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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:
not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted prior to advent of GLP.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1979
Reference Type:
publication
Title:
Unnamed
Year:
1979

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
epichlorohydrin, lot #TB 09157-3, >99.8% purity

Chromatographic Analysis:
Propylene dichloride 0.03% (wt.)
cis-1,3-dichloropropene 0.08% (wt.)
2,3-dichloropropene 0.07% (wt.)
B-chloroallyl alcohol 0.01% (wt.)

Test animals

Species:
other: rat and mice
Strain:
other: rats- Fischer 344 and Sprague-Dawley; mice- B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
Fischer 344 rat: 9-11 weeks old from Charles River Laboratories, Wilmington, Massachusetts, males 205-219 g, females 125-139 g.
Sprague-Dawley rat: 9-11 weeks old from Spartan Research Animals, Haslett, Michigan, males 357-376 g, females 236-245 g.
B6C3F1 mouse: 7-9 weeks old from Charles River Laboratories, Wilmington, Massachusetts, males 20-27 g, females 20-22 g.
(age of the animals is age at time of initial exposure to epichlorohydrin)

Upon receipt, all animals were randomized from a single lot for each sex, species and/or strain, using a statistical randomization procedure generated from a digital computer program (GRAND. CLIST, Computations Research Laboratory, Dow Chemical U.S.A.). For each species and strain, twenty males and twenty females were assigned to each of three exposure groups and a control group. All animals were acclimated to the laboratory environment for approximately three weeks prior to exposure.

Mice were individually housed in suspended wire mesh cages. Rats were housed in groups of 3 or 4 per cage in suspended wire mesh cages. All animals were housed in the exposure, chambers during nonexposure periods. Solid food (Laboratory Animal Chow, Ralston Purina) was not available during exposure but was otherwise available ad libitum. Water was available at all times from an automatic watering system. All exposure chambers were maintained on a twelve-hour light and dark cycle.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
Whole-body vapor exposures were carried out in 14.5 cubic meter chambers. Atmospheres were generated by metering liquid through a pump into a warmed vaporization flask (120C), then sweeping the vapors into the chamber with compressed air. Total airflow through the chambers was approximately 3000 liters/minute.

The chambers are designed to operate under dynamic airflow conditions with temperature maintained at approximately 70-75°F and relative humidity maintained at approximately 40-60%. The exposures were carried out between the hours of 8 a.m. and 5 p.m. on Monday through Friday except during holidays.

The nominal concentrations of epichlorohydrin vapor in the exposure chambers were determined daily from the ratio of the rate at which the liquid was dispensed to the rate of airflow through the chambers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber air was assayed for vapor concentration at least 3 times during each 6-hour exposure period. Epichlorohydrin in all chambers was assayed by gas chromatography.

One-milliliter air samples were injected onto a 6'x1/8" O.D. stainless steel column packed with 10% UCW-98 on 80/100 Mesh Chromosorb P. The column and gas sample loops were housed in a Model 2440 gas chromatograph (Varian Associates, Palo Alto, California) equipped with hydrogen flame ionization detectors. The gas sample loops and column were maintained at 115°C, while the detector temperature was 230°C. Standards for the analysis were prepared by injecting a known volume of liquid epichlorohydrin into a Saran bag filled with a measured amount of filtered compressed air. Standard curves bracketing the entire range of exposure concentrations were prepared weekly. A single calibration standard (25 ppm) was run each exposure day.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week for 87 days (males) or 88 days (females)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 5, 25, 50 ppm (0, 18.9, 94.5, 189 mg/m3)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 5.01 +/- 0.42, 24.90 +/- 0.94, 50.10 +/- 1.60 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
20/sex/dose of each species and or strain
Control animals:
yes, concurrent no treatment
Details on study design:
For each species and strain, groups of 20 animals per sex per dose level were exposed to 0, 5, 25, or 50 ppm six hours daily, five days per week, for approximately 12 weeks (87 days, males; 88 days, females).

Additional male and female Fischer 344 rats were simultaneously exposed and used for supplementary studies. Ten rats/sex/exposure level and
control were used for bone marrow cytogenetic evaluation (Dow Chemical Co., 1979a). An additional five male Fischer 344 rats and B6C3F1 male mice/exposure level and control were added for pharmacokinetic/metabolism studies following repeated exposure (Dow Chemical Co., 1979b). These rats and mice were removed from the study at various time intervals during the 90-day portion of the study.

Dow Chemical Co., (1979a). Epichlorohydrin - Subchronic Studies. III. Cytogenetic evaluation of bone marrow cells from rats exposed to epichlorohydrin for four weeks. Sponsored by Manufacturing Chemists Association.

