Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 203-466-5 | CAS number: 107-13-1
Carcinogenicity
Administrative data
Description of key information
The carcinogenicity of acrylonitrile has been investigated in a large number of studies in rats and mice, using oral (gavage, drinking water) and inhalation exposure. The results of the studies indicate that acrylonitrile is a multi-site carcinogen in rodent species.
Key value for chemical safety assessment
Carcinogenicity: via oral route
Link to relevant study records
- Endpoint:
- carcinogenicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Proprietary study similar to guidelines; also published more recently.
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Principles of method if other than guideline:
- 2 year combined toxicity and carcinogenicity study
- GLP compliance:
- not specified
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- The animals were male and female Fischer 344 rats., obtained from Charles River Breeding Laboratories, Inc., Wilmington. The rats were quarantined for at least 35 days, and were 9-10 weeks at the start of the study. The rats were housed in individual stainless steel suspended wire mesh cages, and were given free access to Purina Certified Rodent Chow and bottled water. The temperature of the animal rooms was maintained at 70-75°F, relative humidity between 40 and 60%, and a 12 hour light/dark cycle. Individuals were identified by metal ear tags. All rats underwent a pretest physical examination and ophthalmoscopic examination to ensure their suitability as study participants.
- Route of administration:
- oral: drinking water
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- Rats were exposed to acrylonitrile in the drinking water (distilled) for 2 years, at doses of 0, 1, 3, 10, 30 and 100 ppm. Fresh dosing solutions were introduced twice weekly via glass water bottles with fitted rubber stoppers and stainless steel sipper tubes.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Stability of the test substance in the dosing solution over the period used was verified analytically using a gas chromatograph equipped with a dual flame ionisation detector and a glass column containing Carbowax or Carbopak. Periodic checks using GC methods (weekly from initiation through week 14, biweekly from weeks 16 through 26, and approximately monthly thereafter) confirmed correct preparation of the dosing solutions.
- Duration of treatment / exposure:
- The intended duration was 2 years, however the study was terminated at 23 months in females because of low survival rates. The males were exposed for 26 months.
- Frequency of treatment:
- Daily - ad libitum in drinking water.
- Post exposure period:
- There was no post exposure period.
- Dose / conc.:
- 0 ppm
- Remarks:
- Control: untreated drinking water
- Dose / conc.:
- 1 ppm
- Remarks:
- In drinking water
- Dose / conc.:
- 3 ppm
- Remarks:
- In drinking water
- Dose / conc.:
- 10 ppm
- Remarks:
- In drinking water
- Dose / conc.:
- 30 ppm
- Remarks:
- In drinking water
- Dose / conc.:
- 100 ppm
- Remarks:
- In drinking water
- No. of animals per sex per dose:
- 100 rats/sex/group. There were two untreated control groups each with 100 males and 100 females.
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- Rats were assigned to study groups using a random number table to achieve approximately equal bodyweights among all study groups of the same sex. An additional 100 rats/sex were included in the untreated control group. Controls received water bottles containing distilled or deionised water only. Groups of 10 rats/sex/group (including controls) were sacrificed at intervals of 6, 12 and 18 months, and at study termination. Final sacrifice of all test groups, by sex, was scheduled to insure that at least 10 animals/sex/group would be available for assessent.
- Positive control:
- A positive control was not included.
- Observations and examinations performed and frequency:
- Animals were observed twice daily for overt signs of toxicity and death. Detailed cage-side examinations, including palpations to identify growths, were performed weekly. Ophthalmoscopic examinations were carried out at pretest, 6, 12, 18, 23 (females) and 26 (males) months. Individual body weights were recorded weekly through the first 14 weeks of the study, bi-weekly between weeks 16 to 26, and monthly thereafter. Food and water consumptions (collected over a 3 day period) were determined from approximately 25 rats/sex/group at the same intervals that body weights were recorded. Test substance intake was calculated and expressed as mg/kg/day.
Ten rats/sex/acrylonitrile group and 5 rats/sex from the control group were selected for periodic haematology, clinical chemistry and urinalysis pretest, at 6, 12, 18 months and at study termination. Blood was drawn via the dorsal aorta from rats fasted overnight. Haematological and clinical chemistry parameters measured were: haemoglobin, haematocrit, erythrocyte count and morphology, reticulocytes, prothrombin time, total and differential leukocytes, serum glutamic pyruvic transaminase, alkaline phosphatase, blood urea nitrogen and fasting glucose. Urinalysis (gross appearance, specific gravity, pH, protein, glucose, ketones, bilirubin and occult blood) was also performed on groups of ten rats/sex from the high dose group and control group at similar study intervals following overnight sample collection in stainless-steel metabolism cages. - Sacrifice and pathology:
- Interim necropsies were performed at 6, 12, and 18 months (10/sex/dose group and 20/sex/control). Necropsies were performed at study termination (females; 23 months and males; 26 months). Examinations included organ weights and microscopic evaluation of tissues and organs.
At each interim sacrifice period and at study termination, the following organs were removed from groups of approximately 10 rats/sex/acrylonitrile group and 5 rats/sex from the control group: brain, pituitarym adrenal, gonads, heart, kidney and liver. Absolute weights were measured and relative organ weight ratios were calculated.
All animals, whether found dead or euthanised in a moribund condition or at the end of the study, underwent a full necropsy. The following tissues and organs were preserved from all animals: adrenal, sternum, brain, ear canal, oesophagus, eye, gonads, heart, colon, duodenum, ileum, kidney, lung, liver, lymph node, mammary gland, pancreas, pituitary, prostate, salivary gland, skeletal muscle, skin, spinal cord, spleen, stomach, thyroid, trachea, urinary bladder, uterus, and all gross lesions and tissue masses. The eye and testis were fixed in Bouin's solution, all other tissues were preserved in 10% neutralised formalin. Haematoxylin and eosin stained sections of tissue were prepared for microscopic examination. Tissues were routinely examined from the high dose and control animals. Potential target organs like brain, ear canal, stomach and spinal cord were examined from all animals on test. All tissue masses or gross lesions observed at necropsy were examined microscopically.
At study termination, all surviving animals were sacrificed. Approximately 40 tissues were examined from 10 rats/sex of the high dose group and 5 rats/sex of the two control groups. Potential target organs and suspicious lesions were examined from all animals. - Other examinations:
- No other examinations reported.
- Statistics:
- Body weights, food and water consumption and organ weights were evaluated using Dunnett's test for pair-wise group comparison. Haematology and clinical chemistry parameters were evaluated using an F-test and when appropriate, Cochran's modified Student's T-test. Histopathology was analysed using 2x2 contingency tables. Fisher's exact test was used to test for an increase in incidence or mortality proportion.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- : reduced surival at 100 ppm
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- : reduced surival at 100 ppm
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- lower bodyweight at 100 ppm
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- : reduced absolute but not relative food consumption at 100 ppm
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- : reduced absolute but not relative water consumption at 100 ppm
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- : slight effects on red blood cell parameters at 100 ppm
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- : slightly elevated AP activity at 100 ppm
- Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- : increased SG at 100 ppm
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- : mainly secondary to bodyweight effects at 100 ppm
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- : hyperplasia/hyperkeratosis in squamous cells of the forestomach at 3 pppm and above
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- : increased incidences of mammary gland carcinoma, astrocytoma, forestomach tumours, Zymbal's gland tumours
- Details on results:
- Mortality
Mortality of males and females receiving 100 ppm was markedly greater than controls, while mortality in the males receiving 10 ppm and the females receiving 3 and 30 ppm was also significantly greater than control.
Bodyweight
Bodyweights for the males and females receiving 100 ppm were consistently lower (p < 0.01) than the controls, while body weights for the males only receiving 30 ppm were significantly lower (p < 0.01) than the controls. The body weights for the animals in the other treatment groups were generally comparable to controls throughout the study.
Food consumption
Food consumption by the females at 100 ppm was consistently slightly lower than controls on a grams/week basis, while this pattern was notable for the males of this group only following the first year of the study. On a grams/kg/day basis, however, food consumption for both males and females at 100 ppm was considered generally comparable to or slightly greater than controls as a result of the lower body weights for these animals. Differences from controls in food consumption for the other groups were sporadic and not indicative of a relationship to treatment.
Water consumption
Water consumption for the males and females at 100 ppm was generally lower (p <0.01) than controls on a ml/3 days basis; however, on a ml/kg/day basis, differences from the controls were less marked for the females and comparable to or greater than controls for the males. Sporadic differences from controls noted for the other groups were not considered to be treatment related.
Haematology
Small but generally consistent reductions in haemoglobin (occasionally achieving statistical significance of p <0.05), haematocrit and erythrocyte counts were noted for the females receiving 100 ppm throughout the study. These parameters were considered comparable to controls for males at this dose level.
Clinical chemistry
Slight increases in alkaline phosphatase activity (p <0.05) were noted for the females receiving 100 ppm from 12 months onwards (to termination), while values for the males in this group were elevated (p <0.01) at 18 months onwards (to termination). Slight elevations (p < 0.05) in the alkaline phosphatase activity were also noted in females receiving 10 and 30 ppm, at termination only.
Urinalysis
Urine specific gravity was increased in males receiving 100 ppm at 18 months and at termination.
Organ weights
Consistent, but not always statistically significant, elevations in the mean relative (to body weight) liver and kidney weights were noted (p <0.01) for animals receiving 100 ppm at most necropsy intervals, while the mean absolute weights for these organs were generally comparable to the controls or slightly elevated. The mean relative heart weights were also elevated (p <0.05) for this group at 18 months and termination. The increases in the mean relative weights of these organs at most necropsy intervals in animals receiving 100 ppm were considered treatment related effects. In addition, at the terminal sacrifice the mean absolute and relative liver and heart weights were elevated (p <0.05) for females at 30 ppm, while their mean body weight was comparable to controls. Other organ weight differences were noted, but were considered attributable to body weight differences or else they did not occur in a pattern suggestive of a relationship to treatment. Elevated (p <0.05) mean organ/brain weight ratios were noted for heart and liver in the females receiving 30 ppm at termination. Other differences were sporadic and not treatment-related. For 100 ppm animals the mean absolute weights of the liver, kidney and heart as well as the brain, were not markedly different from the control animals.
Palpable masses
A dose-related increased incidence of palpable masses on the head (around the ears and eyes and in the cervical region), was noted in the males and females receiving 30 and 100 ppm which died or were sacrificed after 12 months. The masses observed in the area of the ear were characterised as subcutaneous and necrotising or purulent, and were associated with the ear canals. An increased incidence of masses in the mammary region was also noted in females receiving 100 ppm and in males receiving 3 and 10 ppm.
Pathology
Histopathology showed that the number of malignant tumour-bearing rats was increased in the male and female rats at 10, 30 and 100 ppm, when compared to controls. This was due to an increased incidence of astrocytomas of the central nervous system (brain and/or spinal cord) and squamous cell carcinomas of the Zymbal gland, as well as mammary gland carcinomas in the female at 100 ppm. The increases in the incidences of these neoplasms were noted predominantly in animals dying, killed in a moribund condition or sacrificed at scheduled intervals after the first year of the study. The incidence of neoplasms in the rats at 1 and 3 ppm was considered comparable to controls. Other neoplastic and non-neoplastic lesions occurred sporadically in various tissues and organs but were not considered attributable to treatment. - Relevance of carcinogenic effects / potential:
- Evidence of carcinogenicity was seen under the conditions of this study. The main tumours observed in rats exposed to acrylonitrile were microscopic brain tumours and Zymbal gland tumours.
