Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study conduct was based on the procedure described by Sachsse et al. (1973, 1976). The initially highest selected concentration level of 1.3 mg/L air had resulted in mortalities, and the test concentration was changed/lowered from the second day of treatment to 0.301 mg/L air until test ending. Because of the increased mortility rate observed from day 2 to 15 and since the animals exposed to 0.301 mg/L air were not changed but were the same animals that had been first exposed to 1.3 mg/L air, it is not clear whether the observed mortalities were due truly to the refined concentration or were consequent to the initial applied concentration. Similarly in the mid-dose group the exposures were reduced from 0.227 mg/L air on Day 1 to 0.115 mg/l air thereafter. Assigning effect levels with these 2 groups are not reliable because of the changed exposure concentrations and the resulting uncertainty of the what exposure effected the observed toxic signs. Moreover, the study showed some principle deviations compared to the requirement of OECD TG 412, including (1) the reduced daily exposure duration of 2 hours instead of the required 6 hours, and (2) the test duration was 21 days instead of 28 days At the time the study was conducted, GLP was not mandatory. The test material was insufficiently characterized in the report, and no analytical purity was given. However, according to " Triclosan supplement I to EU dossier submitted 18 August 2009", the purity of CIBA-produced Triclosan exceeded 99%, and for FAT 80023/A, a degree of purity of 99.3% was reported. The triclosan test substance was dissolved into ethanol for aerosolization; this is not a representative exposure system for human hazard assessement because triclosan is a solid, powdered substance. Exposures during manufacturing or formulation activity would be a dust, if any, and not to a fully respirable aerosol of triclosan in ethanol. Thus, the study is considered as not reliable for hazard assessment and classification.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1974
Report date:
1974

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
test duration 21 days, daily exposure duration 2 hours
Principles of method if other than guideline:
Inhalation toxicity was tested according to the method of Sachsse et al. (1973, 1976):
- Sachsse K et al. (1973) Measurement of inhalation toxicity of aerosols in small laboratory animals. In: Proceedings of the Europ. Soc. for the Study of Drug Toxicity. Vol. XV, pp. 239-251, Zurich.
- Sachsse K et al. (1976) Toxikologische Prufungen von Aerosolen im Tierexperiment: Aus "Chemische Rundschau" 29 , Nr. 38: 1-4
GLP compliance:
no
Remarks:
GLP was not compulsory at the time the study was conducted
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Triclosan
EC Number:
222-182-2
EC Name:
Triclosan
Cas Number:
3380-34-5
Molecular formula:
C12H7Cl3O2
IUPAC Name:
5-chloro-2-(2,4-dichlorophenoxy)phenol
Details on test material:
- Name of test material (as cited in study report): FAT 80023/A (trade name: Irgasan DP 300)
- Physical state: white solid
- Analytical purity: purity not specified in the study report, however, according to " Triclosan supplement I to EU dossier submitted 18 August 2009", the purity of CIBA-produced Triclosan exceeded 99%, and for FAT 80023/A, a degree of purity of 99.3% was reported.
- No further data given.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: animals raised on the premises
- Age at study initiation: about 6 weeks old
- Weight at study initiation: 165 - 175 g
- Fasting period before study: no
- Housing: nine per cage, males and females separated, in Makrolon cages typ 4
- Diet (e.g. ad libitum): rat food (NAFAG, Gossau SG, Switzerland), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Photoperiod (hrs dark / hrs light): 14 hrs dark/10 hrs light

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: The particle size distribution of the aerosol in the vicinity of the animals was determined after 30 and 90 minutes during the daily exposure of 2 hours using a Cascade Impactor.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
A 10 % suspension of FAT 80023/A in ethanol was generated and injected by a motor driven syringe at a rate of 0.9, 3 and 6 (0.9, 6 and 30 for the first experimental day) mL/hr into a stream of compressed air flowing through a spray nozzle at a rate of 10 L/min. The aerosol mist thus produced was discharged into the exposure chamber. The control rats were treated with an aerosol generated from ethanol (6 mL/hr). The output of the spray device and the dimensions of the inhalation chamber were adjusted to each other so that the solvent evaporates on the way from the nozzle orifice to the rat containers.

CONCENTRATION OF TEST MATERIAL IN THE ATMOSPHERE
The concentration of the aerosol in the vicinity of the animals was monitored at regular intervals throughout the aerosol exposure. The concentration was determined gravimetrically; the aerosol was sampled in the immediate vicinity of the animals, 30, 60 and 90 minutes after the beginning of the daily exposure, which had a duration of 2 hours.

