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Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 November 1986 to 27 April 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
few details available on some parameters and on the substance identity (name). Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.
Reason / purpose for cross-reference:
reference to same study
Objective of study:
toxicokinetics
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
one dose only; no identification/quantification of excreta; few details on husbandry and environmental conditions
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
yes
Species:
rat
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Margate, Kent
- Age at study initiation: 6 weeks
- Weight at study initiation: 200 to 208 g
- Fasting period before study: useless as dermal route
- Housing: not reported
- Individual metabolism cages: yes
- Acclimation period: 5 days

Route of administration:
dermal
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: 9 cm²
- Type of wrap if used: aluminium foil secured with waterproof adhesive dressing

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes with cotton wool swabs moistened in ethanol
- Time after start of exposure: 6 hours (or 3 hours for animals to be sacrificed at this time)

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 5 mg/kg bw
- concentration (if solution): 1 mg/mL

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Duration and frequency of treatment / exposure:
6 hours (or 3 hours for animals to be sacrificed at this time); one application
Dose / conc.:
5 mg/kg bw (total dose)
No. of animals per sex per dose / concentration:
12 male rats at one dose level
Control animals:
no
Positive control reference chemical:
no
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, blood, plasma, serum or other tissues, cage washes, bile
- Time and frequency of sampling:
- Other:
Pairs of rats were at 3, 6, 12, 24, 48 or 72 hours after dosing.
Urine was collected at intervals 0-6, 6-24, 24-48 and 48-72 hours and faeces at 24-hour intervals, only from the rats to be sacrificed at 24, 48 and 72 hours.
Expired air was also collected, in traps containing ethanolamine : 2-ethoxyethanol (1:3, v/v) from the rats sacrificed at 24, 48 and 72 hours. Radioactivity in whole-blood and in plasma was measured at terminal sacrifice.

The following organs/tissues were sampled: liver, kidney, muscle, fat, testes, heart, eyes, brain, lungs, adrenals, bone marrow, lymph nodes, pancreas, spleen, thymus, thyroid, gastro-intestinal tract, treated skin and untreated skin
The dressing covering the treated skin and swabs used to removed unabsorbed dose were also taken for measurement of residual radioactivity.
Type:
excretion
Type:
distribution
Details on absorption:
after the 6-hour period, it was possible to remove a mean of 47.89% dose from the skin of the 6 rats. Means of 0.19% dose was measured in the treated skin of these 6 rats after removal of the excess dose.
Details on distribution in tissues:
Proportions of the applied dose in tissues were greatest at 3-6 hours in the gastro-intestinal tract GIT (2-7% dose), at 3 hours in liver and fat (0.6 - 0.9% dose) and at 3 hours in muscle (0.3-0.4% dose). Radioactivity in all other tissues measured was <0.1% dose at 3-6 hours except in the kidneys and plasma (<0.2% dose). At 12 hours, amounts of dosed radioactivity had decreased slightly in the GIT (2-4% dose), liver (0.08-0.2% dose) and fat (0.1-0.3% dose). At this time radioactivity in all other tissues measured was either Peak concentrations of radioactivity in tissues of the 12 rats were measured at 3-6 hours after dose application. At 24 hours, concentrations were not appreciably lower in the GIT but were substantially lower in liver, kidneys and fat. At 72 hours, concentrations had declined appreciably in the GIT, and had decreased further in liver, kidneys and fat.
The tissue:plasma concentration ratios showed higher concentrations than in plasma during 3-48 hours in the GIT, liver, kidneys, fat, lymph notes and pancreas.
Details on excretion:
Mean overall recovery of 76.4% attributed to the loss of some material by volatilisation during application of the dose to the skin.
Total urinary excretion until the time of sacrifice accounted for means of 8?04, 12.36 and 9.91% dose in the pairs of rats sacrificed at 24, 48 and 72 hours recpectively.
Total excretion of radioactivity in faeces accounted for 1.17, 2.29 and 2.42% dose in rats sacrificed at 24, 48 and 72 hours respectively.
Radioactivity in the expired air traps accounted for means of 17.55, 15.77 and 14.78% dose until the times of sacrifice at 24, 48 and 72 hours respectively. Most of this radioactivity was un the first expired trap (overall mean of 12.57% dose) with a smaller proportion in the second trap (overall mean of 3.47% dose). Radioactivity in the expired air traps may mostly be attributed to 14C-limonene volatilised directly from the site of application.
Metabolites identified:
not measured
Conclusions:
Interpretation of results: low bioaccumulation potential based on study results.
An appreciable proportion of the dermally applied dose (48%) was recovered in the dose washings. The radioactivity in urine (10% dose) and in faeces (2% dose) showed that these proportions were absorbed throught the skin. Radioactivity in the expired air traps may mostly be attributed to volatilisation from the site of application. However, there may also be a contribution of absorbed and exhaled air.
Renal excretion of limonene and/or its metabolite is expected to be the major route (as confirmed by studies using the oral route). The radioactivity in the faeces, gastro-intestinal tract and liver indicate some hepatic elimination.
Executive summary:

