Linalool

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: US FDA Toxicological Principles for the Safety Assessment of Food Ingredients, Redbook 2000, IV.C. 4. a.

Version / remarks:

2007

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Linalool

EC Number:

201-134-4

EC Name:

Linalool

Cas Number:

78-70-6

Molecular formula:

C10H18O

IUPAC Name:

3,7-dimethylocta-1,6-dien-3-ol

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CRL Sprague-Dawley CD® IGS rats

Details on species / strain selection:

The Sprague-Dawley® rat is the system of choice because, historically, it has been a preferred and commonly used species for dietary toxicity tests. The current state of scientific knowledge does not provide acceptable alternatives to the use of live animals to accomplish the objective of this study. PSL is AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) accredited and certified in the appropriate care of all live experimental animals and maintains current staff training, ensuring animals were handled humanely during the experimental phase of this study, and meet all guideline standards.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Females: Yes
- Age at study initiation: Animals were approximately eight weeks at initiation
- Weight at study initiation: The weight variation did not exceed ± 20% of the mean weight for each sex.
- Housing: The animals were individually housed in suspended stainless steel cages which conform to the size recommendations in the latest Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Litter paper placed beneath the cage was changed at least three times/week.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY
Routine analysis consisting of each lot of feed used in this study was received from Envigo Teklad, Madison, WI. Water analysis is conducted periodically and the records are kept on file at Product Safety Labs. Water analysis was conducted by Precision Analytical Services, Inc., Toms River, NJ and South Brunswick Municipal Water Supply, South Brunswick, NJ.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 22-74 (Humidity was below the targeted lower limit for 11 days and above the targeted upper limit for 1 day during the study. A portable dehumidifier/humidifier was used to lower/raise the humidity levels during this time.)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 95 (females) or 96 (males)

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was used because it is recommended in the referenced guidelines (Section 8.C.), and because human exposure may occur via this route.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: All diets were prepared approximately weekly or more frequently.
- Mixing appropriate amounts with: 2016 Certified Envigo Teklad Global Rodent Diet®
- Storage temperature of food: All diets were refrigerated following preparation, unless presented to the test animals on the same day as diet preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of Test Substance: At the initial (Day 0), middle (Day 42), and final (Day 84) diet preparation, a sample of the test substance (neat) was retained for stability analysis. The neat test substance was found to be stable under the conditions of storage at PSL over the course of the study.

Stability in Dietary Matrix: Samples to verify the stability of the test substance in the dietary matrix were collected from the initial and final diet preparation, targeting the lowest and highest concentrations at initial and final collection time points. Samples from the respective dietary concentration were collected at the first presentation of the diet, and 4, 7, and 10 days after preparation. The results of the dietary stability samples were 57.0, 45.3, 41.5, and 44.1% on Day 0 and 42.5, 68.1, 61.5, and 68.7% on Day 10 of the nominal concentrations of 0.0671, 0.740, 0.132, and 1.478% of Linalool for Groups 2 and 4, respectively. These samples represent the highest and lowest concentrations presented over the course of the study. Day 7 samples were compared to the initial samples for overall in-room stability of the test substance in the dietary matrix, as the most accurate assessment of a weekly dietary feeding interval. Based on the 7-Day stability results, animals were considered to have received 68.1-77.6% of the target dietary concentrations of Linalool at 750 mg/kg/day, the highest dose tested.

Homogeneity: Samples to verify the homogeneity of the test substance in the dietary matrix were collected from the initial (Day 0) and final (Day 84) diet preparation, targeting the lowest and highest concentrations prepared at initial (Day 0) and final (Day 84) collection time points. Samples were taken from approximately the top, middle, and bottom strata of the diet mixer. Basal control diets were sampled from the middle strata of the mixer only. Analysis indicates that the test item was homogeneously distributed.

Concentration Verification: Samples were collected from representative animal diets of the initial (Day 0; as part of the homogeneity assessment), middle (Day 42), and final (Day 49) diet preparations during the study. The mean of the Day 42 samples were 102.8, 94.3, and 94.2% and the Day 49 samples resulted in 90.1, 101.7, and 96.4% of the target concentrations of 0.0671, 0.740, 0.132, and 1.478% for Groups 1-4, respectively.

