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carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
other information
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP study, not according to test guideline, however well documented study, meets generally accepted scientific standards. Adopted from OECD SIDS.
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
Initial study in rats evaluating the effects of 1,4-dichlorobutene-2 (DCB) on the respiratory tract.
Mullin LS, Chiu T, Kennedy Jr. GL
Bibliographic source:
Drug Chem. Toxicol. 25, 227-230.
Reference Type:
secondary source
1,4-Dichlorobut-2-ene - CAS No: 764-41-0
Bibliographic source:
SIDS Initial Assessment Report for 22th SIAM, UNEP Publications.
Reference Type:
study report

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Groups of 140 rats (CD® BR) per sex were exposed for 6 hours per day, five days per week (two days of no exposure per week) to graded concentrations of 1,4-dichlorobutene-2 (DCB). Exposures at the lower concentration, 0.5 ppm, continued for two years. The high concentration tested was 5ppm which was lowered to 2.5 ppm after 7 months. DCB exposures in this group were discontinued at 12 months (five months exposure to 2.5 ppm) and 1-year maintenance period followed. A control group was run concurrently.
GLP compliance:
not specified

Test material

Details on test material:
1,4-dichlorobutene-2 (DCB) is a colourless liquid.
The test material was prepared by distillation from 3,4-dichlorobutene-1 as a mixture of cis-and trans-isomers which met the following specifications:

35 +/- 10 % cis-1,4-dichlorobut-2-ene
65 +/- 10 % trans-1,4-dichlorobut-2-ene
< 0.5 % 3,4-dichlorobut-1-ene
< 500 ppm 1,3,4-trichlorobut-2-ene
< 500 ppm high boilers

Samples were prepared fresh monthly and frozen until used. Chamber concentrations were monitored hourly.

Test animals

other: Crl:CD(SD)BR
Details on test animals and environmental conditions:
- Source: Charles River, Breeding Laboratories, Wilmington, MA colony.
- Housing: during non-exposure periods, all study rats were pair-housed in suspended, stainless steel, wire-mesh cages in rooms separate from exposure rooms.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.

- Temperature (°C): approximately 24
- Humidity (%): 50 ±10

Administration / exposure

Route of administration:
Details on exposure:
Four quadrangular 4.6 m³ stainless steel and glass inhalation chambers, housed in a single room, were used. The chambers and all surfaces that DCB contacted prior to exhausting from the chamber were constructed of materials that neither reacted with nor absorbed DCB. The chambers were operated in a one-pass, flow-through mode with an air flow rate of approximately 1500 L/min. Chamber temperatures and relative humidity ranged from 18-27 °C and 30-65%, respectively. Rats were exposed six hours per day, five days a week (except holidays) for approximately 86 weeks. All exposures were conducted during the same 8-hour period of the day to minimize the potential effects of diurnal physiological variations. At the end of each exposure, test groups were left in their respective exposure chambers for approximately 20 minutes to allow clearance of test material from chamber atmospheres before transfer of rats to housing facilities. Cage position within housing and exposure chambers were randomized daily during unloading and loading procedures. Control rats were treated exactly as test rats, except that the control chamber atmosphere did not contain test material.

DCB chamber atmospheres were generated by bubbling nitrogen through liquid DCB maintained between 8 and 10 °C in a constant temperature bath. Chamber atmospheres were analyzed for DCB at least hourly using a Hewlett Packard Model 5880A gas chromatograph (G.C.) equipped with 15mL sampling loop and a flame ionization detector. Chamber atmospheres were collected for analysis by drawing chamber air through the G.C. sampling loop with a Neptune Dyna vacuum pump.
The G.C. column was 5% OV-7 in Chromosorb® G-HP 1000/120. G.C. oven temperature was approximately 110 °C and detector temperature ws 250°C. Peak areas were measured with those produced from known standards prepared daily in Teflon® sampling bags.