Twelve male rats received one dermal dose of 5 mg/kg bw for 6 hours (or 3 hours for those sacrificed at this time). Pairs of rats were sacrificed at 3, 6, 12, 24, 48 and 72 hours after dosing. Urine, faeces, plasma and various tissues were sampled and their radioactivity content measured.

Almost half the administered dose was removed by skin washing at the end of the application time. The renal route is the main one but faeces and exhaled air are also involved.

Endpoint:
basic toxicokinetics
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Type:
excretion
Type:
distribution
Details on absorption:
after the 6-hour period, it was possible to remove a mean of 47.89% dose from the skin of the 6 rats. Means of 0.19% dose was measured in the treated skin of these 6 rats after removal of the excess dose.
Details on distribution in tissues:
Proportions of the applied dose in tissues were greatest at 3-6 hours in the gastro-intestinal tract GIT (2-7% dose), at 3 hours in liver and fat (0.6 - 0.9% dose) and at 3 hours in muscle (0.3-0.4% dose). Radioactivity in all other tissues measured was <0.1% dose at 3-6 hours except in the kidneys and plasma (<0.2% dose). At 12 hours, amounts of dosed radioactivity had decreased slightly in the GIT (2-4% dose), liver (0.08-0.2% dose) and fat (0.1-0.3% dose). At this time radioactivity in all other tissues measured was either Peak concentrations of radioactivity in tissues of the 12 rats were measured at 3-6 hours after dose application. At 24 hours, concentrations were not appreciably lower in the GIT but were substantially lower in liver, kidneys and fat. At 72 hours, concentrations had declined appreciably in the GIT, and had decreased further in liver, kidneys and fat.
The tissue:plasma concentration ratios showed higher concentrations than in plasma during 3-48 hours in the GIT, liver, kidneys, fat, lymph notes and pancreas.
Details on excretion:
Mean overall recovery of 76.4% attributed to the loss of some material by volatilisation during application of the dose to the skin.
Total urinary excretion until the time of sacrifice accounted for means of 8?04, 12.36 and 9.91% dose in the pairs of rats sacrificed at 24, 48 and 72 hours recpectively.
Total excretion of radioactivity in faeces accounted for 1.17, 2.29 and 2.42% dose in rats sacrificed at 24, 48 and 72 hours respectively.
Radioactivity in the expired air traps accounted for means of 17.55, 15.77 and 14.78% dose until the times of sacrifice at 24, 48 and 72 hours respectively. Most of this radioactivity was un the first expired trap (overall mean of 12.57% dose) with a smaller proportion in the second trap (overall mean of 3.47% dose). Radioactivity in the expired air traps may mostly be attributed to 14C-limonene volatilised directly from the site of application.
Metabolites identified:
not measured
Conclusions:
Interpretation of results: low bioaccumulation potential based on study results.
An appreciable proportion of the dermally applied dose (48%) was recovered in the dose washings. The radioactivity in urine (10% dose) and in faeces (2% dose) showed that these proportions were absorbed throught the skin. Radioactivity in the expired air traps may mostly be attributed to volatilisation from the site of application. However, there may also be a contribution of absorbed and exhaled air.
Renal excretion of limonene and/or its metabolite is expected to be the major route (as confirmed by studies using the oral route). The radioactivity in the faeces, gastro-intestinal tract and liver indicate some hepatic elimination.
Executive summary:

Twelve male rats received one dermal dose of 5 mg/kg bw for 6 hours (or 3 hours for those sacrificed at this time). Pairs of rats were sacrificed at 3, 6, 12, 24, 48 and 72 hours after dosing. Urine, faeces, plasma and various tissues were sampled and their radioactivity content measured.