- Dose descriptor:
- NOAEL
- Effect level:
- 1 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increased tumour incidences
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- NOAEL
- Effect level:
- 1 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Local gastric irritation
- Remarks on result:
- other: Effect type: toxicity
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 10 ppm
- System:
- central nervous system
- Organ:
- brain
- spinal cord
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 10 ppm
- System:
- ear
- Organ:
- zymbal gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Clear evidence of carcinogenicity was seen under the conditions of this study.
- Executive summary:
In this study, acrylonitrile was administered in the drinking water for approximately 2 years to groups of 100 male and 100 female F344 rats at nominal concentrations of 1, 3, 10, 30 and 100 ppm. Two additional groups, each of 100 males and 100 females, were used as untreated controls. The average daily intake was 0, 0.1, 0.3, 0.8, 2.5 or 8.4 mg/kg bw/d respectively, for males and 0, 0.1, 0.4, 1.3, 3.7 or 10.9 mg/kg bw/d respectively for females. Clinical biochemistry, interim necropsies, organ weights and microscopic evaluation of tissues and organs were performed on 10 rats/sex/group following treatment for 6, 12 and 18 months and at study termination. Females were sacrificed after treatment for 24 months; males after treatment for 26 months. A consistent decrease in survival, lower body weight and reduced water intake and small reductions in haematological parameters were observed in both sexes at 100 ppm. Increased numbers of early deaths were observed in males at 10 ppm and females at 30 ppm. Relative organ weights at various study intervals were consistently elevated in the high dose group; findings are attributable to lower body weights. At the same intervals, mean absolute weights were either comparable to controls or only slightly elevated and few changes in weight ratios were seen when organ weights were compared with brain weights. No biochemical changes suggested a treatment-related effect. An increase in urine specific gravity in 100 ppm male rats reflected the reduced water consumption by this group. The only significant non-neoplastic finding observed histologically was a dose-related increase in hyperplasia/hyperkeratosis in squamous cells of the forestomach in male and female rats at concentrations of 3 ppm and higher. This observation correlated with the induction of treatment-related squamous cell tumours (papillomas and carcinomas) of the forestomach seen primarily in rats in these groups. Mammary gland carcinomas were increased in female groups; a non dose-related increase was seen in the low dose male groups. Both sexes given 10 ppm acrylonitrile or more had astrocytomas of the brain/spinal cord and adenomas/carcinomas of the Zymbal's gland
- Endpoint:
- carcinogenicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 3rd March 1997 to 5th March 1999
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: NTP protocol
- Principles of method if other than guideline:
- Assessment of carcinogenicity following lifetime exposure using gavage administration to the mouse
- GLP compliance:
- yes
- Remarks:
- FDA GLP
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Male and female B6C3F1 mice were obtained from Taconic Laboratory Animals and Services (Germantown, NY). Mice were quarantined for 12 (males) or 11 (females) days before the beginning of the study. Five male and five female mice were randomly selected for parasite evaluation and gross observation of disease. Animals were approximately 6 weeks old at the beginning of the study. The health of the animals was monitored during the study according to the protocols of the NTP Sentinel Animal Program. Males were housed individually and females were housed five per cage. Feed and water were available ad libitum. Cages were changed once (males) or twice (females) weekly and rotated every 2 weeks; racks were changed and rotated every 2 weeks. Individuals were identified by tail tattoos. Irradiated NTP-2000 pelleted diet was provided, except during urine collections when mean feed was used. Tap water was provided via an automatic watering system ad libitum. Temperature in the animal room was 72±2oF. Relative humidity 50±15%. Fluorescent light was provided 12 hours per day and there were 10 air changes per hour.
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- Acrylonitrile was administered in deionised water by gavage. The dosing volume was 10ml/kg.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The dose formulations were prepared every 4 weeks by mixing acrylonitrile with deionised water. Stability studies of a 0.8145mg/ml formulation were performed using gas chromatography. Stability was confirmed for at least 35 days for dose formulations stored in sealed glass vials with Teflon®-lined caps at temperatures up to 28° C. Periodic analyses of the dose formulations of acrylonitrile were conducted using gas chromatography. The dose formulations were analysed approximately every 8 to 12 weeks. All of the dose formulations analysed were within 10% of the target concentrations.
- Duration of treatment / exposure:
- Two years.
- Frequency of treatment:
- 5 days per week for 104 to 105 weeks.
- Post exposure period:
- No post exposure period.
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- Gavage dose: vehicle control
- Dose / conc.:
- 2.5 mg/kg bw/day
- Remarks:
- Gavage dose
- Dose / conc.:
- 10 mg/kg bw/day
- Remarks:
- Gavage dose
- Dose / conc.:
- 20 mg/kg bw/day
- Remarks:
- Gavage dose
- No. of animals per sex per dose:
- 50 mice per sex per dose.
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Animals were distributed randomly into groups of approximately equal initial mean body weights. Doses were chosen based on the results obtained in the 14 week preliminary toxicty study.
- Positive control:
- No positive control.
- Observations and examinations performed and frequency:
- All animals were observed twice daily. Clinical findings were recorded on day 29, every 4 weeks, and at the end of the study. Five mice of each sex from each dose group were evaluated at 2 weeks, 3, 12 and 18 months for urinalysis parameters (see below). Animals were weighed initially, approximately every 4 weeks, and at the end of the study.
- Sacrifice and pathology:
- Animals were sacrificed by carbon dioxide asphyxiation. Complete necropsies were performed on all animals. Complete histopathology was performed on all mice (see below).
- Other examinations:
- No additional examinations.
- Statistics:
- The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose related trends. All reported P values for the survival analyses are two sided.
The incidence of neoplasms or non-neoplastic lesions was presented as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined micropscopically. The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence.
Body weight data were analysed with Dunnet and Williams tests. Urinary data were analysed with Shirley and Dunn tests. Jonckheere’s test was used to assess the significance of the dose related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose related trend (Dunnett’s or Dunn’s test). - 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:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- effects observed, treatment-related
- 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:
- There were no clinical findings related to acrylonitrile exposure. Mean body weights of 20 mg/kg males and females were generally less than those of the vehicle controls throughout most of the study; however, those of the surviving 20 mg/kg females were similar to the vehicle controls during the last 25 weeks of the study.
There were dose-related increases in urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine concentrations in all dosed groups of mice at 2 weeks and at 3, 12 and 18 months. However, there were apparent increases in the excretion of each metabolite at 12 and 18 months compared to 2 weeks and 3 months.
Forestomach pathology: The incidences of squamous cell papilloma, squamous cell carcinoma, and squamous cell papilloma or carcinoma (combined) of the forestomach occurred with positive trends in males and females, and the incidences in 10 and 20mg/kg mice were generally significantly greater than those in the vehicle controls. The incidences of these lesions in all dosed groups exceeded the historical ranges in controls (all routes) given NTP-2000 diet and in water gavage controls given NIH-07 diet. Some squamous cell carcinomas metastasised, primarily to the liver but also to the pancreas, spleen, kidney, lung, mesenteric lymph nodes, prostate gland, and adrenal gland. The incidences of mild focal or multifocal epithelial hyperplasia (combined) in 20mg/kg males and females were significantly greater than those in the vehicle controls; the incidence of mild diffuse or focal hyperkeratosis (combined) in 20mg/kg males was significantly increased. Sporadic cases of multifocal ulcerations of the forestomach were observed in 2.5 and 10mg/kg males; the ulcerations were generally accompanied by focal chronic active inflammation.
- Harderian Gland pathology: The incidences of harderian gland adenoma and adenoma or carcinoma (combined) occurred with positive trends in males and females. The incidences in 2.5mg/kg males and in 10 and 20mg/kg males and females were significantly increased and exceeded the historical ranges in controls (all routes) given NTP-2000 diet and in water gavage controls given NIH-07 diet. The incidences of harderian gland hyperplasia in dosed groups of males and 10 and 20mg/kg females were greater than those in the vehicle controls, and the incidence in 10mg/kg males was significantly increased. Large proliferative lesions of the harderian gland result in grossly observable compression, protrusion (proptosis), and collapse of the eye. Secondary changes were noted in those eyes that were compressed by the harderian gland neoplasms.
- Ovary pathology: The incidence of benign or malignant granulosa cell tumor (combined) in the ovary of 10mg/kg females was greater than that in the vehicle control group. The incidence in this group exceeded the historical range in controls (all routes) given NTP-2000 diet, in controls (various routes) given NIH-07 diet, and in corn oil gavage controls given NIH-07 diet. One malignant granulosa cell tumor invaded the surrounding fat. The incidences of atrophy and cyst in the ovary of 2.5(cyst only), 10, and 20mg/kg females were significantly increased; in dosed females, the severity of atrophy was marked and the severity of cyst was mild. Atrophy was characterized by a partial to complete lack of histologically evident follicular and corpus luteum development and a predominance of interstitial tissue. There were decreased incidences of cystadenoma in the ovary of dosed females; however, the incidence in the vehicle controls was higher than in any other controls given NTP-2000 diet and exceeded the historical range in water gavage controls given NIH-07 diet.
- Lung pathology: The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in 10mg/kg females was significantly greater than that in the vehicle controls. The incidences of alveolar/bronchiolar carcinoma in dosed groups and the incidences of alveolar/bronchiolar adenoma or carcinoma (combined) in 10 and 20mg/kg females generally exceeded the historical ranges for controls (all routes) given NTP-2000 diet and for water gavage controls or feed controls given NIH-07 diet. The lower incidences of primary alveolar/bronchiolar adenomas and carcinomas in 20mg/kg females may have been related to reduced survival in this group. Incidences of alveolar epithelial hyperplasia were similar in vehicle control and dosed groups of females. The incidences of alveolar/bronchiolar neoplasms in male mice were similar to those in the vehicle controls.
- Liver pathology: The incidence of hepatocellular adenoma in 2.5mg/kg males was significantly greater than that in the vehicle control group (vehicle control, 23/50; 2.5mg/kg, 32/50; 10mg/kg, 29/50; 20mg/kg, 14/50;), and the incidence exceeded the historical ranges in controls (all routes) given NTP-2000 diet [195/659 (30.4% ± 8.9%), range 12%-46%] or water gavage controls given NIH-07 diet (26/50). The incidences of hepatocellular adenoma in 10mg/kg males and of hepatocellular adenoma or carcinoma (combined) (32/50, 36/50, 37/50, 17/50) in vehicle control, 2.5, and 10mg/kg males also exceeded the historical ranges; however, the incidences of these neoplasms in these groups were not significantly increased or dose-related. - Relevance of carcinogenic effects / potential:
- Under the conditions of this 2-year gavage study, there was clear evidence of carcinogenic activity (as defined by the NTP) of acrylonitrile in male and female B6C3F1 mice based on increased incidences of forestomach and harderian gland neoplasms. Neoplasms of the ovary and lung in female mice may have been related to administration of acrylonitrile.