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analytically determined chamber concentrations (in ppm):
Nominal conc./Mean conc. (+/- SD)/Conc. range
0.5 / 0.43 +/- 0.08 / 0.17 - 0.70
2.5 / 2.26 +/- 0.31 / 1.14 - 3.46
5.0 / 4.24 +/- 0.92 / 1.33 - 7.40
Duration of treatment / exposure:
24 months (0.0026 mg/L), 12 months (0.026/0.013 mg/L)
Frequency of treatment:
6 h/day, 5 days/week
Post exposure period:
Post-exposure period: 1 year (only at 0.026/0.013 mg/L)

Doses / concentrations
Doses / Concentrations:
0.5, 5/2.5 ppm = 0.0026, 0.026/0.013 mg/l (see remarks)
nominal conc.
No. of animals per sex per dose:
Control animals:
Details on study design:
Post-exposure period: 1 year (only at 0.026/0.013 mg/L)
Positive control:


Observations and examinations performed and frequency:
Body weights and clinical signs were measured throughout the study. Clinical laboratory and pathological evaluations were conducted on subsets from each group at 3, 12, 18, and 24 months after study initiation.
Sacrifice and pathology:
The experiment was terminated at 24 months when all surviving rats were sacrificed.
Daily exposure concentrations were estimated by calculation of the mean analytical values measured hourly for each chamber. Body weight and relative and absolute organ weight data were analyzed by one way analysis of variance. When the test for differences among test groups means (F-test) was significant, pairwise comparisons were made with the least significant difference (LSD) test. Barlett´s test for homogeneity of variances and a test for linear trend were conducted on the organ weight data. Significance was judged at the alpha =0.05 level.
The probability of survival was estimated by the Kaplan-Meier method. Fisher´s Exact test was used to compare control and test group mortality at the final sacrifice. Significance was judged at the alpha =0.05 level. Estimates of lifetime tumour incidence were made by the Kaplan-Meier procedure. Tumour rates were analyzed using the method of Peto et al. 1980*, were all tumours are assumed to be incidental. For purposes of analysis, adenomas and papillary adenomas were combined into the category of benign nasal tumours. The following designations of nasal neoplasms were combined into the category of malignant nasal tumours: unclassified sarcoma, spindle cell sarcoma, rhabdomyosarcoma, adenocarcinoma, carcinosarcoma, squamoadenocarcinoma, and mixed carcinoma. Incidences of pulmonary tumours were also analyzed by the method of Peto et al. 1980*.

* Peto et al. 1980, Carcinogens: Critical Approach, Supplement 2. pp. 311-426. World Health Organization, Geneva, 1980.

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):
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:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Result (carcinogenicity): positive
Mortality was increased in both male and female rats in the 5/2.5 ppm groups. This continued to increase during the one-year maintenance period. The nasal effects produced by exposure to DCB at these concentrations were considered responsible for enhanced mortality.
The body weight gains of male rats in the 5/2.5 ppm group were 7 to 10% lower than those of the control group rats. Body weights of females in the high exposure groups declined, primarily in the last six months of the study so that the finals body weights in this groups were 18% lower than those of the females rats in the control group. Body weights in this group during the exposure phase and first half of the maintenance period were similar to those of the controls. Body weights of both sexes in the 0.5 ppm groups compared favourably to those of the controls. No unusual signs of response were seen in either of the DCB exposed group rats with observation in the test rats being similar to those in the control group rats.
No compound-related effects in clinical laboratory measurements were observed in the study.
Compound-related changes were observed in the nasal tissues of all DCB-exposed groups and in the trachea of the 5.0/2.5 ppm level groups.
At necropsy, the nasal lesions showed masses of various sizes protruding outward in the nasal bone area. The nasal masses were soft to semi-firm, pale yellow or gray-white to tan, and either in solid tissue or caseous appearing tissue. Histologically, the nasal tumours were classified as adenomas, adenocarcinoma/carcinoma, squamous cell carcinoma, mixed carcinoma, rhabdomyosarcoma and carcinosarcoma. Some tumours were in an early stage of development. In those classified as mixed carcinoma or carcinosarcoma, two or three cell types were found intermingling in the same area or in separate areas. Nasal tumours in the high level groups were predominately malignant, while those in the low level groups were predominately benign. The low level male group had a higher incidence of malignant nasal tumours than the low level female group. All nasal tumours of the low level groups were epithelial types.
Most of the malignant nasal tumours were highly invasive. Besides a local invasion of the skull and the surrounding tissue, about half of those rats with tumours in the high level groups and one low level male had brain invasion.
Many tumours in the high level groups also had metastasized to the cervical lymph nodes and few had metastasized to the lungs. There were three pulmonary squamous cells carcinomas in the 5.0/2.5 ppm males, and one nasal hemangioma in the 0.5 ppm females.
In summary, in the 5.0/2.5 ppm, 88.3% malignant tumours (88.4% in males, 88.3% in females) and 2.7% benign adenomas (1.6% in males, 3.9% in females) were observed at the two-year evaluation. Many of the malignant tumours observed at this exposure concentration had metastasized to cervical lymph nodes and lungs. Malignant tumours, although poorly differentiated, were observed in the high-level groups as early as one year after study initiation.
In the rats exposed to 0.5 ppm at the two-year evaluation, benign nasal adenomas (25.4% in males; 18.0% in females) and malignant nasal tumours (8.5% in males, 1.6% in females) were observed. Similarly differentiated adenomas were observed in the low-level male group at the 18-month evaluation.
The compound-related, non-neoplastic lesions were flattening and metaplasia of the nasal and tracheal mucosal epithelium was observed. Increased incidences of rhinitis, tracheal luminal exudate, atelectasis, suppurative pneumonia and hyperplastic bone marrow in the light level groups were interpreted assecondary changes to the primary lesions of the respiratory system induced by the test compound. The remaining lesions, with a higher incidenc in the DCB-exposed groups, were considered to be non-specific or the results of intercurrent diseases.

