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Diss Factsheets

Administrative data

sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
16th January to 16th April 1992
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with an appropriate OECD test guideline and in compliance with GLP, using a closely related test substance.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Reference substance name:
EC Number:
Cas Number:
Constituent 2
Reference substance name:
EC Number:
EC Name:

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River (UK) Limited, Manston, Kent

- Age at study initiation: 6 -7 weeks

- Weight at study initiation: Males: 174 - 235 g ; Females: 148 - 198 g

- Fasting period before study:

- Housing: Except during the exposure period, animals were housed in groups of 5 by sex in polypropylene grid-floor cages, suspended over trayes lined with absorbent paper.

- Diet: SQC Rat and Mouse Diet No. 1 expanded ad libitum except during exposure

- Water: Mains water from polycarbonate bottles ad libitum except during exposure

- Acclimation period: 14 days


- Temperature (°C): 19 - 24

- Humidity (%): 40 - 55

- Air changes (per hr): 15

- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 16th January 1992 To: 15th April 1992

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
other: unchanged (no vehicle)
Details on inhalation exposure:

- Exposure apparatus: 30 litre cylindrical exposure chambers (ADG Instruments Ltd, Hitchin, Herts.)

- Method of holding animals in test chamber: Each animal was held in a polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed with a rubber O-ring.

- Source of air: Compressed air from GAST-2HBB-10-P25Y oil-free compression pump.

- Method of conditioning air: Air was passed through a water trap and respiratory-quality filters

- System of generating particulates/aerosols: Glass concentric jet nebulisers (Radley's, Sawbridge, Herts.) connected to a glass syringe, attached to a modified infusion pump to provide a continuous supply of test material, and to a metered compressed air supply. The nature of the test substance was such that production of an aerosol in the test chamber resulted in instantaneous volatilisation and the resulting atmosphere was shown to contain no aerosol particles.


- Brief description of analytical method used: Gas chromatography

- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Chamber air was continuously sampled through impinger bottles containing methanol over the entire 6-hour exposure period. The resulting samples were collected twice daily, at the middle and end of the exposure period. Analysis by gas chromatography was carried out twice weekly.

Samples were diluted with methanol where necessary, to obtain a test concentration of approximately 0.1 mg/l. These were analysed by gas chromatography using a flame ionisation detector and an external standard.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
5 days per week
Doses / concentrationsopen allclose all
Doses / Concentrations:
0.22 mg/l
analytical conc.
Doses / Concentrations:
0.75 mg/l
analytical conc.
Doses / Concentrations:
2.54 mg/l
analytical conc.
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
Details on study design:
- Dose selection rationale: range finding study

- Rationale for animal assignment: Random

- Rationale for selecting satellite groups: None

- Post-exposure recovery period in satellite groups: None


Observations and examinations performed and frequency:

- Time schedule: Continuously during exposure

- Cage side observations: appearance, respiratory and behavioural patterns.


- Time schedule: 3 hours after start of exposure and on removal from chamber.


- Time schedule for examinations: Day 0 and weekly intervals thereafter.


- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


- 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


- Time schedule for examinations: Weekly for food consumption; 7-day period during weeks 1, 6 and 12 for water consumption.


- Time schedule for examinations: Commencement and termination of exposure

- Dose groups that were examined: Control and high dose groups


- Time schedule for collection of blood: Prior to necropsy on Day 91

- Anaesthetic used for blood collection: Yes (Halothane B.P.)

- Animals fasted: Yes

- How many animals: All control and dose group animals.

- Parameters checked in Table 1 were examined.


- Time schedule for collection of blood: Prior to necropsy on Day 91

- Anaesthetic used for blood collection: Yes (Halothane B.P.)

- Animals fasted: Yes

- How many animals: All control and dose group animals.

- Parameters checked in Table 1 were examined.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 2)

HISTOPATHOLOGY: Yes (see Table 2)
Other examinations:
Absolute and relative organ weights, haematological and blood chemistry data were analysed by one-way analysis of variance incorporation "F-max" test of homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney U-test.

Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:

1) Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 5 or more;
2) Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity of grades for the more frequently observed graded conditions.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no treatment-related deaths during the study. One male in the low-dose group was killed in extremis on Day 49. No clinical signs of toxicity were observed in control or test group animals throughout the study. Incidents of red/brown staining around the eyes, together with wet fur, were detected in all dose groups throughout the treatment period. These are typical findings associated with the restraint procedure and are not indicative of toxicity. One low-dose male showed signs of hunched posture, piloerection and lethargy from day 44 to day 49. This animal was killed in extremis during the exposure period on day 49. Other isolated signs were noted in low and intermediate dose groups including hunched posture, piloerection, lethargy, decreased respiratory rate and ptosis. These were not dose related and, as such, were not considered to be toxicologically significant.

BODY WEIGHT AND WEIGHT GAIN: No adverse effects on bodyweight were detected that could be attributed to treatment. Slight reductions in bodyweight gains were observed in all dose groups, including controls, throughout the latter part of the study. This was considered to be associated with the restraint procedure and since there were no intergroup differences in the incidence and severity of effects these were considered to be of no toxicological significance.

FOOD CONSUMPTION: There was no apparent adverse effect on food consumption at any dose level.

FOOD EFFICIENCY: Food efficiency in test animals was comparable to controls.

WATER CONSUMPTION: There was no apparent adverse effect on water consumption at any dose level.

OPHTHALMOSCOPIC EXAMINATION: There were no treatment-related ocular effects. The incidental findings recorded were those normally encountered in laboratory animals of this strain and age.

HAEMATOLOGY: There were no treatment-related changes in the haematological parameters examined. A statistically significant increase in mean corpuscular haemoglobin was detected in intermediate and high dose females together with an increase in high dose female neutrophil counts. All values were entirely within the normal ranges for rats of this strain and age and, in isolation, the increases were considered to be fortuitous and of no toxicological significance. The other minor statistically significant difference between test and control groups was limited to intermediate female mean corpscular haemoglobin and was not dose-related.

CLINICAL CHEMISTRY: There were no blood chemistry changes that were considered to be treatment-related. A statistically significant increase in plasma urea in intermediate and high dose males was observed, together with a reduction in high dose male glucose. All values were entirely within the normal ranges for rats of this strain and, in the absence of any associated changes, were considered to be fortuitous and of no toxicological significance. A statistically significant reduction in alkaline phosphatase was seen in high dose animals of both sexes but a reduction in the level of this enzyme cannot be regarded as toxicologically important. Inorganic phosphate was significantly reduced in all female treatment groups, but no convincing dose-response relationship was elicited.

ORGAN WEIGHTS: There were no changes in organ weights that could be attributed to treatment with the test material. A statistically significant reduction in absolute lung weight in high dose females was detected in comparison to controls. No change in relative weight was detected and consequently the reduction was not considered to be of toxicological significance. High dose males showed a statistically signficant increase in relative liver weight, however with no blood chemical or histopathological evidence to support hepatic effects, the increase was considered unlikely to be of toxicological significance. Other minor, statistically significant differences between test and control groups were confined to low-dose males and, as such, were not dose-related.

GROSS PATHOLOGY: No treatment-related gross abnormalities were detected. The decedent from day 49 showed accentuated lobular pattern and patchy pallor of the liver, unusually pink pancreas and epithelial sloughing and thickening of the forestomach. A large pus-filled mass was noted dorsal to the seminal vesicles and bladder which appeared to have caused intestinal blockage. One intermediate dose male showed a similar, but smaller, mass adjacent to the prostate gland at necropsy. Other incidental findings recorded at necrospy were consistent with those normally expected for laboratory maintained rats.

HISTOPATHOLOGY:No treatment-related changes were observed. All morphological changes were those commonly observed in laboratory-maintained rats of the strain and age used in the study. There were no differences in incidence or severity between test and control groups therefore all were considered to be without toxicological significance.

Effect levels

Dose descriptor:
Effect level:
>= 2.54 mg/L air (analytical)
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

In a 90-day nose-only vapour inhalation study with Sprague Dawley rats, the No Observed Effect Level was determined to be >=2.54 mg/l (measured concentration), which was the highest dose tested. The test concentrations were selected on the basis of a range-finding study.