1,2-dichloropropane

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Publication does not state that the study was conducted according to guidelines but was conducted according to GLPs. The report contains sufficient data for interpretation of study results.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2010

Materials and methods

Principles of method if other than guideline:

Publication does not state whether any guidelines were followed. Animals were exposed to test material for 13 weeks. Animals were weighed weekly and blood obtained for hematology and clinical chemistry determinations (specific tests not stated in publication) at necropsy. A complete gross necropsy was performed and histopathological examination of tissues conducted (only nasal tissues specified in methods section of publication although results from other tissues were reported in the results section).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dichloropropane

EC Number:

201-152-2

EC Name:

1,2-dichloropropane

Cas Number:

78-87-5

Molecular formula:

C3H6Cl2

IUPAC Name:

1,2-dichloropropane

Test material form:

other: liquid

Details on test material:

DCP of analytical grade (>99.5% pure) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Each lot of DCP used in the present studies was analyzed for its purity and stability by gas chromatography before and after its use. No gas chromatographic peak other than DCP was detected in the inhalation exposure chambers.

Test animals

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

F344/DuCrj (SPF) rats of both sexes were obtained at 4 wk of age from Charles River Japan, Inc. (Kanagawa, Japan). After 2 wk quarantine and acclimation, the animals were allocated by a stratified randomization procedure into body-weightmatched, DCP-exposed, and clean air-exposed groups. The 13-wk study consisted of five DCP-exposed groups and one control group, each comprising 10 rats of each sex. The animals were housed individually in stainless-steel wire hanging cages (150 mm [W] × 216 mm [D] × 176 mm [H]) in stainless-steel inhalation exposure chambers maintained at a temperature of 23 ± 2°C and at a relative humidity of 55 ± 15% with 12 ± 1 air changes/h. Fluorescent lighting was controlled automatically to give a 12-h light/dark cycle. All rats had free access to sterilized water and γ-irradiationsterilized commercial pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan).

The animals were cared for in accordance with the guide for the care and use of laboratory animals (National Research Council, 1996).

Reference
National Research Council (NRC). 1996. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press, Institute of Laboratory Animal Resources Commission on Life Sciences, NRC.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: Not applicable, test material exposure was via vapor.

Details on inhalation exposure:

Airflow containing DCP at designated target concentrations was prepared by a vaporization technique. The saturated vapor–air mixture was generated by bubbling clean air through the DCP liquid in a temperature-regulated glass flask (25°C), and by cooling it through a thermostatted condenser at 18°C. The airflow containing the saturated vapor was diluted with clean air, and then warmed to 23°C in a thermostatted circulator, which served to stabilize the vapor concentration by complete gasification of the DCP. The flow rate of the vapor–air mixture was regulated with a flowmeter, further diluted with humidity- and temperature-controlled clean air in a spiraling line mixer, and then supplied to an inhalation exposure chamber. Six inhalation exposure chambers of 1060 l, each accommodating 20 individual cages for 10 male and 10 female rats, were used for the 13-wk study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of DCP were monitored by gas chromatography every 15 min throughout the entire exposure periods, and maintained at 125.3 ± 0.7 (mean ± SD), 250.8 ± 1.0, 500.5 ± 2.6, 1000.4 ± 3.4, and 2001.3 ± 5.9 ppm for the 13-wk study

Duration of treatment / exposure:

6 h/day

Frequency of treatment:

5 days/wk for 13 wk.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 (clean air control), 125, 250, 500, 1000, or 2000 ppm (v/v)
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
125.3 ± 0.7 (mean ± SD), 250.8 ± 1.0, 500.5 ± 2.6, 1000.4 ± 3.4, and 2001.3 ± 5.9 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

10 males and 10 females per dose level

Control animals:

yes

Details on study design:

For the 13-wk study, groups of 10 rats of each sex were exposed to DCP at 0 (clean air control), 125, 250, 500, 1000, or 2000 ppm (v/v) for 6 h/day, 5 days/wk for 13 wk.

