Litsea cubeba, ext.

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

repeated dose toxicity: inhalation, other

Remarks:

inhalation during developmental toxicity study (GD 6-15)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Developmental toxicity study comparable to guideline study (OECD Guideline 414) with sufficient documentation, well documented

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: See remarks:

Remarks:

Although this investigation has only been published as an abstract, additional data on the test conditions are available from a valid developmental toxicity study (Gaworski et al., 1992) from the same working group. The results on maternal toxicity in the developmental toxicity study confirm the test results of the repeated dose inhalation toxicity study. Acceptable restrictions in documentation are: number of exposed animals not given, no details on test results given; study acceptable for derivation of a NOAEL for local and systemic effects based on weight of evidence approach

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

F344/N rats were expo 6 hrs/day for either 21 consecutive days at concentrations of 10 & 34 ppm, or 68 ppm, or for 13 weeks at concentrations of 1, 3, or 10 ppm. No mortalities occurred during the 21 day study. Investigated endpoints were mortality, body weight, organ weigths (13 week study only), signs of ocular, nasal and oral irritation, and histological examination of nose tissues, larynx, trachea and lungs. Reversibility was investigated after a 5-week recovery period.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

not specified

Route of administration:

other: exposure to vapours for low concentrations; exposure to vapour/aerosol mixtures to achieve higher concentrations

Type of inhalation exposure:

whole body

Vehicle:

other: nitrogen

Remarks on MMAD:

MMAD / GSD: no data

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION (Information taken from Gaworski et al., 1992)

- Exposure apparatus: Rochester-type stainless steel inhalation chambers of 1 cubic meter volume
- Method of conditioning air: filtered through coarse particulate and HEPA filters and an activated carbon cartridge
- System of generating particulates/aerosols: aerolization of liquid citral with a DeVilbiss Model 41 nebulizer using nitrogen as a carrier gas to prevent degradation, which had been observed during nebulization in the presence of air
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: not data
- Air change rate: 15 +- 3 changes/hr
- Method of particle size determination: cascade impactor; MMAD 4.2 µm, geometric standard deviation 1.9; about 90% of the aerosol with aerodynamic diameter < 10 µm
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography with flame ion detection
- Samples taken from breathing zone: not specified

VEHICLE
- Justification for use and choice of vehicle: to prevent degradation of citral
- Composition of vehicle: nitrogen

Analytical verification of doses or concentrations:

yes

Remarks:

(Information taken from Gaworski et al., 1992)

Details on analytical verification of doses or concentrations:

Analysis of vapour and aerosol samples indicated that citral purity degraded less than 5% with nitrogen nebulization, with only a slight entrichment of the neral isomer (< 3%) in the vapour phase
Chamber vapour atmospheres sampled by cryogenic trapping. Sampling was performed at a flow rate of 400 mL/min for 25 min through two all-glass and teflon traps immersed in a dry-ice methanol bath. The tubes were rinsed with 10 mL of isopropanol containing n-octanol as internal standard. Analysis performed by gas chromatography and flame ion detection.

Duration of treatment / exposure:

21 days or 13 weeks

Frequency of treatment:

Daily for 21 days, 6 h/d
13 w, 5 d/w, 6 h/d
additional group with 5 week recovery period after the 13 week treatment

Dose / conc.:

10 ppm

Remarks:

63 mg/m3
21 Days exposure

Dose / conc.:

34 ppm

Remarks:

215 mg/m3
21 Days exposure

Dose / conc.:

68 ppm

Remarks:

430 mg/m3
21 Days exposure

Dose / conc.:

1 ppm

Remarks:

6 mg/m3, 13 week exposure

Dose / conc.:

3 ppm

Remarks:

19 mg/m3, 13 week exposure

Dose / conc.:

10 ppm

Remarks:

63 mg/m3, 13 week exposure

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: not specified

FOOD CONSUMPTION: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Key result

Dose descriptor:

NOAEC

Remarks:

subchronic inhalation

Effect level:

34 ppm

Based on:

test mat.

Sex:

not specified

Remarks on result:

other: 215 mg/m3

Key result

Dose descriptor:

LOAEC

Effect level:

68 ppm

Based on:

act. ingr.