- Dose descriptor:
- LOAEL
- Effect level:
- 2.5 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Increased tumour incidences (Harderian gland) at 2.5 mg/kg bw/d
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- NOAEL
- Effect level:
- < 2.5 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Increased tumour incidences (Harderian gland) at 2.5 mg/kg bw/d
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- NOAEL
- Effect level:
- 2.5 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Increased tumour incidences at 5 mg/kg bw/d and above
- Remarks on result:
- other: Effect type: carcinogenicity
- Key result
- Critical effects observed:
- yes
- System:
- eye
- Organ:
- other: Harderian gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 10 mg/kg bw/day (actual dose received)
- System:
- respiratory system: lower respiratory tract
- Organ:
- bronchi
- lungs
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 10 mg/kg bw/day (actual dose received)
- System:
- gastrointestinal tract
- Organ:
- stomach
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Acrylonitrile demonstrated clear evidence of carcinogenicity in this study.
- Executive summary:
Male and female B6C3F1mice were administered acrylonitrile in deionised water by gavage at doses of 0, 2.5, 10 or 20 mg/kg bw/d, 5 days per week for 104 to 105 weeks (2 years). Complete necropsies and complete histopathology were performed on all mice. Urine from five male and five female mice from each group was collected at 2 weeks and at 3, 12 and 18 months. Survival of 20 mg/kg bw/d mice was significantly less than that of the vehicle control groups. Mean body weights of 20 mg/kg bw/d males and females were generally less than those of the vehicle controls throughout most of the study. Dose related increases in urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine concentrations occurred in all dosed groups at 2 weeks and at 3, 12 and 18 months. The incidences of squamous cell papilloma, squamous cell carcinoma, and squamous cell papilloma or carcinoma (combined) of the forestomach occurred with positive trends in males and females, and were present in 50% or greater of mice administered 10 or 20 mg/kg bw/d acrylonitrile. The incidences of mild focal or multifocal epithelial hyperplasia (combined) of the forestomach in 20 mg/kg bw/d males and females and of mild diffuse or focal hyperkeratosis (combined) in 20 mg/kg bw/d males were increased. The incidences of harderian gland adenoma and adenoma or carcinoma (combined) were significantly increased in all dosed groups of males and in 10 and 20 mg/kg bw/d females, and the incidence of harderian gland hyperplasia was significantly increased in 10 mg/kg bw/d males. The incidence of benign or malignant granulosa cell tumor (combined) in the ovary of 10 mg/kg bw/d females was greater than that in the vehicle controls. The incidences of atrophy and cyst in the ovary of 10 and 20 mg/kg bw/d females were significantly increased. The incidence of alveolar /bronchiolar adenoma or carcinoma (combined) in 10 mg/kg bw/d females was significantly increased. The authors concluded that under the conditions of this 2-year gavage study, there was clear evidence of carcinogenic activity of acrylonitrile in male and female B6C3F1 mice.
- Endpoint:
- carcinogenicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Chronic rat carcinogenicity study specifically investigating brain tumour incidence and histogenesis.
- GLP compliance:
- no
- Remarks:
- : published literature study
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- The animals were male and female Fischer 344 rats, aged 6 weeks old at the start of the study. Rats were randomly assigned to treatment groups.
- Route of administration:
- oral: drinking water
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- Rats were exposed to acrylonitrile in the drinking water at 0, 100 or 500 ppm for 540 days.
- Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- No information available
- Duration of treatment / exposure:
- 18 months
- Frequency of treatment:
- Daily - ad libitum in drinking water
- Post exposure period:
- Not applicable
- Dose / conc.:
- 0 ppm
- Remarks:
- Control: untreated drinking water
- Dose / conc.:
- 100 ppm
- Remarks:
- In drinking water
- Dose / conc.:
- 500 ppm
- Remarks:
- In drinking water
- No. of animals per sex per dose:
- Group 1 (500 ppm): 153 males and 147 females
Group 2 (500 ppm): 50 females and 50 males
Group 3 (100 ppm): 50 females and 50 males
Group 4 (controls): 49 males and 51 females - Control animals:
- yes, concurrent no treatment
- Details on study design:
- Rats were exposed to acrylonitrile in the drinking water for 18 months to examine the neuro-oncogenic effects resulting from exposure. Rats were randomly allocated to treatment groups as follows:
Group 1: This group contained 153 females and 147 males exposed to 500 ppm acrylonitrile. The animals from this group were used for studies of tumour morphology, biology and karyotype. Complete autopsies were performed on all animals that died spontaneously or were killed for tumour examination.
Group 2: Comparative survival and clinical symptomology studies were made on this group, which consisted of 50 females and 50 males exposed to 500 ppm acrylonitrile.
Group 3: As for group II comparative survival and clinical symptomology studies were made on this group, which was exposed to 100 ppm acrylonitrile and consisted of 50 female and 50 male rats.
Group 4: This control group received no acrylonitrile and consisted of 49 females and 51 males. As above the group was used in comparative survival and clinical symptomology studies. - Positive control:
- Not examined: not required for this study type
- Observations and examinations performed and frequency:
- Animals from all groups were observed daily, and in greater detail during weekly weighing for neurological signs.
- Sacrifice and pathology:
- Complete autopsies were carried out on all Group 1 (500 ppm) animals that died spontaneously or were killed for tumour examination. Brains were examined for tumours macroscopically and microscopically using haematoxylin and eosin (H&E) staining.
- Other examinations:
- None reported.
- Statistics:
- No information available.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased mortality, clinical signs indicative of neurotoxicity
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Increased mortality, clinical signs indicative of neurotoxicity
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Reduced weight gain
- 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:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- Clinical signs indicative of neurotoxicity
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased incidences of brain tumours, skin, stomach and Zymbal's gland tumours
- Details on results:
- Mortality
A clear-cut dose-response effect in mortality was observed in both sexes. Females at both 500 and 100 ppm dose levels died slightly earlier than males, whereas only a few controls (Group 4) of either sex died during the first 18 months of the study.
Bodyweight
There was a significant decrease in mean body weight within 2-3 weeks after the commencement of administration of acrylonitrile at 500 ppm to male rats. Females showed a similar pattern at 500 ppm but with a slightly longer period before the mean weight clearly diverged from that of the controls. Throughout chronic administration of acrylonitrile, this mean weight difference was observed in both sexes at the 500 ppm dose level. At 100 ppm (Group 3) the divergence of the mean weight curves from those of the controls began about 2 months after the start of administration in males but was not apparent in females until well into the second year of administration.
Clinical signs
Neurological effects frequently seen included paralysis, head tilt, circling and seizures. Other more non-specific signs, sometimes associated with brain tumours but also seen in their absence, included precipitate weight loss and huddling in a cage corner with decreased activity. The incidence of neurological signs (observed within 12-18 months) was closely related to the acrylonitrile dose. The proportion of animals affected were 20/300 and 16/100 in the two groups (Group 1 and 2) dosed at 500 ppm acrylonitrile, compared to 4/100 in the 100 ppm dose level group and 0/100 in the controls.
Pathology
A total of 215 brains were examined from the rats (Group 1) exposed to 500 ppm acrylonitrile. Most of these animals died or were killed for tumour examination between 12 and 18 months after the beginning of exposure. Out of these 215 rats, 49 primary brain tumours were found. Tumours were observed in the cortex (approximately 75%), brain stem and cerebellum. When the tumours were differentiated according to size, 10/49 (20%) were found to be larger than 5 mm in greatest diameter, 28/49 (58%) were between 1 and 5 mm in diameter and were detectable by visual examination of an H&E stained slide without a microscope, leaving 11/49 (22%) that could only be detected microscopically. Despite the variation in their size and regardless of their location in the brain, all 49 primary tumours were described as being remarkably similar in their cellular and morphological features. The lesions were densely cellular in the centre with diffusely infiltrative margins. The cells were consistently uniform in size with round or oval nuclei and moderate amounts of pink or clear cytoplasm. Multinucleated giant cells were not seen. Very rarely, tumours contained focal necrosis surrounded by palisading nuclei, but endothelial proliferation was not present in any of the 49 brain tumours. Infiltrating cells at the periphery of lesions often accumulated around small blood vessels, forming perivascular cuffs. Neuronal stellitosis by tumour cells was also observed frequently. Tumour cells gathered in the subpial regions and invaded the ventricles and subarachnoid space in lesions where these spaces were accessible.
The tumours found were noted to be to be similar to, and probably indistinguishable from, a subset of spontaneously occurring rat brain tumours that have been generally classified as astrocytomas or anaplastic astrocytomas by light microscopic evaluation of H&E stained slides. Despite the superficial similarity to astrocytomas, karyotypic analysis did not provide definite evidence to identify any of the neoplastic cells as astrocytic. No glial fibrillary acidic protein (GFAP) was detectable in the tumour cells, despite prominent staining of reactive and normal astrocytes in the same section. Electron microscopy revealed no distinctive intermediate filaments or junctions, nor was there evidence of differentiation of the neoplastic cells. Findings are in conflict with the hypothesis that the neoplastic cells found in this study are astrocytic in origin. - Relevance of carcinogenic effects / potential:
- Clear evidence of a carcinogenic effect was seen in rats administered acrylonitrile in this study.
- Dose descriptor:
- NOAEL
- Effect level:
- < 100 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increased mortality, bodyweight effects, signs of toxicity
- Remarks on result:
- other: Effect type: toxicity
- Dose descriptor:
- LOAEL
- Effect level:
- 100 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increased mortality, bodyweight effects, signs of toxicity
- Remarks on result:
- other: Effect type: toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- < 100 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increased incidences of brain tumours
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- LOAEL
- Effect level:
- 100 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increased incidences of brain tumours
- Remarks on result:
- other: Effect type: carcinogenicity
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 100 ppm
- System:
- ear
- Organ:
- zymbal gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 500 ppm
- System:
- central nervous system
- Organ:
- brain
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Chronic administration of acrylonitrile to rats in the drinking water resulted in signs consistent with neurorotoxicity and increased incidences of brain tumours. Increased incidences of tumours of other tissues (Zymbal's gland, stomach and skin) were also noted. Brain tumours were morphologically similar to astrocytic tumours, however specific investigations do not indicate that tumours were of astrocytic origin.
- Executive summary:
In a study specifically investigating the induction and origin of brain tumours, acrylonitrile was administered to groups of 50 male and 50 female F-344 rats from 6 weeks of age at dose levels of 0, 100 and 500 ppm in the drinking-water. A fourth group of 300 rats (147 males, 153 females), was also administered 500 ppm acrylonitrile. Clinical signs indicative of neurotoxicity were observed in rats at 100 and 500 ppm within 12-18 months. Increased mortality was apparent in treated groups, with females dying slightly earlier than males; a high proportion of the rats in the 500 ppm treatment groups had died by 18 months, with few deaths in the control group. A total of 49 brain tumours were found in rats exposed to 500 ppm acrylonitrile, 11 of which were only detectable microscopically, 28 were 1-5 mm in diameter and 10 were greater than 5 mm. Despite the variation in size, all the tumours were similar in cellular and morphological features. The tumours were noted to be densely cellular, with occasional areas of focal necrosis, and were infiltrative at the margins. Further investigations noted that the tumours stained negative for the presence of glial fibrillary acidic protein (GFAP). Ultrastructurally, the tumour cells showed intermingling cytoplasmic processes but no glial filaments and no neurosecretory granules or specialised cell contacts. Samples of tumour tissue were successfully grown in culture, but transplantation of samples from these cultures (observed for up to 12 weeks) was unsuccessful. However, a direct intracerebral transplantation from a large tumour was successful.
