4-chlorobutyryl chloride

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)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

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)

Remarks:

testing lab.

Test material

Test material information

Constituent 1

Reference substance name:

4-chlorobutyryl chloride

EC Number:

225-059-1

EC Name:

4-chlorobutyryl chloride

Cas Number:

4635-59-0

Molecular formula:

C4H6Cl2O

IUPAC Name:

4-chlorobutanoyl chloride

Details on test material:

Name of the test substance used in the study report: 4-chlorobutyryl chloride
Purity: 99.4 %

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Mean body weights: approx. 228 g (males), 166 g (females) on the day of the first exposure.
Feed: standard diet and tap water ad libitum.
Housing: During exposure the animals were housed individually in the holders. Immediately after the exposure, the rats were housed 5 per sex and cage in the animal room.
Environment: temperature: 19-23.5°C, relative humidity 47-72%, 10 air changes per hour, 12-hour dark-light cycle.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Details on inhalation exposure:

Nose-only inhalation polypropylene/PVC chamber, volume 50 l, with 20 ports which allowed exposing 5 male and 5 female rats, and also test atmosphere sampling and monitoring.

Atmosphere generation: Dynamic vapor generation, using 2 methods:

i) Using roller pumps metered amounts of test material were passed to an airless ultrasonic nebulizer. The aerosolized
material was passed into a heated, all glass mixing chamber where it was mixed in two steps with very dry (<1 % humidity) compressed air. The atmosphere was directed to the inhalation chamber and additionally mixed with very dry air before it was directed towards the animals.

ii) Due to the corrosivity of the test material it was necessary to stop the ultrasonic method and continue with the method that had been used in the 5-day range finding study, where very dry (humidity less than 1 % in order to prevent hydrolysis) compressed air was bubbled through a glass evaporator filled with TS. The atmosphere was then mixed with an adjustable main stream of compressed air before the atmosphere entered the inhalation chamber. The whole system was placed under a hood.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical measurement of atmosphere concentration: 2 samples per hour were examined using a Total Carbon Analyzer (TCA) that was connected to one of the ports of the exposure system. GC/FID was used to calibrate the TCA. Sampling: 50 l of the test atmosphere were passed through 2 impinger placed in series filled with n-pentane at a rate of 0.8 l/min. Determination of actual concentration: calculation using the calibration graph obtained by plotting the concentration measured by GC analysis against the total carbon output.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 2.1, 11.8, and 59.1 mg/m³, i.e. 0, 0.002, 0.012, and 0.059 mg/l
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:

Observation period: 28 days; none after last exposure.
Observations: Twice daily during exposure and twice daily in the animal room for signs of toxicity on exposure days, and daily during the weekends. Body weights were taken pre-exposure during acclimatization, just before the first exposure (day 0), weekly thereafter and at termination. Food intake per cage was measured weekly. Efficiency of food utilization was calculated and expressed in gram weight gain per gram food consumed.
Hematology: blood samples were taken at termination from surviving, unfasted rats. Examinations included hemoglobin concentration (hb), red blood cell count (rbc), reticulocytes (control and high-dose group only), white blood cells (wbc), differential wbc count, prothrombin time, thrombocyte count. Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated.

Clinical chemistry: alkaline phosphatase (ALP), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), gamma glutamyl transferase (GGT), total protein, albumin, urea, creatinine, total bilirubin, glucose (fasted animals, in week 4), sodium, potassium, calcium, chloride, and inorganic phosphate were examined.

Sacrifice and pathology:

Animals were sacrificed at termination, weighed and examined for gross pathological changes. Organs were weighed (adrenals, heart, liver, kidneys, lungs with trachea and larynx, spleen, testes), and tissue samples were preserved from these organs and from nose (nasal cavity) and gross lesions.
Histopathology: All preserved organs from control and high-dose animals were examined following staining with hematoxylin and eosin. Samples from the nasal cavity, larynx, trachea and lung were further cut at several levels. As there were changes in the nasal cavity, larynx, trachea and lungs these were also examined in the low- and mid-dose group.

Statistics:

Body weights and body weight gain, organ weights, hematological and clinical chemistry data were statistically examined by one-way analysis of covariance (COVAR) or analysis of variance (ANOVA) followed by Dunnett's multiple comparison when group means were significantly (p<0.05) different. Relative hematological data were analyzed using Kruskal-Wallis non-parametric analysis of variance followed by the Mann/Whitney U-test. The Fisher exact probability test was used to analyze incidences of histopathological changes.

Results and discussion

Results of examinations

Details on results:

Mortality: None of the animals died.

Clinical signs: The only sign was sniffing in all high-dose rats during the last week of exposure and in a few rats of the mid-dose group.

