Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2003-11-28 until 2004-05-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Minor deviations which did not affect the quality or integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS

- Source: Charles River Deutschland, Sulzfeld, Germany

- Age at study initiation: 7 - 8 weeks

- Weight at study initiation: Mean body weights at the start of treatment (day 0) were 194 and 155 g for male and female animals, respectively.
The initial body weight variation did not exceed ± 20% of the mean weight for each sex.

- Housing: During exposure, the rats were housed individually in animal holders. Immediately after each exposure, the animals were returned to their
living cages. The rats were housed under conventional conditions in animal room 6.0.06, in macroIon cages with a bedding of wood shavings (Espen E-001; ABEDD, Koflach, Austria), five rats per cage, separated by sex. During urine collection, animals were kept individually in stainless-steel metabolism cages.

- Diet: (Rat & Mouse No. 3 Breeding Diet RM3) from SDS Special Diets Services, Witham, England. EEach batch of this diet was analysed by the supplier for nutrients and contaminants. The certificate of
analysis pertaining to the batch used in this study (Batch no. 3273) is included in the report report, ad libitum, except during exposure

- Water: Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EEC Council Directive 98/831EEC) was supplied by N.V. Hydron Midden-Nederland, ad libitum, except during exposure

- Acclimation period: The duration of the acclimatization period until the experimental start date was 4 days, but animals were kept in quarantaine in the same animal room for 2 days (= total of 6 days).


ENVIRONMENTAL CONDITIONS

- Temperature (°C): maintained at a temperature of 22 ± 3°C, except on one occasion when temperature exceeded 25°C (13 January 2004: maximum of 25.1 0c). Because this peak was coincident with room cleaning activities and lasted for less than 1 hour, it was most likely caused by spillage of warm water near the temperature-sensor.

- Humidity (%): aRelative humidity was at least 30%, but exceeded the 70% upper limit occasionally during short periods of time, due to wet cleaning of the animal room (maximum of 100%). In addition, relative humidity exceeded the 70% upper limit on a few occasions which could not be attributed to wet cleaning or meteorological circumstances. However, relative humidity was between 30 and 70% for the vast majority of the time.

- Air changes (per hr): 10

- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
other: not applicable
Remarks on MMAD:
MMAD / GSD: N/A
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

Animals were exposed to the test atmosphere in nose-only inhalation units, modifications of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom. Each unit consisted of a cylindrical column, surrounded by a transparent cylinder. The column consisted of a top assembly with the inlet of the test atmosphere, two rodent tube sections and at the bottom the base assembly with the exhaust port.
Several empty ports were used for test atmosphere sampling, particle size analysis (only measured once), temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column (males and females alternated). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. In our experience, the animal's body does not exactly fit in the animal holder which always results in some leakage from high to low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose and dilution
oftest atmosphere at the nose of the animals is prevented. The units were illuminated externally by normal laboratory TL-lighting.


TEST ATMOSPHERE

Before the start of the fIrst exposure of the range-fInding study (i.e. on 30 November 2003) one container containing the test material was taken from the freezer and warmed
in water (ca. 15°C). Small portions were taken (ca. 10 ml each) and put into 20 ml vials under nitrogen atmosphere. Thereafter these portions were kept in the freezer until use.
This procedure was repeated with three other containers (portions of 10-15 ml) on 4 and 19 December 2003, and on 15 January 2004.
The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. The test atmosphere for each exposure level was generated by passing small amounts of dry air through glass evaporators containing the test material to carry a (nearly) saturated vapour of the test item to the main air stream. This evaporation method was chosen due to the relatively low concentrations to be generated.
The glass evaporators were fIlled daily with fresh test material. The air flows through these evaporators were regulated by mass flow control units (Bronkhorst Hi Tee, Ruurlo, The Netherlands). The mixture was diluted by mass flow controlled humidifIed main air flows and the resulting test atmospheres were directed to the exposure units. At the bottom of the units the test atmospheres were exhausted (see also Figure 1). Both the humidifIer and the glass evaporators were kept in thermal baths kept at 20°C. The read-out of the mass flow control units were recorded at regular intervals (ca. once per two hours, i.e. three times a day). Both during the range-fInding and main study the mass flow control units for the main air flow read 100% for the low- and midconcentration test atmosphere and 50% of the nominal values for the high-concentration test atmosphere. In order to obtain the required concentrations, the mass flow control units for the air flow through the evaporators were adapted accordingly.



