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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 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:
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 postadministration 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.

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
Reason / purpose:
reference to same study
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
Radiolabelling:
yes
Remarks:
[14C]β-OTNE
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals 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
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
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%
Time point:
96 h
Dose:
55 mg/kg bw (covered)
Parameter:
percentage
Absorption:
14 %
Key result
Time point:
96 h
Dose:
550 mg/kg bw (covered)
Parameter:
percentage
Absorption:
15 %
Time point:
96 h
Dose:
55 mg/kg bw (uncovered)
Parameter:
percentage
Absorption:
33 %
Remarks on result:
other: significant absorption due to oral grooming
Time point:
96 h
Dose:
550 mg/kg bw (uncovered)
Parameter:
percentage
Absorption:
72 %
Remarks on result:
other: significant absorption due to oral grooming

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.

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.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 23, 2000 to November 7, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was not conducted according to a guideline (not available), but was performed under GLP conditions. The study design is described well and the results are presented clearly.
Objective of study:
other: milk transfer
Qualifier:
no guideline available
Principles of method if other than guideline:
The transfer of test compound across the placenta and into milk of rats during and after pregnancy following repeated oral administration was investigated.
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Remarks:
14C
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
Sprague-Dawley female animals, 10-15 wks old upon arrival, 250-455 g in body weight, were utilized. They were pre-mated and housed singly in polypropylene solid bottom cages. Food and water were supplied ad libitum. Room temperature was maintained at 21±2ºC and relative humidity, 55±15%. The lighting regime was 12 hours light/12 hours dark cycle.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
No data
Duration and frequency of treatment / exposure:
Daily from Day 14 of the gestation period and continued up to Day 7 of parturition
Remarks:
Doses / Concentrations:
2 and 20 mg/kg bw/day
No. of animals per sex per dose:
18
Control animals:
yes, concurrent no treatment
Positive control:
Not relevant
Details on study design:
The study was conducted using OTNE labelled with carbon-14. For the main study, 14C-OTNE was formulated as a solution in corn oil and was administered orally to two groups of 18 pregnant rats at low and high dose levels of 2 mg/kg bw/day and 20 mg/kg bw/day, respectively. Two additional animals were included at each dose level to be used as spares. Two animals also were added to the low-dose group to be used for whole-body autoradiographic procedures.

Doses were selected based on a pilot study:
14C-OTNE was formulated as a solution in corn oil and was administered orally to 4 pregnant rats at 25 mg/kg bw/day from Day 14 of the gestation period and continued up to Day 3 of parturition. At 3 hours prior to collection of the milk samples, pups were removed from the mothers, anesthetized and sacrificed by injection of sodium pentobarbitone. Milk samples were taken at 4 hours on Day 3 of parturition. Prior to milk sampling (~5 minutes), oxytocin was administered to the mothers by intraperitoneal injection. Milk samples (~0.1–0.5 ml) were collected manually over a period of 2-5 minutes by gently pressing several nipples. Concentrations of radioactivity in milk were determined by liquid scintillation counting.
Details on dosing and sampling:
Animals were administered 14C-OTNE daily for 5 days beginning on Day 14 of the gestation period. All other animals were administered consecutive daily oral doses of 14C-OTNE from Day 14 of the gestation period and continued up to Day 7 of parturition. At 3 hours prior to collection of the milk samples, pups were removed from the mothers, anesthetized and sacrificed by injection of sodium pentobarbitone. Milk and blood samples from three rats at each dose level were taken at 4, 8 and 24 hours on Days 3 and 7 after parturition. Prior to milk sampling (~5 minutes), oxytocin was administered to the mothers by intraperitoneal injection. Milk samples (~0.1–0.5 ml) were collected manually over a period of 2-5 minutes by gently pressing several nipples. Immediately after milk collection, each rat was anesthetized and a terminal blood sample (~4 ml) was collected from the retro-orbital sinus into a heparinised tube. Blood was centrifuged and the plasma separated. Concentrations of radioactivity in milk and plasma were determined by liquid scintillation counting and concentrations of OTNE in milk were determined by HPLC method. Animals to be used for autoradiography were killed by carbon dioxide asphyxiation at 4 or 24 hours after the last dose. Sections were taken at six different levels to include the foetuses.
Statistics:
No data
Preliminary studies:
Total radioactivity concentrations in milk samples taken 4 hours post dosing ranged from 3.75-7.97 ppm. Dose levels of 2 and 20 mg/kg bw/day were selected for the main study.
Details on absorption:
The data show that the substance is orally well absorbed.
Details on distribution in tissues:
The distribution of radiolabelled OTNE is similar in blood and milk indicating an equal distribution in the these tissues. Doses of 2 mg/kg bw result in ca 0.2 mg radiolabel of OTNE in plasma or milk. At 20 mg/kg bw this is almost 10 times as high.
Key result
Transfer type:
other: milk transfer
Observation:
distinct transfer
Details on excretion:
The DT50 in plasma and milk is ca 20 hours.
Metabolites identified:
yes
Remarks:
Qualitatively
Details on metabolites:
In the HPLC chromatograms from the milk samples it can be seen that the retention time of OTNE is around 7.5 minutes while the major peaks are between 3-4 minutes. This indicates that the metabolites have shorter retention times and are more hydrophilic.
At 2 mg/kg bw, mean plasma radioactivity concentrations after Day 3 of parturition declined from 0.296 to 0.109 ppm between the 4-hour and 24-hour sampling time. After Day 7 of parturition, mean plasma radioactivity concentrations peaked at 0.256 ppm at 4 hours and declined to 0.050 ppm at 24 hours. Mean milk radioactivity concentrations after Day 3 of parturition were 0.227 ppm at 4 hours and declined at a similar rate to total plasma radioactivity concentrations. Concentrations of radioactivity were similar in the Day 7 parturition samples. OTNE was not detected in extracted samples of milk at any of the sampling times for both Days 3 and 7 indicating that OTNE was completely metabolised. Of the two additional animals that were sacrificed at 4 and 24 hours after the last dose and taken for whole-body autoradiography, radioactivity levels were barely detectable in the foetus at both sacrifice times.