Almost half the administered dose was removed by skin washing at the end of the application time. The renal route is the main one but faeces and exhaled air are also involved.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Old study but a lot of details available. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.
Reason / purpose for cross-reference:
reference to same study
Objective of study:
other: excretion and metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
The metabolism of d-limonene was investigated in various animal species. Isolation and characterisation of several metabolites was done.
GLP compliance:
no
Radiolabelling:
yes
Species:
other: rat, hamster, guinea-pig, rabbit, dog
Strain:
other: Wistar, Syrian, Hartley, albino, mongrel
Sex:
male
Route of administration:
other: oral gavage for rat, rabbit, hamster and guinea pig; oral capsule for dog and human
Duration and frequency of treatment / exposure:
one administration only
Dose / conc.:
800 mg/kg bw (total dose)
Remarks:
rat, guinea pig, hamster and rabbit
Dose / conc.:
400 mg/kg bw (total dose)
Remarks:
dog
Dose / conc.:
1 600 other: mg
Remarks:
(57 and 60 kg bw)
No. of animals per sex per dose / concentration:
3 rats, 3 guinea pigs, 4 hamsters, 3 rabbits, 2 dogs and 2 human beings
Control animals:
no
Details on dosing and sampling:
For the excretion study and quantitative determination of acidic urine metabolites, [14C]d-limonene was administered orally.
For quantitative determination of neutral urine metabolites, non-labelled d-limonene was administered to another group of animals.
Animals were placed in metabolism cages, and urine and faeces were collected separately during 2 to 3 days.
Humans took the compound in hard gelatin capsules with water, and urine only was collected during 2 days.
Preliminary studies:
In Igimi H. and Nishimura M. (1974) Studies on the metabolism of d-limonene (p-mentha-1,8-diene) I. The absorption, distribution and excretion of d-limonene in rats, Xenobiotica, vol 4(2), 77-84: the absorption, distribution and excretion of d-limonene were investigated in rats. About 60% of administered radioactivity was recovered in urine, 5% from faeces and 2% from expired CO2 within 48h. In bile duct cannulated rats, about 25% of the dose was escreted in bile within 24h.
In Kodama R., Noda K. and Ide H. (1974) Studies on the metabolism of d-limonene (p-mentha-1,8-diene) II. The metabolic fate of d-limonene in rabbits, Xenobiotica, Vol 4 (2), 85-95: following oral administration to 3 male rabbits, about 72% and 7% of the dose was excreted in urine and faeces during 72 hours respectively.
Type:
excretion
Type:
metabolism
Details on absorption:
rapid and almost complete on the basis of the urinary excretion level, with most excretion occuring within the first 24 hours.
In the case of man, absorption may also be rapid and complete as the urinary excretion was about 85% during 48 hours (about 80% within 24 hours) in subject 1 (relatively low urinary excretion in subject 2 might be due to diarrhoea which occurred at 2 hours after administration).
Details on excretion:
The main route of excretion of d-limonene was via urine, 75-95% of administered radioactivity being excreted in the urine during 2-3 days.
Faecal excretion amounted to less than 10% in animals during 2-3 days.
Metabolites identified:
yes
Details on metabolites:
The reactions in the d-limonene biotransformation include the oxidation of methyl groups to hydroxyl and further to carboxylic acid derivatives, hydroxylation at the C-6 position, oxidation at the 8,9-double bond, and glycine and glucuronide conjugation. Hydroxylation at the C-6 position leads to the formation of p-mentha-1,8-dien-6-ol (M-X).
The 8,9-diol metabolites such as M-II and M-IV are likely to be derived through the epoxide intermediate. The structure of 2-hydroxy-p-menth-8-en-7-oic acid (M-VII) can be derived by hydration of the 1,2-double bond of perillic acid (M-III).
The major metabolite of d-limonene in the urine was M-IV in rat and rabbit, M-IX in hamster, M-II in dog and M-VI in guinea pig and man.

List of the metabolites of d-limonene:

M-I: p-mentha1,8 -dien-10 -ol

M-II: p-menth-1-ene-8,9 -diol

M-III: perillic acid

M-IV: perillic acid-8,9 -diol

M-V: p-mentha-1,8 -dien-10 -yl-beta-D-glucopyranosiduronic acid

M-VI: 8 -hydroxy-p-menth-1 -en-9 -yl-beta-D-glucopyranosiduronic acid

M-VII: 2 -hydroxy-p-menth-8 -en-7 -oic acid

M-VIII: perillylglycine

M-IX: perillyl-beta-D-glucopyranosiduronic acid

M-X: p-mentha-1,8 -dien-6 -ol

M-XI: p-menth-1 -ene-6,8,9 -triol

Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
The rate and amount of urinary excretion in rat, rabbit, hamster, guinea pig, dog and man suggest rapid elimination with no significant accumulation of compounds related to d-limonene in these species.
Executive summary:

The excretion and metabolism of d-limonene was studied in various species (rat, rabbit, hamster, guinea pig, dog and human after one oral administration (gavage or capsule according to species). Excretion was measured in urine and/or faeces during 2 to 3 days after administration.