- Rationale for the selection of the starting concentration: The concentration levels of the sub-acute study were selected based on the results obtained during the range-finding study,
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the range-finding study, a total carbon analyser (RS55S, Ratfisch, Poing, Germany) was used to measure the concentration of the test item in the low-, mid- and high-concentration inhalation units. The total carbon analyser (TCA) alternately sampled test atmosphere from each of the inhalation units (low-, mid- and high concentration test atmosphere) each hour for approximately 5 min. The remaining periods (45 min each) were used to measure the control atmosphere. This frequency was chosen because of the expected damage the test item could induce to the TCA (the burning of the test item was expected to result in SiOz which would sooner or later deposit on sensitive parts of the flame ionisation detector of the TCA).

ICPAES analysis was also performed, however, without success.

Monitoring the concentration of the test item during the main study was performed in two ways:

1. total carbon analysis once per day for 5 min for each concentration in order to prevent damage to the TCA as much as possible. The first day was measured at approximately one hour after start, the second day at about two hours after start, until 5 hours after start on the fifth day, and so on.
2. nominal concentration. At regular intervals this concentration was checked and generation air flows were slightly adapted in order to obtain the required
concentrations.
Duration of treatment / exposure:
31 days, with a total of 20 exposure days.
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1.8, 5.8, 16 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 1.6 ± 0.7, 4.9 ± 0.8, 18.1 ± 3.1 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period:
Satellite groups of 5 male and 5 female animals each, similarly exposed to target concentrations of 0 or 15 mg/m3 of the test item, were kept for an additional recovery period of 14 days.

- Dose selection rationale:
The concentration levels used during the 31-day study were based on a preceding 7-day range-finding study, with a total of 5 exposure days. In this study groups of 5 rats/sex were exposed to target concentrations of 0, 5, 25, and 100 mg/m3.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A group-wise observation was made halfway during exposure. On working days, all cages were checked again in the afternoon. At weekend days and public holidays only one check per day was carried out. All abnormalities or reactions to treatment were recorded.

BODY WEIGHT: Yes
The body weight of each animal was recorded once during the acclimatization period (day -1), at initiation of treatment (day 0), and once per week thereafter. The animals were also be weighed on the day before overnight fasting (main study groups: nominal day 30, recovery groups: nominal day 44), and on their scheduled sacrifice date (in order to calculate the correct organ to body weight ratios).


FOOD CONSUMPTION:
Food consumption was measured per cage, over successive periods of 7 days (and a 2 day period at the end of the exposure and recovery period), by weighing the feeders. The results were expressed in g per animal per day. The efficiency of food utilization was calculated and expressed in g weight gain per g food consumed.


HAEMATOLOGY: Yes /
At scheduled necropsy at the end of the treatment period, blood samples were taken from the abdominal aorta of all rats of the main study groups, whilst under Nembutal anaesthesia. The rats were fasted overnight before necropsy. Kz-EDTA was used as anticoagulant. In each sample the following deterrninations were carried out:
haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell count
prothrombin time
thrombocyte count
The following parameters were calculated:
mean corpuscular volume (MCY)
mean corpuscular haemoglobin (MCR)
mean corpuscular haemoglobin concentration (MCRC)
Because of changes observed in the number of trombocytes in females of the highconcentration
group at the end of the exposure period, the following haematological
parameters were determined in female animals of the recovery groups at the end of the
recovery period:
haemoglobin
packed cell volume
red blood cell count
total white blood cell count
thrombocyte count
The following parameters were calculated:
mean corpuscular volume (MCY)
mean corpuscular haemoglobin (MCR)
mean corpuscular haemoglobin concentration (MCRC)

CLINICAL CHEMISTRY: Yes
Clinical chemistry determinations were conducted on all rats of the main study groups. The rats were fasted overnight before necropsy. At necropsy at the end of the treatment period, blood was collected from the abdominal aorta in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma:
alkaline phosphatase activity (ALP)
aspartate aminotransferase activity (ASAT)
alanine aminotransferase activity (ALAT)
gamma glutamyl transferase activity (GGT)
total protein
albumin
ratio albumin to globulin
urea
creatinine
fasting glucose
bilirubin total
cholesterol
triglycerides
phospholipids
calcium (Ca)
sodium (Na)
potassium (K)
chloride (Cl)
inorganic phosphate
Because treatment-related changes were not observed in clinical chemistry parameters at the end of the exposure period, additional clinical chemistry parameters were not measured in animals of the recovery groups.

