Bergamot, ext.

Endpoint summary

Description of key information

Repeated dose toxicity via oral route (WoE):
- Subacute oral toxicity study in rats (similar to OECD 407, read-across): NOAEL = 117 mg/kg bw/day
- Subchronic (6-month) toxicity study in dogs (similar to OECD 409, read-across): NOAEL = 100 mg/kg bw/day
- Subchronic toxicity study in rats (similar to OECD 408, read-across): NOAEL = 600 mg/kg bw/day
- Subchronic toxicity study in mice (similar to OECD 408, read-across): NOAEL = 500 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

28 June 1988 - 27 January 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to an equivalent of OECD guideline 407 and under GLP conditions. Klimisch 2 reliability has been assigned in accordance with (ECHA Practical Guide #6) due to the read-across purpose of this study.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: approx. 43 days
- Weight at study initiation:
Males: 206.2-249.3 g
Females: 142.8-191.2 g
- Housing: Individually under standard laboratory conditions
- Diet (e.g. ad libitum): Ad libitum, rodent chow
- Water (e.g. ad libitum): Ad libitum, tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 +/- 6
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 12 July 1988
To: 11 August 1988

Route of administration:

oral: gavage

Vehicle:

other: 1% methylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Calculated amount of test material was weighed into a beaker of appropriate size. The amount was then transferred into a pre-calibrated container using a rinsing process. Vehicle (1% methylcellulose) was added to achieve the appropriate volume and the container was placed on a magnetic stirrer and stirred for five minutes while being sonicated. Test mixtures were prepared fresh weekly.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not relevant
- Amount of vehicle (if gavage): 10 ml/kg B10

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Based on peak area for selected constituents test material is stable in 1% methylcellulose (Memo Lorillard, 9 August 1988)

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
160, 400, 1000 mg coriander oil/kg/day, equivalent to 117, 292, 729 mg linalool/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: mortality and morbundity

DETAILED CLINICAL OBSERVATIONS: Yes, including physical examination
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to initiation of study and at termination
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes, overnight
- How many animals:
Initiation: 10 animals/sex
Termination: all animals
- Parameters checked:

Leukocyte count (WBC)
Erythrocyte count (RBC)
Hemoglobin (HGB)
Hematocrit (HCT)
Platelet count (PLATELET)
Leukocyte differential count
Cell morphology
Myeloid/erythroid ratio (M/E)-(Groups 1 and 4 only at Week 5)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to initiation of study and at termination
- Animals fasted: Yes, overnight
- How many animals:
Initiation: 10 animals/sex
Termination: all animals
- Parameters checked:

Sodium (SODIUM)
Potassium (POTAS)
Chloride (CHLORIDE)
Total protein (T PROT)
Blood urea nitrogen (BUN)
Creatinine (CREAT)
Glucose (GLUCOSE)
Alanine aminotransferase (AL1)
Albumin (ALBUMIN)
Calcium (CALCIUM)
Total carbon dioxide (T C02)
Total bilirubin (T BILI)
Aspartate aminotransferase (AST)
Gamma glutamyltransferase (GGT)
Alkaline phosphatase (ALK P)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, in all animals

External surfaces
All orifices
Cranial cavity
Carcass
Nasal cavity and paranasal sinuses
Cervical tissues and organs
External surface of the brain and spinal cord (at necropsy) ; the cut surfaces of the brain were examined at the time of tissue trimming
Thoracic, abdominal and pelvic cavities and their viscera

ORGAN WEIGHTS: Yes, in all animals. Fat was first removed.

Brain (including brainstem)
Spleen
Liver
Heart
Kidneys
Testes with epididymides
Thyroid with parathyroids
Adrenals
Ovaries
Pituitary

HISTOPATHOLOGY: Yes, tissues from all control and high-dose animals as presented below. Heart, liver, stomachs and lesions from all low- and mid-dose animals were also examined.

Femoral bone marrow
Lung (with mainstem bronchi)
Ovaries
Lesions
Kidneys
Adrenals
Testes with epididymides
Duodenum, Jeiunum, ileum
Brain with brainstem (medulla/pons cerebellar cortex, cerebral cortex)
Pancreas
Urinary bladder
Pituitary
Uterus
Thyroid (parathyroids)
Heart
Liver
Spleen
Colon, cecum, rectum
Stomach
Mesenteric lymph node

Following tissues from control and high-dose animals were preserved but not examined:

Thymus
Esophagus
Sciatic nerve w/skeletal muscle
Cervical, thoracic and lumbar spinal cord
Salivary gland (mandibular)
Trachea

Other examinations:

A femoral bone marrow smear was prepared from all sacrificed animals and preserved in methanol and stained with Wright stain.

