Linalool

Immunotoxicity

Description of key information

Antibody Plaque Forming Cells (PFC) Assay and Host Resistance Assay (no guideline): NOAEL 375 mg/kg bw/day (intragastrically exposure)

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

immunotoxicity

Remarks:

acute

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was not conducted according to an official OECD guideline, but is comparable to the US FDA program. The design of the study and the results are clearly described. No individual data is included, but basic data is acceptable.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Antibody Plaque Forming Cells (PFC) Assay and Host Resistance Assay (Listeria monocytogenes bacterial challenge according to:
1) Hinton D. M. (1992) Testing guidelines for evaluation of the immunotoxic potential or direct food additives. Critical Reviews in Food Science and Nutrition 32, 173-190.
2) Luster M. I..et al(1988) Development of a testing battery to assess chemical-induced immunotoxicity: National Toxicology Program's guidelines for immunotoxicity evaluation in mice. Fundamental and Applied Toxicology 10, 2-19.
3) Thomas P., et al (1985) Evaluation of host resistance and immune function in cadmium-exposed mice. Toxicology and Applied Pharmacology 80, 446-456.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI, USA).
- Age at study initiation: approx. 6-8 weeks
- Housing: Group housed in polypropylene cages with hardwood chip bedding (Sani-Chips, Murphy Forest Products, Montville, N J, USA)
- Diet: Certified Purina Rodent Chow 5002 (Ralston Purina, St Louis, MO, USA) - ad libitum
- Water: tap water ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 10-70
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

other: intragastrically

Vehicle:

other: methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: Solubility
- Concentration in vehicle: 10 mL/kg
- Amount of vehicle: 1%

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

5 days

Frequency of treatment:

once daily for 5 consecutive days

Dose / conc.:

94 mg/kg bw/day

Dose / conc.:

188 mg/kg bw/day

Dose / conc.:

375 mg/kg bw/day

No. of animals per sex per dose:

30 mice/dose group (10 for PFC asssay and 20 for host resistance assay/dose group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels for the immunotoxicity assays were chosen based on the results of acute toxicity tests (5-day repeated oral dose range-finding studies) conducted with Linalool (NOAEL 375 mg/kg bw/day)

Observations and clinical examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice each day (a.m. and p.m.)

BODY WEIGHT: Yes
- Time schedule for examinations: PFC assay: at the time of dosing initiation, exposure day 5, and at autopsy

Sacrifice and pathology:

Not performed

Cell viabilities:

SPLEEN: Yes
- Method: Spleen cells were counted and viability was assessed by trypan blue exclusion
- Dose groups: Vehicle control, test article treated, naïve (untreated) and positive control groups
- No. of animals: 10 mice/dose group (PFC Assay)

THYMUS: No

BONE MARROW: No

Humoral immunity examinations:

ANTIBODY PLAQUE FORMING CELLS (PFC) ASSAY: Yes
- Method: The anti-SRBC PFC response to sheep red blood cells (SRBCs) was performed according to the method of Cunningham and Szenberg (Cunningham A. J. and Szenberg A. (1968) Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14, 599), as modified by Thomas et al. (Thomas P., et al (1985) Evaluation of host resistance and immune function in cadmium-exposed mice. Toxicology and Applied Pharmacology 80, 446-456).
- Dose groups: Vehicle control, three test article treated, naïve (untreated) and positive control groups
- No. of animals: 10 mice/dose group (5 mice/positive control group)

Specific cell-mediated immunity:

OTHER ASSAYS
- Method: Host resistance assay (Listeria monocytogenes bacterial challenge) - Mortality
- Dose groups: Vehicle control, three test article treated, naïve (untreated) and positive control groups
- No. of animals: 20 mice/dose group

Positive control:

For each PFC assay, five animals were injected ip with cyclophosphamide (80 mg/kg) 24 hr prior to assay.