At 20 mg/kg bw, mean plasma radioactivity concentrations after Day 3 of parturition were 2.70 ppm at 4 hours, approximately 10 times those measured in the low dose animals. Mean concentration of plasma radioactivity declined to 1.17 ppm at 24 hours. A similar pattern was observed in the Day 7 parturition samples. Total radioactivity concentrations in milk at this dose were 10-19 times those measured at the 2 mg/kg level and showed a similar pattern of decline with time. OTNE was not detected in extracted samples of milk at any of the sampling times for both Days 3 and 7 indicating that test compound was completely metabolised.

At 4 and 24 hours post dosing, whole-body autoradiographs showed barely detectable radioactivity in placenta and foetus with highest levels in small intestinal contents and preputial glands, followed by stomach, liver, large intestinal contents, thyroid, and bladder.
Conclusions:
During the 4-24 hours post dosing, plasma radioactivity concentrations at the low and high doses declined by 26-80% and 32-72%, respectively. Radioactivity in milk decreased by >79% at both dose levels by 24 hours post dose indicating a more rapid decline than that of plasma. At the high dose, total radioactivity in plasma and milk was 8-19 times greater than at the low dose. OTNE appeared to be completely metabolised since no detectable levels were found in milk samples taken at 4 and 8 hours.
Executive summary:

In a milk transfer study, groups of pregnant rats were gavaged with 2 or 20 mg/kg bw/day of 14C-OTNE in corn oil from Day 14 of the gestation period and up to Day 7 of parturition. Two animals also were added to the low-dose group to be used for whole-body autoradiographic procedures and were administered 14C-OTNE daily for 5 days beginning on Day 14 of the gestation period. Milk and blood samples were taken at 4, 8 and 24 hours on Days 3 and 7 after parturition. Concentrations of radioactivity in milk and plasma and concentrations of OTNE in milk were determined. Animals to be used for autoradiography were killed at 4 or 24 hours after the last dose. During the 4-24 hours post dosing, plasma radioactivity concentrations at the low and high doses declined by 26-80% and 32-72%, respectively. Radioactivity in milk decreased by >79% at both dose levels by 24 hours post dose indicating a more rapid decline than that of plasma. At the high dose, total radioactivity in plasma and milk was 8-19 times greater than at the low dose. OTNE appeared to be completely metabolised since no detectable levels were found in milk samples taken at 4 and 8 hours.