Statistics:

Mean body weight changes (Weeks 0-4), total food consumption (Weeks 1-4), clinical pathology data (except cell morphology), absolute organ weight and organ-to-body weight ratios of the control group were compared statistically to the data from the same sex of the treated groups.

Transformation of heterogeneous data was performed. If homogeneity of variances was not succesful analyses was performed on rank-transformed data. Group comparisons performed at 5% two-tailed probability level.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
One high-dose female was found dead on day 2 of the study and was replaced by another animal (as directed by the study director).
One high-dose male was found dead on day 9 of the study, cause of death was attributed to a handling incident.

Isolated incidences of alopecia and sores were noted in both males and females of several groups (also control group). One animal of the control group showed a small movable head mass at week 1.

CLINICAL CHEMISTRY
For males of the high and mid-dose group the glucose and albumin levels were significantly lower than that of control. Total protein was higher than that of control males in these groups. For males of the high-dose group also calcium levels were higher than that of control.

In females, only total protein and albumin were found higher in the high-dose group than that of controls.

ORGAN WEIGHTS
Absolute kidney weight was higher in males and females of the high-dose group, but relative kidney weight was higher in males of the mid- and high-dose group and females of the high-dose group. For males, this can be confined to the histopathological effects.

Absolute liver weight was higher in males of the mid- and high-dose group and in females of the low-, mid- and high-dose group. Relative liver weight was significantly different in the mid- and high-dose group of both males and females as compared to controls.

Absolute thyroid/parathyroid weight was lower in males of the low- and high-dose group, while relative thyroid/parathyroid weight was only significant different in low-dose males as compared to controls. In females, only absolute thyroid/parathyroid weight was significantly different from controls in the low-dose group. These findings were without clear-dose-response relationship.

GROSS PATHOLOGY
Prominent liver effects (mainly prominent reticular pattern in M+F, mottled liver in F) were found in males and females of the mid- and high-dose groups. No other prominent effects were observed when compared to controls.

Kidney effects were largely confined to males of the mid- and high-dose group (pale area). Females showed no prominent effects, while the control group did not show any effect.

Stomach effects (thickened mucosa, dark area) were observed in both males (high-dose) and females (mid- and high-dose). Control group showed no effect. It is unclear if this effect is caused by administration of the test article by gavage in the animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
Increased periportal cytoplasmic vacuolization was observed in the liver of females of the low-, mid- and high-dose group. This was not observed in males or the control group. The cytoplasmic vacuolization was graded as slight in all affected animals, and is not expected to have an overall health of the animals or hepatic function. This is supported by ALT and AST levels being comparable to controls. Therefore this is not considered an adverse effect.

Regeneration and necrosis of tubules in the kidney were observed in males of the high-dose group. The effects seen were microscopically similar to those seen in the kidneys of male rats treated with a variety of hydrocarbons (due to interaction with the in abundance produced alpha-2-u globulin protein), which is a male rat-specific effect. In females, no prominent effects were observed as compared to controls.

Inflammation and acanthosis of the stomach was observed in females of the mid- and high-dose group. This was not seen in males and the control group. It is unclear if this effect is caused by administration of the test article by gavage in the animals.

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: stomach and kidney effects (clinical chemistry and pathology)

Dose descriptor:

NOAEL

Remarks:

linalool

Effect level:

117 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: stomach and kidney effects (clinical chemistry and pathology)

Critical effects observed:

not specified

Conclusions:

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) was established to be 160 mg/kg bw/day. This corresponds with 117 mg/kg bw/day linalool. Based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC, linalool does not need to be classified for oral repeated dose toxicity.

Executive summary:

This report presents the findings of a 28-day oral toxicity Study designed to evaluate the toxicity of B10 (72.9% linalool) in rats when administered daily by gavage to achieve dosage levels of 160, 400, and 1000 mg/kg of body weight per day . A concurrent control group received only the vehicle. Criteria evaluation for signs of compound effect included survival, clinical observations, body weights, food consumption, clinical pathology, gross pathology, organ weights, and histopathology.

No treatment-related effects on survival, clinical observations, body weights, or food consumption were observed. Treatment-related increases in total protein and- serum albumin were observed in the mid- and high-dose males and the high-dose females. Serum calcium was also increased in these same treated groups, apparently as a secondary response to the increase in albumin, its major serum binding protein. The pathogenesis of these increases, however, is unknown. Treatment-related lesions were noted histopathologically in the kidney of the high-dose males, in the nonglandular region of the stomach in the mid- and high-dose females, and in the liver of the high-dose females. Similar lesions of the liver were also noted in the low- and mid-dose females, but at a lower incidence. The findings in liver of females were considered to be slight and unlikely to influence liver function. Liver enzymes like AST and ALT were not changed and the histopathological effect was therefore considered rather adaptive than an adverse effect. Kidney lesions were seen in males only and are related to alpha-2u-globulin nephropathy which is not of relevance for human. Stomach lesions are considered to be result of bulk administration of an irritant substance via gavage.