Up to 11 metabolites were isolated and characterized.

The main route of elimination of d-limonene administered orally was via the urine in animals and man, 75 -95% of the administered radioactivity being excreted in the urine during 2 -3 days. Faecal excretion accounted for less than 10% of the dose in animals during 2 -3 days.

Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Preliminary studies:
In Igimi H. and Nishimura M. (1974) Studies on the metabolism of d-limonene (p-mentha-1,8-diene) I. The absorption, distribution and excretion of d-limonene in rats, Xenobiotica, vol 4(2), 77-84: the absorption, distribution and excretion of d-limonene were investigated in rats. About 60% of administered radioactivity was recovered in urine, 5% from faeces and 2% from expired CO2 within 48h. In bile duct cannulated rats, about 25% of the dose was escreted in bile within 24h.
In Kodama R., Noda K. and Ide H. (1974) Studies on the metabolism of d-limonene (p-mentha-1,8-diene) II. The metabolic fate of d-limonene in rabbits, Xenobiotica, Vol 4 (2), 85-95: following oral administration to 3 male rabbits, about 72% and 7% of the dose was excreted in urine and faeces during 72 hours respectively.
Type:
excretion
Type:
metabolism
Details on absorption:
rapid and almost complete on the basis of the urinary excretion level, with most excretion occuring within the first 24 hours.
In the case of man, absorption may also be rapid and complete as the urinary excretion was about 85% during 48 hours (about 80% within 24 hours) in subject 1 (relatively low urinary excretion in subject 2 might be due to diarrhoea which occurred at 2 hours after administration).
Details on excretion:
The main route of excretion of d-limonene was via urine, 75-95% of administered radioactivity being excreted in the urine during 2-3 days.
Faecal excretion amounted to less than 10% in animals during 2-3 days.
Metabolites identified:
yes
Details on metabolites:
The reactions in the d-limonene biotransformation include the oxidation of methyl groups to hydroxyl and further to carboxylic acid derivatives, hydroxylation at the C-6 position, oxidation at the 8,9-double bond, and glycine and glucuronide conjugation. Hydroxylation at the C-6 position leads to the formation of p-mentha-1,8-dien-6-ol (M-X).
The 8,9-diol metabolites such as M-II and M-IV are likely to be derived through the epoxide intermediate. The structure of 2-hydroxy-p-menth-8-en-7-oic acid (M-VII) can be derived by hydration of the 1,2-double bond of perillic acid (M-III).
The major metabolite of d-limonene in the urine was M-IV in rat and rabbit, M-IX in hamster, M-II in dog and M-VI in guinea pig and man.

List of the metabolites of d-limonene:

M-I: p-mentha1,8 -dien-10 -ol

M-II: p-menth-1-ene-8,9 -diol

M-III: perillic acid

M-IV: perillic acid-8,9 -diol

M-V: p-mentha-1,8 -dien-10 -yl-beta-D-glucopyranosiduronic acid

M-VI: 8 -hydroxy-p-menth-1 -en-9 -yl-beta-D-glucopyranosiduronic acid

M-VII: 2 -hydroxy-p-menth-8 -en-7 -oic acid

M-VIII: perillylglycine

M-IX: perillyl-beta-D-glucopyranosiduronic acid

M-X: p-mentha-1,8 -dien-6 -ol

M-XI: p-menth-1 -ene-6,8,9 -triol

Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
The rate and amount of urinary excretion in rat, rabbit, hamster, guinea pig, dog and man suggest rapid elimination with no significant accumulation of compounds related to d-limonene in these species.
Executive summary:

The excretion and metabolism of d-limonene was studied in various species (rat, rabbit, hamster, guinea pig, dog and human after one oral administration (gavage or capsule according to species). Excretion was measured in urine and/or faeces during 2 to 3 days after administration.

Up to 11 metabolites were isolated and characterized.

The main route of elimination of d-limonene administered orally was via the urine in animals and man, 75 -95% of the administered radioactivity being excreted in the urine during 2 -3 days. Faecal excretion accounted for less than 10% of the dose in animals during 2 -3 days.