- Endpoint:
- carcinogenicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Oral 2 year carcinogenicity study in rats; drinking water administration.
- GLP compliance:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- The animals were male Sprague-Dawley derived CD rats.
- Route of administration:
- oral: drinking water
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- Male rats were exposed to acrylonitrile in the drinking water for 2 years at dose levels of 0, 20, 100 and 500 ppm.
- Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- No information available
- Duration of treatment / exposure:
- 2 years
- Frequency of treatment:
- Daily / ad libitum in drinking water
- Post exposure period:
- Not examined
- Dose / conc.:
- 0 ppm
- Remarks:
- Controls: untreated drinking water
- Dose / conc.:
- 20 ppm
- Remarks:
- In drinking water
- Dose / conc.:
- 100 ppm
- Remarks:
- In drinking water
- Dose / conc.:
- 500 ppm
- Remarks:
- In drinking water
- No. of animals per sex per dose:
- 20 male rats per group
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- Rats were randomly allocated to treatment groups.
- Positive control:
- Not examined; not required
- Observations and examinations performed and frequency:
- Mortality was recorded. Rats were weighed weekly. At monthly intervals for periods of 1 week, food and water consumption were measured daily, with mean consumption calculated for each group of animals.
- Sacrifice and pathology:
- Necropsy was performed on all animals either at death or the 2 year terminal sacrifice. Histopathological examinations were also carried out.
- Other examinations:
- None reported.
- Statistics:
- No information available.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- : increased mortality at 500 ppm
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- : increased mortality at 500 ppm
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- : reduced weight gain at 100 and 500 ppm
- Food consumption and compound intake (if feeding study):
- no effects observed
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- : no significant effects, a trend to reduced water consumption at 500 ppm
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- : increased tumour incidences (Zymbal's gland) and forestomach papillomas
- Details on results:
- Mortality
Animals receiving the highest concentration of acrylonitrile (0.05% or 500 ppm) had earlier mortality, and the last rats from this group died just before the 2-year terminal killing. Survival in the other treated groups (20 and 100 ppm) was similar to controls.
Bodyweight
The average bodyweight of the controls and the 20 ppm group was virtually identical throughout the course of this study. The animals receiving 100 ppm or 500 ppm of acrylonitrile showed a slower body weight gain than the controls in the first year of the study and a greater decrease in body weight gain than the controls during the second year.
Food and water consumption
No statistically significant differences in food and water intake were observed, but a trend towards decreased water consumption in animals terated with 500 ppm of acrylonitrile was noted.
Pathology
There were no histopathological changes reported in this study which were indicative of chronic toxicity, as opposed to neoplastic effects, following exposure to acrylonitrile. The necroscopy results revealed no tumours in the heart, brain, liver, lungs, kidneys, adrenals, or testes of experimental animals or controls, with the exception of a few primary or metastatic tumours.
The only conclusive dose-related lesions were those found in the forestomach, pituitary and Zymbal gland. Papillomatous proliferation of the squamous epithelium of the forestomach was observed in 4 animals receiving 500 ppm acrylonitrile. Although one of these preneoplastic/neoplastic lesions (from a single animal) showed cytological atypia, invasion of the submucosa by proliferating epithelium was not seen. Zymbal gland tumours were associated with acrylonitrile exposure in a dose-related manner in animals exposed to 100 or 500 ppm acrylonitrile. All of these lesions were centred around the ear canal, and most were locally destructive and histologically poorly-differentiated squamous carcinomas with numerous abnormal mitotic figures. One metastatic lesion which morphologically resembled the primary tumour was observed in the lung. In some cases growth of the tumours restricted mouth opening and contributed to the death of the animal. Pituitary adenomas were found in 5 of 18 control animals (28 %). The incidence decreased among the animals receiving increasing concentrations of acrylonitrile. These tumours expanded locally but were not noted to be invasive or metastatic; however, they were a major cause of mortality among the control and low-dose groups. Although cytological atypia was often pronounced, in the absence of other features of malignancy, it was clear that these lesions represented benign neoplasms. In some adenomas, multinucleated giant cells were seen. Immunocytological staining revealed the presence of prolactin in the cytoplasm of several scattered adenoma cells. This lower incidence of pituitary tumours in acrylonitrile-treated rats is interesting. Increased mortality among the high-dose animals possibly contributes to the apparent protective effect noted in that group by prematurely reducing the number of animals at risk. Most of the animals with pituitary adenomas in the other groups died between 16 and 22 months. This, however, together with the dose-response relationship, suggests that the effects observed were not simply due to attrition among the acrylonitrile treated rats. The high-dose rats dying during that period were examined microscopically for tumours, and none were found. - Relevance of carcinogenic effects / potential:
- Increased tumour incidences were noted in Zymbal's gland; pre-neoplastic papillomatous lesions were also noted in the forestomach of treated rats, consistent with local irritation. The value of the study is limited by the small group size.
- Dose descriptor:
- NOAEL
- Effect level:
- 20 ppm
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Zymbal's gland tumours at 100 and 500 ppm
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- LOAEL
- Effect level:
- 100 ppm
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Zymbal's gland tumours at 100 and 500 ppm
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- NOAEL
- Effect level:
- 20 ppm
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Reduced weight gain & food consumption at 100 and 500 ppm
- Remarks on result:
- other: Effect type: toxicity
- Dose descriptor:
- LOAEL
- Effect level:
- 100 ppm
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Reduced weight gain & food consumption at 100 and 500 ppm
- Remarks on result:
- other: Effect type: toxicity
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 100 ppm
- System:
- ear
- Organ:
- zymbal gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Chronic administration of acrylonitrile resulted in increased incidences of Zymbal's gland tumours under the conditions of this study.
- Executive summary:
Male Sprague-Dawley rats (20/group) were exposed to acrylonitrile at levels of 0, 20, 100 or 500 ppm in the drinking water for up to 2 years. Bodyweights, food and water consumption were determined at regular intervals. Complete necropsy was performed on rats that died and on surviving rats killed at the end of 2 years. Accelerated mortality was noted at 500 ppm acrylonitrile; this group showed a trend toward decreased water consumption. Bodyweights of animals treated with 100 ppm and 500 ppm increased more slowly in the first year and decreased more in the second year, relative to controls. Increased Zymbal's gland carcinomas were observed in the treated groups; decreased incidences of benign pituitary adenomas were also apparent. Most of the Zymbal's gland tumours were noted to be histologically poorly differentiated squamous carcinomas and were locally destructive; one tumour produced a metastatic lung lesion. Forestomach papillomatous changes were noted in the 500 ppm group almost certainly due to the irritant action of acrylonitrile. Tumours in other systems e.g. the central nervous system, respiratory tract, or urogenital tract, were not related to chronic exposure to acrylonitrile,however the small group size may have limited the power of the study to detect carcinogenicity.
- Endpoint:
- carcinogenicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1975-1977
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Proprietary GLP study, also summarised in a more recently published paper.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Principles of method if other than guideline:
- 2 year oral toxicity and oncogenicity study
- GLP compliance:
- yes
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Male and female Sprague-Dawley rats, 6-8 weeks of age, were obtained from Spartan Research Animals. They were randomly placed in pairs in suspended wire bottom cages and identified by numbered metal ear tags. The housing room was maintained at a temperature of 70-75°F, relative humidity of 40-60%, with 12-15 air changes per hour and a daily 12 hour light: 12 hour dark cycle. The rats were acclimatised for approximately 3 weeks before treatment commenced. Rats were 8-12 weeks when acrylonitrile treatment started. The study was conducted in animal facilities accredited by the American Association for Accreditation of Laboratory Animal Care.
Ground laboratory chow (Ralston-Purina Company) and either untreated water (controls) or acrylonitrile-containing water were available ad libitum throughout the study. Water was supplied from bottles with sipper tubes. - Route of administration:
- oral: drinking water
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- Rats were exposed to acrylonitrile in their drinking water for 2 years. The doses used were 0, 35, 85, or 210 ppm for the first 21 days, and thereafter for the remainder of the study the doses were 0, 35, 100 or 300 ppm.
Water was supplied from 8 oz glass bottles, the bottles were refilled twice each week from stock solutions prepared the previous day. The large bottles were used to minimise loss of acrylonitrile into the headspace. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The stock solutions and the drinking water bottles were analysed for acrylonitrile content 18 times during the study. During the first 12 months of the study acrylonitrile drinking water samples were analysed using gas chromatography. The chromatograms were compared with those from standard aqueous solutions of acrylonitrile to confirm the test solution concentrations. During the latter half of the study the drinking water samples were analysed using gas chromatography-mass spectrometry (to enhance the quality of the data).
- Duration of treatment / exposure:
- 2 years
- Frequency of treatment:
- Daily / continuous - ad libitum in drinking water
- Post exposure period:
- Not examined
- Dose / conc.:
- 0 ppm
- Remarks:
- Controls: untreated drinking water
- Dose / conc.:
- 35 ppm
- Remarks:
- In drinking water: throughout the study. Equivalent to 3.4/4.4 mg/kg bw/d
- Dose / conc.:
- 100 ppm
- Remarks:
- In drinking water: increased from 85 ppm on Day 22. Equivalent to 8.5/10.8 mg/kg bw/d
- Dose / conc.:
- 300 ppm
- Remarks:
- In drinking water: increased from 210 ppm on Day 22. Equivalent to 21.2/25.0 mg/kg bw/d
- No. of animals per sex per dose:
- There were 48 rats/sex/group, with 80 controls per sex. One 35 ppm male rat was found to have been mis-sexed (female) and was removed from the study on day 56. Gross and histopathologic examination of tissues from this rat were unaffected by treatment, therefore, data is presented for only 47 male rats remaining in this group.
- Control animals:
- yes
- Details on study design:
- A preliminary 90 day subchronic study was conducted to determine dose rates for the 2 year study. Doses of 0, 35, 85, 210 or 500 ppm were used. Water consumption was markedly decreased in a dose-related fashion with females more affected than males. In addition to decreased water consumption, the 210 ppm and 500 ppm groups also exhibited decreased food consumption and body weight. A number of treatment related effects were observed in the 85 ppm group, however this dose appeared to be an acceptable high dose level for the 2 year study.
Dose levels were changed from day 21 of the 2 year study at the request of the Manufacturing Chemists Association, in response to the FDA, based upon their interpretation of an insufficient adverse effect at 210 ppm prior to day 21.
Satellite groups were included for interim sacrifice at 1 year to assess the early onset of toxicity and possible oncogenicity. - Positive control:
- Not examined
- Observations and examinations performed and frequency:
- Food consumption was determined for rats in 15 cages/sex/dose (2 rats/cage), insofar as their survival permitted. Feed weights were measured weekly for the first 3 months of the study, and for 1 week of each of the following months: 4, 5, 6, 7, 9, 11, 12, 15, 18, 21 and 24.