Duration of treatment / exposure:

95 days (females) and 96 days (males)

Frequency of treatment:

Feeding study, continous treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Females, mean overall daily intake: 740.2 mg/kg bw/day;
Females, overall mean adjusted intakes (corrected for stability): 532.1 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Males, mean overall daily intake: 731.1 mg/kg bw/day;
Males, overall mean adjusted intakes (corrected for stability): 497.9 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Females, mean overall daily intake

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Males, mean overall daily intake

Doses / concentrations 5

Dose / conc.:

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

Remarks:

Females, mean overall daily intake

Doses / concentrations 6

Dose / conc.:

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

Remarks:

Males, mean overall daily intake

Doses / concentrations 7

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Males and females, mean overall daily intake

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: According to the sponsor, in consultation with the study director, based on a 14-day palatability/toxicity study and based on the results of a short-term oral gavage study according to OECD guideline 407 (supporting study) where the NOAEL was found to be 117 mg/kg bw/day.
- Fasting period before blood sampling for clinical biochemistry: At least 15 hours prior to blood collection

Positive control:

Not included

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations: Changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Likewise, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling), or bizarre behavior (e.g., self-mutilation, walking backwards) were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Day 0, prior to the first treatment with the test substance, and approximately weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: At least two times during acclimation, on Day 0 (prior to study start) and approximately weekly thereafter (intervals of 7 days ± 1).

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimation period and prior to study termination.
- Dose groups that were examined: All animals were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Via sublingual bleeding under isoflurane anesthesia during approximately Week 12 of the test period. Blood samples used to determine the prothrombin time and activated partial thromboplastin time (coagulation) were collected via the inferior vena cava under isoflurane anesthesia at terminal sacrifice.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animales were examined.
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Via sublingual bleeding under isoflurane anesthesia during approximately Week 12 of the test period.
- Animals fasted: Yes
- How many animals: All animals were examined.
- Parameters checked in table 2 were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: Once at the end of the dosing phase of the study.
- Animals fasted: Not specified
- How many animals: Not indicated.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once towards the end of the exposure period (no earlier than Week 11)
- Dose groups that were examined: All animals were tested.
- Battery of functions tested: Excitability, autonomic function, gait, and sensorimotor coordination (open field and manipulative evaluations), reactivity and sensitivity (elicited behavior), and other abnormal clinical signs, including but not limited to convulsions, tremors unusual or bizarre behavior emaciation, dehydration, and general appearance. In addition to the above observations, Forelimb and Hindlimb Grip Strength and Foot Splay measurements was obtained and recorded. Motor activity (MA) was monitored using an automated Photobeam Activity System®.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4+5)

HISTOPATHOLOGY: Yes (see table 6). Histological examination was performed on the preserved organs and tissues of the animals from both the control and high dose groups (Groups 1 and 4, respectively) as well as the animal that died (Animal Number 7062). In addition, gross lesions of potential toxicological significance noted in any test group at the time of terminal sacrifice were examined.

Optional endpoint(s):

Optional endpoints: Yes

ESTRUS CYCLICITY
-Time schedule for examinations: During Weeks 7 and 8 and again during Weeks 12 and 13.
- Parameters investigated: Vaginal smears for determination of estrus cycle stages

SPERM ANALYSIS
-Time schedule for examinations: On the day of necropsy
- Parameters investigated: Sperm Motility Evaluation, Total Sperm Count Determination, Sperm Morphology

Statistics:

Male and female rats were evaluated separately. Statistical analysis was conducted by using one or more of the following software applications: Provantis® version 9, Tables and Statistics, Instem LSS, Staffordshire, UK; and Pristima® version 7, Reporting, Xybion Corporation, Lawrenceville, NJ.

In-life endpoints that were identified as multiple measurements of continuous data over time (e.g., body weight, food consumption, etc.): Treatment and control groups were compared using a two-way analysis of variance (ANOVA), testing the effects of both time and treatment, with methods accounting for repeated measures in one independent variable. Significant interactions observed between treatment and time as well as main effects were further analyzed by a post hoc multiple comparisons test (e.g., Dunnett’s test) of the individual treated groups to control.

If warranted by sufficient group sizes, all endpoints with single measurements of continuous data within groups (e.g., organ weight, relative organ weight, etc.) were evaluated for homogeneity of variances (Bartlett) and normality (Shapiro). Where homogeneous variances and normal distribution were observed, treatment and control groups were compared using a one-way ANOVA. When one-way ANOVA was significant, a comparison of the treated groups to control was performed with a multiple comparisons test (e.g., Dunnett’s test). Where variances were considered significantly different, groups were compared using a non parametric method (e.g., Kruskal-Wallis non-parametric analysis of variance). When non-parametric ANOVA was significant, a comparison of treated groups to control was performed (e.g., Dunn’s test).