Description of key information

Background

Orange oil is a substance of Unknown or Variable composition, Complex reaction products or Biological material (UVCB substances), or more specifically anNCS (Natural Complex Substance). As such, orange oil is part of the particular category of essential oils, extracts, fractions and distillation products of the citrus species of chapter 1.2, which are all variants of the botanicalRutacaefamily. A justification for read across within this category can be found in the “Reporting format for Natural Complexe Substances of the Citrus essential oils of the Rutacae family”. The other members of this category are lemon oil, mandarin oil, lime oil, grapefruit oil, bitter orange oil, and tangerine oil. The category is based on the part of the plant from which the NCSs are produced (pericarb of the fruit), the common methods of production, the same dominant constituent (limonene), and the same and/or similar constituents.

 

The toxicokinetics assessment of the citrus oils will be based on the toxicokinetic assessment of dominant constituent limonene. For the other constituents, physical/chemical parameters will be used to determine whether absorption via the oral, inhalation, and dermal route is expected.

 

Limonene

For limonene, the main route of excretion of d-limonene is via urine (75-95% of the dose orally administered). Faecal excretion accounts for less than 10% of the dose. When dermally administered, urine is again the main route of excretion, but almost half the administered dose is recovered by skin washing after exposure.

Furthermore, in a review done by the National Institute of Occupational Health in(Karlberg A.T. and Lundell B., 1993, Limonene, Beije B., Lundberg P. (eds). Criteria documents from the Nordinc Expert group. Arbete och Habsa, 35, 1 -254, the following information is mentioned:

- distribution: after absorption, d-limonene disappears rapidly from blood. This is mainly due to distribution to different tissues and the biotransformation. The blood clearance in human after exposure has been determined to 1.1 L/kg x hour. A high solubility of d-limonene in olive oil (calculated partition coefficient oil/blood = 140) as well as a long half-life in blood, in the slow elimination phase, indicate affinity to adipose tissues. Species differences in renal disposition and protein binding of d-limonene were observed.

- biotransformation: there are some different pathways in the metabolism of d-limonene among the various species explored, including oxidation at the 8,9 -double bond or the 1,2-double bond, oxidation of the methyl groups to hydroxyl and further to carboxylic acid derivatives, ring hydroxylaion at the C-1 and C-6 position, and glycine or glucuronide conjugation. Species differences in plasma and urinary metabolites were observed.

- excretion: 3 different phases of elimination of d-limonene were observed in human blood. The half-lifes in blood after exposure by inhalation for 2 hours to 450 mg d-limonene/m3 were ca. 3 min, 33, min and 750 minutes. D-limonene seems to be metabolised to a very large extent.

 

Other constituents

Physical/chemical parameters indicating whether absorption via the oral, inhalation and dermal route is expected, are for example molecular weight, water solubility, log Kow, and vapour pressure. Please find below a table containing these parameters for the other constituents of orange oil.

Constituent

Molecular weight

Water solubility (mg/l at 25°C)

Log Kow

Vapour pressure (Pa at 25°C)

α-Pinene

136.24

3.4834

4.27

536

α-Terpineol

154.25

1767.3

3.33

2.62

Citral

152.24

1101

3.45

12.2

Citronellal

154.25

514.22

3.53

33.9

Decanal

156.27

85.498

3.76

31.4

Linalool

154.25

709.26

3.38

11.1

Myrcine B

136.24

17.814

4.88

320

Octanal

128.22

831.88

2.78

199

Although, based on vapour pressure, all constituents have a low volatility (<0.5 KPa), exposure via inhalation is included based on the use of orange oil as a fragrance. Oral absorption and absorption via inhalation is expected for most substances, based on molecular weights <500, moderate to high water solubility, and log Kow between 1-4.α-Pinene and myrcine B may be less well absorbed orally due to their log Kow >4 and relatively low water solubility.

Dermal absorption is not favoured based on the molecular weights of the substances, neither can it be ruled out. As most of the substances have a log Kow <4 combined with a relatively high water solubility, dermal absorption is expected. Forα-pinene and myrcine B, which have a log Kow >4 and relatively low water solubility, dermal absorption is expected to be low to moderate.

 

Conclusion

The major constituent of orange oil, limonene, is expected to be readily absorbed by the oral and inhalation route. Dermal absorption is assumed to be lower, however, as no information is available on the amount of dermal absorption, it is assumed to be similar to oral absorption as a worst case. For the other constiuents of orange oil, the absorption via the oral, inhalation and dermal route is expected to be moderate to high.

For risk assessment, oral and dermal absorption are assumed to be similar (100%). In absence of information on the inhalation route, a default absorption of twice the oral absorption is assumed for inhalation absorption.

Key value for chemical safety assessment

Additional information