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Repeated dose toxicity: inhalation

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short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
Limit test:

Test material

Details on test material:
CTF (2-chloro-5-trifluoromethyl pyridine) CTL Reference No. Y00846/003/002 was supplied by Imperial Chemical Industries Limited, Mond Division, Runcorn, Cheshire UK, Mond Division Reference No. MD757. The chemical is a white crystalline solid, which is 99% pure, the major impurity being 0.5% 2-chloro-3-trifluoromethyl pyridine.

Test animals

other: Alpk/AP (Wistar Derived)
Details on test animals and environmental conditions:

Male and female, specific pathogen free, Wistar derived albino rats were supplied in 4 batches of 20 males and 20 females from the colony maintained at Alderley Park, Cheshire, UK. The bodyweight range requested was 90 - 110 g, which is reached at approximately 5 weeks of age. The 64 males and 64 females selected for the test (actual weight on selection 88-112g) were divided equally by number and sex into four treatment groups and individually numbered by means of metal ear tags. Lost ear tags were either replaced or the animal concerned marked with its individual number by tail tattoo. Each animal was individually numbered by means of an ear tag.


Except during exposure periods and urine collection, animals were housed four per cage (sexes separately) in cages with internal measurements: length 38.0cm, width 32.5cm, height 21.0cm, which were suspended over collecting trays lined with absorbent paper. The cages were contained on single-sided mobile rat racks. The animals received Porton Combined Diet (PCD) supplied by BP Nutrition Limited, Witham, Essex UK and tap water via an automatic system ad libitum, except during the exposure period. Attached to each cage was a card identifying the contained animals, their treatment group and experiment to which they belonged. The animals were acclimatized for 8 days before the study started. The temperature within the animal room was controlled to approximately 22°C with a relative humidity in the range 22-64%. Daily measurements of both were made during the experiment. Twelve hours of light (soft white fluorescent) starting at 06.00 hrs was alternated with twelve hours of darkness.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Details on inhalation exposure:
Exposure System

On each day of treatment rats were transferred to Perspex exposure chambers. These were stacked vertically in pairs with a single lid on the upper chamber on all days when more than one batch of animals was exposed. The rats were housed one or two to each section and alternated (on a daily basis) between the upper and lower chambers. The temperature within the chambers was recorded each day.

Atmosphere Generation

Saturated Vapours of CTF were generated using a jacketed glass bubbler maintained at 33°C using a Churchill thermocirculator (Churchill Instrument Co Ltd, Perivale, Middlesex UK)
These atmospheres were diluted as required by adding clean dry air, dried using a Birlec autosorber system (model BZ25DNP, Rimmer Birlec, Cardiff, UK) and using flow meters as initial indicators of concentration. After dilution, the atmosphere was passed directly into the central column of the Perspex exposure chamber.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
A standard curve of absorbance against atmospheric concentration of CTF was generated by cumulatively injecting CTF in carbon disulphide into a closed loop optical system attached to an infra red gas analyser. A Miran IA with 20cm cell (Foxboro Analytical) was used. At least one point on the standard curve was checked on each exposure day.

The atmospheres were analysed by semi-continuous sampling of the chambers for periods of approximately 15 minutes. The output from the analyser was recorded on a flatbed recorder. The control chamber was monitored once each day.
Duration of treatment / exposure:
4 Weeks
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrations
Doses / Concentrations:
Exposures 0 (control), 0.25 mg/l (33 ppm), 1.0 mg/l (132 ppm), 4.0 mg/m3 (529 ppm)
analytical conc.
No. of animals per sex per dose:
16 Males and 16 Females. 4 males and 4 females per group were given a 2-week recovery period.
Control animals:
yes, concurrent no treatment
Details on study design:
The day following their arrival, the rats were allocated to four experimental groups. The groups were randomly arranged (male and females on separate racks) according to computer generated random numbers to decide the positions within each replicate (ie row) containing one cage from each group. From the 20 males and 20 females in each batch, 16 males and 16 females were selected by excluding animals at the extreme ends of the weight range received. The rats were then allocated to the four groups using random numbers. After the final exposure, 4 males and 4 females per group were given a 2-week recovery period.


Observations and examinations performed and frequency:
Clinical observations

Animals were observed before, during and after each exposure. Detailed clinical examinations particularly to evaluate neurotoxicity were carried out for Batch D, pre-experimentally, immediately following exposure on days 1, 7, 14, 21 and 28 and on days 35 and 42 during the recovery period.