PARTICLE SIZE DISTRIBUTION
The particle size distribution of the aerosol in the vicinity of the animals was determined after 30 and 90 minutes during the daily exposure of 2 hours using a Cascade Impactor.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Two hours daily
Frequency of treatment:
5 days a week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.05, 0.227 and 1.3 mg/L air
Basis:
other: concentrations used on day 1 of treatment
Remarks:
Doses / Concentrations:
0.05, 0.115 and 0.301 mg/L air
Basis:
other: concentrations used from day 2 to day 15 of treatment
No. of animals per sex per dose:
Group 1(control): 9 / sex (4/sex were kept fot the 17 day recovery period following the 21 day exposure period)
Group 2 (0.05 mg/L air). 9 /sex
Group 3 (0.227 and 0.115 mg/L air): 9/ sex (4/sex were kept fot the 17 day recovery period following the 21 day exposure period)
Group 4 (1.3 and 0.3 mg/ L air): 9/sex
Control animals:
yes
Details on study design:
For inhalation the rats were kept in separate PVC tubes positioned radially around the exposure chamber such that snout and nostrils of the animals only were exposed to the aerosol. After the daity 2-hour inhalation the rats were returned to their cages.

Examinations

Observations and examinations performed and frequency:
MORTALITY AND CLINICAL OBSERVATIONS
The animals were daily examined/observed for appearance, behaviour, appetite, excretion, symptoms of toxicity and mortality.
symptoms, bodyweight, food consumption and mortality.

BODY WEIGHT
Body weights were recorded daily.

FOOD CONSUMPTION
Food consumption was recorded daily.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmic examinations were performed weekly.

HAEMATOLOGY AND CLINICAL CHEMISTRY
To reduce variations due to circadian rhythmus blood sampling for haematology and blood chemistry was between the hours of 7.30 and 9.00 a.m. from the orbital sinuses. No anesthetic was used. Blood samples from each animal with the respective anticoagulant were aliquoted into individual vials.
Haematologic and blood chemistry measurements were carried out by standard methods on 54 rats (9 males / 9 females per group) from the control, low and intermediate concentration groups at 18 experimental days. Measurements at the highest concentration were in 7 rats (5 males / 2 females) only. 16 Rats (4 males / 4 females per group) from the control and intermediate concentration groups were examined after a 17-day recovery period.
Following haematological parameters were considered: haemoglobin, methaemoglobin, erythrocytes count, packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin, reticulocytes, Heinz bodies, thrombocytes, prothrombin time, leucocytes (total and differential count).
Following clinical chemical parameters were considered: glucose, blood urea nitrogen (BUN), glutamate-pyruvate transaminase (GPT), lactate deshydrogenase (LDH), alkaline phosphatase (AP), total protein, protein electrophoresis.
Sacrifice and pathology:
NECROPSY
The surviving rats of the control and three treated groups were anaesthetized with ether and bled after 21 days of treatment. The animals of the recovery group (controls and 0.115 mg/L air) were sacrificed after the 17 days recovery period. The body weight of each animal was determined after sacrifice.

GROSS PATHOLOGY
Complete autopsy of all rats was performed.

ORGAN WEIGHT
The following organs were weighed: brain, heart, lungs, liver, kidneys, adrenals, gonads. Organ to bodyweight and organ to brainweight ratios were calculated for each of these organs.

HISTOPATHOLOGY
Tissue samples of pituitary, oesophagus, nasal cavity, trachea, thyroid, thymus, lymph node, heart, aorta, lungs, liver, kidneys, adrenals, spleen, pancreas, stomach (fundic and pyloric), small and large intestine, bladder, skin, prostate, testes, epididymis or ovaries and uterus were fixed in Bouin fixative. Additional tissue samples of the brain and eye ball were fixed in buffered 4 % neutral formalin.
The fixed tissue samples from all control and treated animals were embedded in paraffin wax and sectioned, and the sections sections were stained with haematoxylin and eosin. Brain sections were stained with cresyl violet. Sections from the liver and spleen were stained also for iron with the Perl method. Periodic acid-Schiff reaction for mucopolysaccharides was performed on the sections from kidneys.
Additional frozen sections of liver and adrenals were specifically stained for fat with Sudan III.

Gross autopsy and histopathological examination were performed also on rats which died during the experimental period unless advanced autolysis prevented it.
Statistics:
Student's "t" and the analysis of variance were employed to assess the significance of difference between concentration groups and controls whenever indicated.

