2-Hydroxypropyl-β-cyclodextrine ethers

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

.beta.-Cyclodextrin, 2-hydroxypropyl cycloheptaamylose

IUPAC Name:

.beta.-Cyclodextrin, 2-hydroxypropyl cycloheptaamylose

Constituent 2

Reference substance name:

-

EC Number:

420-920-1

EC Name:

-

Cas Number:

128446-35-5

Molecular formula:

Hill formula: (C42H70-nO35)(C3H7O)n; n(mittel)=5,25

IUPAC Name:

5,10,15,25-tetrakis(hydroxymethyl)-40,44,47,49-tetrakis(2-hydroxypropoxy)-20,30,35-tris[(2-hydroxypropoxy)methyl]-2,4,7,9,12,14,17,19,22,24,27,29,32,34-tetradecaoxaoctacyclo[31.2.2.2³,⁶.2⁸,¹¹.2¹³,¹⁶.2¹⁸,²¹.2²³,²⁶.2²⁸,³¹]nonatetracontane-36,37,38,39,41,42,43,45,46,48-decol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): hydroxypropylated .beta.-cyclodextrin
- Physical state: white powder
- Melting Point: 120-160°C
- Storage condition of test material: at ambient temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 225g
- Housing: individually in restraining tubes (during exposure); suspended, stainless steel cages fitted with wire mesh floor and front (after exp.)
- Diet (e.g. ad libitum): powder/commercially available rodent diet; no access during exposure
- Water: ad libitum; no access during exposure
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 - 22.0°
- Humidity (%): 47- 76
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light):12 hours light/12 hours dark

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: MMAD: 1.9, 2.3 and 2.8 µm
gsd: 2.3, 2.4 and 2.4
for the low, mid and high concentration test atmospheres

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only inhalation units
- Method of holding animals in test chamber: The rodent tube section had 20 ports fo28r animal exposure. Animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column.
- Source and rate of air: humidified air
- Method of conditioning air: high concentration test atmosphere was generated by passing test material to an eductor using a dry material feeder
- System of generating particulates/aerosols: eductor (Fox Mini, type 060, Spraybest Europe BV, Zwanenburg, The Netherlands) using a dry material feeder (Gericke GMD 60, Gericke AG, Regensburg-Ziirich, Switzerland )
- Pressure in air chamber: 0.8-1.0 bar
- Air flow rate: 63, 15, 19 and 109 l/min (control, low, mid and high concentration test); 20-25 l/min (low and mid concentration test)
- Method of particle size determination: Every week, one particle size distribution measurement was carried out on each of the three test atmospheres using a 10-stage Andersen cascade impactor (Andersen, Atlanta, USA). The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated (Lee, 1972).

TEST ATMOSPHERE
- Brief description of analytical method used: The filters were weighed before and immediately after sampling. The actual concentrations were calculated by dividing the amount of test material present on the filter by the volume of of the respective sample taken.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A-control goup: 0mg/m³ concentration in the air
B-Iow concentration group: 10mg/m³ concentration in the air
C-mid concentration group: 100mg/m³ concentration in the air
D-high concentration group: 1000mg/m³ concentration in the air

Gravimetric analysis
The concentration of the test material in the test atmosphere of each unit was determined by gravimetric analysis. Test atmosphere samples were obtained by passing measured amounts of test atmosphere at a sampling speed of approximately 5 l/min on fibre glass filters (Sartorius). The filters were weighed before and immediately after sampling. The actual concentrations were calculated by dividing the amount of test material present on the filter by the volume of of the respective sample taken. The volumes sampled were 10,50 and approximately 300 L, for exposure units D, C and B, respectively. Generally four samples per exposure day were taken from each exposure unit times (i.e. B, C and D).

Nominal concentration:
The nominal concentration of the high concentration test atmosphere was calculated each day by dividing the weight of the test material used that day by the total amount of air passed on that day through the high concentration exposure unit. Since the test atmospheres of the mid and low concentration were derived from the high concentration test atmosphere, their nominal concentrations could not be derived.

