[(2-chlorophenyl)methylene]malononitrile

Carcinogenicity

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982-1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

GLP compliance:

not specified

Specific details on test material used for the study:

CS2 is 94% o-chlorobenzalmalononitrile (CAS No. 2698-41-1) formulated in a mixture of 5% Cab-0-Sil@ colloidal silica and 1% hexamethyldisilizane (CAS No. 999-97-3). No more details are specified.
No impurities were detected by either thin-layer chromatographic system. Gas chromatographic system 1indicated three unresolved impurities after the major peak, with combined areas of 0.09%relative to the major peak area. Gas chromatographic system 2 indicated two impurities, one before and one after the major peak, with a combined relative area of 0.08%.

Species:

rat

Strain:

Fischer 344

Remarks:

F344/N rats

Details on species / strain selection:

Animal source : Frederick Cancer Research Facility (Frederick, MD)
Study Laboratory : Battelle Pacific Northwest Laboratories

Sex:

male/female

Details on test animals or test system and environmental conditions:

Method of Animal Identification :Ear tags and cage numbers
Time Held Before Study : 21 days
Age When Placed on Study : 8-9 weeks
Method of Animal Distribution : Distributed to weight classes and thenassigned to groups by tables of random numbers
Feed : NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA); available ad libitum during nonexposure periods
Water : Automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
Chambers : Stainless steel (Hazleton Systems, Inc., Aberdeen, MD)
Animals per cage : 1
Chamber Environment : Temp--67°-81° F; hum--31%-84%; fluorescent light 12 h/d; 20 air changes/h. Ammonia levels in the chambers in the morning varied be- tween 2 and 56 ppm

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

not specified

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
- Method of holding animals in test chamber:
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber:
- Air flow rate:
- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes/no

VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:

Duration of treatment / exposure:

22/12/82 to 28/12/84

Frequency of treatment:

6 hours/day, 5 days/week for 105 weeks

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

0.075 mg/kg bw/day (nominal)

Dose / conc.:

0.25 mg/kg bw/day (nominal)

Dose / conc.:

0.75 mg/kg bw/day (nominal)

No. of animals per sex per dose:

50 males and 50 females

Control animals:

yes

Observations and examinations performed and frequency:

Type and frequency of Observation : Observed 2 X day ; weighed 1 X week for 12 weeks and then 1 X month

Sacrifice and pathology:

Necropsy :
Age of the rats when Killed : 115-116 weeks
Necropsy Dates : 01/07/85-11/10/85

Clinical examination : Necropsy performed on all animals
The following tissues examined histologically for control and high dose groups: adrenal glands, brain, bronchial lymph nodes, cecum, colon, duodenum, epididymis/prostate/testes or ovaries/uterus, esophagus, eyes, gross lesions and tissue masses with regional lymph nodes, heart, ileum, jejunum, kidneys, larynx, liver, lungs and mainstem bronchi, mammary gland, mandibular lymph nodes, nasal passage and turbinates, pancreas, parathyroid glands, pituitary gland, preputial or clitoral gland, rectum, salivary glands, skin, spleen, sternebrae including marrow, stomach, thymus, thyroid gland, trachea, and urinary bladder.
The following tissues were examined for the lower dose groups: adrenal glands, liver, lungs, nasal passage, preputial gland, spleen, and thyroid gland for male ; liver, lungs, lymph nodes, nasal passage, ovary, and spleen for female

Statistics:

Survival Analyses : Probability of survival estimated by the product-limit procedure of Kaplan and Meier. If the death is other than natural : Animals censored. If natural death : not censored. Statistical analyses for a possible dose-related effect on survival used the method of Cox for testing two groups for equality and Tarone’s life table test for a dose-related trend. P values are two-sided.

Calculation of Incidence : Incidence of neoplastic/nonneoplastic lesions = ratio of number of animals bearing such lesions at a specific anatomic site/number of animals in which that site was examined.

Analysis of Tumor Incidence: Logistic regression analysis, which assumed that the diagnosed tumors were discovered as the result of death from unrelated cause and thus did not affect the risk of death. Tumor prevalence modeled as a logistic function of chemical exposure and time. Linear and quadratic terms in time were incorporated initially, and quadratic term was eliminated if it did not significantly enhance the fit of the model. Dosed and control groups compared on the basis of likelihood score test for the regression coefficient of dose. Alternative methods of statistical analysis were used : the life table test appropriate for rapidly lethal tumors, and the Fisher exact test and the Cochran-Armitage trend test, procedures based on the overall proportion of tumor-bearing animals. Tests of significance include pairwise comparisons of each dosed group with controls and a test for an overall dose-response trend. P values are one- sided.

Analysis of Continuous Variables: Organ weight data analysed by nonparametric multiple comparison procedures of Dunn or Shirley to assess the significance of pairwise comparisons between dosed and control groups. Jonckheere’s test was used to evaluate the significance of dose-response trends and to determine whether Dunn’s and Shirley’s test was more appropriate for pairwise comparison.