Therefore, the No Observed Adverse Effect Level (NOAEL) was established to be 160 mg/kg/day, which corresponds to a NOAEL of 117 mg/kg bw/day linalool. Based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC, linalool does not need to be classified for oral repeated dose toxicity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

117 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Four reliable studies (all assigned Klimisch 2) are available for this endpoint and therefore the quality of the database is considered sufficient. One of the four studies is chosen accordingly as most relevant study with regard to the NOAEL.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No repeated dose toxicity studies are available with Bergamot oil itself. As an alternative, a weight of evidence approach was chosen based on the available repeated dose toxicity studies in different species for the major constituents of Bergamot oil: d-limonene (CAS nr 5989-27-5), linalyl acetate (CAS nr. 115-95-7) and linalool (CAS nr. 78-70-6). Using these data results in a coverage of >80% of the composition of Bergamot oil, which ensures that the same data requirements are covered as for mono-constituent substances.

Data for d-limonene and linalool are available, as further summarized below. The data for linalool also cover linalyl acetate, as this substance is quickly metabolized to linalool and read-across is therefore justified.

 

Selected data for derivation of the DNEL for Bergamot oil

The following NOAELs en LOAELs were available for the major constituents d-limonene and linalool.

 

Constituent	Study	Animal	Type of study	NOAEL (mg/kg bw/day)	LO(A)EL (mg/kg bw/day)	Critical effect LOAEL
d-limonene	Webb, 1990	Beagle dogs	6-month, oral gavage	100	1000	Increased kidney weight
	Jameson, 1990	B6C3F1 mice	90-d, oral gavage	500	1000	clinical signs, decreased bodyweights in males
	Jameson, 1990	F344/N rats	90-d, oral gavage	600*	1200	clinical signs, decreased bodyweight gains
linalool	Serota, 1990	rats	28-day oral gavage	117	292	effects on stomach and kidney

Table 1 relevant NOAELs/LOAELs for DNEL derivation *Observed nephrotoxic effects in male rats were not considered relevant for humans (see Meek M.E. et al. (2003) A Framework for Human Relevance Analysis of Information on Carcinogenic Modes of Action, 33(6): 591 -653).

 

All studies were taken into account to evaluate the point of departure for derivation of the DNEL. Hydrocarbons including d-limonene are known to result in nephropathy in male rats. From the study with d-limonene in dogs (Webb, 1990) it was concluded that d-limonene should be classified as a male-rat specific nephrotoxin and that these effects are species- and sex-specific and not relevant for other species including humans (see also Meek et al., 2003). When considering the non relevance of the nephrotoxic effects for humans, the NOAEL in the 90-day oral gavage study in rats (Jameson, 1990) was adjusted to 600 mg/kg bw/day (see Table 1), based on decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day.

The most critical NOAEL is 100 mg/kg bw/day, based on a 6-month study in dogs with d-limonene (Webb, 1990). This NOAEL may not be the most appropriate value to base the DNEL on given the fact that the next dose level (1000 mg/kg bw/day) only produced effects on kidney weight which were regarded non-adverse effects. The second most critical NOAEL is 117 mg/kg bw/day, based on a 28-day oral gavage study with linalool in rats. The observed effects may be the result of bulk administration via gavage, but in the absence of definitive evidence to the contrary, these findings must be considered as treatment-related. This NOAEL of 117 mg/kg bw/day based on study with linalool in rats was taken as point of departure for derivation of the DNEL

Repeated dose toxicity of d-limonene

Available studies

Three repeated dose toxicity studies were available for limonene. The studies were performed in different species, and are described below.

The 6-month subchronic toxicity study in dogs (Webb, 1990) was performed similar to OECD Guideline 409. The test substance d-Limonene was administered through gavage to groups of adult beagle dogs (5/sex/dose) at dose levels of 0 (tap water), 0.12 or 1.2 mL/kg bw/day (which equals 0, 100 or 1000 mg/kg bw/day) for 180 days. Feed consumption and body weight were unaffected by treatment. Clinical signs of toxicity noted were excretion of soft faeces, dose-related occasional mild discomfort during defaecation, sporadic episodes of emesis and diarrhoea. No treatment-related differences other than an increase in serum cholesterol (35%) and serum alkaline phosphatase levels (two-fold increase) at 1000 mg/kg bw/day were observed in male and female dogs. Linear regression analyses indicated a positive and significant dose-related increase in absolute and relative female kidney weight and relative male kidney weight. There were no histopathological changes in the kidneys that could be associated with the organ-weight changes. Furthermore, there was no evidence of hyaline droplet accumulation or of any other sign of hydrocarbon-induced nephropathy typical of those seen in male rats treated with d-limonene. It was concluded that d-limonene did not induce any adverse renal changes in dogs. Under the test conditions, the NOAEL and LOAEL for beagle dogs were considered to be 100 and 1000 mg/kg bw/day respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight.