Body weights: Statistically significantly decreased body weights were seen in both high-dose groups and in the male mid-dose group over the entire exposure period. The terminal mean body weights of the high-dose groups recovered to some extend but remained significantly (p<0.01) below that of the other animal groups. Compared to the control group, food intake of the high dose male group was decreased over the entire exposure period and in female rats of this group over the first 3 weeks.

Hematology: Hematology revealed significant (p<0.01) increases of red blood cells (rbc), hemoglobin (hb), and packed cell volume (PCV) in high dose males. Similar but less pronounced effects were noted in females, only the increase of rbc and PCV reached a level of significance (p<0.05). The differential white blood cell count revealed significantly (p<0.05) increased absolute and relative numbers of neutrophils and decreased absolute and relative numbers of lymphocytes in the high-dose groups.

Clinical chemistry revealed several scattered changes that were either insignificant or were not dose-related. Findings of relevance comprised significant (p<0.05) increases of alkaline phosphatase in all treated groups, and the increased (p<0.01) sodium and total protein content in high-dose males.

Pathology: Significant (p<0.05 or p<0.01) relative organ weight increase were noted for adrenals, kidneys, and lungs in both high-dose groups. In the mid dose only the relative kidney weight was increased. Absolute weight changes were noted for adrenals (females only) and lungs (increase), and liver and spleen (decrease). No statistically significant weight changes was noted in testes, but small testes, either unilateral or bilateral, were noted in 4 males and this finding was considered to be treatment-related.
Swollen lungs in all high-dose animals and in 5/10 mid-dose animals was the only findings during gross pathology.

Histopathology: There was no target organ other than the respiratory tract. Changes were noted only the nasal cavity in low dose rats; in nasal cavity,larynx and trachea in mid-dose rats; and in the entire respiratory tract in high-dose rats.
The effects (severity, localization, extension) in the nasal cavity depended on the dose level. In low-dose animals it was described as very slight to slight squamous metaplasia and hyperplasia of the transitional epithelium, and very slight focal hyperplasia of the respiratory epithelium. In mid-dose animals, the localization of effects on the transitional epithelium was comparable to that seen in low-dose rats; the severity ranged from slight to moderate. The transitional epithelium was almost entirely hyperplasic and included mucous cell hyperplasia. Very slight to slight suppurative rhinitis was seen. In high-dose rats, almost the entire transitional and ciliate respiratory lining of the nasal cavity was affected by squamous metaplasia, hyperplasia, and suppurative rhinitis, which included focal intraepithelial microabcesses and focal epithelial erosion.
Epithelial hyperplasia was seen in the larynx of 9/10 high-dose, and 3/10 mid-dose rats. Occasionally, very slight inflammatory cell infiltrates, epithelial erosion and intraluminal cell debris with mucous were observed. Focal hyperplasia predominated in mid-dose animals; a more diffuse, disorganized atypical hyperplasia was mainly noted in high-dose rats.
Epithelial hyperplasia of the trachea and extrapulmonary bronchi was found in animals of the mid- and high-dose groups. The tip of the carina at the bifurcation was a sensitive site. The lesion progressed from focal, organized hyperplasia with mucous cell hyperplasia mainly in mid-dose rats to diffuse, more disorganized, atypical hyperplasia mainly in high-dose animals.

Histopathological changes in lungs were seen in high-dose rats only. The lesions consisted of epithelial hyperplasia lining the bronchi/bronchioli, a increased number of polymorphonuclear cells around blood vessels and bronchi/bronchioli, perivascular edema, and an increase in size and activation of peribronchial/peribronchiolar lymphoid tissue. The epithelial hyperplasia ranged from an atypical hyperplasia in the upper bronchi to mucouscell hyperplasia in the lower bronchi/bronchioli. The epithelium of the terminal bronchioli was not involved.

Effect levels

Dose descriptor:

LOAEC

Effect level:

0.002 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: Slight effects on transitional and respiratory epithelium at the lowest concentration.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Rats exposed to 0.002, 0.012, and 0.059 mg/l 4-chlorobutyryl chloride for 4 weeks showed clinical symptoms linked to respiratory tract inflammation/ irritation. Growth retardation, changes in blood parameters clinical chemistry, and organ weights (liver, kidney, spleen, and lung, adrenals) are predominantly noted at the high dose level and can be explained to be secondary due to severe effects on the respiratory tract. They were noted in a dose-related manner, regarding both severity and localization. The entire respiratory tract was severely affected in all high-dose animals, whereas only very slight effects on the transitional and respiratory epithelium were in animals exposed to the lowest dose of 0.002 mg/l, which may be regarded as a lowest observed adverse effect level.