Bergamot, ext.

Repeated dose toxicity: oral

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

8008-52-4

Cas Number:

8008-52-4

IUPAC Name:

8008-52-4

Details on test material:

- Name of test material (as cited in study report): B10 (Coriander oil)
- Physical state: Liquid
- Storage condition of test material: Protected from light in the refrigerator
- Stability under test conditions: Stable

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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)

2

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)

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

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.