Relevance of carcinogenic effects / potential:
Malignant and non-malignant tumours of the nasal tissues were seen in both test groups with the incidence and proportion of malignant tumours being much higher in the 5.0/2.5 ppm rats.

Effect levels

Dose descriptor:
Effect level:
2.6 mg/m³ air
Based on:
test mat.
Basis for effect level:
other: Benign Nasal Tumors (Adenomas) and Malignant Nasal Tumors (adenocarcinoma/carcinoma, squamous cell carcinoma, mixed cell carcinoma, carcinosarcoma, rhabdomyosarcoma).
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Any other information on results incl. tables

1. Incidence of mortality in rats exposed for two years to either 0, 0.5, or 5.0/2.5 ppm DCBa

 Concentration (ppm)  Sex  Number of deaths total/sacrificed in extremis  % Mortality
 0  M  54/40  39
   F  54/45  39
 0.5  M  48/37  34
   F  48/37  34
 5.0/2.5  M  115*/100*  82
   F  117*/104*  84

a All rats were found dead except where noted (scheduled sacrifice rats not included). * p<0.05. Table 2. Number and percent incidence of nasal tumours in rats exposed to either 0, 0.5, or 5.0/2.5 ppm DCB
   Concentration (ppm)  Number of examined nasal cavities  Benign Tumours b  Malignant Tumours c
 Males  0  127  0 (0%)d  0 (0%)
   0.5  130  33 (25.4%)*  11 (8.5%)*
   5.0/2.5  129  2 (1.6%)  114 (88.4%)*
 Females  0  128  0 (0%)   0 (0%)
   0.5  128  23 (18%)*   2 (1.6%)
   5.0/2.5  128 5 (3.9%)*  113 (88.3%)*

a Includes examination of nasal cavities of some rats found dead or sacrificed in extremis.

b Benign tumours consist of adenomas and one hemangioma.

c Malignant tumours consist of adenocarcinoma/carcinoma, squamous cell carcinoma, mixed cell carcinoma, carcinosarcoma, and rhabdomyosarcoma.

d The number in parentheses is percent incidence.

* Significantly different (p<0.05) from controls by Fisher´s Exact test.

Applicant's summary and conclusion

Executive summary:

Mullin (2002)

Rats were exposed by inhalation to either 0.5 ppm 1.4-dichlorobuten-2 (DCB) for two years or to 5.0 ppm for seven months, 2.5 ppm for five months, and no further exposure for 12 months prior to sacrifice.

Malignant and non-malignant tumours of the nasal tissues were seen in both test groups with the incidence and proportion of malignant tumours being much higher in the 5.0/2.5 ppm rats. Under the conditions of this study, DCB is carcinogenic in rats of both sexes.