The body weights of all surviving rats were determined monthly throughout the study. The weight of at least one rat in each cage involved in the weekly food consumption determination was measured for the first 4 months to assess the negative effects of exposure on early growth.
Water consumption was determined for the same rats that were used for food consumption measurements. Water bottles were weighed when filled and again before refilled. Bottles were refilled twice weekly, and consumption was measured for at least one of those periods each week for the first 3 months. Thereafter, water consumption was determined a total of 11 times, during the same week of each month when food consumption was measured.
Rats were observed daily for general appearance, demeanor, moribundity and death. In addition, when rats were weighed their oral cavity was examined for malocclusion. Once each month all rats were given a detailed clinical examination by the author to evaluate their general well being and to detect any palpable masses.
Haematology was conducted on 10 rats/sex of the control and highest dose rats on days 45, 87, 180 and 355. In addition, these determinations were performed on 10 rats/sex (or all surviving rats when less than 10/group) for controls and all treatment groups on day 544 (males) and day 545 (females), and at study termination on day 724 for males and females. Blood samples were collected from the tail vein and used for PCV, RBC, HGB, WBC, differential WBC and morphology.
Urinalysis was conducted on the same rats and at the same time as used for haematology. An additional urinalysis was performed on 10 rats/sex/group on test day 181. Determinations included pH, specific gravity, sugar, protein, ketones, bilirubin, occult blood, urobilinogen and colour.
BUN concentration, AP activity, and SGPT activity were measured in 10 rats/sex of the control and highest dose on days 46 and 356, and on 10 rats/sex from all groups on days 88, 180, 550, and from all survivors on day 746. The blood samples for these determinations were obtained by orbital sinus punture for all days except day 746, when samples were obtained from the severed cervical vessels at necropsy.
Both eyes of all rats were examined using a moistened glass slide technique applied to the cornea under fluorescent illumination at necropsy. Rats which were removed from the study moribund or found dead had their eyes fixed in neutral phosphate buffered 10% formalin, while those from rats at scheduled necropsy were preserved in Zenker's fixative. - Sacrifice and pathology:
- A satellite group of 10 rats/sex/group were used for 1 year interim necropsy. Moribund and dead rats were removed from the study and necropsied to obtain a complete set of tissues for final histopathologic examination.
A complete gross pathologic examination was performed on each rat from the 12 and 24 month study groups. A representative sample from the following major organs and tissues was saved in neutral phosphate buffered 10% formalin fixative: oesophagus, salivary glands, stomach, small intestine, large intestine, pancreas, liver, kidneys, urinary bladder, prostate, accessory sex glands, epididymides, testes, ovaries, oviducts, uterus, caervix, vagina, mammary tissue, brain, spinal cord, sciatic nerve, pituitary gland, tongue, trachea, lungs, head (including nasal turbinates and Zymbal gland), sternum and sternal bone marrow, spleen, mediastinal lymphoid tissue, mesenteric lymph node and mesenteric tissues, heart, aorta, skeletal muscle, adrenal glands, thyroid gland, parathyroid glands, adipose tissue, skin, and any gross lesions or masses.
All rats surviving until scheduled termination on day 746 were fasted overnight, weighed and submitted for necropsy. The rats were decapitated and fasted blood samples obtained for clinical chemistry determinations. Wet organ weights for brain, heart, liver, kidneys, and testes were obtained immediately. The lungs of all rats from the scheduled necropsy, and for most of those removed from study dead or moribund, were inflated to their approximate normal inspiratory volume with neutral phosphate buffered 10% formalin via a handheld syringe. Brain tumour tissue was saved from several rats with a macroscopic tumour at the time of necropsy.
Tissues for histopathology were embedded in paraffin and stained with haematoxylin and eosin. Microscopic evaluation of a complete set of tissues from control and 300 ppm rats of the 12 and 24 month necropsies were performed. Based upon statistically significantly identified microscopic findings from the high-dose rats in the 24 month group, 22 tissues were selected and examined from nearly all 35 and 100 ppm rats. The tissues were: liver, spleen, heart, brain stem, cerebellum, cerebral cortex, pituitary gland, sciatic nerve, spinal cord, adrenal glands, kidneys, stomach, lungs, thyroid gland, parathyroid glands, oesophagues, trachea, mediastinal fat, mediastinal lymph node, mediastinal vessels/aorta, thymus and eyes. All tumours or tumour like lesions were specifically selected for examination from all rats. - Other examinations:
- A more thorough histopathologic evaluation of the CNS was implemented for all rats from the 2 year study; a minimum of nine CNS sections were routinely obtained.
- Statistics:
- ANOVA, Dunnett's test, Fisher's Exact Probability Test. Significance was accepted at the 5% level.
- 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):
- effects observed, treatment-related
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- 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:
- no effects observed
- 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:
- The first death in this study occurred during the 4th month and by the end of the first year losses amounted to 33 (14 males and 19 females). The mortality of females in all treatment groups was considerably higher than their controls. The increased early mortality rate was directly correlated to increasing concentrations of acrylonitrile in the water. Early mortality was observed only in the 300 ppm group of males when compared to their respective controls. The total number of animals dead or removed from the study prior to the time of necropsy on day 746 was 206 males and 199 females (405 total = 90.4 %). There were no 300 ppm male or female rats alive during the last 2 months of the study, and at study termination only one female in the 100 ppm group and 4 in the 35 ppm group survived until necropsy. Mortality data are shown in Table 1.
The results of haematology, urinalysis and clinical chemistry determinations indicated that acrylonitrile did not have a primary adverse effect on bone marrow, kidney or liver function in either male or female rats. Minor effects seen were thought to be secondary effects of other treatment-related effects, e.g. nutritional anaemia.
There was a dose related decrease in food and water consumption, and reduced body weight gain in all treatment groups, with females being more severely affected than males. Clinical observations showed that treated animals were unthrifty (reduced weight gain), exhibited early mortality compared to controls and had an earlier onset of tumours, many of which were detectable on external examination and palpation. These observations were intially noted in the highest dose level rats, however the same observations occurred at the lower doses as the study progressed.
Gross and microscopic examination of tissues revealed a variety of pathological findings in treated rats which occurred with statistically significant increased or decreased frequency compared to the respective control animals. Certain non-neoplastic age-related changes, for example chronic nephropathy, were less frequent in the treated animals compared to controls. This can be interpreted to be due to the early mortality and decreased food and water consumption in treated animals. An increased incidence of endocardial fibrosis was noted only in males at the 300 ppm level.
Both male and female acrylonitrile-treated rats exhibited a statistically significant increased incidence of various tumour types. A statistically significant increase was seen in the incidence of tumours of the CNS, ear canal (Zymbal) gland. These effects were detected first at the highest dose level (300 ppm) and later in the lower dose groups (100-35 ppm). Tumours of the subcutaneous tissue, mammary region, and pinna of the ear were not significantly different in treated and control rats. The occurrence of the various tumour types are summarised in Table 2.
Histopathological observations revealed that a significantly increased incidence of CNS tumours, characterised as astrocytomas, was observed in rats in all dose groups. In addition, a significantly increased incidence of a focal or multifocal glial cell proliferation suggestive of an early tumour of the same cell type was observed in the 35 and 300 ppm groups. In each category of the two identified proliferative changes in the CNS, it was observed most frequently in the cerebral cortex, followed by the brain stem in the region of the cerebellum, and less frequently in the cerebellum and the thoracic spinal cord. In general, the changes of a proliferative type in the cerebral cortex sections were most frequently observed in the section obtained from the middle of the cerebral hemisphere.
Histopathological examination of the tongue showed a statistically significant increase in incidence of squamous cell tumours and for the nonglandular portion of the stomach (forestomach) the increase in incidence was in squamous epithelial tumours. On gross examination there were many rats with multiple papillomas present in this region of the stomach. Upon microscopic examination of these stomach tumours some were found to be papillomas only, others were carcinomas only, and yet other rats had both a papilloma and a carcinoma present. The earliest tumours were papillomas, while later in the study carcinomas were also frequently observed. Stages of the lesion progressed from hyperplasia and hyperkeratosis, to papilloma, and ultimately carcinoma (papillary and ulcerating) formation, with some overlap in the sequence of lesion development. These observations were dose related in severity at the 100 and 300 ppm groups. There were greater numbers of rats with a carcinoma in the stomach at the highest dose level, and they also showed a decreased latency period compared to the lower dose groups. The carcinomas present in the nonglandular stomach were predominantly papillary in type, with only a small proportion of the rats with a carcinoma having the ulcerating type. Only a single ulcerating carcinoma of the nonglandular stomach invaded through the wall of the stomach and extended locally into the mesentery. - Relevance of carcinogenic effects / potential:
- Exposed rats experienced early mortality and increased incidence of tumours of the CNS and Zymbal gland
- Dose descriptor:
- NOAEL
- Effect level:
- < 35 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increased tumour incidences in all treated groups including the lowest dose level of 35 ppm (equivalent to 3.4 and 4.4 mg/kg bw/d in males and females respectively)
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- NOAEL
- Effect level:
- < 35 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Reduced bodyweight and food consumption in all treated groups including the lowest dose level of 35 ppm (equivalent to 3.4 and 4.4 mg/kg bw/d in males and females respectively)
- Remarks on result:
- other: Effect type: toxicity
- Dose descriptor:
- LOAEL
- Effect level:
- 35 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increased tumour incidences in all treated groups including the lowest dose level of 35 ppm (equivalent to 3.4 and 4.4 mg/kg bw/d in males and females respectively)
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- LOAEL
- Effect level:
- 35 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Reduced bodyweight and food consumption in all treated groups including the lowest dose level of 35 ppm (equivalent to 3.4 and 4.4 mg/kg bw/d in males and females respectively)
- Remarks on result:
- other: Effect type: toxicity
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 35 ppm
- System:
- central nervous system
- Organ:
- brain
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 35 ppm
- System:
- gastrointestinal tract
- Organ:
- stomach
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 300 ppm
- System:
- gastrointestinal tract
- Organ:
- tongue
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 35 ppm
- System:
- gastrointestinal tract
- Organ:
- duodenum
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- yes
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 35 ppm
- System:
- endocrine system
- Organ:
- mammary gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 35 ppm
- System:
- ear
- Organ:
- zymbal gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Exposure to acrylonitrile in the drinking water for 2 years resulted in higher mortality and an increased incidence of certain types of tumour. A NOAEL could not be identified.
- Executive summary:
Male and female Sprague-Dawley rats were exposed to acrylonitrile in their drinking water for 2 years. The doses used were 0, 35, 85, or 210 ppm for the first 21 days, and thereafter for the remainder of the study the doses were 0, 35, 100 or 300 ppm (at the request of the FDA). The equivalent mean dosages of acrylonitrile converted to mg/kg/ bw/d were estimated to be 3.4, 8.5 and 21.2 in male rats and 4.4, 10.8 and 25.0 in female rats.