Dow Chemical Co., (1979b). Pharmacokinetics of epichlorohydrin (EPI) administered to rats by gavage or inhalation. Toxicology Research Laboratory,
Sponsored by Manufacturing Chemists Association.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
Animals were observed daily for signs of toxicity and morbidity/mortality. Body weights were collected twice weekly for the first two weeks of exposure, weekly for weeks three and four, and every two weeks thereafter.

Feed/water consumption were not monitored during the study. Ophthalmoscopic examinations were not performed during the study.
Sacrifice and pathology:
One to two weeks prior to necropsy (rats) or at necropsy (mice), blood samples were collected for hematologic analysis. Parameters examined were packed cell volume, red blood cell count, hemoglobin concentration, and white blood cell count. Urinalysis samples from rats were collected when blood samples were collected; parameters examined were specific gravity, pH, sugar, protein, ketones, bilirubin, occult blood, urobilinogen.

At necropsy, blood samples were collected for analysis of clinical chemistry parameters: blood urea nitrogen, glutamic pyruvic transaminase activity, alkaline phosphatase activity, glutamic oxaloacetic transaminase activity, and glucose concentration.

A complete necropsy examination was conducted on each animal, during which organ weights for brain, heart, liver, kidneys, testes, spleen, and thymus were recorded. With the exception of testes and epididymides of all male rats and mice which were in Bouin's solution and eyes from 5 rats and 5 mice/sex/groupat each necropsy which were preserved in Zenker's fixative, all tissues were fixed in buffered 10% formalin. Approximately 40 organs/tissues (esophagus, stomach, small intestine, pancreas, mediastinal lymphoid tissue (thymus, mediastinal lymph nodes), urinary bladder, heart, testes, ovaries, uterus, gall bladder, pituitary gland, salivary glands, lungs, vertebral bone and bone marrow, spinal cord, spleen, kidneys, prostate, epididymides, skeletal tissue, oviducts, parathyroid gland, brain, skin, eyes, trachea, nasal turbinates, large intestine, liver, lymph nodes, seminal vesicles, aorta, adrenal gland, thyroid gland, mammary gland, adipose tissue, peripheral nerve) from each animal were examined histologically for the control and high dose groups; possible target organs were examined for low and middle dose groups. For the nasal turbinates, the nasal turbinates and vertebral bone were decalcified and three to four transverse sections were obtained from the nasal turbinate region. Sections of regions lined primarily by respiratory epithelium, transitional epithelial region and olfactory epithelium were examined.

After thirty days on test, an interim sacrifice was conducted on 10 animals per sex per dose level. Parameters evaluated were hematology, urinalysis (rats only), clinical chemistry, gross pathology, organ weights, and histopathology. Histopathology was conducted on five animals per sex per dose level from the control and high dose groups.
Other examinations:
not applicable
Statistics:
Body weights, hematology, urinalysis, clinical chemistries, and organ weights were analyzed via ANOVA with Dunnetts Test, p< 0.05.

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 examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
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:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Terminal white blood cell counts in male Fischer 344 rats were statistically decreased from controls only at 5 ppm. At 5 ppm, interim and terminal kill white blood cell counts in female Sprague Dawley rats were statistically identified as different from controls. Terminal SGOT values for male Fischer 344 rats at 5 ppm only were statistically lower than controls. Interim kill BUN values in male B6C3F1 mice and AP values in female B6C3F1 mice were statistically different from controls at 5 ppm, but were not different from controls at the terminal kill. Urine specific gravity was statistically decreased in female Fischer 344 rats at 5 ppm on test day 73, but not at study termination. Interim (day 30) absolute kidney and testes weights in male Fischer 344 rats at 5 ppm were statistically increased, but relative kidney and testes weights were unaffected. Terminal absolute kidney weights in male Sprague-Dawley rats at 5 ppm were statistically increased, but relative kidney weights were unaffected. Interim (day 30) absolute and relative liver weights in male and female B6C3F1 mice at 5 ppm were statistically decreased; this effect was not seen at study termination. Terminal spleen weights in female B6C3F1 mice at 5 ppm were statistically increased; this effect was not seen at higher exposure concentrations. Since neither dose-response nor temporal patterns were consistently present, all of the differences described above were not considered treatment-related.

ACTUAL DOSE RECEIVED:
Analysis of chamber air for epichlorohydrin found the concentrations (mean +/- SD) to be 5.01 +/- 0.42, 24.90 +/- 0.94, 50.10 +/- 1.60 ppm.
No treatment-related deaths were observed at any dose level.

