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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 Feb 1993 to 1 Nov 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed in conformance with the OECD guideline 413 and with US EPA 798.2450 and was GLP-compliant (GLP UK, EC, OECD, TSCA and MITI)
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.2450 (90-Day Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Analytical purity: 90.2% (GC-MS)
- Impurities (identity and concentrations): 9.4 % dimer of methacrolein (3,4-dihydro-2,5-dimethyl-2H-pyran-2-carboxaldehyde)
- Lot/batch No.: 9200914
- Supplier: Aldrich Chemical Co Ltd, Gillingham, Dorset, England, UK
- Expiration date of the lot/batch: no data; re-analysis after end of study: 90.5% (GC-MS)
- Stability under test conditions: adequate for the study
- Storage condition of test material: refrigerated (4 °C)
- Total of 4 test material analyses during the study: 29 Apr 1993 (Week -1, 90.2%), 1 Jun 1993 (Week 5, 90.6%), 1 Jul 1993 (Week 9, 90.2%), 3 Aug 1993 (Week 14, 90.5%)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Manston Road, Margate, England
- Age at study initiation: approx. 6 weeks
- Weight at study initiation (allocation to the groups): means of 196.8 g - 197.7g (males), 164.1 g - 165.3 g (females)
- Fasting period before study: none
- Housing:group caging (5/cage), suspended cages with stainless steel sides and stainless steel mesh floors, 53 cm x 35 cm x 25 cm (LxWxH). Plastic trays, lined with absorbent paper, were placed below each cage to collect animal excreta. The paper was changed daily and clean cages were introduced at intervals throughout the study.
- Diet (ad libitum): SDS Rat and Mouse No.1 modified diet, Special Diets Services, Witham, Essex, England, ad libitum
- Water (ad libitum): Tap water was available from moulded polypropylene water bottles at all times while the rats were in the cages. The water bottles were rinsed and refilled daily and thoroughly cleaned at intervals during the study.
- Acclimation period: 9 days


ENVIRONMENTAL CONDITIONS (acclimation and between exposures):
- Temperature (°C): 18.0 - 25.5 °C
- Humidity (%): 31 - 58%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs light, 12 hrs dark


IN-LIFE DATES: From: 21 April 1993 (arrival of animals) To: 27 August 1993 (terminal kill of withdrawal group animals)

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: nor applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure chambers were constructed of stainless steel and glass. Each of the 4 chambers used was approximately 0.75 cubic meter in volume. The chambers were of square plan section fitted with a pyramidal base and top. A square-shaped 3 inch diameter tubular perforated exhaust plenum was fitted in the base of each chamber below exposure cage level. A perforated dispersion plate was fitted at the point of air entry. A 1.5 inch drainage duct fitted with a ball valve was present in the centre base of the chamber and was connected with a drainage system.
- Method of holding animals in test chamber: Exposure cages, constructed of stainless steel mesh, were suspended on a framework, arranged on four levels. Each level held four cages each capable of individually housing 4 rats. This gave a total potential animal exposure capacity of 64 rats. However, in this investigation a maximum of 8 cages were used, per group.
- Atmosphere generation: Vapour was produced by metering the liquid test substance from a glass gas-tight syringe mounted on an infusion pump (Precidor type 5003) to a sintered glass frit contained in a glass vessel. Air was fed through each vaporiser at 150 L/min. The air was warmed by passage through a coiled copper tube immersed in a water bath maintained at 80 °C. The vapour/air mixture produced passed from the all glass vaporiser into the base of a glass elutriation columnn and from there to the chamber inlet duct. After passing through the chamber, the chamber atmosphere was removed via an extract duct using an extract fan. Different vapour concentrations appropriate to each exposure group were achieved by varying the rate the test substance was metered to the glass frit.
- System of generating particulates/aerosols: not relevant
- Temperature, humidity, pressure in air chamber (means): 25.6 °C/38 % (controls), 25.4 °C/33 % (low conc.), 25.0 °C/36 % (int. conc.), 25.8 °C/47 % (high conc.), chamber pressure 10 mm of water below ambient. The air entered the chamber through the inlet duct and dispersion plate. Airflows were monitored by tapered tube flow meters, mounted at the front of a purpose-built stainless steel trolley. The generation apparatus was also mounted on the trolley. A magnehelic pressure gauge (0 - 100 mm water gauge) was connected with each chamber by a nylon tube. This was also mounted on the trolley and was used to monitor chamber internal.pressure.
- Air flow rate: 150 l/min
- Air change rate: approx. 12/h
- Method of particle size determination: not relevant
- Treatment of exhaust air: Removal of the test atmosphere from each chamber was accomplished by means of individual air handling units containing coarse and fine filtration media, together with activated charcoal. The flow was adjusted using gate valves, mounted in the exhaust ducting between the chamber and filters, and the internal pressure within each chamber was set to 10 mm water gauge below ambient when operational.