Clinical Pathology: Barlett’s test for homogeneity and Shapiro-Wilk test for normality. If preliminary test was not significant, One-way analysis of variance followed with Dunnett's test. If preliminary test was significant, Log transformations of the data to achieve normality and variance homogeneity were used.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

750 mg/kg bw: Yellow ano-genital staining, slight alopecia of the head and neck, and superficial eschar of the head, hair loss, staining, and broken teeth (male animal that was found dead on day 45), broken upper incisors (1/9 males), hair loss (2/10 females), slight to moderate alopecia of the left/right forelimb (2/10 females)

250 mg/kg bw: Slight swelling of the right ear (1/10 males), Superficial wound on the nose/snout (1/10 males), hair loss (1/10 males), red ocular discharge of the right eye (1/10 females), superficial eschar of the nose/snout (1/10 females), Red nasal discharge (1/10 females), exophthalmos (1/10 females)

80 mg/kg bw: Swelling of the right eye (1/10 females), exophthalmos (1/10 females), hair loss (2/10 females), Slight to moderate alopecia of the left/right forelimb (1/10 females)

Mortality:

mortality observed, non-treatment-related

Description (incidence):

750 mg/kg bw: One animal was found dead on Day 45. This death was considered incidential.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

750 mg/kg bw: Transient but significant decrease in mean daily bodyweight gain on Days 84-91 (females)

250 mg/kg bw: Transient but significant decrease in mean daily bodyweight gain on Days 35-42 and on Days 56-63 (males)

80 mg/kg bw: Transient but significant decrease in mean daily bodyweight gain on Days 56-63 (males)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

750 mg/kg bw: Transient but significant decrease in food consumption on Days 21-42 (females)

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

750 mg/kg bw: Transient but significant decrease in food efficiency on Days 56-63 (males)


250 mg/kg bw: Transient but significant decrease in food efficiency on Days 56-63 (males)

80 mg/kg bw: Transient but significant decrease in food efficiency on Days 56-63 (males)

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Any changes in clinical chemistry were considered unrelated to test substance administration, including that attained statistical significance, because they occurred sporadically, were considered due to biological variance among rats as magnitude of variation was minimal.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Any changes in thyroid hormones were considered unrelated to test substance administration, including that attained statistical significance, because they occurred sporadically, were considered due to biological variance among rats as magnitude of variation was minimal.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

750 mg/kg bw: No tail pinch response (1/9 males)

250 mg/kg bw: No tail pinch response (1/10 males), insignificant increases (p < 0.05-0.01) in motor activity at intervals 1, 2, and 6

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

750 mg/kg bw: Statistically significant increases (p < 0.01-0.001) in mean absolute, relative-to-body, and relative-to-brain kidney and relative liver-to-body weights (males) and statistically significant decreases (p < 0.05-0.01) in absolute brain and relative-to-body kidney and liver weights (females)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

750 mg/kg bw (male animal that was found dead on day 45): Enlarged urinary bladder containing 15 mL of reddish/yellow fluid, bilaterally enlarged kidneys with solid consistency, marked necrosis and moderate inflammation at the urinary bladder, bilateral moderate pelvic dilatation, moderate acute pelvic inflammation, and bilateral moderate fibrosis at the kidneys, bilateral diffusely hyperplastic urothelium, moderate chronic active inflammation of the prostate gland, adrenal cortical hypertrophy, mild atrophy of lymphoid tissues including the thymus and mandibular lymph nodes, and moderate atrophy of the splenic white pulp, moderate diffuse hyperplasia of the granulocytic cell series (white blood cell lineage) in the bone marrow which were interpreted as a results from chronic stimulation due to urinary tract inflammation.

It was concluded that the urinary bladder and kidney findings were indicative of an ascending pyelonephritis and chronic stress secondary to the urinary tract disease, rather than an effect of test substance administration.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw: Dose-dependent accumulation of alpha 2u-globulin in the renal cortical tubules (males and females), exacerbation of the background finding renal tubular basophilia (males)

250 mg/kg bw: Dose-dependent accumulation of alpha 2u-globulin in the renal cortical tubules (males and females)

80 mg/kg bw: Dose-dependent accumulation of alpha 2u-globulin in the renal cortical tubules (males and females)

All of the test substance-related kidney findings were not considered to be adverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Estrus Cyclicity: No effects observed.