Duration of treatment / exposure:

6 hours a day, 5 days a week for a period of 28 days

Frequency of treatment:

daily apart from weekends

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/m³ air (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/m³ air (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/m³ air (nominal)

No. of animals per sex per dose:

12 per dose (6x12 males)

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily in the morning hours and in the afternoon (shortly after exposure); weekenddays once daily

BODY WEIGHT: Yes
- Time schedule for examinations:4 days before start of 1st exposure; just prior befor start of 1st exposure; once/week thereafter

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g and body weight gain data: Yes

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of their exposure period (day 28)
- Anaesthetic used for blood collection: Yes (identity)
- Animals fasted: No data
- How many animals: 6 rats/group
- Parameters checked in table were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: no data
- Animals fasted: No data
- How many animals: 6/group (same as at HAEMATOLOGY)
- Parameters checked in table were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased on day 7 in the animals of the high concentration group

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

significantly decreased in 1st exp. week in high exp. group, sig. increased in last exp. week in all exp. groups compared to control group

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

lung weights increased in mid+high concentration group; weights of testes+liver increased in high concentration group

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

olfactory mucosa of the epithelium and lamina proria, squamous epithel in the nasal cavity and trachea

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical signs and survival:
The group-wise observation carried out about halfway through each day's exposure did not reveal abnormalities.
Individual observations in the mornings, i.e. before the start of each day's exposure did neither reveal treatment-related abnormalities. The changes observed, sparsely haired skin, microphtalmia and, dermal encrustations, were seen at a low incidence and only in the control group. They were therefore not considered to be related to the exposure. All rats survived until their scheduled necropsy.

Body weights:
Body weight was significantly decreased on day 7 in the animals of the high concentration group. Although body weight was numerically less in the latter group compared to the control during the exposure period, the differences were slight and were not necessarily considered to be related to exposure.
No differences in body weight gain were observed during the recovery period

Food consumption and food conversion efficiency:
Differences in food intake were not observed during exposure, except for a slight, statistically insignificant, lower overall intake in animals of the high concentration group. In the second week of the recovery period food intake was significantly increased in animals of the high exposure recovery group.
Food conversion efficiency was significantly decreased in the first exposure week in animals of the high exposure group and significantly increased in the last exposure week in all exposure groups compared to the control group. Most probably, food conversion efficiency was low in control animals during the last week of exposure rather than that it was increased due to the exposure.
Food conversion efficiency was increased during the first week after exposure, both in the control and the high exposure group. The increase, however, was significantly larger in the high exposure (recovery) group (p<0.00l).
Haematology: At the end of the exposure period, changes in red blood cell parameters were limited to a decrease in thrombocytes in the animals of the mid concentration group. Because it was not concentration-related, it was considered a fortuitous finding unrelated to treatment.
Statistically significant changes in the absolute or relative numbers- of white blood cells were not seen at the end of the exposure period.

Clinical chemistry: At the end of the exposure period, significant changes in clinical chemistry parameters were not seen.

Organ weights:
At the end of the exposure period, absolute and relative lung weights were increased in a concentration-related fashion in animals of the mid and high concentration groups. In addition, the absolute and relative weight of the testes and the relative weight of the liver were increased in animals of the high concentration group. In the other organs significant weight differences between control and exposed animals could not be detected.
An increase in absolute and relative lung weight was still present in animals of the high concentration group at the end of the recovery period but to a clearly lower extent. No changes were observed in the weight of the other organs.

Pathology:
Macroscopic examination: Gross examination at necropsy at the end of the exposure period did not reveal exposure-related changes. One low-concentration male exhibited red appearance of the thymus; one mid-concentration male showed uni-lateral red patches on the thymus. Both changes are quite common for rats of this strain and age.
Gross examination at necropsy after the recovery period did not reveal exposure related changes. Several changes were observed in control males and one change was found in a high-concentration male. These changes were considered to be common for rats of this strain and age.

Microscopic examination: Histopathological examination was performed on the complete respiratory tract of all animals of all groups (including the animals of the recovery groups). Liver, kidneys and testes were examined in animals of the main control (group A) and the high concentration group (group D) only.
Inhalation of hydroxypropylated .beta.-cyclodextrin induced histopathological changes in the nasal tissues, the trachea and the lungs of exposed animals. In the larynx the relationship between the observed changes and exposure to the test compound was questionable.