TEST ATMOSPHERE
- Brief description of analytical method used: flame ionisation chromatography using external standards
- Samples taken from breathing zone: yes


VEHICLE: air

PROCEDURE
The water baths were switched on and set to 70 °C. The animals were removed from their holding cages, and placed within the wire mesh exposure cages in the chamber appropriate to each group. The chamber doors were sealed after fitting of a wet and dry bulb thermohygrometer. Syringes were filled with the test substance and mounted on Precidor infusion pumps. The initial volume of each syringe was recorded. The airflow to the vaporisers was turned on and the internal pressure of the chambers adjusted to 10 mm water gauge using the gate valves. Exposure commenced when the infusion pumps were switched on and operating at the determined rate. Samples for the determination of total chamber concentration were taken during each six-hour exposure. Records of chamber temperature and relative humidity were made, together with the reaction, if any, by exposed rats. After six hours exposure the infusion pumps were switched off and the volume of methacrolein remaining in the syringes recorded. The vaporiser air supplies were turned off. The rats were unloaded from the chamber into their respective holding cages following a period of chamber clearance of at least 20 - 30 minutes for test group animals.

CHAMBER SPATIAL DISTRIBUTION
The spatial distribution of methacrolein in the exposure chambers was determined for each concentration level during preliminary studies. The spatial distribution of the test vapour was considered satisfactory.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of methacrolein present in the exposure chambers used for the test material groups was determined on at least 3 occasions during each exposure. Samples were taken at approximately 1, 3 and 5 hours from the start of exposure. Samples of test atmosphere were withdrawn at 2 L/min through charcoal adsorption tubes (Lot 120, NIOSH approved, SKC Inc, PA, USA). The contents of the tubes were eluted into accurately measured 2 mL aliquots of carbon disulphide. The samples were analysed by flame ionisation chromatography using external standards.
For analytical method see Appendix 2.
Duration of treatment / exposure:
Six hours a day for 13 weeks
Frequency of treatment:
5 days/ week
Doses / concentrations
Remarks:
Doses / Concentrations:
1; 5 and 15 ppm = 0.003, 0.014 and 0.044 mg/L
Basis:
nominal conc.
No. of animals per sex per dose:
Group 1 (air control):
vehicle only , 20* males/20* females
Group 2 (low concentration):
1 ppm (nominal conc.), 1.0 ppm (analytical conc.), 10 males/10 females
Group 3 (intermediate concentration):
5 ppm (nominal conc.), 4.9 ppm (analytical conc.), 10 males/10 females
Group 4 (high concentration):
15 ppm (nominal conc.), 15.3 ppm (analytical conc.), 20* males/20* females

*) 10 animals of each sex were further observed for a 4-week observation period after the end of 13 weeks exposure.
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: 4 weeks
- Dose selection rationale: Exposure levels were selected in consultation with the Sponsor, following a 2-week inhalation study (HRC Report No. BGH 40/920648) where rats were exposed to 77, 19 and 5 ppm. Deaths occurred following the first exposure at 77 ppm and exposure of surviving rats was discontinued. Treatment-related effects of exposure at 19 ppm were evident, including reduced bodyweight gain and food consumption together with increased water consumption. Treatment-related changes in the nasal turbinates and larynx were also seen in rats exposed to 19 ppm methacrolein. The no-effect-level in this preliminary study was established at 5 ppm. On the basis of these findings, exposure levels of 0 (Control), 1, 5 and 15 ppm were selected, in order to elicit clear response at the High exposure concentration and with 1 ppm expected to be a no-effect exposure concentration.
- Rationale for animal assignment (if not random): computer program to achieve approx. equalised group mean body weights
- Rationale for selecting satellite groups: to investigate possible recovery from test material related effects
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): no data
Positive control:
not applicable

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to loading and immediately following unloading

BODY WEIGHT: Yes
- Time schedule for examinations: at allocation to groups, then on each day commencing 1 week before the start of exposures and continuing throughout the study. In addition, the weight of each rat at necropsy was recorded.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Food consumption per cage recorded daily commencing 1 week prior to the start of exposures until the end of the study