Sperm Analysis: No effects observed.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 532.1 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 497.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study and based on the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for administration of the test item in the diet was determined to be of 497.9 and 532.1 mg/kg/day for male and female Sprague-Dawley rats, respectively, based on adjusted intake levels in the dietary matrix of the highest dose tested.

Executive summary:

The potential subchronic toxicity of the test item was evaluated in a GLP compliant study according to OECD guideline 408 in male and female rats after continuous administration in the diet for at least 90-days. Four groups of adult CRL: Sprague-Dawley CD® IGS rats (10/sex/group) were maintained on diets calculated to provide target dietary intakes of 0 (basal diet), 80, 250, and 750 mg/kg/day of the test item for at least 90 days (Groups 1-4, respectively). The neat test substance was found to be stable under the conditions of storage at PSL over the course of the study. Stability of the test substance in the diet was evaluated by analyzing dietary levels of Linalool on Days 0, 4, 7, and 10 following initial (Day 0) and final (Day 84) preparation. Test substance homogeneity in the diet was assessed by evaluating dietary levels of test substance in the top, middle, and bottom strata of the diet preparations at the beginning (Day 0) and end (Day 84) of the study. Diet preparation samples were analyzed to verify the concentration of the test item in the diets over the course of the study. Analysis indicates that the test item was homogeneously distributed. Based on the 7-Day stability results, animals were considered to have received 68.1-77.6% of the target dietary concentrations of the test item at 750 mg/kg/day, the highest dose tested. Body weight and food consumption measurements collected throughout the study were used to calculate the mean overall daily intake of the test item. The mean overall (Days 0-91) daily intake of the test item in rats fed dietary concentrations of 80, 250, and 750 mg/kg/day was 78.4, 344.3, and 731.1 mg/kg/day, respectively, for the males and 78.8, 245.9, and 740.2 mg/kg/day, respectively, for the females. Based on the stability data, weekly diet refreshment and measured dietary intakes, the overall mean adjusted intakes targeting the highest dose level of 750 mg/kg/day were at a minimum of 497.9 and 532.1 mg/kg/day for male and female rats, respectively. There were no test substance-related mortalities during the course of the study. There were no clinical observations, ophthalmological or other findings attributable to the dietary administration of the test item. There were no test-substance-related functional observation battery findings. Mean quantitative measurements of grip strength and foot splay in the test substance-treated groups were comparable to the respective control group. There were no changes in motor activity attributed to the dietary administration of the test item. There were no test substance-related changes in overall body weight and food consumption parameters. Statistically significant decreases observed in mean daily body weight gain for Groups 2 and 3 male and Group 4 female rats as well as mean daily food consumption for Group 4 female and mean food efficiency for Groups 2-4 male rats were transient and of no toxicological significance. Administration of the test item  in the diet for at least 90 consecutive days at targeted dietary levels of 0, 80, 250 and 750 mg/kg/day to male and female rats did not induce any test substance-related changes in hematology, coagulation, clinical chemistry, thyroid hormones or urinalysis. There was one early death in this study, stemming from an incidental cause. There were no macroscopic findings due to test substance administration. There were no test substance-related organ weight changes associated with the administration of Linalool. There were test substance-related microscopic findings in the kidneys of all treated male groups given 80, 250 or 750 mg/kg/day of the test substance. Findings consisted of accumulation of alpha-2u-globulin in the renal cortical tubules, which increased in magnitude associated with dose level in the male kidneys at all dietary levels. This was confirmed with positive Mallory-Heidenhain histochemical staining. Additionally, exacerbation of the background finding renal tubular basophilia was noted in the Group 4 males given 750 mg/kg/day. This finding is consistent with age-related chronic nephropathy, a condition specific to male rats historically, and of no relevance to humans. All of the test substance-related kidney findings were not considered to be adverse under the conditions of this study. Dietary administration of the test item to rats at target dietary levels of 0, 80, 250 or 750 mg/kg/day resulted in no statistically significant or test substance-related changes in percent motile sperm, epididymal sperm count, homogenization-resistant spermatid (HRS) count or percent abnormal sperm. Morphological abnormalities observed across all groups included amorphous heads, small heads, coiled tails and bent tails. None of these findings were specifically attributed to administration of the test item. Under the conditions of the study and based on the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for administration of the test item in the diet was determined to be of 497.9 and 532.1 mg/kg/day for male and female Sprague-Dawley rats, respectively, based on adjusted intake levels in the dietary matrix of the highest dose tested.