TOXIC RESPONSE/EFFECTS:
Clinical Observations (Fischer 344 and Sprague-Dawley rats, B6C3F1 mice): During the hours of exposure, the rats appeared to show conjunctival redness and palpebral closure without subsequent evidence of ocular involvement. These effects appeared to be transient, with recovery occurring overnight. Slight decreases in cage movements were observed in rats exposed to 25 or 50 ppm primarily during the first ten days of exposure and intermittently thereafter. These effects were not observed in mice.

FISCHER 344 RATS
Body Weights: Female Fischer 344 rats exposed to 50 ppm showed statistically decreased body weights on day zero which tended to remain lower throughout the study. Female Fischer 344 rats exposed to 25 ppm occasionally showed decreased body weights during the first month of exposure, but not at study termination.

Clinical Chemistry: No treatment-related changes in clinical chemistries were found in Fischer 344 rats.

Hematology: No treatment-related changes in hematology were found in Fischer 344 rats.

Urinalysis: No treatment-related changes in urinalysis parameters were found in Fischer 344 rats.

Organ Weights: Absolute and/or relative kidney weights were increased in male and female Fischer 344 rats at 25 and 50 ppm at interim and terminal examinations. Various other changes in organ weights were judged to be unrelated to treatment.

Gross Pathology: No lesions were observed which were considered treatment-related.

SPRAGUE-DAWLEY RATS
Body Weight: Male Sprague-Dawley rats exposed to 50 ppm showed statistically decreased body weights during the first month which tended to remain lower throughout the study. There were no treatment-related changes in body weights for female rats.

Clinical Chemistry: No treatment-related changes in clinical chemistries were found in Sprague-Dawley rats.

Hematology: No treatment-related changes in hematology were found in Sprague-Dawley rats.

Urinalysis: No treatment-related changes in urinalysis parameters were found in Sprague-Dawley rats.

Organ Weights: Absolute and/or relative kidney weights were increased in male and female Sprague-Dawley rats at either 25 or 50 ppm at interim and terminal examinations. Various other changes in organ weights were judged to be unrelated to treatment.

Gross Pathology: Kidneys in male Sprague-Dawley rats exposed to 25 or 50 ppm were slightly pale in color and increased in size at study termination. Livers in male Sprague-Dawley rats exposed to 50 ppm exhibited a slight accentuated lobular pattern with a pale color at study termination. No other lesions were observed which were considered treatment-related in either male or female Sprague-Dawley rats.

B6C3F1 MICE
Body Weight: Male and female B6C3F1 mice exposed to 50 ppm showed a trend toward decreased weight during the last few weeks of the study, occasionally identified as statistically significant.

Clinical Chemistry: No treatment-related changes in clinical chemistries were found in B6C3F1 mice.

Hematology: No treatment-related changes in hematology were found in B6C3F1 mice.

Organ Weights: Interim (day 30) absolute and relative liver weights in male mice at 5 and 50 ppm were statistically decreased; this effect was not seen at study termination. Male mice at 50 ppm had statistically significantly increased relative brain weights at study termination. Interim (day 30) absolute and/or relative liver weights in female mice at 5, 25, and 50 ppm were statistically decreased; this effect was not seen at study termination. Absolute heart weight was also decreased in female mice at 50 ppm; this effect was not seen at study termination.

Gross Pathology: At study termination, male mice (and one female mouse) at 50 ppm had decreased intraabdominal fat, consistent with decreased body weight. No other lesions were observed which were considered treatment-related in either male or female mice.

Histopathology (Fischer 344 and Sprague-Dawley rats, B6C3F1 mice): Histopathological examinations showed that the nose was the most sensitive organ to exposure. At the higher concentrations microscopic examination revealed hyperplasia, metaplasia and inflamed cell infiltration in the nasal turbinates. In addition, exposure at 25 or 50 ppm caused slight non-progressive kidney effects in rats but not mice, as evidenced by increased kidney weights (relative and/or absolute). Slight non-degenerative liver effects were noted in rats of both strains as well as the mice exposed to 50 ppm. Male rats of both strains at 50 ppm had slight effects on the adrenal glands, possibly mediated by stress. In male Sprague-Dawley rats at 50 ppm, there were minimal changes in the contents of the epididymides.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
5 ppm
Sex:
male/female
Basis for effect level:
other: For all species and strains.
Dose descriptor:
LOAEL
Effect level:
25 ppm
Sex:
male/female
Basis for effect level:
other: For all species and strains pathological changes were noted in the nasal epithelium.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

FISCHER 344 RATS The most consistent and readily recognized treatment-related effect observed in both male and female Fischer 344 rats was in the nasal turbinates. There were inflammatory, degenerative, and reactive changes in the epithelium in rats exposed to 25 or 50 ppm epichlorohydrin. The lesions within the nasal turbinates were exposure concentration related in severity in both male and female rats. However, the changes in the nasal turbinates of males were generally more severely involved than the females, at both the interim and the terminal kill. These changes were interpreted to be a result of irritation caused by inhaling the test material. There were no treatment-related changes detected in the nasal turbinates of rats exposed to 5 ppm epichlorohydrin.