At 4 and 24 hours post dosing, whole-body autoradiographs showed barely detectable radioactivity in placenta and foetus, indicating minimal transfer across the placenta.
Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not stated
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to a relevant FDA guideline and under GLP conditions.
Qualifier:
according to
Guideline:
other: Human skin in vitro and the FDA/AAPS guidelines (Skelley et al., 1987 Pharmaceutical Research 4:265-267)
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Remarks:
14-C
Species:
human
Sex:
female
Details on test animals and environmental conditions:
Subcutaneous fat from four individual donors was removed from full-thickness human female breast and abdominal skin and portions of the skin were immersed in water at 60ºC. The skin was placed dermis-side down and pinned to a corkboard and the epidermis gently removed from the underlying dermis. The epidermal membranes were removed, dried, and stored flat at -20ºC. Membrane integrity was assessed prior to dosing by measuring the permeation rate of tritiated water. On the day of use, the membranes were mounted as a barrier between the halves of horizontal Franz-type diffusion cells and trimmed to size. The stratum corneum was made to face the donor chamber and the area available for diffusion was about 1.0 cm2. The skin surface temperature was maintained at 32.0 +/- 1ºC and the receptor chambers were maintained at 37.0 +/- 0.5ºC throughout the experiment by immersing the diffusion cells in a constant temperature water bath. The receptor chamber contents were continuously agitated.
Type of coverage:
open
Vehicle:
ethanol
Duration of exposure:
48 hours
Doses:
~200 µg/cm2
Details on study design:
OTNE was prepared as a 1% (w/v) solution in 25 ml ethanol and added to a volumetric flask, the radiolabelled spike was added, and the flask was made to volume with ethanol. Twelve diffusion cells were dosed with OTNE and an additional two control cells were run. Permeation was measured by taking 200 µl samples from the receptor chamber at 2, 8, 24, 36 and 48 hours, placing them in scintillation fluid and analyzing for 14C with a liquid scintillation counter. Following the 48-hour receptor phase sample, diffusion cells were dismantled; fragrance remaining on the skin surface was wiped and analysed for radiolabel content. Each epidermal membrane was tape stripped 10 times and the radiolabel content of the strips and remaining epidermis determined.
Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Total recovery:
Because of the volatility of the fragrance material, overall recovery was relatively low at 53.3 +/- 1.4% of the applied dose. The amount of test compound recovered decreased with skin depth.
Dose:
198.4 µg/cm2
Parameter:
percentage
Absorption:
0.5 %
Remarks on result:
other: 2 hr
Remarks:
of the applied dose permeating into the receptor phase. Results indicated that the percutaneous absorption levels of the test material were significant.
Dose:
198.4 µg/cm2
Parameter:
percentage
Absorption:
2.9 %
Remarks on result:
other: 8 hr
Remarks:
of the applied dose permeating into the receptor phase. Results indicated that the percutaneous absorption levels of the test material were significant.
Dose:
198.4 µg/cm2
Parameter:
percentage
Absorption:
9 %
Remarks on result:
other: 24 hr
Remarks:
of the applied dose permeating into the receptor phase. Results indicated that the percutaneous absorption levels of the test material were significant.
Dose:
198.4 µg/cm2
Parameter:
percentage
Absorption:
12.3 %
Remarks on result:
other: 36 hr
Remarks:
of the applied dose permeating into the receptor phase. Results indicated that the percutaneous absorption levels of the test material were significant.
Key result
Dose:
198.4 µg/cm2
Parameter:
percentage
Absorption:
15.3 %
Remarks on result:
other: 48 hr
Remarks:
of the applied dose permeating into the receptor phase. Results indicated that the percutaneous absorption levels of the test material were significant.
Key result
Dose:
198.4 µg/cm2
Parameter:
percentage
Absorption:
1.21 %
Remarks on result:
other: 48 hr
Remarks:
of the applied dose permeating into the epidermis.

Evaporation was assessed as the main cause of the low recovery of OTNE. Total recovery of applied fragrances from PTFE surfaces and the cell donor chamber at 48 hours was 57%, and the estimated loss through evaporation was 43%.

Conclusions:
The percutaneous absorption levels of OTNE were significant. The permeation rate of OTNE was linear with no significant plateauing. In the receptor fluid 16.5% of the applied dose was recovered. A conservative value of 28.95% is calculated as it is corrected for the loss of the substance.
Executive summary:

The percutaneous penetration of radiolabelled OTNE in 1% ethanol was studied in anin vitrohuman skin model. Twelve cells were dosed with OTNE and an additional two control cells were run. Permeation was measured by taking samples from the receptor chamber at 2, 8, 24, 36 and 48 hours, and analysing for14C with a liquid scintillation counter. Following the 48-hour receptor phase sample, skin was removed, wiped and analyzed for radiolabel content. Each epidermal membrane was tape stripped to determine the radiolabel content of the strips and remaining epidermis. Evaporative loss from the surface of epidermal membranes was assessed using polytetrafluoroethylene (PTFE) sheets mounted in horizontal Franz-type diffusion cells (n=6) under similar conditions. Following dosing, one cell was dismantled at 1, 2, 4, 8, 24 and 48 hours. The PTFE sheet was removed and radiolabel content was determined. Because of the volatility of the fragrance material, overall recovery was relatively low at 53% of the applied dose. At 48 hours, absorption was 16.5% of the applied dose, from which 15.3% was permeated into receptor fluid and 1.2% was permeated into the epidermis. When this value is corrected taking into account the loss of the substance due to volatillisation, a dermal absorption of 28.95% is calculated. The percutaneous absorption levels of OTNE were significant. The permeation rate of OTNE was linear with no significant plateauing.

Description of key information

OTNE is absorbed via the oral route and dermal route for 86 and 15%, respectively, based on experimental in vivo toxico-kinetic information using radiolabel. Using the precautionary principle for the inhalation route 100% inhalation absorption will be selected. The radiolabelled substance is fully metabolised into more hydrophilic metabolites, as is shown in HPLC chromatograms and it is reasoned that these are (partly) glucuronic conjugates of the substance. The DT50 of the substance after oral exposure is 34.3 hours (20 mg/kg bw), after dermal exposure it is 70 (55 mg/kg bw) and 40 (550 mg/kg bw) hours after application, respectively. Via oral exposure the main the main excretion pathways are via urine and bile. Via dermal exposure the urine is the main excretion pathway.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
86
Absorption rate - dermal (%):
15
Absorption rate - inhalation (%):
100

Additional information

The toxico-kinetic assessment of OTNE for humans and air breathing mammals

In this toxico-kinetic assessment the experimental and theoretical consideration of the fate of OTNE in humans and mammals will be presented. There is oral and dermal kinetic information available from several kinetic and repeated dose toxicity studies. First the overall toxico-kinetic assessment is presented and thereafter the executive summaries of the experimental studies.

 

Introduction

The toxico-kinetic assessment of OTNE, with its constituents and impurities (it is a reaction mass) is presented below.

Structure description: The test material OTNE has three main constituents which all have a double hexyl-ring, two methyl groups are attached to one C atom in the first ring. The second ring has one C atom with one methyl group and one C atom with a methyl group and a ketone-methyl group. Also the impurities have a similar structure. There is one impurity where the right ring is open instead of closed. The double bond is either between the two rings or on the left or right ring.

Relevance of information derived from radiolabelled OTNE: The toxico-kinetic experiments are carried out with radiolabelled OTNE. In view of OTNE being a multi-constituent it is considered appropriate to derive the kinetic values on this radioactivity because the assessment then includes all constituents and impurities. It is also considered conservative because also metabolites and residual carbons will be included in this assessment.

Physico-chemical properties: The substance is a liquid with a molecular weight of 234.4, a volatility of (0.233 Pa), a water solubility of (2.68 mg/l) and a log Kow of 5.65.

Absorption:

Oral: the absorption of OTNE was studied by the NTP (2014) using [14C]OTNE (β-isomer) in male F344 rats following gavage administration of 20 mg/kg bw, resulting in an oral absorption of 86%, which will be forwarded to the risk characterisation.

Skin: Key information is an in an in vivo skin absorption study with male F344 rats following a single dermal application of 55 or 550 mg/kg bw [14C]β-OTNE in ethanol to covered sites after 24, 48, or 96 hours. The dermal absorption of 15% is used (derived from 550 mg/kg bw) because the recovery was highest for this dose. The information from the supporting in vitro study is in line with this 15% value showing 16.5% skin absorption, but with a lower recovery compared to the in vivo study. Therefore the in vivo study is selected as key and 15% dermal absorption is selected for the risk characterisation.

Lungs: Absorption via the lungs for humans and air breathing mammalsis also indicated based on the physico-chemical properties. Though the inhalation exposure route is considered to be of minor relevance, because of its low volatility (0.233 Pa), the potential for lung absorption is assessed. The blood/air (BA) partition coefficient is thought to be relevant for humans and mammals because when the substance enters the airways the first partitioning step is between blood and air. This coefficient is also considered more relevant compared to the Koctanol / air (Koa) partitioning coefficient because the substance first has to enter the blood stream before it can enter the body. Buist et al. 2012 have developed BA model for humans using the most important and readily available parameters:

Log PBA = 6.96 – 1.04 Log (VP) – 0.533 Log (Kow) – 0.00495 MW.