Results and discussion

Results of examinations

Details on results:
MORTALITY
Mortality occurred in group 4, i.e. the group treated with the highest test concentration of initially 1.3 mg/L air (day 1) and then of 0.301 mg/ L air (day 2 to 15); Two females died on day 1, 5 females and 4 males died on day 2, and one male died on day 18.

CLINICAL SYMPTOMS
During the first week of treatment, the rats of group 4 showed dyspnoe, nasal discharge, muscle spasms, pallor and diarrhoea. These symptoms were particularly severe on the first day after the exposure to 1.300 mg/L air and this necessitated reduction of the concentration to 0.301 mg/L air. A total of 11 rats of this group died during the first two days of the experiment. The symptoms became less pronounced in the course of the first week; after the 7th day the animals were free of general symptoms.

BODY WEIGHT AND FOOD INTAKE
The food intake and the bodyweight gain of the male and female rats of group 4 (0.301 mg/L ) was markedly reduced during the first week of treatment. Both parameters improved till the end of the experiment when compared with that of the controls. The food intake and the bodyweight gain of the animals of group 3 (0.115 mg/L air was only slightly reduced during treatment, when compared with that of the controls and group 2 (0.050 mg/L air). At the end of treatment or of the recovery period, the mean bodyweight of both the males and the females was largerly comparable with that of the controls.

EYE EXAMINATION
Throughout the treatment period irritation of the conjunctivae was seen in all rats of group 4.

HAEMATOLOGY AND BLOOD CHEMISTRY
In group 3, there was a slight tendency of total leucocyte counts to be above control values at day 18. This was somewhat more pronounced in the surviving animals of group 4. Concomitant with the leucocytosis, the percentage of neutrophils increased and the percentage of lymphocytes tended to decrease. These findings were of a rather small degree of magnitude and were considered to be associated with inflammatory changes of the respiratory tract.
Serum glutamic-pyruvic transaminase (GPT) activity was significantly (p=< 0.01) elevated in the surviving animals of the highest concentration group. A higher activity of alkaline phosphatase (AP) was also recorded for this group and for the males of the intermediate concentration group (p =< 0.01 and p =<0.05 respectively). After the recovery period, the blood chemistry findings in the animals of the 0.115 mg/L air were considered within physiological limits and were comparable to the controls.
There was no significant difference in the alkaline phosphatase values between the animals of group 2 (0.05 mg/L air) and the controls.

GROSS PATHOLOGY
Twelve treated rats on the highest concentration died in the course of the inhalation experiment. These animals showed severe acute congestion and numerous haemorrhages in all organs. Apart from these findings gross pathological changes were seen neither in the treated nor in the control rats.

ORGAN WEIGHT
The absolute and relative organ weights of the rats of groups 1 to 3 were comparable to each other. The data of group 4 reflected the low body weight of the animals.

HISTOPATHOLOGY
Histopathologically, in all spontaneously died rats of the highest concentration (0.301 mg/L air) group, acute purulent inflammation with focal ulceration of the mucous membrane in the nasal cavity and in the trachea were seen. The lungs of these animals showed severe acute congestion, edema, recent haemorrhages. In two rats foci of lobular pneumonia were seen.
In 2 out of 6 rats of this group, which survived till the end of the test period only slight focal inflammatory changes in the mucous membrane of the nasal cavity or of the trachea were observed. Similar slight inflammatory changes in the nasal cavity or in the trachea were seen in 2 rats of the 0.115 mg/L air group. No other histopathological changes were observed that were related to the treatment. The histopathological appearances in all treated rats of the 0.050 mg/L air group were comparable to those of the controls.

Effect levels

open allclose all
Dose descriptor:
NOEC
Effect level:
0.05 mg/L air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: (no differences when compared to controls)
Dose descriptor:
LOEC
Effect level:
0.115 mg/L air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see below
Dose descriptor:
NOAEC
Effect level:
0.115 mg/L air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEC
Effect level:
0.301 mg/L air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: (effects similar as at 0.115 mg/L air, however more severe, mortalities seen at this level)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The experimental design of this study was not appropriate to assess human health hazard of triclosan from repeated inhalation exposures because triclosan exposure would not be to an aerosol of solubilized, respirable sized triclosan. As a powdered, solid substance triclosan would at worst be a dust but also due to low vapor pressure would give only limited amounts of volitilized material into the atmosphere. Further, the reduction of day 1 exposures and subsequent administration to the same animals increases the uncertainty of the effective exposure concentration inducing the observed efffects. The study should be diregarded and considered as not reliable for hazard determination of triclosan.