Decreased water consumption, food consumption, and concomitant body weight suppression occurred soon after study initiation and persisted throughout the study in all treatment groups. An early onset of Zymbal gland tumours in high dose rats, and in the mammary gland of all treated groups of females, was detected in-life. There were no significant effects of treatment on haematology, clinical chemistry of urinalysis parameters. Organ weights at study termination were also unaffected by treatment. Mortality was high in all female treated groups, with no surviving male or female 300 ppm rats during the last 2 months of the study. The primary non-neoplastic lesions detected at histopathologic examination were found in the forestomach and central nervous system of rats of both sexes and involved all treatment groups. A statistically significant increased incidence of tumours in one or more dose levels of either sex occurred in the central nervous system, Zymbal gland, forestomach, tongue, small intestine, and mammary gland. In general, rats ingesting the highest dose of acrylonitrile (300 ppm) showed the earliest onset and greatest number of tumours which infrequently metastasised. Female rats exhibited a slightly greater toxic and tumorigenic response than males, which was concluded to be the result of the higher dose of acrylonitrile (mg/kg/day) consumed by the females than males. A NOAEL could not be identified for this study.
- Endpoint:
- carcinogenicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- 1 year oral carcinogenicity study
- GLP compliance:
- no
- Remarks:
- : older, non-standard published literature study
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Male and female Sprague-Dawley rats
- Route of administration:
- oral: gavage
- Vehicle:
- olive oil
- Details on exposure:
- Acrylonitrile was administered orally by gavage in olive oil, at a single daily dose of 5 mg/kg bw 3 times weekly for 52 weeks.
- Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- No information available
- Duration of treatment / exposure:
- 52 weeks
- Frequency of treatment:
- 3 times weekly for 52 weeks
- Post exposure period:
- After the treatment period the animals were monitored until spontaneous death.
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- Vehicle controls
- Dose / conc.:
- 5 mg/kg bw/day
- Remarks:
- Gavage: three times weekly for 52 weeks
- No. of animals per sex per dose:
- 40 male and 40 female treated rats, plus 75 male and 75 female controls.
- Control animals:
- yes
- Details on study design:
- Acrylonitrile was administered orally by gavage in olive, at a single daily dose of 5 mg/kg bw 3 times weekly for 52 weeks. The rats were then observed until spontaneous death occurred.
- Positive control:
- Not examined.
- Observations and examinations performed and frequency:
- The rats were examined weekly and weighed every 2 weeks during the exposure period, and monthly after treatment was over, until spontaneous death.
- Sacrifice and pathology:
- Rats were subject to a complete autopsy. Histological examination of the Zymbal glands, interscapular brown fat, salivary glands, tongue, lungs, liver, kidney, spleen, stomach, different segments of the intestine, bladder, brain, and any other organs with pathological lesions was performed.
- Other examinations:
- None reported.
- Statistics:
- No information available.
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- 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:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- : increased incidences of mammary gland and forestomach tumours
- Details on results:
- Mortality
There were no effects of treatmnet on the survival of the test animals
Bodyweights
Bodyweights were unaffected by treatment
Pathology
No treatment-related histological changes were observed in the liver, kidneys and lung. Acrylonitrile administration did not affect the percentage of animals bearing benign and malignant tumours, the number of animals bearing malignant tumours only, the number of total malignant tumours per 100 animals or the incidence of Zymbal gland carcinomas, extrahepatic angiosarcomas, hepatomas and encephalic gliomas.
The only increase in incidence of tumours was in the mammary gland and forestomach of female rats. - Relevance of carcinogenic effects / potential:
- Increased incidences of mammary gland and forestomach tumours were seen in female rats.
- Dose descriptor:
- LOAEL
- Effect level:
- 5 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: Effect type: carcinogenicity (migrated information)
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 5 mg/kg bw/day (actual dose received)
- System:
- gastrointestinal tract
- Organ:
- stomach
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 5 mg/kg bw/day (actual dose received)
- System:
- endocrine system
- Organ:
- mammary gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Increased incidences of mammary gland and forestomach tumours were seen in female rats.
- Executive summary:
Acrylonitrile was administered orally to male and female Sprague-Dawley rats by gavage in olive, at a single daily dose of 5 mg/kg bw 3 times weekly for 52 weeks. The rats were then observed until spontaneous death occurred. There were no effects on survival or body weight of the test animals. No treatment-related histological changes were observed in liver, kidneys and lung. Acrylonitrile administration did not affect the percentage of animals bearing benign and malignant tumours, the number of animals bearing malignant tumours only, the number of total malignant tumours per 100 animals or the incidence of Zymbal gland carcinomas, extrahepatic angiosarcomas, hepatomas and encephalic gliomas. The only increase in incidence of tumours was in the mammary gland and forestomach of female rats.
- Endpoint:
- carcinogenicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1977-1979
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Proprietary study, also published more recently.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Principles of method if other than guideline:
- 2 year combined toxicity and carcinogenicity study
- GLP compliance:
- not specified
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- The animals were male and female Spartan Sprague-Dawley rats, obtained from Spartan Research Animals, Inc., Michigan. They were 4-5 weeks old on receipt, and following a 28 day acclimatisation period were placed onto the study at 8-9 weeks of age.
The rats were housed singly in elevated stainless steel cages. Purina Lab Chow was provided ad libitum; fresh food was presented weekly. Distilled water or relevant acrylonitrile solutions were provided ad libitum via water bottles. Light was provided on a 12 hour light/dark cycle, and temperature of the animal room was monitored twice daily.
Animals underwent a pretest physical examination and ophthalmoscopic examination to ensure their suitability as test animals prior to study initiation. Individuals were identified with metal ear tags. - Route of administration:
- oral: drinking water
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- Acrylonitrile was administered in the drinking water at dose levels of 0, 1 and 100 ppm. Appropriate amounts of test substance were mixed with distilled water weekly, animals received fresh dosing mixtures twice weekly.
- Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- No information available.
- Duration of treatment / exposure:
- The intended duration was 2 years, however the study was terminated at 19 months in females and 22 months in males because of low survival rates.
- Frequency of treatment:
- Daily - ad libitum drinking water
- Post exposure period:
- Not examined
- Dose / conc.:
- 0 ppm
- Remarks:
- Control: untreated drinking water
- Dose / conc.:
- 1 ppm
- Remarks:
- In drinking water
- Dose / conc.:
- 100 ppm
- Remarks:
- In drinking water
- No. of animals per sex per dose:
- 100 rats/sex/dose
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- Acrylonitrile was administered in the drinking water to groups of rats (100/sex/group) at doses of 0, 1 or 100 ppm. Rats were randomly assigned to treatment groups.
Interim necropsies were performed on 10 rats/sex/group at 6, 12 and 18 months. - Positive control:
- Not examined.
- Observations and examinations performed and frequency:
- Animals were observed for mortality and gross signs of toxicological/pharmacological effects twice daily. A detailed examination for signs of local or systemic toxicity, pharmacological effects and palpation for tissue masses was conducted weekly. Ophthalmoscopic examinations were carried out at pretest, 6, 12, 18, 19 (females) and 22 (males) months.
Body weights were recorded weekly pretest through 14 weeks, then biweekly 16 through 26 weeks, then monthly thereafter. Food consumption was determined for 25 rats/sex/group weekly from 1 week prior to treatment through 14 weeks, biweekly 16 through 26 weeks, and monthly thereafter. Water consumption was measured in 25 rats/sex/group over a three day period; weekly from one week prior to treatment through 14 weeks, biweekly 16 through 26 weeks and monthly thereafter. Test substance intake was also determined in 25 rats/sex/group at the same intervals, and was calculated from water consumption data and expressed as mg/kg/day.
Blood was obtained via the dorsal aorta under light anaesthesia for haematology and clinical chemistry. Animals (10/sex/group) were selected randomly and were fasted overnight prior to blood collection. Blood was collected at 6, 12, 18, 19 (females) and 22 (males) months. The following haematology parameters were evaluated: haemoglobin, haematocrit, erythrocyte count, reticulocytes, prothrombin time, total and differential leukocytes, erythrocyte morphology. The following clinical chemistry parameters were determined: serum glutamic pyruvic transaminase, alkaline phosphatase, blood urea nitrogen, fasting glucose.
Urine was also collected for evaluation of the following parameters: gross appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, occult blood. - Sacrifice and pathology:
- All animals (dying or killed in a moribund condition) were subject to a complete gross postmortem examination. Necropsies were carried out on 10 rats/sex/group at 6, 12 and 18 months. Early terminal sacrifices (all survivors) were carried out at 19 months (females) and 22 months (males).
The following organs were weighed (10 rats/sex/group): brain, heart, pituitary, kidneys, adrenals, liver and gonads. Tissues preserved from all animals were: adrenals, bone marrow, brain, ear canal, oesophagus, eyes, gonads, heart (with coronary vessels), intestine (colon, duodenum, ileum), kidneys, liver, lungs, mesenteric lymph node, mammary gland, pancreas, pituitary, prostate, salivary gland, skeletal muscle, skin, spinal cord, spleen, stomach, thyroid, trachea, urinary bladder, uterus, gross lesions, tissue masses. Tissues were preserved in 10% neutral buffered formalin except the eyes and testes which were preserved in Bouin's solution. Preserved tissues were stained with haematoxylin and eosin. All tissues listed were examined for 10 rats/sex for the control and high dose groups and each necropsy time point. The following tissues were examined for the low dose group: brain, ear canal, spinal cord and stomach. - Other examinations:
- None reported.
- Statistics:
- Comparison of means.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- : reduced at 100 ppm
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- : reduced at 100 ppm
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- : reduced at 100 ppm
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- : reduced at 100 ppm
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- : reduced at 100 ppm
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- : small, sometimes significant, reductions in haemoglobin, haematocrit, and erythrocyte counts at 100 ppm
- 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
- Description (incidence and severity):
- : absolute kidney wieght increased in 100 ppm females
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- : increase incidence of subcutaneous masses associated with the ears in 100 ppm males and females
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- : forestomach hyperplasia at 100 ppm
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- : increased tumour incidences at 100 ppm
- Details on results:
- MORTALITY
Mortality was greater in both the high-dose males and females, compared to controls, during the first 19 months of the study. Increased numbers of deaths among the high dose groups animals prompted the early end of the study after 20 months. The surviving males and females were sacrificed after 22 and 19 months, respectively. Mortality in the low-dose males and females was comparable to the controls throughout the study.
CLINICAL SIGNS
None observed.
PALPABLE MASSES
An increase in the incidence of animals with palpable masses on the head (the area around the ears and eyes and the cervical region) was noted in the high-dose animals. An increased incidence of masses in the mammart region was noted in the high-dose females.
BODYWEIGHTS, FOOD AND WATER CONSUMPTION
Body weights for the high-dose rats were consistently lower than controls: the differences from control for males was generally less than 10%, while differences for the females were generally less than 8%. Food consumption for the high-dose rats was generally lower than controls on a weekly basis, but on a g/kg/day basis, food consumption was comparable between groups as a result of the lower body weights for the high dose animals. Marked reductions in water consumption were noted in the high dose group.
HAEMATOLOGY, CLINICAL CHEMISTRY AND URINALYSIS
Small, sometimes significant, reductions in haemoglobin, haematocrit, and erythrocyte counts were observed in high dose animals. Slight elevations in alkaline phosphatase activity were noted for the high-dose females from 12 months to termination, however the differences were not significant. There were no differences from control values in any of the urinalysis parameters evaluated.