Sex:

not specified

Basis for effect level:

body weight and weight gain

clinical signs

Remarks on result:

other: 430 mg/m3

Critical effects observed:

not specified

Table: Overview on findings in a subacute and a subchronic inhalation study

Exposure time	Concen-   tration (ppm)	Mortality	Body weight gain	Organ weights	Signs of irritation	Histological findings
						Nasal respiratory epithelium	Other
21 d	10^a	no	No effect	No data	No effect	Dose related increase of chronic active inflammation, hyperplasia, squamous metaplasia and goblet cell atrophy^c	No data
	34^a	no	No effect	No data	No effect		No data
	68^b	no	Significant reduction	No data	Severe nasal, oral and ocular irritation; corneal inflammation and ulceration		Signs of irritation in trachea and lungs
13 w	1^a	no	No effect	No effect	No effect	No effect	No effect
	3^a	no	No effect	No effect	No effect	No effect	No effect
	10^a	no	No effect	No effect	No effect	No effect	Minimal hyperplasia and squamous metaplasia of the laryngeal epithelium
13 w + 5 w recovery	10						Effect in laryngeal epithelium fully reversible

^avapour concentration

^bconcentration of vapour/aerosol mixture

^cno information given from which concentration up effects have to be considered as significant and biologically relevant

Conclusions:

Based on the results of the present study the NOAEC for Citral is set to 34 ppm or 215 mg/m3 based on evident local irritation and systemic effects, i.e. body weight changes, observed at 68 ppm or 430 mg/m3.

Executive summary:

To evaluate the potential toxic effects of inhaled citral, F344/N rats were expo 6 hrs/day for either 21 consecutive days at concentrations of 10 & 34 ppm, or 68 ppm, or for 13 weeks at concentrations of 1, 3, or 10 ppm. No mortalities occurred during the 21 day study. Rats exposed to 68 ppm citral displayed signs of severe ocular, oral & nasal irritation and had significantly reduced body weight gains compared to controls. Treatment related lesions consisted of dose-related chronic-active inflammation, hyperplasia, squamous metaplasia & goblet cell atrophy of the nasal respiratory epithelium. Animals exposed to 68 ppm citral also developed changes indicative of irritation in the tracheas and lungs, as well as corneal inflammation & ulceration. Exposure to citral for 13 wk at concentrations up to 10 ppm did not produce mortality or treatment related signs of tox. Body weight gains, clinical pathology indices and organ weight were not adversely affected by exposure. Rats exposed to 10 ppm citral developed minimal hyperplasia and squamous metaplasia of the laryngeal epithelium; however, these changes were completely reversed during a 5-wk recovery period. No significant lesions were observed in the rats exposed to 1 or 3 ppm citral.

Data source

Reference

Reference Type:

publication

Title:

Developmental toxicity evaluation of inhaled citral in Spraque-Dawley rats.

Author:

Gaworksi C.L., Vollmuth T.A., York R.G., Heck J.D., Aranyi C.

Year:

1992

Bibliographic source:

Fd Chem. Toxic. 30, 269-275

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD414

Deviations:

yes

Remarks:

Mortalities, clinical signs of intoxication, body weight gains and gross lesions were investigated as signs of maternal toxicity during a developmental toxicity study.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-3,7-dimethylocta-2,6-dienal

EC Number:

203-379-2

EC Name:

(Z)-3,7-dimethylocta-2,6-dienal

Cas Number:

106-26-3

Molecular formula:

C10H16O

IUPAC Name:

(Z)-3,7-dimethylocta-2,6-dienal

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc. (Raleigh, NC, USA)
- Age at study initiation: 9-11 w
- Weight at study initiation: females ca. 230 g
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum during non-exposure periods
- Water: ad libitum during non-exposure periods
- Acclimation period: 2 w

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 1
- Humidity (%): 31 +- 8
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: nitrogen

Remarks on MMAD:

no data

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Rochester-type stainless steel inhalation chambers of 1 cubic meter volume
- Method of conditioning air: filtered through coarse particulate and HEPA filters and an activated carbon cartridge
- System of generating particulates/aerosols: aerolization of liquid citral with a DeVilbiss Model 41 nebulizer using nitrogen as a carrier gas to prevent degradation, which had been observed during nebulization in the presence of air
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: not data
- Air change rate: 15 +- 3 changes/hr
- Method of particle size determination: cascade impactor; MMAD 4.2 µm, geometric standard deviation 1.9; about 90% of the aerosol with aerodynamic diameter < 10 µm
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography with flame ion detection
- Samples taken from breathing zone: not specified