There were slight decreases in body weight gain noted in the 50 ppm males and the 25 ppm females. The 50 ppm females frequently showed a statistically significantly decreased body weight compared to the controls. However, this difference was present from day 0 onward, and the relative difference in weight between the controls and 50 ppm group did not change during the course of the study. These minimal effects upon the weight gain of the male and female rats may be a result of the irritation in the nasal turbinates resulting in decreased food consumption. These observations were noted to be accentuated when rats and mice were exposed to 100 ppm epichlorohydrin by the inhalation route.

Treatment-related effects were observed in the liver and kidneys of male and female rats from the 25 or 50 ppm groups at either the interim or terminal kill. These were observed as either minimal changes in organ weight or organ to body weight ratios. Based upon gross pathology and histopathology there were treatment-related effects detected in these organs only at the 50 ppm level. The changes observed in these organs were of a minimal degree and did not result in alterations of hematology, clinical chemistry or urinalysis parameters which were considered treatment related. In addition, the changes affecting these organs did not increase in severity from the 30 to 90-day kill which suggests a lack of progression of the effects upon repeated exposure. Clinical observations which would suggest treatment-related toxicity were not detected during the course of the study.

The adrenal glands of some 50 ppm male rats from the terminal kill showed slight microvacuolation of cells in the zona fasciculata, a possible stress response.

There were no toxicologically significant treatment-related effects detected in the 5 ppm group of male or female Fischer 344 rats. The following parameters were evaluated in this study and were interpreted to be unaffected at the 5 ppm level of exposure: clinical observations, body weight, hematology, urinalysis, clinical chemistry, fasted body, weight, organ weights, organlbody weight ratios, gross pathology, and histopathology.

SPRAGUE-DAWLEY RATS The most consistent and readily recognized treatment-related effect in both male and female Sprague-Dawley rats was in the nasal turbinates. There were inflammatory, degenerative, and reactive changes in the epithleium in rats exposed to 25 or 50 ppm epichlorohydrin. The lesions within the nasal turbinates were exposure concentration related in severity in both male and female rats. However, the nasal turbinates of males were generally more severely involved than the females, at both the interim and terminal kill. These changes were interpreted to be a result of irritation caused by inhaling the test material. In general, the inflammatory and degenerative changes noted in the turbinates of the Sprague-Dawley rats appeared more severe than in the Fischer 344 rats. There were no treatment-related changes detected in the nasal turbinates of rats exposed to 5 ppm epichlorohydrin.

There was a slight decrease in body weight gain in the 50 ppm male rats during the first month of the study. No other groups of male or female rats showed any significant changes in weight gain. These minimal effects upon weight gain of 50 ppm males may be a result of irritation in the nasal turbinates resulting in decreased food consumption. These observations were noted to be accentuated when rats and mice were exposed to 100 ppm epichlorohydrin by the inhalation route.

Treatment-related effects were observed in the liver and kidneys of male and female rats from the 25 or 50 ppm groups at either the interim or terminal kill. These were observed as either minimal changes in organ weight, prgan to body weight ratios, or gross pathology. Based upon histopathology there were treatment-related effects detected in these organs only at the 50 ppm level. The changes observed in these organs were of a minimal degree and did not result in alterations of hematology, clinical chemistry or urinalysis parameters which were considered treatment related. In addition, the changes in these organs were less remarkable at the terminal kill than at the interim kill which suggests a lack of progression of the effects upon repeated exposure. Clinical observations which would suggest treatment-related toxicity were not detected during the course of the study.

The adrenal glands of some 50 ppm male rats from the terminal kill showed slight microvacuolation of cells in the zona fasciculata, a possible stress response. In addition, in the epididymides of several male rats from the 50 ppm group there was normal sperm content present with either increased numbers of nucleated cells and/or amorphous eosinophilic staining material within the lumen.