Individual bodyweights were recorded on days –7, 1, 3, 8, 15, 22, 29, 36 and 43 and during the four week exposure period and were always measured prior to the relevant day’s exposure

Food Consumption

Food consumption per cage was measured at weekly intervals during the experiment, but mean values per rat were calculated. Food utilization has also been calculated based on (food consumption per rat) / (mean weight gain per rat).

Clinical chemistry

Urinalysis: Individual urine samples were collected overnight on day 28 from six males and six females deprived of food and water. Samples were also collected from four males and four females per group on day 7 of the recovery period. Samples were analysed for volume, pH, specific gravity, protein, glucose, urobilinogen and ketones.

Blood chemistry: The day following exposure, cardiac blood samples were collected from six males and six females per group (the same animals on which urinalysis was carried out) while tail vein blood samples were collected from 4 males and 4 females per group after 7 days recovery. These blood samples were analysed for plasma urea, glucose, alanine transaminase (ALT), aspartate transaminase, alkaline phosphatase (ALP), Albumin, total protein and triglycerides. Plasma sodium, potassium, calcium, magnesium and phosphorous were measured only at the end of the exposure period.


Cardiac blood samples were taken from 6 males and six females per group at the end of the exposure period only and examined for changes in cytological and haemostatic profiles using the following parameters: haemoglobin (Hb), haematocrit (Hct), total white cell count (WBC), red cell count (RBC), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), platelet count (PCT), differential white count kaolin-cephalin time (KCT) and prothrombin time (PT). Bone marrow samples were also prepared.
Sacrifice and pathology:

All animals including one found dead were given a post mortem examination. Surviving animals were killed either on the day following the last exposure or at the end of the recovery period as appropriate, by an overdose of halothane BP and immediately bled by cardiac puncture. This was to provide uniformity of conditions for organ weighing, whether or not the blood samples were being retained. All major organs and tissues were examined microscopically. The heart, lungs, liver, spleen, adrenals and gonads were trimmed and weighed. Samples of brain, spinal chord, sciatic nerves, heart, lungs (inflated with 1.5ml – 2ml formol saline/100g bodyweight) trachea, liver, kidneys, adrenals, ovaries or testes and any abnormal tissue were preserved in formol saline. All the tissues taken at the end of the exposure period from 10 males and 10 females in groups 1 and 4 and gross abnormalities only from 10 males and 10 females in groups 2 and 3 were embedded in paraffin wax. Sections 5um thick were cut and stained with haematoxylin and eosin and examined histologically. Livers from all groups, however, were processed and examined histologically. Additional frozen liver sections from groups 1 and 4 at the end of exposure and group 4 at the end of the recovery period were also stained with Oil Red O for fat. The following organs were preserved in formol saline (except where stated for future reference if required: eyes (Davidson’s, pituitary, nasal, sinuses, salivary glands, cervical lymph nodes, thyroid, thymus, thymic lymph nodes, aorta, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, mesenteric lymph nodes, seminal vesicle, epididymis, and prostate or uterus and cervix, muscle, skin (Bouin’s) and mammary tissue.
Where appropriate, mean values from test groups have been compared with those from the control group using Student’s t test.

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):
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Atmospheric concentrations

The overall mean values (with standard deviations) were as follows:

Low level 0.27 ± 0.14 mg/l

Middle level 0.95 ± 0.16 mg/l

High level 3.87 ± 0.30 mg/l

It was found that it was difficult to keep constant the concentration of the high exposure level: the same flowmeter settings gave apparently different concentrations on different days and there were also frequently considerable variations within a given day. It should also be noted that the animals of groups 2 and 3 (batches C and D only) were inadvertently transposed prior to exposure on day 34 and were therefore exposed to mean concentrations of 0.95 and 0.24mg/l respectively.

Clinical effects

General effects: during exposure, it was generally noted that animals in groups 1-3 were normal but that signs of eye and nasal irritation were seen in animals exposed to 4.0mg/l. These animals appeared to be slightly worse during their first day of exposure, but on most days similar observations were made i.e. they were restless, their eyes were partially closed, they had salivation and/or nasal discharge, they were often rubbing at their eyes and noses and grooming themselves or each other, although having a poorly groomed appearance. Tremors were also seen in some animals. The absorbent tray paper under the exposure chambers was wetter for the 4.0mg/l group than for the other groups, suggesting increase urination. The results of the more detailed clinical examinations carried out after exposure are described below but it was noted when returning animals to their cages after exposure that, especially at the top dose, they were wet, ill-groomed and had piloerection. They were also frequently hunched, subdued, vocalised when handled and had tail erection. A few animals exposed to 1.0mg/l were affected especially in the earlier stages of the experiment. The controls and animals exposed to 0.25mg/l appeared normal apart from male 439 which was noted to have partial paralysis of the hind limbs after its first exposure. It was subsequently found that the left limb only was affected and persisted for a further three days. On the first exposure of batch B animals, one animal exposed to 0.25mg/l (which included male 439) had bee trapped in the exposure chamber but it had not been possible to identify it when it was released. It was probable, however, that it was male 439. One animal, male 505 (group 4 batch C) was found dead after its first exposure.