For OTNE the B/A partition coefficient would result in:

Log P (BA) = 6.96 – 1.04*Log (0.233) – 0.533*5.65 – 0.00495*234.4 = 3.44

This means that the substance has a tendency to go from air into the blood. It should, however, be noted that this regression line is only valid for substances which have a vapour pressure > 100 Pa. From these calculations we would expect absorption via inhalation to be 100%.

Distribution for humans and air breathing mammals

In the oralin vivo toxico-kinetic studies in rats (NTP, 2014) it is shown that the oral DT50 is 34.3 hours (at 20 mg/kg bw). Tmax via oral exposure is at 8 hours and Cmax is almost 1 mg/l blood after 20 mg/kg bw exposure, (843 ng/g tissue) again based on all radioactivity.

In the dermalin vivo toxico-kinetic study in rats (NTP, 2014) the DT50 values are 70 and 40 hours, for the low and high dose of 55 and 550 mg/kg bw, respectively. Tmax via dermal exposure is reached within 24 hours and Cmax is 0.9 mg/l at the low dose (55 mg/kg bw). OTNE is distributed in all organs blood/tissue concentrations. Via oral dosing blood/tissue concentration after 24 hours are 1:10 in liver, bladder and pancreas. In adipose tissue it is 1:3, in testes it is 1: 0.5, in brain it is 1: 0.2. In all other organs the distribution is roughly 1:1 based on radioactivity. In the in vivo dermal studies the substance was distributed as follows: 1:17 in skin; 1:14 in bladder; 1:7 in kidney, liver, pancreas (roughly) also in adipose tissue; in brain it is 1:0.5. In all other tissues it is 1:1 based on radioactivity.

Adipose tissue: This shows that in adipose tissue the distribution is 1:3 and 1:7 after 24 hours via oral and dermal route respectively showing that it is limitedly distributed in body fat. In addition, in the NTP studies no parent OTNE could be found. The determination of the peaks showed that the likely metabolites were glucuronic conjugates of OTNE, indicating full metabolism of OTNE.

Milk transfer: In a RIFM study (2001) the transfer of OTNE to milk and placenta is studied during and after pregnancy using radiolabelled OTNE. The parent compound, OTNE, was not detected in extracted milk samples at both dose levels but radioactivity. Exposure to 2 and 20 mg/kg bw resulted roughly in 0.2 and 2 ppm radioactivity in the milk. From the HPLC chromatograms of the milk samples it can be seen that the metabolites have lower retention times and therefore are more hydrophilic . This indicates that radioactivity detected is related to metabolites of OTNE. In a Chinese study OTNE has been detected at low levels in mother’s milk (3.9 ng/g lipid ca 0.175 ug/l milk assuming 3.5% fat in milk) (Yin et al., 2016). The exposure in this paper is related to exposure via cosmetics.

Whole body autoradiographs: OTNE related radioactivity has been measured in whole-body autoradiographs showed barely detectable radioactivity in placenta and foetuses at 4 and 24 hours post dosing, indicating negligible transfer of parent OTNE or metabolites across the placenta. Highest levels of radioactivity were observed in the content of the small intestine and in preputial glands, followed by stomach, liver, large intestinal contents, thyroid, and bladder.

Metabolism for humans and air breathing mammals

OTNE is extensively metabolized as no parent OTNE is detected in several in vivo toxico-kinetic studies. The substance is extensively conjugated as is measured in male rats using bile cannulated rats. The alpha-2u hydrocarbon nephropathy seen in the kidneys show that the substance is partly metabolized in the lysosomes. Due to the low pH in the lysosomes, the ketone becomes reduced to a secondary alcohol which is then conjugated with alpha-2u globulin and transported to the kidneys.

The metabolism of the substance is also assessed using the OECD Toolbox liver metabolism simulator. Hydroxylation can occur at several sites on the molecule. No reactive metabolites are popping up and the substance is detoxified. This is because these metabolites are expected to be more water soluble, have a lower log Kow and will therefore be more easily excreted (see Figure in attached document).

There is no possibility of the formation of a gamma-diketone, because OTNE has no ethyl groups at the side of the ketone functional groups. This gamma-diketone is the likely metabolite of AETT causing neurotoxicity at low doses (see neurotoxicity section).

 

 

Fig. 1 The sites of beta-OTNE where oxidation and reduction can occur using e.g. OECD Toolbox.