The National Toxicology Program (NTP) performed two 90-day repeated dose toxicity studies in mice and in rats (Jameson, 1990). These studies were performed according to a method similar to OECD Guideline 408 and in compliance with GLP.

• In the study in mice, B6C3F1 mice (10 animals/sex/dose ) were administered d-limonene at dose levels of 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day (in corn oil) through oral gavage for 90 days (5 days/week). One of 10 males and 2/10 females that received 2000 mg/kg bw/day and 1/10 females that received 500 mg/kg bw/day died before the end of the study. Several animals in other groups died as a result of gavage error. Clinical signs including rough hair coats and decreased activity were observed at the two highest doses. Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the male vehicle controls and 2% lower than that of the female vehicle controls. An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day. Under the conditions of this study, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
• In the study in rats, F344/N rats (10 animals/sex/dose) were administered d-limonene at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day (in corn oil) through oral gavage for 90 days (5 days/week). Five of 10 males and 9/10 female rats that received 2400 mg/kg bw/day died during week 1. Final mean body weights of male rats in the 600, 1200 or 2400 mg/kg bw/day dose groups were respectively 6%, 12% or 23% lower, than that of the male vehicle controls. Final body weight of the female rats that received 2400 mg/kg bw/day was 11% lower than the that of the female vehicle controls. Clinical signs including rough hair coats, lethargy and excessive lacrimation were observed at 1200 or 2400 mg/kg bw/day. No treatment-related histopathologic lesions were observed in female rats. Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups. Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. This mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans (see Meek et al., 2003).

Under the conditions of this test, the NOAEL for female rats was considered to be 600 mg/kg bw/day. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats could be identified in this study. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. When considering the non relevance of the nephrotoxic effects for humans (see Webb 1990 study in dogs and Meek et al., 2003), the NOAEL for male rats would be 600 mg/kg bw/day, based on decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day.

 

Repeated dose toxicity of linalool (also covering linalyl acetate)

Available studies

One repeated dose toxicity study was available for coriander oil which contains 72.9% linalool. In a 28-day oral toxicity study, rats (10 animals /sex/dose) were administered coriander oil (containing 72.9% linalool) at dose levels of 160, 400, and 1000 mg/kg bw/day (in 1% methylcellulose) through oral gavage for 28 days (7 days/week). No treatment-related effects on survival, clinical observations, body weights, or food consumption were observed. Treatment-related histopathological lesions were noted in the kidney of the high-dose males, in the nonglandular region of the stomach in the mid- and high-dose females, and in the liver of the high-dose females. Similar lesions of the liver were also noted in the low- and mid-dose females, but at a lower incidence. The findings in liver of females were considered to be slight and unlikely to influence liver function. Liver enzymes like AST and ALT were not changed and the histopathological effect was therefore considered rather adaptive than an adverse effect. Kidney lesions were seen in males only and are related to alpha-2u-globulin nephropathy which is not of relevance for humans. Stomach lesions are possibly the result of bulk administration via gavage, but in the absence of definitive evidence to the contrary, these findings must be considered as treatment-related.

Under the conditions of this test, the NOAEL was established to be 160 mg/kg bw/day, which corresponds to a NOAEL of 117 mg/kg bw/day linalool, based on observed effects in the stomach in both sexes (thickened mucosa, inflammation and acanthosis) and kidney in males (regeneration and necrosis of tubules, alpha-2u-globulin nephropathy). Although hepatocellular cytoplasmic vacuolisation and increased liver weight were observed in treated females, these effects are probably the result of metabolizing enzyme induction and can therefore be considered rather an adaptive change than an adverse effect. The LOAEL was established to be 400 mg/kg bw/day, which corresponds to a LOAEL of 292 mg linalool/kg bw/day.

 

 

 

 

 

 

 

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
No repeated dose toxicity studies are available with Bergamot oil itself. As an alternative, a weight of evidence approach was chosen based on the availability of repeated dose toxicity studies in different species for the major constituents of bergamot oil: d-limonene, linalyl acetate and linalool.
Selected study with linalool provides the most critical NOAEL relevant for humans.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: stomach; urogenital: kidneys

Justification for classification or non-classification

Evaluation of repeated dose toxicity studies of Bergamot oil is based on the major constituents of bergamot oil: d-limonene, linalyl acetate and linalool in a weight of evidence approach. The most critical NOAEL that is considered relevant for humans originates from a study with linalool in rats and was established to be 117 mg/kg bw/day. Based on this evaluation and the criteria outlined 1272/2008/EC (CLP/EU-GHS), Bergamot oil does not have to be classified with regard to repeated dose toxicity.