Positive control:

No data.

Examinations

Observations and examinations performed and frequency:

The animals were observed daily for clinical signs and mortality. Body weight and food consumption were measured once a wk in the 13-wk study.

Sacrifice and pathology:

All rats, including those found dead or moribund, received complete necropsy. For hematology and blood biochemistry, blood was collected under etherization at terminal necropsy after overnight fasting. The blood sample was analyzed with an automatic blood cell analyzer (ADVIA120, Bayer Co. NY, USA) and an automatic analyzer (Hitachi 7080, Hitachi, Ltd., Ibaraki, Japan) for blood biochemistry.

Organs were removed, weighed, and examined for macroscopic lesions at necropsy. All organs and tissues and the entire respiratory tract including nasal cavity, pharynx, and larynx were examined for histopathology in all the animals. The organs and tissues were fixed in 10% neutral buffered formalin. The nasal cavity was decalcified in formic acid-formalin solution before trimming, and was transversely trimmed at three levels according to the procedure described in our previous paper (Nagano et al., 1997): at the level of the posterior edge of the upper incisor teeth (Level 1), at the incisive papilla (Level 2), and at the level of the anterior edge of the upper molar teeth (Level 3). The tissues were embedded in paraffin, and 5 μm-thick sections were prepared and stained with hematoxylin and eosin (H&E).

Reference:
Nagano K, Katagiri T, Aiso S, Senoh H, Sakura Y, Takeuchi T. 1997. Spontaneous lesions of nasal cavity in aging F344 rats and BDF1 mice. Exp Toxicol Pathol 49:97–104.

Other examinations:

No additional information available.

Statistics:

The Chi-square test was used to evaluate the statistical significance of the differences in the incidences of pre and nonneoplastic lesions between DCP-exposed groups and the clean air-exposed control group. Survival curves were plotted according to the method of Kaplan and Meier (1958), and the log-rank test (Peto et al., 1977) and Fisher’s exact test were used to test for statistically significant differences in survival rates between any DCP-exposed rat group of either sex and the clean air-exposed control group. Body and organ weights were analyzed by Dunnett’s test. Two tailed tests were used for all statistics. In all cases, a P-value of 0.05 was used as the level of significance.

References:
Kaplan EL, Meier P. 1958. Nonparametric estimation from incomplete observations. Am Stat Assoc J 53, 457–481.

Peto R, Pike MC, Day NE, Gray RG, Lee PN, Parish S, Peto J, Richards S, Wahrendorf J. 1980. Guidelines for simple, sensitive significance tests for carcinogenic effects in long-term animal experiments. IARC Monogr Eval Carcinog Risk Chem Hum Suppl:311–426.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No deaths occurred in any of the DCP-exposed male rat groups. One female exposed to 2000 ppm died during the 12th wk of the exposure period. There were no DCP-related clinical signs in any of the DCP-exposed groups of either sex.

Mortality:

mortality observed, treatment-related

Description (incidence):

No deaths occurred in any of the DCP-exposed male rat groups. One female exposed to 2000 ppm died during the 12th wk of the exposure period. There were no DCP-related clinical signs in any of the DCP-exposed groups of either sex.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Growth rates were clearly suppressed in both male and female rats exposed to 1000 and 2000 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was lowered in both male and female rats exposed to 2000 ppm

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hemolytic anemia occurred in both male and female rats exposed to 500 ppm and above, as indicated by decreases in some of the erythrocyte parameters including red blood cell count together with concomitant increases in reticulocytes and platelets

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Total bilirubin and γ-GTP activity significantly increased in the male rats exposed to 2000 ppm and in the female rats exposed to 1000 and 2000 ppm.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relative weights of the spleens significantly increased in both male and female rats exposed to 2000 ppm. The absolute and relative weights of the livers significantly increased in female rats exposed to 500 ppm and above

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Thirteen weeks of inhalation exposure to DCP affected the nasal cavity, liver, hematopoietic system, and adrenal gland.