Histopathological findings in animals of the main groups:
High-concentration group animals
The nasal lesions occurred in the nasal squamous epithelium (SE), transitional (non-ciliated) epithelium (TE) and olfactory epithelium (OE). The SE changes consisted of hyperplasia of the septal squamous epithelium in the vestibulum of the nose (cross level 1), and of polymorphonuclear inflammatory cells infiltrating the SE of the ventral meatus at cross levels 1, 2, 3 and/or 4. The TE changes consisted of hyperplasia of the septal transitional epithelium at level 1, and occurred in association with the hyperplasia of the SE. Degeneration was observed in the entire OE lining, at cross levels 3, 4, 5 and 6. It was manifested by a decrease in nuclear density of the sensory cell layer rather than by a thinning of the epithelium. The loss of nuclei was especially prominent towards the topical site of the epithelium. As a result, the intact nuclei of the sustentacular cells were very conspicuous. There was some nuclear debris and mitotic figures were seen occasionally. The nerves in the underlying lamina propria appeared slightly affected, i.e. a decrease in diameter, a coarse aspect which suggested vacuolation of the axons, and/or an increase in nuclei in the larger nerves. These changes were collectively reported as 'neural change'.
The epithelium of the trachea was slightly thickened, due to hypertrophy of the lining epithelial cells and hyperplasia. It is highly conceivable that the increase in cells consisted mainly of mononuclear inflammatory cells which infiltrated the epithelium.
Accumulation of inflammatory cells in the alveolar lumina was the most prominent treatment-related observation in the lungs and could very well explain the increase in lung weight. The inflammatory cells consisted of a mixture of large macrophages with foamy cytoplasm and granulocytes. The respiratory epithelium lining the bronchi and• larger bronchioli was• slightly hypertrophic and hyperplastic.
Minimal respiratory cell hypertrophy/hyperplasia was also observed in the larynx of half of the top-concentration animals, and in the larynx of one control. In addition, a small intraepithelial cyst and a mixed inflammatory cell infiltrate were observed in a single top-concentration animal. The relationship between these changes and exposure to the test compound is questionable because either the incidence and degree of these changes did not differ distinctly between the test animals and controls or they occurred in a single animal and were not of a remarkable nature.
The remaining changes observed in the aforementioned organs and tissues were not considered to be treatment-related. In the other organs examined (liver, kidneys and testes) no exposure-related changes were observed.

Mid-concentration animals
The nasal lesions occurred in the OE and consisted of very slight to slight degeneration.
Accumulation of inflammatory cells in the alveolar lumina occurred in the lungs of all mid-concentration animals. The infiltrate was mixed in only one animal, in all others it consisted almost exclusively of alveolar macrophages. Respiratory epithelial hypertrophy/hyperplasia was found in one mid-concentration animal only.
No exposure-related changes were observed in the trachea and larynx.

Low-concentration animals
Accumulation of alveolar macrophages was focal instead of diffuse as observed in the high- and mid-concentration animals.
No exposure-related changes were observed in the nasal cavity, trachea and larynx.

Histopathological findings in animals of the recovery groups:
Microscopic examination of animals at the end of the recovery period revealed exposure-related histopathological changes in the nasal tissues and the lungs only. The nasal lesions occurred in the nasal transitional/ciliated epithelium (TE/CE) and the olfactory epithelium (OE). The TE/CE lesion consisted of squamous metaplasia on the septum at cross level 2, and was observed in 2 out of 6 high concentration animals. The OE of all high-concentration animals showed areas of degeneration at cross levels 3, 4, 5 and 6. The degeneration was of a somewhat lesser degree than observed at the end of the exposure period. One high concentration animal exhibited cytoplasmic eosinophilic inclusions in the OE. This change is sometimes found in the OE of old rats. Because of this and the occurrence in a single animal, the relationship with the exposure is not clear. Accumulation of alveolar macrophages was observed in the lungs of all high concentration animals. The majority of macrophages was smaller than those of the high-concentration animals in the main study. Also, the accumulation of macrophages was more focal. These focal accumulations were associated with increased septal cellularity. Interstitial mononuclear inflammatory cell infiltrations, an increase in type 11 pneumocytes and alveolar macrophages between or adjacent to the epithelial cells were the major contributors to the increased septal cellularity. Bronchial/bronchiolar respiratory epithelial hypertrophy/hyperplasia was not observed.
No exposure-related changes were found in the larynx and trachea.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

>= 1 000 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

2

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

10 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

3

Key result

Dose descriptor:

LOAEC

Remarks:

local effects

Effect level:

100 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/m³ air

System:

respiratory system: lower respiratory tract

Organ:

alveoli

bronchi

trachea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/m³ air

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Further, additional numbers of animals were included in the present study. These were exposed to the test atmospheres as described above, and part of these were also kept for a post-treatment period of 14 days. At necropsy, the lungs of these animals were lavaged and the bronchoalveolar lavage fluid obtained was examined for total and differential white blood cell numbers, viability and cell differentials, and for a few biochemical parameters.

Applicant's summary and conclusion

Conclusions:

From the results of this study it was concluded that inhalation exposure to hydroxypropylated .beta.-cyclodextrin for four weeks resulted in local toxicity at levels of 100 and 1000 mg/m³, consisting of histopathological changes of the respiratory tract and a concentration-related increase in lung weight. Although changes were less severe at a concentration of 1000 mg/m³ after a recovery period of 14 days, reversibility of these effects was not complete. At a level of 10 mg/m3 no such effects were observed, microscopical changes in the lung were limited to particle driven adaptive focal macrophage accumulation. 10 mg/m3 was therefore considered a No-Observed-Effect Concentration (NOAEC) for local toxicity. The severity of effects on the olefactory epithelium at 100 mg/m3 was at maximum slight (2 with very slight and 4 slight degeneration out of 6 animals) and effects tended at 1000 mg/m3 to recover.
Very slight growth retardation and reduced food intake and food conversion efficiency were observed at a level of 1000 mg/m³. These changes were considered to be indirect effects due to a lack in well-being (respiratory tract lesions). Since no systemic toxicity was observed at any of the concentrations tested, the NOAEL for systemic toxicity was >=1000 mg/m³.