In all groups CTF produced symptoms consistent with effects on both the central and autonomic nervous systems, which persisted after exposure had ended. Although dose related, the clinical effects were minimal in the group exposed to 0.25mg/l. The autonomic effects were consistent with both androgenic and cholinergic stimulation, but it is unlikely that CTF works post synaptically in both cholinergic and androgenic nerves. A more likely explanation is that its action is on ganglia or on the higher nervous system probably by false transmissions or mediator release. The effects of CTF on the central nervous system in this study suggest an anaesthetic or depressive action.


Weigh gains of animals exposed to 0.25mg/l and 1.0mg/l were comparable with those of the control group. Males and females exposed to 4.0mg/l, however, lost weight after two exposures although at the end of the exposure period, body weights of the females were similar to those of the other groups. Weight gain in males was reduced although was comparable with that in the other groups during the second week of the recovery period.

Food consumption

Food consumption was reduced for males exposed to 4.0mg/l during the whole of the exposure period but was comparable with the control group during the recovery period (although measured for one cage of animals only). Food consumption for females exposed to 4.0mg/l was reduced during the first week of exposure but was similar to that of the controls during the rest of the exposure period and was apparently increased during the recovery period. No correction has been made for those animals of groups 2 and 3 which were housed in each other’s cages during a three day period.

Food utilisation

Food utilisation during the exposure period showed no reduction in efficiency in the treated groups compared with the controls.


A statistically significant difference was found between the control group and the test group means at 28 days for the following parameters:

a) Haemoglobin. The mean haemoglobin level was slightly reduced in the females exposed to 4.0mg/l.

b) Red cell count. The mean red cell count was slightly reduced in the females exposed to 4.0mg/l.

c) Mean cell haemoglobin concentration. MCHC was slightly reduced in all the female test groups but only at the top dose mean was significantly lower than the control.

d) Lymphocyte count. The mean lymphocyte count was increased in the females exposed to 1.0mg/l. In addition, kaolin-cephalin time was slightly reduced in all female groups but was not statistically significant.

The majority of the above changes were sporadic and even where all test groups were affected (MCHC, female) the change was too small to be of haematological significance.

Clinical chemistry


a) Terminal. There were slight decreases in the glucose levels of the males and females exposed to 4.0mg/l. There were also increases in cholesterol levels of males and females exposed to 4.0mg/l and of females exposed to 1.0mg/l with associated increases in triglyceride levels of males and females exposed to 4.0mg/l. No other effects were observed.

b) Seven-day recovery. No difference between control and treated groups were seen.


a) Terminal. There was a small decrease in the volume excreted together with an increase in specific gravity of the females exposed to 4.0mg/l. A dose-related decrease in pH was seen in females exposed to 1.0 and 4.0mg/l. All male treated groups had some evidence of ketones present. The protein level of the males exposed to 4.0mg/l was slightly lower than that of the other groups.

b) Seven day recovery. The protein level of the males which had been exposed to 4.0mg/l was still lower than that of the other group and there was still some evidence of ketones present in males which had been exposed to 1.0 and 4.0mg/l and females which had been exposed to 4.0mg/l.


Macroscopic Findings: The male exposed to 4.0mg/l (no. 505) which was found dead had froth in its trachea and an ulcer in the pyloric region of the stomach (described as depressed area in mucosa, entire surface of pyloric region irregular and altered blood present in the ileum). There were no other findings related to treatment either at the end of the exposure or recovery period.

Organ weights

At the end of the exposure period, liver, kidneys, and adrenals showed statistically significant increases in both organ weights and organ/bodyweight ratios. These were usually found in both sexes and appeared to be dose-related. In addition, spleen weights were decreased. At the end of the recovery period there were isolated statistically significant differences but all except for the heart in females exposed to 4.0mg/l were decreases. There appeared, therefore, not to be any relationship with the changes found at the end of the recovery period and those found at the end of the exposure period.