Histopathological findings: neoplastic:

not specified

Details on results:

In the nasal cavity, hyperplasia of the respiratory epithelium and atrophy of the olfactory epithelium occurred in both male and female rats exposed to 125 ppm and above, and the averaged severity scores of these two lesions increased in a concentration-related manner. Hyperplasia of the respiratory epithelium was characterized by an increased number of ciliated columnar epithelial cells and accompanied by goblet cell hyperplasia. The hyperplasia was located diffusely in the dorsal or septum region of Level 1. Atrophy of the olfactory epithelium was characterized by decreases in epithelial thickness and the number of olfactory sensory cells and often accompanied by necrosis of the olfactory sensory cells and respiratory metaplasia of the olfactory epithelium. Atrophy was located in the dorsal region of Levels 2 and 3. Inflammation of the respiratory epithelium significantly increased only in the male rats exposed to 1000 and 2000 ppm. In the liver, swelling of centrilobular hepatocytes was observed in both male and female rats exposed to 2000 ppm. Increased hematopoietic activity in the spleen and bone marrow, a compensatory response to hematotoxicity, was noted in the 1000 and 2000 ppm-exposed rats of both sexes. Increased hemosiderin deposition resulting from hemolysis of erythrocytes was observed in the spleen of male rats exposed to 1000 and 2000 ppm and female rats exposed to 500 ppm and above. Fatty change in the adrenal gland was significant in the female rats exposed to 2000 ppm. No exposure-related lesions were observed in any other organs in the DCP-exposed rats of either sex.

Effect levels

Dose descriptor:

LOAEC

Effect level:

125 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Hyperplasia and inflammation noted in the respiratory epithelium of the nasal cavity. The exposure level is the lowest concentration of DCP tested.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Hematological and blood biochemical parameters of the rats exposed by inhalation to DCP or clean air for 13 wk.

Group (ppm)	0 (Control)	125	250	500	1000	2000
Male
No. of animals examined	10	10	10	10	9^a	10
Red blood cell (10^6/uL)	9.31 + 0.21	9.36 + 0.19	9.33 + 0.16	8.95 + 0.17**	8.00 + 0.22**	7.58 + 0.36**
Hemoglobin (g/dL)	15.9 + 0.4	16.0 + 0.4	15.8 + 0.4	15.4 + 0.3*	14.7 + 0.2**	14.6 + 0.5**
Hematocrit (%)	45.6 + 1.2	46.1 + 1.1	46.0 + 0.7	45.2 + 0.8	43.4 + 0.8**	43.7 + 1.2**
Platelet (10^3/uL)	780 + 57	804 + 39	809 + 53	816 + 67	925 + 59**	959 + 64**
Reticulocyte (%)	1.9 + 0.1	1.8 + 0.2	1.9 + 0.2	2.3 + 0.2	5.5 + 0.6**	10.5 + 3.0**
No. of animals examined	10	10	10	10	9^a	10
γ-GTP(IU/L	2 + 1	4 + 5	3 + 1	2 + 1	2 + 1	6 + 10*
Total bilirubin (mg/dL)	0.13 + 0.02	0.13 + 0.01	0.13 + 0.01	0.13 + 0.01	0.14 + 0.01	0.18 + 0.02**
Female
No. of animals examined	9^a	10	10	10	10	9^b
Red blood cell (10^6/uL)	8.60 + 0.21	8.59 + 0.20	8.44 + 0.24	8.13 + 0.27**	7.77 + 0.24**	7.18 + 0.39**
Hemoglobin (g/dL)	15.9 + 0.5	15.8 + 0.4	15.7 + 0.4	15.4 + 0.6	15.1 + 0.4**	14.3 + 0.8**
Hematocrit (%)	44.3 + 0.9	44.4 + 1.0	44.2 + 1.0	43.7 + 1.2	43.7 + 1.1	42.5 + 1.4**
Platelet (10^3/uL)	817 + 64	783 + 56	825 + 58	863 + 78	874 + 54	932 + 114**
Reticulocyte (%)	1.9 + 0.2	1.9 + 0.3	2.5 + 0.3	3.5 + 0.4*	6.4 + 2.7**	11.5 + 4.5**
No. of animals examined	9^a	10	10	10	10	9^b
γ-GTP (IU/L)	3 + 1	2 + 1	3 + 1	3 + 1	5 + 2**	10 + 2**
Total bilirubin (mg/dL)	0.16 + 0.02	0.16 + 0.03	0.15 + 0.03	0.16 + 0.02	0.20 + 0.03*	0.25 + 0.06**