URINALYSIS: Yes
The day before necropsy, all rats of the main study groups were deprived of water and
food and kept overnight in stainless-steel metabolism cages (one rat per cage) to collect
urine (nominal day 31). The concentrating ability of the kidneys was investigated by
measuring the volume and density of the individual samples. In the urine collected, the
following determinations were carried out in individual samples:
volume
density
appearance
pH
glucose
occult blood
ketones
protein
bilirubin
urobilinogen
electrolytes (Na, K, Cl)
creatinine
microscopy ofthe sediment (pooled samples 5/sex/group).
Because of changes observed in urine volume in males of the high-concentration group at the end of the exposure period, urine was also collected in males of the recovery groups at the end of the recovery period (nominal day 45). The following urine parameters were examined in male animals ofthe recovery groups:
volume
density

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On day 31, after overnight fasting, all animals of the main study groups were killed in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under Nembutal anaesthesia and then examined grossly for pathological changes. On day 45, after overnight fasting, all animals of the recovery groups were killed in the same way, also in such a sequence that the average time of killing was approximately the same for each group. All animals were also subjected to a complete gross necropsy.
The following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying:
adrenals
brain
heart
kidneys
liver
spleen
testes
lungs with trachea and larynx

Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde:
adrenals
brain
heart
kidneys
liver
spleen
testes
complete respiratory tract including nasal passages
all relevant gross lesions

HISTOPATHOLOGY: Yes
The tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 um and stained with haematoxylin and eosin. Histopathological examination (by light microscopy) was performed on the organs listed above of all animals of the control group and the high-concentration group. The nose was examined at 6 levels, the larynx at 3 levels, the trachea at 3 levels (including the bifurcation), and the lung lobes at 1 level each.
Since treatment-related histopathological changes were observed in the nasal passages/cavities, larynx, trachea and lungs in animals of the high-concentration group, histopathology on these organs/tissues was extended to animals of the low-and midconcentration groups, and to animals of the recovery groups. In addition, due to changes observed in adrenal- and liver weights in animals of the high-concentration group at the end of the recovery period, histopathology examination was extended to the liver and adrenals of recovery animals as well.
Statistics:
The statistical procedures used in the evaluation of data were as follows:
body weight: one-way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests. Body weights in the recovery
groups were evaluated by one-way analysis of variance (Anova) followed by Student's t-tests;
food consumption/efficiency: no statistics because of the low number of experimental units per group;
red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, urine volume and
density, organ weights: one-way analysis of variance (Anova) followed by Dunnett's multiple comparison tests;
reticulocytes, relative differential white blood cell counts, urinary parameters except for volume and density: Kruskal-Wallis non-parametric Anova followed by
Mann-Whitney V-tests;
histopathological changes: Fisher's exact probability test.

All tests were two-sided. Probability values ofp<0.05 were considered significant.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
Treatment-related abnormalities consisted of slight breathing abnormalities during
exposure in animals of the high-concentration group between day 7 and day 17.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight gain was reduced in male and female animals of the mid- and highconcentration
group during the course of the exposure period. Body weight gain
recovered completely in female animals during the 14-day recovery period, whereas
recovery of body weight gain was not yet complete in males.

FOOD CONSUMPTION & FOOD EFFICIENCY
Food consumption was reduced in males of the high-concentration group, whereas food
conversion efficiency was low in males of the mid- and high-concentration groups.
Such changes were not observed in females. Food intake and food conversion efficiency
recovered during the 14-day recovery period.

HAEMATOLOGY
No treatment-related changes in haematology were observed except for a significantly decreased thrombocyte count in females of the high
concentration group at the end of the exposure period. Such a change was not present anymore at the end of the 14-day recovery period.

CLINICAL CHEMISTRY
No treatment-related changes in clinical chemistry were observed.

