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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD guideline 412. However, summary in public literature available, no summary and individual data of all results available.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
No analytical results available, however, analysis of test concentrations was performed.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch No.: not specified
Purity: 98+% pure

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Sprague-Dawley (SD)
- Weight at study initiation: 184±2 g for females and 240±2 g for males
- Housing: Animals were individually identified with ear notches and randomly assigned by a computer-assisted method that provided homogeneity of variance and equality of initial body weights for all groups.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±1°C
- Humidity (%): 50%±15%

No additional data

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Not applicable. Animals were exposed to vapour.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Sage Instrument co. syringe pump (model 220).
- Method of holding animals in test chamber: Animals were exposed in 2.50 m3 stainless steel chambers.
- Air flow rate: 500 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography
- Samples taken from breathing zone: yes

No additional data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The inhalation chambers were continuously monitored throughout the exposure by gas chromatography. Concentration of the test substance in air was deteremined for each chamber every 6 minutes by using an automatic sampling system and a Bendix gas chromatograph. The monitory was conducted by comparion with a standard sample chromatographed on a CSP-20M stainless steel column under the following conditions: injector and electron capture detector temperature, 175°C, oven temperature, 100°C, flow rate for the carrier gas nitrogen, 120mL/min, hydrogen, 32.3 mL/min, and air: 300 mL/min. The retention time of the test substance was found to be 260 seconds.
Duration of treatment / exposure:
Animals were exposed for 6 hours/day for 14 days.
Frequency of treatment:
Animals were exposed for 14 consecutive days.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 750 and 1000 ppm
Basis:
no data
No. of animals per sex per dose:
14 animals/sex/concentration
Control animals:
yes
Details on study design:
The study was performed in accordance with OECD guideline 412.
Rats from the control and each test substance exposure group were necropsied 72 hours after the last exposure.
Positive control:
No positive control was included.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: after 2, 8 and 14 exposures

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken at necropsy from the vena cava.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 6

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken at necropsy from the vena cava.
- Animals fasted: No data
- How many animals: 6

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER:

No additional data
Sacrifice and pathology:
At necropsy, a gross pathological examination was performed on all animals. The rats found moribund after exposure were immediately necropsied to prevent autolysis of tissues. Seven animals/sex/dose were necropsied 72 hours after the last exposure, the remaining animals were subjected to whole body perfusion and prepared for future electron microscopy examination.
The following tissues were excised and collected for histopathological examination: adrenals, brain, heart, kidneys, liver, lungs, small and large intestines, larynx, rhinopharynx, spleen, stomach, trachea, testes or ovaries, thyroid and urinary bladder. The following organs were weighed prior to perfusion: brain, heart, kidneys, livers, lungs and spleen.
During necropsy, special care was taken to avoid damage to the brain and nasal tissues. Tisses were fixed, processed, and embedded in paraffin, sectionated at 6 µm, stained with hematoxy and eosin, and examined by light microscopy.
Nasal tissues of 7 animals of the control, 500 and 1000 ppm groups were first declacified in a formin acid-sodium citrate solution before processing and embedding. Sections were cut to illustrate the respiratory, olfactory and stratified squamous eptihelia.
Other examinations:
Heamatology and clinical biochemistry was performed in 6 animals of the control, 500 and 750 ppm groups and 4 animals of the 1000 ppm group. Blood was taken at necropsy (no time after last exposure indicated).
The following haematological parameters were determined: red blood cell count, white blood cell count, hematocrit, mean corpuscular volume, hemoglobin, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration.
Acetylcholinesterase was determined in red cells, and the following biochemical parameters were determined in serum: albumin, bilirubin, blood urea nitrogen, cholesterol, glucose, inorganic phosphorus, total protein, triglycerides, calcium, chloride, magnesium, potassium, sodium, amylase, cholinesterase, creatine phophokinase, gamma-glutamyl transferase, aspartate aminotransferase, alanine aminotransferase, alpha-hydroxybutyrate dehydrogenase and alkaline phosphatase.
Statistics:
Body and tissue weights and hematology, biochemistry and physiological data were analyzed using a one-way analysis of variance, Duncan's multiple range test, and a modified least significant difference procedure. For parameters fuond to be significantly different (p≤0.05). Student's t-test was used to determine levels of significance between groups.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional 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
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Clinical signs (incl. signs of neurotoxicity e.g. reduced motor activity) and hypothermia in all test substance exposed groups. Mortality at 750 and 1000 ppm. Nasal and ocular irritatation in all test substance exposed groups.

BODY WEIGHT AND WEIGHT GAIN
Reduced weight gain in all test substance exposed males and 1000 ppm females.

HAEMATOLOGY
Increase in monocytes in all test females, slightly reduced red blood cell count, hemoglobin and hematocrit in 1000 ppm females. Increased white blood cell count and neurophils in males at 1000 ppm, a slightly reduced hemaglobin and hematocrit in males at 1000 ppm. A decrease in lymphocytes, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration at 750 and 1000 ppm.

CLINICAL CHEMISTRY
Reduced serum ChE in all test females. An increased in ASAT in all test males and females. A decrease in albumin and total protein in all test females.

NEUROBEHAVIOUR
Tremors, piloerection, diuresis, reduction of breathing rate in all test groups. Animals at 750 and 1000 ppm were extreme sensitive to noise in the room and displayed aggressive behaviour.

ORGAN WEIGHTS
Increase in absolute and relative liver weight in all female test groups.

GROSS PATHOLOGY
No findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Goblet cell metaplasia was seen in all treated males: 4/7 at 500, 750 and 1000 ppm. The goblet cell metaplasia was extensive in the respiratory epithelial lining of the septum, but was minimal in the epithelial lining of the turbinates. The overall change in males was considered mild. Similar, less frequent changes were observed in females. Besides the goblet cell metaplasia, no inflammatory, degenerative, or other alterations were observed in the nasal tissues. Findings in other organs were infrequent and appeared to be incidental background changes regularly observed in this strain of rat.

No additional data

Effect levels

Dose descriptor:
LOAEC
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Physiological monitoring, clinical observations, organ weights.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a subacute inhalation study in rats, a NOAEC could not be derived, since effects of the test substance were noted in all treatment groups. Clinical observations (indicative of neurotoxicity), hypothermia and goblet cell metaplasia were noted at concentrations of 500 ppm and above. Therefore, the LOAEC for systemic and local effects is established at 500 ppm (equivalent to 2.2 mg/L).
Executive summary:

This study was run according to a guideline which is equivalent or similar to OECD Guideline 412 with Sprague-Dawley rat. The clinical signs, mortality, body weight, haematology, clinical chemistry, neurobehaviour,organ weights, gross pathology and non-neoplastic histopathology were observed.

In a subacute inhalation study in rats, a NOAEC could not be derived, since effects of the test substance were noted in all treatment groups. Clinical observations (indicative of neurotoxicity), hypothermia and goblet cell metaplasia were noted at concentrations of 500 ppm and above. Therefore, the LOAEC for systemic and local effects is established at 500 ppm (equivalent to 2.2 mg/L).