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
--- all rats: prior to allocation
--- all rats from Main groups: once during final week of exposure
- Dose groups that were examined: Air control, Low, Intermediate and High concentration (no satellite groups)


HAEMATOLOGY: Yes
- Time schedule for collection of blood: once, on the morning following the final exposure
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight
- Air control, Low, Intermediat and High concentration (no satellite groups), total of 80 animals
- The following parameters were examined:
Erythrocyte count (RBC)
Haemoglobin (Hb)
Haematocrit (Hct)
Mean corp. volume (MCV)
Mean corp. Hb. conc. (MCHC)
Reticulocytes
Total leukocyte count
Neutrophils (differential)
Eosinophils (differential)
Basophils (differential)
Lymphocytes (differential)
Monocytes (differential)
Prothrombine time
Thrombocyte count



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once, on the morning following the final exposure
- Animals fasted: Yes, overnight
- How many animals: Air control, Low, Intermediat and High concentration (no satellite groups), total of 80 animals
- The following parameters were examined:
Calcium
Chloride
Phosphorus (inorganic)
Potassium
Sodium
Albumin
A/G ratio
Bilirubin (total)
Cholesterol (total)
Creatinine
Globulins
Glucose
Protein (total)
Urea nitrogen
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Alkaline phosphatase (ALP)
Gamma-glutamyl-transpeptidase (GGT)
Creatine phosphokinase



URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
-all animals, all groups:
Adrenal glands
Aorta
Brain
Bone marrow (from femur and sternum, histopathology of sternum bone marrow only)
Caecum
Colon
Duodenum
Epididymides
Oesophagus
Eyes (with optic nerve)
Femur (with joint)
Gross lesions/masses
Heart
Ileum
Jejunum
Kidneys
Lacrymal glands
Larynx (2 levels)
Liver
Lungs (all lobes and mainstem bronchi)
Lymph nodes (cervical, mesenteric, tracheobranchial)
Female mammary gland
Muscle, skeletal
Nasal passages (3 levels)
Nerve, periphaeral (sciatic n.)
Ovary and oviduct
Pancreas
Pharynx
Pituitary
Prostate
Rectum
Salivary glands (mandibular, sublingual)
Seminal vesicles
Skin/subcutis
Spinal cord (3 levels, cervical, thoracic, lumbar)
Spleen
Sternum
Stomach
Testes
Thymus
Thyroid/parathyroid
Tongue
Trachea (incl. bifurcation)
Ureter
Urinary bladder
Uterus (corpus and cervix)
Vagina
Head (paranasal sinsuses, oral cavity, nasopharynx, middle ear, teeth, eyelids, Harderian gland, Zymbal's gland)

ORGAN WEIGHT: Yes
-all animals, all groups:
Adrenal glands
Brain
Epididymides (weighed together with testes)
Heart
Kidneys
Liver
Lungs (all lobes and mainstem bronchi)
Ovary and oviduct
Pituitary
Prostate
Spleen
Testes (weighed together with epididymides)
Thymus



HISTOPATHOLOGY: Yes
-all animals from group 1 and group 4.
Adrenal glands
Aorta
Brain
Duodenum
Epididymides
Oesophagus
Eyes (with optic nerve)
Gross lesions/masses
Heart
Ileum
Jejunum
Kidneys
Larynx (2 levels)
Liver
Lungs (all lobes and mainstem bronchi)
Lymph nodes (cervical, mesenteric, tracheobranchial)
Nasal passages (3 levels)
Nerve, periphaeral (sciatic n.)
Ovaries
Pancreas
Pharynx
Pituitary
Salivary glands (mandibular, sublingual)
Spinal cord (3 levels, cervical, thoracic, lumbar)
Spleen
Sternum
Stomach
Testes
Thymus
Thyroid/parathyroid
Trachea (incl. bifurcation)
Urinary bladder
Uterus (corpus and cervix)

-all animals, all groups:
Larynx (2 levels)
Nasal passages (3 levels)
Other examinations:
None
Statistics:
STATISTICAL ANALYSIS

Data relating to food were analysed on a cage basis. For all other parameters the analyses were carried out using the individual animal as the experimental unit. Food consumption data were analysed using cumulative cage weekly average intake per group. Bodyweight data were analysed using weight gains. The following sequence of statistical tests was used for food consumption, bodyweight, organ weight and clinical pathology data:

(I) If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75 %), the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise:

(II) Bartlett's test (1) was applied to test for heterogeneity of variance between treatments; where significant (at the 1 % level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.

(III) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.