 

Excretion for humans and air breathing mammals

The in vivo oral toxico-kinetic study shows the majority of the administered dose was excreted in urine (28%) and faeces (39%) after 48 hours following gavage administration of 20 mg/kg. Approximately 73% of the 20 mg/kg dose was eliminated in bile in 48 hours confirming that the faecal excretion was not due to poor absorption but biliary excretion. Additionally, approximately 80% of the dose in the bile was excreted within the first 4 hours, while the faecal excretion continued through 48 hours suggesting some enterohepatic recirculation of OTNE. The in vivo dermal kinetic study shows that OTNE is mainly excreted via the urine.

Discussion for humans and air breathing mammals

Key information on bioaccumulation including air breathing organisms:

In accordance with the PBT guidance on air breathing organisms (2017) it can be seen that the first criterion for concern may be met: Log Kow >2 and Koa > 5 for OTNE as such. The Koa may not be the best parameter. The blood/air partition coefficient is a better partition coefficient for indication of accumulation via air because blood is the first compartment (when air exposure occurs) from which the substance will enter the body. This coefficient is 3.44. The Koa is a compilation of Henry coefficient and Kow, and water (as is a parameter in the Henry coefficient) is a different compartment compared to blood. In addition, all other criteria indicating an absence of concern for air breathing organisms are also not met: 1) extensive metabolisation is seen in all studies (no parent substance detected, anticipated glucuronic metabolites are found using radiolabel); 2) low distribution in body fat such as mother’s milk and adipose tissue (based on radiolabel and therefore concerning metabolites and residual carbon) and; 3) low DT50 < 1.4 and 3 days (based on radiolabel from in vivo oral and dermal studies respectively); is supported with the experimental DT50 in fish being 1.2 days and a BCF value: 391.

Key information on absorptionwhich will be brought forward to the risk characterisation: The substance is readily absorbed orally up to 86% and via inhalation expected to be up to 100%, based on the available human toxicological information and physico-chemical parameters. Based on the key in vivo dermal absorption study the absorption via this route is 15%.

In view of the available experimental in vivo toxico-kinetic information the IGHRC (2006) document of the HSE and mentioned in the ECHA Guidance Chapter R.8 will be followed only for the inhalation route.

Oral to inhalation extrapolation: Though OTNE is not a volatile liquid some inhalation exposure will be calculated. OTNE is not corrosive to skin and eye and the systemic effect will overrule the effects at the site of contact. In the absence of inhalation absorption data it is most precautionary that 100% of the inhaled vapour is bioavailable. For the oral absorption 86% will be used and 15% for the dermal route when using route to route extrapolation.

Oral to dermal extrapolation: The experimental oral and dermal absorption values will be used: 86 and 15%, respectively.

Conclusion:

OTNE is absorbed via the oral route and dermal route for 86 and 15%, respectively, based on experimental in vivo toxico-kinetic information using radiolabel. Using the precautionary principle for the inhalation route 100% inhalation absorption will be selected. The radiolabelled substance is fully metabolised into more hydrophilic metabolites, as is shown in HPLC chromatograms and it is reasoned that these are (partly) glucuronic conjugates of the substance. The DT50 of the substance after oral exposure is 34.3 hours (20 mg/kg bw), after dermal exposure it is 70 (55 mg/kg bw) and 40 (550 mg/kg bw) hours after application, respectively. Via oral exposure the main the main excretion pathways are via urine and bile. Via dermal exposure the urine is the main excretion pathway.

References

- Buist, H. E., Wit-Bos de, L., Bouwman, T., Vaes, W. H. J., 2012, Predicting blood: air partition coefficient using basis physico-chemical properties, Regul. Toxicol. Pharmacol., 62, 23-28.

- Martinez, M. N., And Amidon, G. L., 2002, Mechanistic approach to understanding the factors affecting drug absorption: a review of fundament, J. Clinical Pharmacol., 42, 620-643.

- IGHRC, 2006, Guidelines on route to route extrapolation of toxicity data when assessing health risks of chemicals, http: //ieh. cranfield. ac. uk/ighrc/cr12[1]. Pdf

- NTP, 2014, Waidyanatha, S., and Ryan, K, Disposition of fragrance ingredient [14C]1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)ethanone in male Fisher rats following oral administration and dermal application Xenobiotica  Vol. 44 , Iss. 8, 749-756.

- Yin, J., Wang, H., Li, J., Wu, Y.,  Shao, B., 2016, Occurrence of synthetic musks in human breast milk samples from 12 provinces in China,Food Additives & Contaminants: Part A,Volume 33 (7), Pages 1219-1227