitted with an 18-gauge gavage needle and spread evenly on the marked dose site; dose formulations contained [14C]β-OTNE (10 µCi/ rat) and an appropriate amount of non-radiolabeled OTNE in ethanol in a dose volume of 1 mL/kg bw. All animals were returned to metabolism cages following dosing. Groups of 4 animals were sacrificed at 24, 48, and 96 h following application.

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: yes: In groups protected from oral grooming, prior to dosing animals, a protective foam appliance was glued onto each rat’s back using tape and medical adhesive (3M, St. Paul, MN). Following application of the dose, a non-occlusive cotton cloth cover was fitted over the appliance and an aluminum grate was placed over the entire appliance to secure the appliance to the rat and to protect the appliance from being chewed.

REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: prior to blood collection, the protective appliance was removed.
- Washing procedures and type of cleansing agent: the dose site skin was washed with 8 gauzes moistened with soapy water, and 4 moistened with water, and rinsates and gauzes were collected.
- Time after start of exposure: 96 h

SAMPLE COLLECTION
- Collection of blood: at 6, 12, 24, 48, 72, or 96 h
- Collection of urine and faeces: 0–4, 4–8, 8–24 h in main study rats, 24–48, 48–72, 72–96 h in the bile collection group, bile, urine and feces were collected separately at 0–4, 4–8, 8–24, and 24–48 h intervals.
- Terminal procedure: at the end of all studies (except the bile duct cannulated group), the animals were euthanized by asphyxiation with CO2 and blood was collected via cardiac puncture into a heparinized syringe.
- Analysis of organs/tissues: skin (ears), dose site skin (dermal groups only), spleen, liver, kidney, brain, heart, lung, pancreas, thymus, testes, bladder, muscle (abdominal and hind leg), adipose (perirenal and epididymal), and stomach, cecum, large intestine, and small intestine (all with contents)

SAMPLE PREPARATION
- Storage procedure: urine and feces were collected separately into flasks cooled over dry ice, in the bile collection group, bile, urine and feces were collected separately into flasks cooled over dry ice. All samples collected were stored at -20°C until analyzed.
- preparation: Blood and tissues from all studies were aliquoted in triplicate. All tissues were minced before sampling ca. 100 mg aliquots for further processing. Tissues, skin, and carcass were digested in 2N ethanolic NaOH prior to analysis. Darker samples such as feces, blood, kidney, spleen, heart, and lungs were bleached. Duplicate aliquots of urine, and triplicate aliquots of cage rinse, feces, skin wash, blood, and tissue and carcass digests were added directly to vials containing Ultima Gold scintillation cocktail and analyzed for radioactivity content by liquid scintillation spectroscopy (LSS) using a Packard 1900CA Tri-Carb Liquid Scintillation Analyzer (Perkin Elmer, Waltham, MA).

ANALYSIS
- Method type(s) for identification: liquid scintillation counting

Results and discussion

Signs and symptoms of toxicity:

no effects

Dermal irritation:

not specified

Absorption in different matrices:

In general, the concentration of radioactive equivalents in tissues (excluding digestive tract tissues) reached a maximum by 24–48 h and declined by 96 h post dosing.
The concentrations were highest in kidney, adipose, pancreas, liver, bladder, and non-dose site skin. The percent dose recovered in tissues 96 h post application to the covered dose site was 0.37% and 0.60% for 55 and 550 mg/kg bw, respectively. Compared to animals with covered dose sites, the percent dose in tissues was increased slightly in groups with uncovered dose sites with 0.57% and 1% of the applied dose recovered at 96 h post application. For further details please refer to the tables in the "other information on results" section.

Total recovery:

- Total recovery (% dose recovered at 96 h):
Uncovered 55 mg/kg bw: 34 +/- 2.8%
Uncovered 550 mg/kg bw: 80 +/- 7.4%
Covered 55 mg/kg bw: 74 +/- 2.8%
Covered 550 mg/kg bw: 90 +/-1.6%

Percutaneous absorption

Percutaneous absorption 1

Time point:

96 h

Dose:

55 mg/kg bw (covered)

Parameter:

percentage

Absorption:

14 %

Percutaneous absorption 2

Key result

Time point:

96 h

Dose:

550 mg/kg bw (covered)

Parameter:

percentage

Absorption:

15 %

Percutaneous absorption 3

Time point:

96 h

Dose:

55 mg/kg bw (uncovered)

Parameter:

percentage

Absorption:

33 %

Remarks on result:

other: significant absorption due to oral grooming

Percutaneous absorption 4

Time point:

96 h

Dose:

550 mg/kg bw (uncovered)

Parameter:

percentage

Absorption:

72 %

Remarks on result:

other: significant absorption due to oral grooming

Any other information on results incl. tables

Table 1. Disposition of radioactivity following a single dermal application of 55 or 550 mg/kg bw [14C]b-OTNE to male Fisher rats and sacrificed 24, 48 or 96 h following application.