ORGAN WEIGHTS
Absolute kidney weights were increased in high-dose females at all sacrifice time points. Mean testicular weights of the high-dose males were elevated at the 12, 18 and 22 month sacrifices. Mean relative liver weights were elevated in high-dose males at 6 and 18 months, and high-dose females at termination, however the differences were not always statistically significant.
PATHOLOGY
There was an increase in the incidence of subcutaneous masses associated with the ears in the high-dose males and females following the first 6 months on test. An increase in the degree of severity of forestomach hyperplasia was observed in all high dose animals. High dose males and females had an increased incidence of astrocytomas of the brain and spinal cord, carcinomas and adenomas of the Zymbal gland, squamous cell carcinomas and papillomas of the forestomach. Other microscopic findings in these studies were considered unremarkable and within normal limits. There were no observable nonneoplastic effects attributable to treatment in the low concentration group (1 ppm in water). - Relevance of carcinogenic effects / potential:
- Clear evidence of carcinogenicity was seen in this study
- Dose descriptor:
- NOAEL
- Effect level:
- 1 ppm
- Sex:
- male/female
- Remarks on result:
- other: Effect type: carcinogenicity
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 100 ppm
- System:
- central nervous system
- Organ:
- brain
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 100 ppm
- System:
- ear
- Organ:
- zymbal gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 100 ppm
- System:
- gastrointestinal tract
- Organ:
- stomach
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 100 ppm
- System:
- endocrine system
- Organ:
- mammary gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Clear evidence of carcinogenicity was seen in this study. High dose males and females had an increased incidence of astrocytomas of the brain and spinal cord, carcinomas and adenomas of the Zymbal gland, squamous cell carcinomas and papillomas of the forestomach. The NOAEL is considered to be 1 ppm.
- Executive summary:
Spartan Sprague-Dawley rats (100/sex/group) were administered acrylonitrile continually at concentrations of 0 (controls), 1 or 100 ppm in the drinking water. The equivalent mean doses of acrylonitrile were 0, 0.09 and 8.0 mg/kg bw/d in males; 0, 0.15 and 10.7 mg/kg bw/d in females. Groups of ten rats/sex were sacrificed at 6, 12 and 18 months and at study termination. Ophthalmoscopic, haematological, clinical biochemistry, urinalysis and full histopathological exams were performed on control and high dose groups and in lower dose groups, as required, to define dose-responses of observed effects. All animals were necropsied and underwent microscopic examination of target tissues, including brain, ear canal, stomach, spinal cord and any grossly observable tissue masses. High dose male and female rats exhibited statistically decreased bodyweights. Food consumption and water intake were also reduced. Due to increased deaths in groups of high dose rats, surviving males and females were terminated after 22 and 19 months, respectively. Small, sometimes statistically significant, reductions in haemoglobin, haematocrit and erythrocyte count were observed in male and female rats at 100 ppm drinking water. Absolute and relative kidney weights were increased in high-dose females compared to controls (at all sacrifice intervals, and mean testicular weights for high-dose males were elevated at the 12 and 18 month and terminal sacrifices, however the differences were not statistically significant. There were no changes in clinical biochemistry. Absolute kidney weights were increased in high dose female rats only. Male and female rats from high dose groups had a higher incidence of palpable masses of the head and the non-glandular stomach and, in females only, the mammary region. In both sexes, treatment-related tumours of the central nervous system (brain, spinal cord), ear canal, and gastrointestinal tract were observed in rats administered 100 ppm.
- Endpoint:
- carcinogenicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Proprietary study, also published more recently.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Principles of method if other than guideline:
- 2 year combined toxicity and carcinogenicity study
- GLP compliance:
- not specified
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- The animals were male and female Spartan Sprague-Dawley rats.
- Route of administration:
- oral: gavage
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- Acrylonitrile was administered by gavage at dose levels of 0, 0.1 and 10 mg/kg bw/d
- Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- No information available.
- Duration of treatment / exposure:
- The intended duration was 2 years, however the study was terminated at 19 months in females and 22 months in males because of low survival rates.
- Frequency of treatment:
- Daily
- Post exposure period:
- Not examined
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- Untreated controls
- Dose / conc.:
- 0.1 mg/kg bw/day
- Remarks:
- Gavage
- Dose / conc.:
- 10 mg/kg bw/day
- Remarks:
- Gavage
- No. of animals per sex per dose:
- 100 rats/sex/dose
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- Acrylonitrile was administered by gavage to groups of rats (100/sex/group) at dose levels of 0, 0.1 or 10 mg/kg bw/d
- Positive control:
- Not examined.
- Observations and examinations performed and frequency:
- Body weight and food and water consumption. Opthalmoscopic, haematologic, clinical biochemistry, urinalysis and full histopathological examinations were performed on control and high dose rats. Similar tests were done on the lower dose group if necessary to establish a dose-response relationship for observed effects.
- Sacrifice and pathology:
- All animals were necropsied and underwent microscopic examination of target tissues (i.e., brain, ear canal, stomach, spinal cord, and any observable tissue masses). Interim necropsies were performed at 6, 12 and 18 months (10/sex/group). Necropsies were performed at study termination (males; 22 months and females; 19 months).
- Other examinations:
- None reported.
- Statistics:
- No information available
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- : reduced at 10 mg/kg bw/d
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- : reduced at 10 mg/kg bw/d
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- : reduced at 10 mg/kg bw/d
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- : small, sometimes significant, reductions in haemoglobin, haematocrit, and erythrocyte counts at 10 mg/kg bw/d
- 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
- Description (incidence and severity):
- : increased liver and adrenal weights at 10 mg/kg bw/d
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- : forestomach hyperplasia at 10 mg/kg bw/d
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- : increased tumour incidences at 10 mg/kg bw/d
- Details on results:
- Mortality
Increased numbers of deaths among the high dose groups animals prompted the early end of the study after 20 months. The surviving males and females were sacrificed after 22 and 19 months, respectively.
Clinical signs
None observed
Bodyweights, food and water consumption
High dose animals had significant reductions in body weight. Food and water consumption were unaffected
Haematology
Small, sometimes significant, reductions in haemoglobin, haematocrit, and erythrocyte counts were observed in high dose animals.
Organ weights
Increased in absolute or relative organ weight ratios for liver and adrenal at 10 mg/kg bw/d.
Pathology
An increase in the degree of severity of forestomach hyperplasia was observed in all high dose animals. High dose males and females had an increased incidence of astrocytomas of the brain and spinal cord, carcinomas and adenomas of the Zymbal gland, squamous cell carcinomas and papillomas of the forestomach and mammary gland tumours (females). Other microscopic findings in these studies were considered unremarkable and within normal bounds. There were no observable nonneoplastic effects attributable to treatment in the low dose group. - Relevance of carcinogenic effects / potential:
- Clear evidence of carcinogenicity was seen in this study
- Dose descriptor:
- NOAEL
- Effect level:
- 0.1 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No effects at this dose level
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- LOAEL
- Effect level:
- 10 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increased tumour incidence
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- NOAEL
- Effect level:
- 0.1 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No effects at this dose level
- Remarks on result:
- other: Effect type: toxicity
- Dose descriptor:
- LOAEL
- Effect level:
- 10 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Mortality, bodyweights, food consumption
- Remarks on result:
- other: Effect type: toxicity
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 10 mg/kg bw/day (actual dose received)
- System:
- central nervous system
- Organ:
- brain
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 10 mg/kg bw/day (actual dose received)
- System:
- endocrine system
- Organ:
- mammary gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 10 mg/kg bw/day (actual dose received)
- System:
- gastrointestinal tract
- Organ:
- stomach
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 10 mg/kg bw/day (actual dose received)
- System:
- ear
- Organ:
- zymbal gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Clear evidence of carcinogenicity was seen in this study. High dose males and females had an increased incidence of astrocytomas of the brain and spinal cord, mammary gland tumours, carcinomas and adenomas of the Zymbal gland, squamous cell carcinomas and papillomas of the forestomach.
- Executive summary:
Spartan Sprague-Dawley rats (100/sex/group) were administered lifetime oral doses of acrylonitrile by gavage at 0, 0.1 or 10 mg/kg bw/d, 7 days per week. The doses selected were designed to approximate the same daily intake of acrylonitrile in the drinking water study by the same authors. Groups of ten rats/sex were sacrificed at 6, 12 and 18 months and at study term. Ophthalmoscopic, haematological, clinical biochemistry, urinalysis and full histopathological exams were performed on control and high dose groups. Similar tests were done in lower dose groups, as required, to define dose-responses of observed effects. All animals were necropsied and underwent microscopic examination of target tissues, including brain, ear canal, stomach, spinal cord and any observable tissue masses. High dose male and female rats exhibited statistically decreased body weights. Food consumption and water intake were unaffected. Due to increased deaths in groups of high dose rats, all test groups were terminated after 20 months of treatment. Small, sometimes statistically significant, reductions in haemoglobin, haematocrit and erythrocyte count were observed in male and female rats in the high dose group. There were increases in absolute or relative organ weight ratios for liver and adrenal in the high dose groups, but could not be correlated with acrylonitrile toxicity in the absence of adverse clinical biochemistry or microscopic findings. Absolute kidney weights were increased in high dose male and female rats. Male and female rats from the high dose group had a higher incidence of palpable masses of the head and the non-glandular stomach and, in females only, the mammary region. In both sexes, treatment-related tumours of the central nervous system (brain, spinal cord), ear canal and gastrointestinal tract, and in females only, the mammary gland were observed in rats administered 10 mg/kg bw/d.
Referenceopen allclose all
Summary of mortality data
Level of acrylonitrile (ppm) |
Mortality at the end of the 2-year dosing perioda |
|
Males |
Females |
|
0 |
48/140 |
29/140 |
1 |
18/70 |
20/70 |
3 |
24/70 |
24/70 |
10 |
33/70 |
20/70 |
30 |
26/70 |
29/70 |
100 |
56/71 |
54/69 |
a Males terminated during month 26; females terminated during month 24. 30 animals were taken out for interim kills, therefore the actual incidence can be related to at least a total population of 70 for treatment groups and 140 for controls.