There were no toxicologically significant treatment-related effects detected in the 5 ppm, group of male or female Sprague-Dawley rats. The following parameters were evaluated in this study and were interpreted to be unaffected at the 5 ppm level of exposure: clinical observations, body weight, hematology, urinalysis, clinical chemistry, fasted body weight, organ weiglits, organ/body weight ratios, gross pathology, and histopathology.

B6C3F1 MICE The most consistent and readily recognized treatment-related effect in both male and female B6C3F1 mice was in the nasal turbinates. There were inflammatory, degenerative, and reactive changes in the epithelium in mice exposed to 25 or 50 ppm epichlorohydrin. The lesions within the nasal turbinates were exposure concentration related in severity in both male and female mice.

Both male and female mice from the 50 ppm group showed a trend towards decreased weight gain. These minimal effects upon weight gain noted may be a result of irritation in the nasal turbinates resulting in decreased food consumption. These observations were noted to be accentuated when rats and mice were exposed to 100 ppm epichlorohydrin by the inhalation route. Evaluation of gross pathology, histopathology, hematology, and clinical chemistry data from the interim and terminal kills do not reveal any treatment-related changes suggestive of liver or kidney toxicity. A slight decrease in liver weight was observed in all groups from the interim kill; however, the liver weight was normal at the terminal kill. The decreased liver weight observed at the interim kill was not associated with histopathology suggestive of hepatotoxicity, but rather it was suggestive of a decreased food intake. These observations for the liver weight were consistent with the.slightly lighter body weight of the exposed mice at the interim kill compared to the controls. One very significant species difference was noted in the kidneys of mice when compared to rats exposed to epichlorohydrin. The mice did not show any kidney weight changes or histopathology compared to the changes noted in both Fischer 344 and Sprague-Dawley rats. There were no toxicologically significant treatment-related effects detected in the 5 ppm group of male or female B6C3F1 mice. The following parameters were evaluated in this study and were interpreted to be unaffected at the 5 ppm level of exposure: clinical observations, body weight, hematology, clinical chemistry, absolute organ weights, organ/body weight ratios, gross pathology, and histopathology.

Applicant's summary and conclusion

Conclusions:
The results of this sub chronic study indicate that both strains of rats as well as mice exposed to 25 or 50 ppm of epichlorohydrin consistantatly had substantial changes in the epithelium nasal turbinates. Lesser effects in the other tissues also occurred at these exposure levels, but with some variation in response. Rats of both strains as well as the mice exposed to 5 ppm of epichlorohydrin had no adverse effects in any of the parameters monitored in these studies.
Executive summary:

The subchronic toxicity of epichlorohydrin was studied in 2 strains of rats (Fischer 344 and Sprague-Dawley) and 1 strain of mouse (B6C3F1). Inhalation exposures of 0, 5, 25, and 50 ppm of epichlorohydrin were conducted 6 hours/day, 5 days/week for 3 months, with an interim kill after 1 month.

Inhalation of 5 ppm of epichlorohydrin did not result in toxicologically significant effects in the rats or mice as measured by clinical observations, body weights, hematology, urinalysis, clinical chemistry, organ weights, gross pathology and histopathology examination of tissues.

However, in both strains of rats and in the mice, inhalation of 25 or 50 ppm of epichlorohydrin caused multiple treatment-related effects; the most consistent and readily detectable of which were degenerative, inflammatory and reactive histopathologic changes in the epithelium of the nasal turbinates. These changes were observed with some degree of species and strain variability. The lesions in the nasal turbinates were most severe in the Sprague-Dawley rats and least severe in the B6C3F1 mice with Fischer 344 rats being intermediate in severity.

Additional treatment-related effects at both these exposure levels included slight nonprogressive kidney effects observed in rats of both strains, but not mice. Slight nondegenerative liver effects were noted in rats of both strains as well as the mice exposed to 50 ppm of epichlorohydrin. Male rats of both strains exposed to 50 ppm of epichlorohydrin also had slight effects in the adrenal gland, possibly mediated via stress. In male Sprague-Dawley rats exposed to 50 ppm of epichlorohydrin there were minimal changes in the content of the epididymides. These toxic effects were accompanied by some slight indications of decreased body weight gain in rats of both strains and the mice exposed to 50 ppm of epichlorohydrin as well as the female Fischer 344 rats exposed to 25 ppm of epichlorohydrin.

In summary, the results of this subchronic study indicate that both strains of rats as well as mice exposed to 25 or 50 ppm of epichlorohydrin consistently had substantial changes in the epithelium of the nasal turbinates. Lesser effects in other tissues also occurred at these exposure levels, but with some variation in response. Rats of both strains as well as the mice exposed to 5 ppm of epichlorohydrin had no adverse effects in any of the parameters monitored in these studies.