Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed similar to OECD TG 413 but not under compliance with GLP. Not all original test data were present and less examinations have been carried out. Only one dose level was used. Exposure was to a fragrance mixture with only low HHCB level.

Data source

Reference
Reference Type:
publication
Title:
Subchronic inhalation studies of complex fragrance mixtures in rats and hamsters.
Author:
Fukayama, M.Y., Easterday, O.D., Serafino, P.A., Renskers, K.J., North-Root, H., Schrankel, K.R.
Year:
1999
Bibliographic source:
Toxicology Letters 111, 175-187.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
use of mixture instead of a single substance
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Reference substance name:
fragrance mixture B
IUPAC Name:
fragrance mixture B
Constituent 2
Reference substance name:
fragrance mixture G
IUPAC Name:
fragrance mixture G
Details on test material:
HHCB (purity not reported) was part of the Fragrance mixture.
mixture B: 5.7 ug/m3 HHCB
mixture G: 132 ug/m3 HHCB

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study:
- Housing: individually
- Diet (e.g. ad libitum): ad libitum (except during times in exposure chamber)
- Water (e.g. ad libitum): ad libitum (except during times in exposure chamber)
- Acclimation period: Yes, 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: particle size were between 0.5 and 7.5 µm, based on this study and similar studies with other fragrance mixtures
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Whole-body exposure chamber system
- Method of holding animals in test chamber: animals were placed in custom made stainless steel inhalation chambers fitted with plexiglass doors in the front and back. Each chamber held 12 cages
- Source and rate of air: Compressed air drawn through an absolute filter. .
- Method of conditioning air:
- System of generating particulates/aerosols: Compressed air nebulizer
- Temperature, humidity, pressure in air chamber:
- Air flow rate: 5 to 10 cubic feet per minute
- Air change rate: 1-2 times per minute
- Method of particle size determination: Particle size distribution of the aerosols created in the inhalation chambers under the conditions described above was determined with an Andersen Cascade Impactor.
- Treatment of exhaust air: The chambers were connected to a central exhaust fan that drew the chamber air out through a combination of pre-filter and absolute filter, and then through a charcoal filter before final discharge.


TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: no


VEHICLE (if applicable)
- Justification for use and choice of vehicle: Special Denaturated Alcohol - 40B, used for dilution of fragrance mixtures.
- Composition of vehicle: SDA-40B
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle: 5% (w/w)
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Several experiments were conducted in which each test material was aerosolized into the chamber and filter samples taken. The weight of the filter was determined and corrected for the volatile components of each formula that were not retainable on the filter. If the resulting chamber concentration did not match the target concentration of 5 or 50 mg/m3, the generating airflow or the chamber exhaust rate was adjusted accordingly.
Duration of treatment / exposure:
SD rats: 13 weeks
Frequency of treatment:
4 hours per day, 5 days per week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5 mg/m3 (mixture B) 5.7 ug.m3 (HHCB)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
50 mg/m3 (mixture G); 132 ug/m3 (HHCB)
Basis:
nominal conc.
No. of animals per sex per dose:
50 mg/m3: 24 females
5 mg/m3: 20 females
Control animals:
yes
Details on study design:
- Dose selection rationale: calculated from Human Lung Burden (LB): EC (Exposure Concentration= (LB (Lung Burden-human) * LW (Lung Weight-animal))/ (ET (Exposure Time-animal) * LMV (Lung Minute Volume-animal) * PD (Pulmonary Deposition of particles-animal))
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
Positive control:
no positive controls were used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: No
- Time schedule:


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: No


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: No


WATER CONSUMPTION: No
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:


HAEMATOLOGY: Yes
- Time schedule for collection of blood:at sacrifice and possibly also interim
- Anaesthetic used for blood collection: Yes , sodium pentobarbital
- Animals fasted: Yes 16 hours prior to sacrifice
- How many animals: all
- Parameters checked in table 1 were examined


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice and possibly also interim
- Animals fasted: Yes 16 hours prior to sacrifice
- How many animals: all
- Parameters checked in table 2 were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: possibly at sacrifice or interim collection
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes 16 hours prior to sacrifice
- Parameters checked in table 3 were examined.


NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Animals were killed by exsanguination via the abdominal aorta or by overdose with intraperitoneal injection of sodium pentobarbital and ob-served grossly for any pathological changes. Various organs including adrenal glands (both), brain, esophagus, eye, heart, muscle with sciatic nerve, kidneys, colon, trachea, lungs, hilar lymph node, liver, pancreas, spleen, stomach, skin, duo-denum, spinal cord, sternum, thyroid, urinary bladder, uterus, ovaries or testes, and any lesions or tissue masses and their regional lymph nodes, were collected for weighing and/or histopathological examination. The list of collected tissues was standard for subchronic studies conducted at the time.

HISTOPATHOLOGY: Yes

Organs examined histologically included the trachea, lungs, adrenals, brain, esophagus, heart, kidneys, liver, pancreas, spleen, sternum and uterus. Bone marrow was evaluated on a sample taken from the femur.

Statistics:
Data from all studies were subjected to statistical analysis by the Student’s t -test. The level of significance chosen for all parameters was P<
0.05. Unless otherwise stated, any value referred to as statistically significant would have shown significance above the 95% confidence level.

Results and discussion

Results of examinations

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:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived to study termination.


BODY WEIGHT AND WEIGHT GAIN
body weights and weight gains of treated animals did not differ significantly from controls.


HAEMATOLOGY
Animals exposed to fragrances for 13 weeks did not exhibit any changes in hematology or clinical chemistry parameters that were considered significant toxicologically.


URINALYSIS
No abnormal findings were noted

BEHAVIOUR
None of the animals in any studies exhibited any abnormal behavior or physical appearance as the result of their exposure to the fragrance mixtures.

ORGAN WEIGHTS
Animals showed no differences in organ weights or organ to body weight ratios that were judged significant toxicologically

GROSS PATHOLOGY
lung discoloration was noted.

HISTOPATHOLOGY: NON-NEOPLASTIC
Changes in respiratory tract tissues, consistent with those seen in animals with chronic murine pneumonia, were noted in treated and
untreated rats exposed to Fragrance G (Study 7 ). None of these findings were considered to have resulted from exposure to the test material.

OTHER FINDINGS
Exposure to the mixture did not result in mortality, skin reactions or effects on body weight, behaviour or physical appearance, haematology and clinical chemistry, organ weights and gross pathology (including uterus and ovaries), or histopathology (uterus but not ovaries examined)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Exposure to mixture G did not result in mortality, skin reactions or effects on body weight, behaviour or physical appearance, haematology and clinical chemistry, organ weights and gross pathology (including uterus and ovaries), or histopathology (uterus but not ovaries examined)
Executive summary:

A group of 24 female SD rats was exposed to 50 mg/m3 for 4 hr per day, 5 days per week for 13 weeks (mixture G). These fragrance mixtures were aerosolised with a compressed air nebulizer (particle size was not determined for this particular study, but was assumed to be between 0.5 and 7.5 µm, based on similar studies with 5 other fragrance mixtures). HHCB (purity not reported) was part of these fragrance mixtures, and the level of HHCB to which the animals were exposed was 132 µg/m3 at highest for mixture G. Exposure to the mixture did not result in mortality, skin reactions or effects on body weight, behaviour or physical appearance, haematology and clinical chemistry, organ weights and gross pathology (including uterus and ovaries), or histopathology (uterus but not ovaries examined)