VEHICLE
- Justification for use and choice of vehicle: to prevent degradation of citral
- Composition of vehicle: nitrogen

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of vapour and aerosol samples indicated that citral purity degraded less than 5% with nitrogen nebulization, with only a slight entrichment of the neral isomer (< 3%) in the vapour phase Chamber vapour atmospheres sampled by cryogenic trapping. Sampling was performed at a flow rate of 400 mL/min for 25 min through two all-glass and teflon traps immersed in a dry-ice methanol bath. The tubes were rinsed with 10 mL of isopropanol containing n-octanol as internal standard. Analysis performed by gas chromatography and flame ion detection.

Duration of treatment / exposure:

6 h/d, gestation day 6-15

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 ppm

Remarks:

nominal conc. corresponding to 63 mg/m3 (=MW*ppm/24.1*1000); MW=152,2 g/mol

Doses / concentrations 2

Dose / conc.:

34 ppm

Remarks:

nominal conc. corresponding to 215 mg/m3 (=MW*ppm/24.1*1000); MW=152,2 g/mol

Doses / concentrations 3

Dose / conc.:

68 ppm

Remarks:

nominal conc. corresponding to 430 mg/m3 (=MW*ppm/24.1*1000); MW=152,2 g/mol

No. of animals per sex per dose:

25

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: highest selected to produce maternally toxic effects

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 2 4, 6, 8 (exposure), 12, 16, 20 (post-exposure)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

FOOD CONSUMPTION: No data

WATER CONSUMPTION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No data

Statistics:

Maternal body weights and body-weight gains were analysed by a one-way analysis of variance (ANOVA), followed by a Dunnett's when applicable ( Steel and Torrie, 1960).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ocular opacity, difficulty in breathing during the exposure phase of the study indicating stress of severe respiratory tract irritation; normal breathing returned in most of affected animals by gestation day 20; other frequently observed clinical signs: nasal discharge, salivation, redness around eyes, discolored facial fur, scrubby hair coat
However, after completion of the exposure period recovery clinical signs of toxicity occurred.

Mortality:

mortality observed, treatment-related

Description (incidence):

68 ppm: moribund condition of 1/25 animals (killed on gestation day 17);

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

68 ppm:
- Body weight loss during exposure period from gestation day (GD) 6 to 15; after exposure period body weight gain was comparable to other groups;
- Overall mean body weight (GD 20) significantly decreased
- Body weight gain (GD0-20) decreased by 39% compared to controls.
However, after completion of the exposure period recovery of body weight occurred.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

34 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponds to 215 mg/m3

2

Dose descriptor:

LOAEC

Effect level:

68 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: severe respiratory tract irritation with reduced body weight gain and clinical signs as secondary effects in pregant rats; corresponds to 430 mg/m3

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The results of the present study revealed no significant maternal toxicity or adverse developmental effects in rats exposed to citral vapour at concentrations up to 34 ppm (NOAEL: 215 mg/m3).

Executive summary:

The repeated dose inhalation toxicity of citral was evaluated based on maternal toxicity data from a OECD TG 414 developmental toxicity inhalation study in rats. Exposure atmospheres contained citral concentrations of 1, 3, 10 and 34 ppm (both as vapour), or 68 ppm (aerosol/vapour mixture) corresponding to 6, 19, 63, 215, 430 mg/m3. The exposure condition at 68 ppm comprised an aerosol/vapour mixture with the intention to produce signs of toxicity. Maternal toxicity was indicated at 68 ppm (430 mg/m3) by decreased body weights and by clinical signs as ocular opacity, breathing difficulty, nasal discharge and salivation. These signs of maternal toxicity were secondary to the stress produced by severe respiratory tract irritation, as recovery of body weight and clinical signs of toxicity occurred after completion of the exposure period. At 10 and 34 ppm, findings were incidental and not siginficantly different from control animals.