Clinical signs:

no effects observed

Description (incidence and severity):

No compound-related clinical signs were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No significant differences in survival were seen between any groups of either sex.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of male rats exposed to 0.75 mg/m3 were 5%-12%lower than those of controls after week 8.
Mean body weights of female rats exposed to 0.75 mg/m3 were 5%-10% lower than those of controls from weeks 9 to 31 and 11%-15% lower thereafter.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nasal Passage - treatment related : The principal toxic lesions associated with inhalation exposure of rats to CS2 were present in the tissues of the nasal passage.The respiratory epithelium, particularly that on the nasal septum and the free margins of the naso- and maxilloturbinates, and the olfactory epithelium lining the dorsal meatus and tips of the ethmoid turbinates were affected. Hyperplasia and focal squamous metaplasia of the respiratory epithelium occurred at increased incidences in rats exposed to 0.75 mg/m3 CS2. Hyperplasia was characterized by increased thickness and slight folding of the respiratory epithelium with increased numbers of goblet cells. Squamous metaplasia consisted of several layers of well-differentiated squamous cells replacing the pseudostratified columnar epithelium . Degeneration with ciliated columnar and/or squamous metaplasia of the olfactory epithelium also occurred at increased incidences at the top concentration. The degeneration was characterized by the loss of olfactory sensory cells and atrophy of the submucosal nerve bundles. Focally, there was replacement of the olfactory epithelium by ciliated columnar cells (metaplasia) or by several layers of squamous cells (squamous metaplasia). Many of the columnar epithelial cells contained a large eosinophilic intracytoplasmic droplet Downgrowth of the columnar epithelium into the Bowman’s glands was associated with these lesions. Inflammation, characterized by focal accumulations of mononuclear inflammatory cells in the submucosa, and proliferation of the periosteum of the turbinate bones also occurred at increased incidences in rats at the top concentration.

Lung - no treatment related : Chronic inflammation and histiocytic cellular infiltrates occurred in male and female rats of all exposure groups, including controls, and the incidences of these lesions were increased in females exposed to 0.75 mg/m3. The chronic inflammation was generally minimal in severity and affected only a few scattered terminal bronchioles, alveolar ducts, and the adjacent alveoli in the histologic sections. It was characterized by small numbers of mononuclear cells and occasional neutrophils in the interstitium around the terminal bronchioles and alveolar macrophages in the alveolar lumina. The histiocytic cellular infiltrates were small, focal accumulations of alveolar macrophages in alveolar lumina in more distal portions of the lung, usually near the pleura. Since the histologic appearance of the lesions in exposed rats was similar to that in controls, the lesions are not considered to be caused by the inhalation of CS2 or of the particles of colloidal silica that might be present in the aerosol. The chronic inflammation may be related to subclinical infection with rat coronavirus/sialodacryo-adenitis virus (RCV/SDA), since positive serologic titers to RCV/SDA were observed in sentinel animals at the various time points sampled. RCV has been shown to replicate in the airways of the lungs and cause inflammatory lesions in the centriacinar regions (terminal bronchioles and alveolar ducts). The reason for the increased incidences of these lesions in female rats at the top concentration has not been determined. However, inhalation of CS2 may have compromised local immune mechanisms and allowed for greater frequency of viral replication and higher incidences of lesions in female rats exposed to 0.75 mg/m3.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Nasal Passage: The adenocarcinoma and the squamous cell car- cinoma that occurred in the nasal passage of a single male rat at 0.75 mg/m3 and the adenoma in the 0.075 mg/m3 female rat were not considered to be caused by exposure to CS2.

Thyroid Gland: The incidences of C-cell adenomas in male rats exposed to 0.075 mg/m3 and of C-cell adenomas or carcinomas (combined) in male rats exposed to 0.075 or 0.25 mg/m3, but not to 0.75 mglm3, were significantly greater than those in controls. Since incidences of these neoplasms did not increase in a dose-related fashion and since the marginal inci- dences in all groups are within the historical control range, the increases in the incidences of these neoplasms are not considered to be related to exposure to CS2 aerosol. There was no in- creased incidence of C-cell neoplasms in any group of exposed female rats compared with that in controls.

Kidney: Renal tubular cell adenomas were seen in two female rats exposed to 0.25 mg/m3. The historical incidence of renal tubular cell neoplasms inchamber control female F344/N rats is 11347 (0.3%)) and the highest observed incidence is 1/50. The historical incidence of renal tubular cell neoplasms in untreated control female F344/N rats is 211,639 (0.1%),and the highest observed incidence is 1/50. The incidences of renal tubular cell hyperplasia in the current study were: control, 3/49; 0.075 mgIm3, 2/37; 0.25 mgIm3, 1/30; 0.75 mgIm3, 1/50. Because the renal tubular cell neoplasms were restricted to the 0.25 mg/m3 exposure group and did not involve the low or high exposure groups, they were not considered to be related to exposure to CS2.

Testis: A marginally significant increase in the incidence of interstitial cell adenomas occurred in the high dose male group compared with that in controls (control, 31/50; 0.075 mg/m3, 38/47;
0.25 mgIm3, 36/50; 0.75 mgtm3, 41/50). The incidence in controls is well below the average historical incidence for untreated controls in NTP studies. The marginal increase was not considered to be chemically related.

Other effects:

not specified

Relevance of carcinogenic effects / potential:

Under the conditions of these inhalation studies, there was no evidence of carcinogenic activity of CS2 for male or female F344/N rats exposed to 0.075,0.25, or 0.75 mg/m3 in air for up to 2 years.

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.75 ppm

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

other: respiratory epithelium / olfactory epithelium

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

yes

Conclusions:

Under the conditions of these inhalation studies, there was no evidence of carcinogenic activity of CS2 for male or female F344/N rats exposed to 0.075,0.25, or 0.75 mg/m3 in air for up to 2 years.

Executive summary:

Under the conditions of these inhalation studies, there was no evidence of carcinogenic activity of CS2 for male or female F344/N rats exposed to 0.075,0.25, or 0.75 mg/m3 in air for up to 2 years.