(IV) Except for pre-exposure data, analyses of variance were followed by Student's 't' test and Williams' test for a dose-related response, although only Williams' test was reported. The Kruskal-Wallis analyses were followed by Shirley's test, the non-parametric equivalent of the 't' and Williams' tests.

Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, the final bodyweight was used as a covariate in an attempt to allow for differences in bodyweight which might affect the organ weights.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During exposures half-closed eyes were observed in Group 3 (5 ppm), (Exposures 1 - 6 only) and in Group 4 (15 ppm) (most exposures throughout the study). In addition, occasional observations of licking die inside of the mouth and wet chins, indicating salivation, were made in Group 4 (15 ppm).
The observations made are indicative of a non-specific response to exposure to an irritant atmosphere.
Mortality:
no mortality observed
Ophthalmological findings:
no effects observed
Details on results:
MORTALITY
There were no unscheduled deaths.

CLINICAL SIGNS
During exposures half-closed eyes were observed in Group 3 (5 ppm), (Exposures 1 - 6 only) and in Group 4 (15 ppm) (most exposures throughout the study). In addition, occasional observations of licking the inside of the mouth and wet chins, indicating salivation, were made in Group 4 (15 ppm).
The observations made are indicative of a non-specific response to exposure to an irritant atmosphere.
Observations made between exposures and at other times: No signs were seen that were considered to be attributable to exposure to methacrolein.

BODY WEIGHT AND WEIGHT GAIN
Recording of bodyweights at Week 13 took place following overnight starvation prior to removal of blood samples, therefore statistical analysis of weight gains following 12 weeks of exposure was also undertaken.
Female rats from Group 4 (15 ppm) gained weight at a reduced rate compared with controls from the commencement to termination of exposures, the reduction was statistically significant (P < 0.01 using Williams' test) from Week 3 until Weeks 12 and 13. Male rats from the Group 4 (15 ppm) gained weight at a similar rate to control rats for the first 7 weeks of exposure. Thereafter, weight gain by Group 4 (15 ppm) males was lower than that of controls. The reduction attained statistical significance (P <0.01) using Williams' test at Week 13 only, however, at Week 12, the degree of significance (P < 0.05) was apparent using Student's 't' test.
During the post-exposure observation period a degree of recovery was observed in all Group 4 (15 ppm) rats such that their weight gain over this period was greater than that of controls.
See Table 4.


FOOD CONSUMPTION
Food consumption measurements at Week 13 were affected by the preceding overnight period of starvation, therefore statistical analysis of cumulative consumption was also undertaken for Week 12 data.
Consumption by female rats from Group 4 (15 ppm) progressively declined relative to control rats from the commencement to the termination of exposures. The reduction attained statistical significance (P <0.01) at Weeks 12 and 13. Consumption by male rats from Group 4 (15 ppm) was similar to that of controls for the first 7 weeks of exposure, thereafter consumption declined relative to controls. The reduction did not achieve statistical significance.
During the recovery period the progressive decline in consumption by Group 4 (15 ppm) animals halted and consumption stabilised albeit at a lower level than that of control rats. Cumulative consumption by Group 4 (15 ppm) females remained significantly (P < 0.05) lower than that of controls.
See Table 5.

OPHTHALMOSCOPIC EXAMINATION
Findings were consistent with the age and strain of the animals examined, there were no treatment-related changes at Week 13.

HAEMATOLOGY
The following statistically significant differences from control values were seen:
Haemoglobin: Higher concentration in Group 4 (15 ppm) females.
Red cells: Greater numbers in Group 4 (15 ppm) males.
Mean corpuscular haemoglobin concentration: Greater in Group 2 (1 ppm), Group 3 (5 ppm) and Group 4 (15 ppm) females.
Mean corpuscular volume: Lower in Group 3 (5 ppm) and Group 4 (15 ppm) females.
The differences seen were small, inconsistent between the sexes and considered not to be of toxicological significance.
See Table 6.

CLINICAL CHEMISTRY
The following statistically significant differences from control values were seen:
Globulin: Lower in Group 4 (15 ppm) females.
Glutamic-pyruvic transaminase: Greater in Group 4 (15 ppm) females.
Electrolytes:
- Lower sodium concentration in Group 3 (5 ppm) and Group 4 (15 ppm) females.
- Lower potassium in Group 4 (15 ppm) females.
- Lower inorganic phosphorus in Group 2 (1 ppm), 3 (5 ppm) and 4 (15 ppm) females.
- Lower chloride in Group 4 (15 ppm) females.
The differences seen were small, inconsistent between the sexes and considered not to be of toxicological importance.
See Table 7.