Covered

Uncovered

55 mg/kg

550 mg/kg

55 mg/kg

550 mg/kg

Sample

24 h

48 h

96 h

24 h

48 h

96 h

24 h

48 h

96 h

24 h

48 h

96 h

Unabsorbed dose

60±4.2

57±6.8

58±2.8

61±3.7

60±4.5

59±3.9

5.3±1.8

1.6±0.7

0.5±0.2

20±7.3

4.6±2.1

0.9±1.0

Dose site skin

9.2±1.6

4.4±1.10

3.1±0.22

18±4.6

16±2.0

11±2.8

8.2±3.1

2.5±0.79

0.72±0.31

8.6±3.9

2.6±1.2

1.4±0.54

Absorbed dose

8.9±1.0

13±1.5

14±1.9

10±1.1

14±1.2

15±9.9

36±12.2

35±6.0

33±2.4

54±12.3

68±6.1

72±6.9

Urineb

1.2±0.3

3.0±0.6

5.1±0.6

0.9±0.2

3.9±0.4

8.1±1.2

6.3±2.6

11±1.8

14±1.2

9.5±2.8

29±3.7

36±3.6

Feces

0.6±0.3

2.0±0.4

4.0±0.3

0.2±0.1

1.4±0.7

5.3±0.3

2.9±1.6

7.4±1.4

12±2.6

1.9±0.9

11±2.8

22±5.9

Tissues

0.6±0.1

0.8±0.5

0.4±0.1

0.6±0.1

0.8±0.2

0.6±0.2

1.6±0.5

1.1±0.3

0.6±0.2

1.9±0.3

1.9±0.4

1.0±0.2

GI tractc

4.3±0.7

4.3±0.7

3.1±0.6

2.7±0.2

3.5±0.4

2.5±0.8

16±4.0

12±2.2

4.6±0.2

9.9±2.5

8.3±0.9

5.1±0.8

Carcass

2.3±0.1

2.5±0.9

1.7±0.5

5.8±1.2

4.1±1.3

2.9±1.2

10±4.0

3.9±0.6

1.5±0.2

31±8.6

18±1.4

7.3±3.3

Total

78±6.2

74±9.0

74±2.8

89±2.1

91±2.1

90±1.6

50±11

39±4.9

34±2.8

84±2.0

80±3.2

80±7.4

 

Table 2. Tissue distribution of radioactivity following a single dermal application of 55 and 550 mg/kg bw [14C]b-OTNE to covered dose sites in male Fisher rats and sacrificed 24, 48 or 96 h following application.

Concentration (ng Equivalents/g Tissue)
	55 mg/kg			550 mg/kg
Tissue	24 h	48 h	96 h	24 h	48 h	96 h
Blood	246±113	258±68	191±23	2924±1367	5374±1689	2349±1618
Adipose	1842±924	1472±555	963±623	21599±3881	25116±5679	19875±8054
Muscle	208±90	657±817	188±134	1984±532	4335±2491	2883±1806
Skin	4217±1536	3127±684	2405±1185	23017±5333	28747±6047	27947±9663
Bladder	3477±3308	2403±1332	2104±1370	8466±2739	23919±16051	10225±3396
Testes	200±81	136±32	92±14	1862±419	2084±512	1592±492
Kidney	2697±434	1533±279	1132±229	32238±2309	28923±7053	18418±1999
Spleen	360±100	508±377	440±262	3595±1531	4404±2247	3451±1921
Liver	1818±262	1874±426	1747±250	14889±3572	19002±4625	15296±2055
Lung	638±121	465±96	347±75	6068±956	6635±1296	4896±1561
Heart	356±75	283±76	247±70	3734±484	4178±1093	3049±902
Brain	128±28	82±18	63±13	1771±122	1717±450	1242±316
Thymus	294±115	312±84	219±45	9031±564	9675±2885	4963±1043
Pancreas	1816±708	2568±1775	1046±370	21942±3681	18080±2458	17389±12555
% Dose in Tissues	0.56±0.14	0.76±0.50	0.37±0.14	0.55±0.09	0.83±0.16	0.60±0.24

 

Table 3. Tissue distribution of radioactivity in male Fisher rats following a single dermal application of 55 and 550 mg/kg bw [14C]b-OTNE to uncovered dose sites and sacrificed 24, 48 or 96 h following application.