The main target organ was the liver. After four weeks exposure to 4.0mg/l, most animals showed mild diffuse hepatic degeneration in the form of granular eosinophilic cytoplasm of hepatocytes, displacement of basophilic granules, swollen hepatocytes and apparent increase in mitotic figures in both sexes. In addition, increased fatty vacuolation occurred in females. Half of the animals exposed to 1.0mg/l showed hepatic degenerative lesions similar to those seen in animals exposed to 4.0mg/l but the changes were mainly focal. Almost half showed slight fatty vacuolation. Of the animals exposed to 0.25mg/l, three only showed focal hepatic degeneration. Almost half showed slight fatty vacuolation. No hepatic degeneration was seen in the control rats but some livers showed slight fatty vacuolation.

After a two week recovery period, half the rats previously exposed to 4.0 mg/l showed focal to diffuse hepatic degenerative lesions and only one male showed moderate fatter vacuolation. Almost half the livers from the middle exposure group showed focal hepatic degenerative lesions and half the male livers showed slight fatty vacuolation. All the livers from the group 2 animals appear to the normal in both sexes.

No degenerative lesions were seen in the controls and only one male rat showed slight fatty vacuolation.

Hepatocyte degeneration was still apparent in some animals in groups 3 and 4. The livers of the group 2 rats were within normal limits.

In addition more that half the animals exposed to 4.0mg/l showed a mild degree of tracheitis while the controls showed no such lesion. Although there were no other treatment-related effects, there was peribronchial lymphocyte infiltration in almost all controls and in animals exposed to 4.0mg/l, which was attributed to a virus infection. The animal that died at the end of its third exposure (male 505, group 4) had acute gastritis and duodenitis but these findings were considered not to be dose related.

Applicant's summary and conclusion

Rats exposed by inhalation to 0.25, 1.0 and 4.0mg/l CTF showed dose-related changes shown clinically, principally by effects on the central and autonomic nervous systems and by degenerative changes in the liver associated with some functional changes. 0.25mg/l CTF was considered to be a minimal effect level for both liver and nervous system effects. Reversal of these effects occurred during the two week recovery period.

Although the exposure level of 0.25mg/l was not a true ‘no effect’ level, it can be regarded as being a minimal effect level and provides sufficient information for setting a hygiene standard.
Executive summary:

Groups of 16 male and 16 female rats were exposed for six hours a day, five days a week for four weeks to 0, 0.25, 1.0, 4.0mg/l (0, 33, 132 or 529ppm) 2-chloro-5-trifluoromethyl pyridine (CTF). The atmospheric concentration of CTF was monitored by infra-red spectroscopy.

The animals were observed during and after exposure and were given detailed clinical observations at intervals during the experiment. Bodyweight and food consumption were measured regularly. Urine samples were taken terminally. The day following the last exposure, 12 males and 12 females per group were killed and, after cardiac blood samples had been taken for clinical chemistry and haematology, each animal was given a post mortem examination. Selected organs were weighed and samples of tissues preserved for histopathological examination. The remaining 4 males and 4 females per group were retained for a two week recovery period. After one week of this period, tail vein blood samples and urine samples were taken for clinical chemistry. At the end of the recovery period, the animals were killed and subjected to a post mortem examination during which again organs were weighed and tissues take for histopathology.

It was found that there were dose-related clinical abnormalities consistent with effects on the central and autonomic nervous systems and, at the top exposure level, irritant effects on mucous membranes. Bodyweight gain was reduced in the animals exposed to 4.0mg/l, particularly in males, and was associated with reduced food consumption. There were no haematological effects but there were biochemical ones, the major changes being an increase in triglyceride and cholesterol levels in the high exposure group and a slight increase in ALT activity in the females of this group.

At post mortem examination, liver weights showed a dose-related increase. Histologically, the livers showed dose-related degenerative changes (without necrosis) which became more diffuse as the dose increased, and fatty vacuolation which similarly became less marked with decreasing dose. Adrenal and kidney weights were also increased at the top dose and mild tracheitis was seen histologically. All effects recovered in the 0.25mg/l group and recovered or showed signs of recovery at higher levels.

It is concluded that CTF is irritant to mucous membranes at high levels, affects the nervous system and is also toxic specifically to the liver, although not severely so at the exposure levels tested. Although there is no true ‘no effect’ level in this study, the 0.25mg/l level is considered to be a minimal effect level.