URINALYSIS
Urinary volume was significantly lower and creatinine content concomitantly
significantly higher in males of the high concentration group at the end of the exposure
period. At the end of the 14-day recovery period, urinary volume was comparable with
that of the control males.

ORGAN WEIGHTS
Relative lung weights were statistically significantly increased in males of the highconcentration
group and in females of the low-, mid-, and high concentration group at
the end of the exposure period. The step-wise increase in females was, however,
relatively low. A significantly increase in relative adrenals weight and a significantly
decrease in absolute liver weight were observed in males of the high concentration
group at the end ofthe 14-day recovery period.

GROSS PATHOLOGY
Macroscopic examination at necropsy did not reveal treatment-related changes

HISTOPATHOLOGY: NON-NEOPLASTIC
Inhalation of the test item induced histopathological changes in the nasal passages, larynx, trachea and lungs:
Effects in the nasal passages were seen in all exposure groups. There was a distinct concentration-related increase in severity (high concentration animals showed the most severe effects) and there was a concentration-related anterior-posterior gradient, i.e. high concentration group animals showed nasal effects at levels 1-4 (of the 6 levels examined) whereas in low concentration group animals changes were seen at the most anterior part (level 1) only. Effects in the nasal passages consisted of focal erosion/ulceration and severe epithelial degeneration in levels 1-4 of animals exposed to 15 mg/m3. Similar changes, i.e. focal erosion/ ulceration, and epithelial hyperplasia/metaplasia, were observed in animals exposed to 5 mg/m3 which were severe in the most anterior part of the nasal cavity (level 1), moderate at levels 2 and 3, and very slight to slight at level 4. The latter changes were seen in single animals only.
Female animals exposed to 1.5 mg/m3 exhibited changes (epithelial hyperplasia) of a minimal nature in the most anterior part ofthe nasal passages (level 1).
Laryngeal effects were observed in animals of the mid- and high-concentration groups. Laryngeal effects at 15 mg/m3 generally consisted of epithelial degeneration, epithelial hyperplasia and squamous metaplasia, and mononuclear cell infiltrates, and varied from slight to moderate. Slight mononuclear cell infiltrate was also observed in one male exposed to 5 mg/m3.
Exposure-related effects in the trachea and lungs were observed in animals exposed to 15 mg/m3 only. Tracheal effects consisted of epithelial hyperplasia, epithelial degeneration/regeneration, and mononuclear cell infiltrates. Effects in the lungs consisted of very slight to slight epithelial degeneration/regeneration at the tip of the bifurcation.
A recovery period of 14 days resulted in a considerable decrease in the severity of the nasal and laryngeal changes and in a full disappearance of tracheal and pulmonary changes.
Histopathological changes were not observed in other organs/tissues examined indicating that the test compound induced local changes, i.e. at the sites of direct contact.

Effect levels

Dose descriptor:
other: MOAEL (Minimal-Observed-Adverse-Effect Level). A No-Observed-Effect-Level (NOEL) could not be established.
Effect level:
1.5 mg/m³ air
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
From the results of the present study in rats, it was concluded that exposure to the test item at levels of 15 mg/m3 induced breathing difficulties during exposure, increased relative lung weights, and histopathological changes in the complete respiratory tract, i.e. from the nose to the lungs. Exposure to 5 mg/m3 resulted in histopathological changes in the nasal passages and larynx. Changes in the nasal passages, however, were also observed in animals exposed to 1.5 mg/m3 , which is considered an adverse reaction to exposure to the test compound. A No-Observed-Effect-Level (NOEL) could, therefore, not be established. However, as this lesion was observed in female animals only and the degree of the lesion was only very slight to slight, the concentration of 1.5 mg/m3 was considered to be a Minimal-Observed-Adverse-Effect Level (MOAEL) for local toxicity.
Executive summary:

In a sub-acute (31 -day) repeated inhalation study in Wistar rats conducted to OECD 412, and to GLP, a NOEL could not be determined as changes in the nasal passages, were observed in female animals only exposed to the lowest dose group of 1.5 mg/m3 , which is considered an adverse reaction to exposure to the test compound. However, as this lesion was observed in female animals only and the degree of the lesion was only very slight to slight the concentration of 1.5 mg/m3 was considered to be a Minimal-Observed-Adverse-Effect Level (MOAEL) for local toxicity.