Executive summary:

The inhalation toxicity of hydroxypropylated .beta.-cyclodextrin was studied in a sub-acute (28-day) study in Wistar rats according to OECD Guideline Study 412. Groups of 6 male rats were exposed to target concentrations of 0 (control), 10, 100 or 1000 mg/m³ hydroxypropylated .beta.-cyclodextrin for six hours a day, 5 days a week during a period of 28 days, with a total of 20 exposure days. The rats were necropsied the day after the last exposure. Two additional groups of 6 rats were exposed to either 0 (control) or 1000 mg/m³ hydroxypropylated .beta.-cyclodextrin as described above and were kept for a post-treatment period of 14 days to examine reversibility of toxic effects. To examine the toxicity of the test material clinical signs, body weights, food consumption, food conversion efficiency, haematology, and clinical chemistry were used. In addition, a full necropsy was performed and a selection of organs including the respiratory tract was examined microscopically.

Further, additional groups of animals (6 male rats/ group) were included in the present study. The animals were exposed to the test atmospheres as described above during a period of 29 days (21 exposure days), and part of them were also kept for a post-treatment period of 14 days. Clinical signs, body weights, food consumption and food conversion efficiency were recorded. At necropsy, the lungs of these animals were lavaged and the bronchoalveolar lavage fluid obtained was examined for total and differential white blood cell numbers, viability and cell differentials, and for a few biochemical parameters.

These results are described in part 2 of this report. The mean actual concentrations (± standard deviation) of hydroxypropylated .beta.-cyclodextrin in the test atmospheres were 10.4 ± 0.8, 102 ± 9 and 1006 ± 74 mg/m³ for the low, mid and high concentration test atmospheres, respectively. The Mass Median Aerodynamic Diameter (MMAD)was 1.9, 2.3 and 2.8 µm, and the geometric standard deviation (gsd) was 2.3, 2.4 and 2.4 for the low, mid and high concentration test atmospheres, respectively.

No treatment-related abnormalities in clinical signs or behavior were seen during the exposure and recovery -period.

Minimal reductions in body weight gain, food intake and food conversion efficiency were observed in animals of the high concentration group during the exposure period.

No treatment-related changes in haematology or clinical chemistry were observed.

A concentration-related increase in absolute and relative lung weights was observed in animals of the mid and high concentration group. In addition, the absolute and relative weight of the testes and the relative weight of the liver were increased in animals of the high concentration group. At the end of the recovery period, increased absolute and relative lung weights were still observed. No changes were observed in weights of other organs.

Inhalation of hydroxypropylated .beta.-cyclodextrin induced histopathological changes in the respiratory tract, i.e nasal cavity, trachea and lungs. Concentration-related changes were seen in the olfactory mucosa of the epithelium and underlying associated lamina propria of animals of the mid and high concentration. Histopathological changes of the transitional (non-ciliated) and squamous epithelium in the nasal cavity, consisting of epithelial hyperplasia and/or presence of polymorphonuclear inflammatory cells were seen in animals of the high concentration group only. Tracheal changes, consisting of epithelial hyperthrophy and hyperplasia, were also observed in animals of the high concentration group only. A concentration-related increase in accumulation of alveolar macrophages was seen in animals of the mid and high concentration group. Granulocytes were also present in all animals of the high concentration group, and in one animal of the mid concentration group. This change was accompanied by hypertrophy and hyperplasia of the respiratory epithelium lining the.bronchi and larger bronchioli. Although macrophage accumulations were also seen in animals of the low concentration group, no granulocytes were observed.

Moreover, these changes were focal rather than diffuse and were not accompanied by epithelial changes. Therefore, they are considered as rather an adaptive response to particle exposure than an substance specific adverse effect.

At the end of the recovery period, changes of the olfactory epithelium were still present but less severe. In lout of 6 high concentration animals, squamous metaplasia of the transitional/ciliated epithelium was observed on the nasal septum. Histopathological lung changes consisted of focal accumulations of alveolar macrophages which were associated with increased septal cellularity but not with bronchial/bronchiolar respiratory epithelial hypertrophy/hyperplasia. As overall changes were less severe at the end of the recovery period, reversibility of the effects occurred but was not completed during the 14-day recovery period.

No treatment-related histopathological changes were observed in the larynx, liver, kidneys and testes.