ORGAN WEIGHTS
The following statistically significant differences from control values were seen:
Thymus: Lower weight in Group 4 (15 ppm) Recovery group males.
Spleen: Lower weight in Group 4 (15 ppm) Main group males.
Kidneys: Greater weight when adjusted for bodyweight as covariate in Group 4 (15 ppm) Recovery group males.
Prostate: Lower weight in Groups 2 (1 ppm), 3 (5 ppm) and 4 (15 ppm) Main group males.
The differences seen were small, inconsistent between the sexes and considered not to be of toxicological significance.
See Table 9.

GROSS PATHOLOGY
There were no changes revealed in rats killed following 13 weeks of dosing or those killed following 4 weeks of withdrawal that were considered to be attributed to exposure to methacrolein.
See Table 8.

HISTOPATHOLOGY

Treatment-related changes

Nasal passages
A range of irritant-related changes characterised by erosion, inflammatory infiltration, atrophy and squamous metaplasia of respiratory and olfactory epithelia of the dorsal meatus and the central septum were seen in the nasal passages of the majority of animals from Group 4 (15 ppm). These changes were not seen in animals in Group 3 (5 ppm) or 2 (1 ppm).
In general the atrophic change was more evident in the anterior levels (ie: A had a higher incidence than C) while inflammatory and metaplastic changes predominated in the more posterior B and C levels. Erosive and inflammatory changes, when present, often obscured areas of atrophy and the changes recorded are best considered as a related spectrum of events rather than discrete pathological entities.
After a period of recovery, clear evidence of attempted repair was seen at the affected sites with a diminution of inflammatory and metaplastic changes.

Larynx
Minor epithelial, primarily hyperplastic, changes were seen in the larynges of Group 4 (15 ppm) animals only. These changes were minor in degree and mostly confined to the anterior aspect of level A and also of the epithelium overlying the arytenoid cartilages as well as some small amounts of squamous metaplastic change of the ventral epithelium at level B.


Incidental changes
The other histological changes that were recorded were all within the normal range of changes seen in animals of this age and strain in this Iaboratory and were not considered to be of toxicological significance.

See Table 10.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
0.014 other: mg/L
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect at this concentration
Dose descriptor:
LOAEC
Effect level:
0.043 other: mg/L
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Treatment-related changes

Nasal passages

A range of irritant-related changes characterised by erosion, inflammatory infiltration, atrophy and squamous metaplasia of respiratory and olfactory epithelia of the dorsal meatus and the central septum were seen in die nasal passages of die majority of animals from Group 4

(15 ppm). These changes were not seen in animais in Group 3 (5 ppm) or 2 (1 ppm). In general the atrophic change was more evident in die anterior leveis (ie: A had a higher incidence than C) while inflanmmatory and metaplastic changes predominated in the more posterior B and C levels. Erosive and inflaninatory changes, when present, often obscured areas of atrophy and die changes recorded are best considered as a related spectrum of events rather than discrete pathological entities.

After a period of recovery, clear evidence of attempted repair was seen at the affected sites with a diminution of inflammatory and metaplastic changes.

Larynx

Minor epithelial, primarily hyperplastic, changes were seen in die larynges of Group 4 (15 ppm) animals only. These changes were minor in degree and mostly confined to the anterior aspect of level A and also of the epithelium overlying the arytenoid cartilages as well as some small amounts of squamous metaplastic change of the ventral epithelium at level B.

Applicant's summary and conclusion

Executive summary:

In this study, rats were exposed by inhalation to methacrolein vapour for 6 hours a day, 5 days a week (except Week 1 exposed for 1 day and Week 13 for 4 days), for 13 weeks. The mean exposure levels were 1, 5 and 15 ppm.

During exposure, clinical signs indicative of exposure to an irritant atmosphere were seen in rats exposed to 5 and 15 ppm. Effects on weight gain and food consumption were seen in rats exposed to 15 ppm, the effect was more marked in females.

Histopathological changes were confined to the nasal passages and larynges of rats exposed to 15 ppm. The changes comprised epithelial inflammatory, atrophic and metaplastic changes seen in the dorsal meatus and dorsal central septum of the nasal passages and, to a lesser degree, in the larynges. These changes are consistent with the inhalation of an irritant substance. Clear signs of repair and recovery of the above changes were seen in rats killed following the 4-week recovery period.

Clinical signs, during exposure, observed in rats exposed to 4.9 ppm were transient (the first 6 exposures only) and of a non-specific nature and in the absence of any other treatment-related effects, the no adverse effect concentration for this study is considered to be 5 ppm.