Statistics:

Dunnett's test and chi-square analysis were used to evaluate mean survival time and mortality data in the Host resistance assay. For continuous response data, analysis of variance (two-tailed) and appropriate post-hoc comparisons using Dunnett's test were performed on natural logarithmic or logit transformed PFC data. For the positive control group, post-hoc comparisons were made to the naive control group using a Student's t-test. Individual groups of data were evaluated for outliers prior to statistical analysis (Dixon W. J. (1953) Processing data for outliers. Biometrics 9, 74-89). The level of significance in all tests was P<=0.05.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Gross pathological findings:

not examined

Cell viabilities:

no effects observed

Humoral immunity examinations:

no effects observed

Specific cell-mediated immunity:

no effects observed

Non-specific cell-mediated immunity:

no effects observed

Other functional activity assays:

not examined

Other findings:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

375 mg/kg bw/day

Sex:

female

Basis for effect level:

other: cell viability (spleen); humoral immunity (PFC assay); cell-mediated immunity (Host resistance assay (Listeria monocytogenes bacterial challenge)-mortality

No effects were reported in both the Host resistance assay and the Antibody Plaque-forming cell assay.

Host resistance assay mortality observations: group mortality (%):

Control: 15 %

Dose level 94 mg/kg/d: 15 %

Dose level 188 mg/kg/d: 21 %

Dose level 375 mg/kg/d: 15 %

Anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) assay observations: specific cellular activity (PFC/10*6 viable spleen cells):

Control: 1309 ± 234

Dose level 94 mg/kg/d: 1283 ± 192

Dose level 188 mg/kg/d: 2083 ± 195

Dose level 375 mg/kg/d: 1242 ± 175

Cyclophosphamide positive control: 77 ± 15

Anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) assay observations: total spleen activity (PFC x 10*4 spleen):

Control: 7.0 ± 1.1

Dose level 94 mg/kg/d: 9.7 ± 1.7

Dose level 188 mg/kg/d: 12.1 ± 1.3

Dose level 375 mg/kg/d: 7.1 ± 1.2

Cyclophosphamide positive control: 0.4 ± 0.1 

Conclusions:

Linalool produced no significant alteration in the PFC response. No increase in mortality after bacterial challenge was observed. No changes in body weight, spleen and thymus weight as well as spleen cellularity were noted. These results indicated that Linalool did not modulate the cell-mediated or humoral immune response.

Executive summary:

A rapid screening protocol incorporating key elements of the US National Toxicology Program's immunotoxicity tier testing strategy was used to evaluate the effect of Linalool on humoral and cell-mediated immune responses. The test compound was administered intragastrically on a daily basis for 5 days at 375, 188, and 94 mg/kg/day to female B6C3F1 mice, 6-8 wk old.

A host resistance assay (Listeria monocytogenes bacterial challenge) was conducted to assess cell-mediated immunity by observation of increase in mortality rate or reduction of survival time.

Humoral immunity was measured by the antibody plaque-forming cell (PFC) response to sheep red blood cells (SRBCs). Body weights, spleen and thymus weights and spleen cellularity were also measured. Cyclophosphamide (80 mg/kg) served as an immunosuppressive positive control agent.

Linalool produced no significant alteration in the PFC response and no increase in mortality after bacterial challenge was observed. No changes in bodyweight, thymus and spleen weight as well as spleen cellularity were noted. These results indicated that linalool did not modulate the cell-mediated or humoral immune response.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

375 mg/kg bw/day

Species:

mouse

Quality of whole database:

Comparable to guideline study with acceptable restrictions

Effect on immunotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In this rapid screening protocol, linalool produced no significant alteration in the PFC response, no increase in mortality after bacterial challenge was observed, and no other changes related to immunologic responses were observed. The results indicate that linalool did not modulate the cell-mediated or humoral immune response.

Justification for classification or non-classification

As no effects were observed, linalool does not need to be classified based on the criteria outlined in Annex I of Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/1182.