Note: Values were expressed as means ± standard deviation.

^a,bNumber of rats examined were 9 instead of 10, because blood sampling failed for one rat^a, and because another rat died before the end of the 13-wk exposure period^b.

Significant difference: *p < 0.05; **p < 0.01 by Dunnett’s test.

γ-GTP, γ -glutamyl transpeptidase.

Table 2. Number of rats bearing the selected histopathological lesions and their severities in the rats exposed by inhalation to DCP or clean air for 13 wk.

Group (ppm)	0	125	250	500	1000	2000
Males
Number of animals	10	10	10	10	10	10
Nasal cavity Hyperplasia respiratory epithelium	0	10** [1.0]	10** [1.3]	10** [1.3]	10** [2.0]	10** [2.0]
Inflammation respiratory epithelium	0	0	2	4	8**	8**
Atrophy: olfactory epithelium	0	10** [1.0]	10** [1.2]	10** [1.5]	10** [2.2]	10** [2.7]
Liver Swelling centrilobular	0	0	0	0	0	9* [1.0]
Spleen Deposition of hemosiderin	0	0	0	1	10**	10**
Increased extramedullary hematopoiesis	0	0	0	0	10**	10**
Bone marrow Increased hematopoiesis	0	0	0	1	10**	10**
Adrenal gland Fatty change	0	0	0	0	0	1
Females
Number of animals	10	10	10	10	10	9^a
Nasal cavity Hyperplasia: respiratory epithelium	0	7** [1.0]	10** [1.0]	9** [1.0]	10** [1.2]	9** [1.1]
Inflammation: respiratory epithelium	0	0	0	0	3	4
Atrophy: olfactory epithelium	0	10** [1.0]	10** [1.0]	10** [1.1]	10** [1.0]	9** [2.1]
Liver Swelling: centrilobular	0	0	0	0	1	6** [1.8]
Spleen Deposition of hemosiderin	0	0	4	10**	10**	9**
Increased extramedullary hematopoiesis	0	0	0	1	8**	9**
Bone marrow Increased hematopoiesis	0	0	0	0	10**	9**
Adrenal gland Fatty change	0	0	0	0	2	9**

Note: The values in brackets indicate the averaged severity grade index of the lesion in affected animals, according to the following equation. [Σ(grade × number of animals with grade)]/number of affected animals. Grade: “slight” scored as 1, “moderate” as 2, “marked” as 3, and “severe” as 4.

^aNumber of female rats examined were 9 instead of 10, because one rat died before the end of the 13-wk exposure period. Significant difference: *p < 0.05; **p < 0.01 by χ2-test.

Applicant's summary and conclusion

Conclusions:

Thirteen-week exposure to DCP induced hyperplasia in the respiratory epithelium and atrophy of the olfactory epithelium at 125 ppm and above. At the higher levels of exposure, hemolytic anemia and lesions of liver and adrenal gland were observed.

Executive summary:

The toxicity of 1,2-dichloropropane (DCP) was examined by inhalation exposure of male and female F344 rats to DCP for either 13 wk. In the 13-wk study, the DCP concentrations used were 125, 250, 500, 1000, or 2000 ppm (v/v). Thirteen-week exposure to DCP induced hyperplasia in the respiratory epithelium and atrophy of the olfactory epithelium at 125 ppm and above. At the higher levels of exposure, hemolytic anemia and lesions of liver and adrenal gland were observed.