Concentration (ng Equivalents/g Tissue)
	55 mg/kg			550 mg/kg
Tissue	24 h	48 h	96 h	24 h	48 h	96 h
Blood	1021±468	739±70	424±58	10950±2092	11546±2226	7483±1009
Adipose	3803±1468	1660±307	784±408	39493±4907	57538±12802	29037±8190
Muscle	621±305	555±438	360±175	10441±240	7477±3033	3605±707
Skin	25997±11358	4270±477	1729±155	311904±21496	352727±76805	100693±38394
Bladder	6080±230	3990±1738	1515±880	64338±21769	50426±57568	25114±26261
Testes	472±164	303±29	129±14	7987±1323	5333±598	2608±759
Kidney	7443±2449	3136±740	1385±129	103291±44534	65017±3982	32110±6845
Spleen	1407±722	777±363	384±28	19961±15094	9629±1536	6039±787
Liver	5910±1916	4909±1111	2730±97	44378±6188	48522±3416	36618±6926
Lung	1747±580	595±160	460±59	19758±2021	15843±1749	8381±2615
Heart	911±289	665±115	345±47	14112±4469	9640±1048	6168±2034
Brain	324±105	149±17	89±6	4827±216	3306±453	1418±513
Thymus	958±354	535±70	342±107	27550±8948	18755±5995	6146±2715
Pancreas	5076±3388	1863±1054	833±158	33364±3211	34451±5020	12218±3613
% Dose in Tissues	1.62±0.50	1.05±0.31	0.57±0.19	1.90±0.35	1.95±0.35	1.00±0.17

 

Effects of grooming and volatilisation

The absorption of b-OTNE was low and was not dose-dependent (14% and 15%, for 55 and 550 mg/kg, respectively). When the dose site was uncovered, the total dose absorbed increased suggesting significant oral grooming (33% and 72%, for 55 and 550 mg/kg, respectively). In addition, the total dose recovered was lower when the dose site was uncovered compared to covered dose sites suggesting rapid volatilization of the dose from the uncovered site.

Excretion after dermal absorption

Excretion following dermal dosing was mainly via urine and feces. The dose excreted in urine 96 h post application for 55 and 550 mg/kg bw, respectively, was ca. 5% and 8% in covered groups and ca.14% and 36% in the uncovered groups. The dose excreted in feces 96 h post application was slightly lower than in urine and was ca. 4% and ca. 5% in covered groups and ca. 12% and ca. 22% in the uncovered groups, for 55 and 550 mg/kg bw, respectively.

Applicant's summary and conclusion

Conclusions:

The percutaneous absorption of OTNE (using radiolabel) was determined to be 14% and 15% after semi-occlusive dermal application of 55 and 550 mg/kg bw, respectively. The reported percutaneous absorption level of 15% is the most conservative value and can be used for risk assessment purposes. The DT50 in at the low and high dose is 70 and 40 hours, respectively.

Executive summary:

Disposition of β-OTNE was investigated following a single dermal (55 or 550 mg/kg) dose of [14C] β-OTNE to male Fisher rats. The study was rated Klimisch 2, since the study was not conducted according to a guideline (not available), but the study design is documented well and the results are presented clearly. Prior to inclusion in the study, male rats were surgically fitted with indwelling jugular vein cannulae at the study laboratory. On the day of dosing, the fur was clipped from the animal’s back and the clipped area on each animal was examined for nicks. The outline of the dosing area of a 3 x 4 cm was inscribed on the back of the animal with a marker and defined as the dose area. Single dermal doses of 55 or 550 mg/kg bw were applied to the marked area in a dose volume of 1 mL/kg. In groups protected from oral grooming, prior to dosing animals, a protective foam appliance was glued onto each rat’s back using tape and medical adhesive. Following application of the dose, a non-occlusive cotton cloth cover was fitted over the appliance and an aluminium grate was placed over the entire appliance to secure the appliance to the rat and to protect the appliance from being chewed. All animals were returned to metabolism cages following dosing. Groups of 4 animals were sacrificed at 24, 48, and 96 h following application. The absorption of β-OTNE was low and was not dose-dependent (14% and 15%, for 55 and 550 mg/kg, respectively). When the dose site was uncovered, the total dose absorbed increased suggesting significant oral grooming (33% and 72%, for 55 and 550 mg/kg bw, respectively). In addition, the total dose recovered was lower when the dose site was uncovered compared to covered dose sites suggesting rapid volatilization of the dose from the uncovered site. b-OTNE was distributed to tissues with bladder, pancreas, liver, adipose, and kidney showing the highest exposure to the test substance. Excretion of the substance was mainly via urine and faeces following both routes of administration. The dose excreted in urine 96 h post application for 55 and 550 mg/kg bw, respectively, was ca. 5% and 8% in covered groups and ca.14% and 36% in the uncovered groups. The dose excreted in faeces 96 h post application was slightly lower than in urine and was ca. 4% and ca. 5% in covered groups and ca. 12% and ca. 22% in the uncovered groups, for 55 and 550 mg/kg bw, respectively. Biliary excretion and enterohepatic recirculation contributed to the high and prolonged excretion of the test substance in faeces. The higher fecal and urinary excretion levels in the uncovered group are also suggesting a significant effect of oral intake due to grooming. The results for this study indicate that a covered dermal exposure provides a reliable estimate of the percutaneous absorption of the test substance, as it prevents oral intake due to grooming, and rapid volatilisation of the substance. The reported percutaneous absorption level of 15% is the most conservative value and can be used for risk assessment purposes. The The DT50 in at the low and high dose is 70 and 40 hours, respectively.