Percentage incidence of palpable masses
|
0 ppm |
1 ppm |
3 ppm |
10 ppm |
30 ppm |
100 ppm |
Head Region |
|
|
|
|
|
|
Males |
3% |
2% |
1% |
3% |
6% |
16% |
Females |
2% |
3% |
0 |
3% |
6% |
10% |
Mammary Region |
|
|
|
|
|
|
Males |
6% |
9% |
10% |
13% |
7% |
9% |
Females |
6% |
8% |
6% |
7% |
8% |
13% |
Incidence of observed tumours
Sex |
Concentration (ppm) |
Brain Astrocytoma |
Zymbal's Gland papilloma, adenoma, carcinoma |
Forestomach papilloma, carcinoma |
Male |
0 |
2/200 |
2/189 |
0/199 |
|
1 |
2/100 |
1/97 |
1/100 |
|
3 |
1/100 |
0/93 |
4/97* |
|
10 |
2/100 |
2/88 |
4/100* |
|
30 |
10/99 |
7/94* |
4/100* |
|
100 |
21/99 |
16/93* |
1/101 |
Female |
0 |
1/199 |
0/193 |
1/199 |
|
1 |
1/100 |
0/94 |
1/100 |
|
3 |
2/201 |
2/92 |
2/100 |
|
10 |
4/95 |
4/90* |
2/97 |
|
30 |
6/100* |
5/94* |
4/100* |
|
100 |
23/98* |
10/86* |
2/97 |
* significantly different from control incidence
Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Acrylonitrile
|
Male B6C3F1 Mice |
Female B6C3F1 Mice |
Doses in deionized water by gavage |
Vehicle control, 2.5, 10 or 20mg/kg |
Vehicle control, 2.5, 10 or 20mg/kg |
Survival rates |
38/50, 42/50, 39/50,14/50 |
39/50, 32/50, 39/50, 23/50 |
Body weights |
20mg/kg group generally less than vehicle control group |
20mg/kg group generally less than vehicle control group |
Nonneoplastic effects |
Forestomach: epithelial hyperplasia, focal (2/50, 4/50, 8/50, 9/50); hyperkeratosis, diffuse or focal (2/50, 3/50, 7/50, 12/50) Harderian Gland: hyperplasia (1/50, 4/50, 7/50, 4/50) |
Forestomach: epithelial hyperplasia, focal or multifocal (2/50, 2/50, 5/50, 7/50) Ovary: atrophy (6/50, 8/50, 45/50, 40/50); cyst (12/50, 20/50, 27/50, 19/50) |
Neoplastic effects |
Forestomach: squamous cell papilloma (3/50, 4/50, 19/50, 25/50); squamous cell carcinoma (0/50, 0/50, 8/50, 9/50); squamous cell papilloma or carcinoma (3/50, 4/50,26/50, 32/50) Harderian Gland: adenoma (5/50, 16/50, 24/50, 27/50); adenoma or carcinoma (6/50,16/50, 27/50, 30/50) |
Forestomach: squamous cell papilloma (3/50, 6/50, 24/50, 19/50); squamous cell carcinoma (0/50, 1/50, 1/50, 11/50);squamous cell papilloma or carcinoma (3/50,7/50, 25/50, 29/50) Harderian Gland: adenoma (10/50, 10/50,25/50, 23/50); adenoma or carcinoma (11/50,10/50, 26/50, 25/50) |
Equivocal findings |
None |
Ovary: benign or malignant granulosa cell tumor (0/50, 0/50, 4/50, 1/50) Lung: alveolar/bronchiolar adenoma or carcinoma (6/50, 6/50, 14/50, 9/50) |
Level of evidence of carcinogenic activity |
Clear evidence |
Clear evidence |
Dose-related effects of acrylonitrile on weight gain and mortality were readily apparent in both sexes, with effects in weight gain appearing earlier in males (significantly decreased), while deaths occurred earlier in females. However it was not determined whether these differential effects between the sexes were due to greater ingestion of acrylonitrile-containing water or to other sex-related factors. There were no reported histopathological changes due to chronic toxicity reported in this study. The incidences of Zymbal gland tumours, stomach and skin papillomas and of brain tumours were higher in acrylonitrile exposed animals than in controls. While the increased incidence of tumours, other than brain tumours is noted, this study specifically deals with the question of biological significance and histogenesis of neuro-oncogenic effects in rats chronically exposed to acrylonitrile. In further investigations, the authors note that samples of tumour tissue were successfully grown in culture, but transplantation of samples from these cultures (observed for up to 12 weeks) was unsuccessful. However, a direct intracerebral transplantation from a large tumour was successful.
Tumour incidence
Site of tumour |
No. animals with tumours at increasing dose levels of acrylonitrile (ppm)a |
|||
0 |
20 |
100 |
500 |
|
Blood (lymphoproliferation) |
1 |
0 |
0 |
0 |
Soft tissue |
1 |
1 |
5 |
1 |
Forestomach |
0 |
0 |
0 |
4 |
Zymbal gland |
0 |
0 |
1 |
9 |
Pituitary |
5 |
3 |
1 |
0 |
Pancreas |
1 |
0 |
2 |
0 |
Kidney |
0 |
0 |
0 |
1 |
Parathyroid |
1 |
1 |
0 |
1 |
Skin |
0 |
0 |
2 |
1 |
a 20 rats per group
Mortality data
Dose level (ppm) |
Sex of animal |
No. animals |
No. dead (% dead) |
0 |
Male |
80 |
73 (91.3) |
35 |
Male |
47a |
42 (89.4) |
100 |
Male |
48 |
43 (89.6) |
300 |
Male |
48 |
48 (100)b |
0 |
Female |
80 |
60 (75.0) |
35 |
Female |
48 |
44 (91.7) |
100 |
Female |
48 |
47 (97.9)b |
300 |
Female |
48 |
48 (100)b |
a In the 35 ppm male group there was one female that was apparently mis-sexed and placed on test in the group (she was removed from the study on day 56 and none of the data from this rat was reported in the study report)
b Significantly increased (p < 0.05)
Tumour occurrence
Organs affected by tumour |
Dose levels showing elevations in tumour incidence |
Central nervous system |
35, 100 and 300* ppm (male & female) |
Zymbal gland |
35, 100 and 300* ppm (female) 300* ppm (male) |
Stomach (non-glandular) |
35, 100 and 300* ppm (male) 100 and 300* ppm (female) |
Tongue |
300* ppm (male & female) |
Small intestine |
35 and 300* ppm (male) 100* and 300* ppm (female) |
Mammary gland: Malignant Total no. rats with mammary gland tumour, malignant and benign combined |
300 ppm (female) 35 and 300 ppm (female) |
* Statistically significant compared to controls (p< 0.05)
None
Percentage incidence of palpable masses
|
0 ppm |
1 ppm |
100 ppm |
Head Region |
|
|
|
Males |
7% |
1% |
16% |
Females |
5% |
2% |
13% |
Mammary Region |
|
|
|
Males |
22% |
21% |
11% |
Females |
38% |
39% |
46% |
Tumour incidence
|
0 ppm |
1 ppm |
100 ppm |
|||
Male |
Female |
Male |
Female |
Male |
Female |
|
Brain |
||||||
No. Tissues |
98 |
99 |
95 |
100 |
97 |
97 |
Astrocytomas |
2 |
0 |
3 |
1 |
23 |
32 |
Spinal Cord |
||||||
No. Tissues |
|
96 |
|
99 |
|
98 |
Astrocytomas |
|
0 |
|
0 |
|
7 |
Brain/Spinal Cord |
||||||
No. Tissues |
|
100 |
|
100 |
|
100 |
Animals with astrocytomas/ animals with brain or spinal cord examined |
|
0 |
|
1 |
|
34 |
Zymbal Gland/Ear Canal |
||||||
No. Tissues |
100 |
99 |
91 |
95 |
93 |
98 |
Total Carcinomas |
1 |
0 |
0 |
0 |
14 |
7 |
Stomach |
||||||
No. Tissues |
98 |
100 |
98 |
99 |
97 |
99 |
Total Carcinomas |
0 |
0 |
1 |
0 |
4 |
0 |
Total Papillomas (benign) |
3 |
1 |
2 |
4 |
8 |
7 |
High dose animals had significant reductions in body weight; food and water consumption were unaffected. The increased number of deaths among the high dose groups animals prompted the early end of the study after 20 months. Small, sometimes significant, reductions in haemoglobin, haematocrit, and erythrocyte counts were observed in high dose animals. Liver and adrenal weights were increased in high dose rats, but without any clinical chemistry or microscopic correlates. An increase in the degree of severity of forestomach hyperplasia was observed in all high dose animals. High dose males and females had an increased incidence of astrocytomas of the brain and spinal cord, carcinomas and adenomas of Zymbal's gland, mammary gland tumours, squamous cell carcinomas and papillomas of the forestomach. Otherwise all clinical and microscopic findings in these studies were considered unremarkable and within normal bounds. There were no observable non-neoplastic effects attributable to treatment in the low dose group.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Study duration:
- chronic
- Species:
- other: rat, mouse
- Quality of whole database:
- A number of mouse and rat studies are available of variable quality and consisitently identify carcinogenic effects in various organs/tissues.
Carcinogenicity: via inhalation route
Link to relevant study records
- Endpoint:
- carcinogenicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Principles of method if other than guideline:
- Two year combined toxicity and carcinogenicity inhalation study
- GLP compliance:
- not specified
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- The animals were male and female Spartan Sprague-Dawley rats
- Route of administration:
- inhalation
- Type of inhalation exposure (if applicable):
- whole body
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- Rats were exposed for 6 hours per day, 5 days per week, during 2 years, to concentrations of 0, 20 and 80 ppm (44 mg/m³ and 176 mg/m³, respectively).
- Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- No information available
- Duration of treatment / exposure:
- 2 years
- Frequency of treatment:
- 6 hours per day, 5 days per week.
- Post exposure period:
- Not examined.
- Dose / conc.:
- 0 ppm
- Remarks:
- Controls
- Dose / conc.:
- 20 ppm
- Remarks:
- Inhalation: 44 mg/m3
- Dose / conc.:
- 80 ppm
- Remarks:
- Inhalation: 176 mg/m3
- No. of animals per sex per dose:
- 100 rats/sex/group. Additional animals were included for interim sacrifice at 6 months (7 rats/sex/group), and at 12 months (13 rats/sex/group.
- Control animals:
- yes, sham-exposed
- Details on study design:
- Rats were exposed to acrylonitrile by inhalation at concentrations of 20 or 80 ppm, 6 hours/day, 5 days/week for 2 years. Controls were exposed to air only. Additional animals were included for interim sacrifice at 6 and 12 months.
- Positive control:
- Not examined.
- Observations and examinations performed and frequency:
- Mortality, clinical signs, body weight, haematology, urinalysis, clinical chemistry.
- Sacrifice and pathology:
- Interim sacrifices were carried out on satellite groups at 6 and 12 months. All rats were subject to necropsy and histopathological examination.
- Other examinations:
- None reported.
- Statistics:
- No information available.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- : statistically significant increase in mortality was observed within the first year in both male and female rats administered 80 ppm and in the females of the 20 ppm group during the last 10 weeks of the study
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- : statistically significant increase in mortality was observed within the first year in both male and female rats administered 80 ppm and in the females of the 20 ppm group during the last 10 weeks of the study
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- : reduced weight gain
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- : changes secondary to tumour incidence
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- : secondary chnages only
- Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- : during the first 6 months of the study, exposed rats excreted lower specific gravity urine than control animals.
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- : nasal irritation
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Details on results:
- Clinical observations detected a variety of toxic effects characterised by decreases in bodyweight, early mortality, clinical appearance, earlier onset of tumours and more frequently observed palpable tumours. These observations were most apparent and occurred earliest in the high-dose group (80 ppm).
Mortality
A statistically significant increase in mortality (p <0.05) was observed within the first year in both male and female rats administered 80 ppm acrylonitrile and in the females of the 20 ppm group during the last 10 weeks of the study. The apparent increase in the reported mortality for the 20 ppm females was principally due to early sacrifice of rats with large, benign, mammary gland tumours. In Sprague-Dawley the tumours are known to occur spontaneously at a high rate, but in this experiment the tumours were observed earlier and more frequently, and became larger in exposed animals. Statistically significant early mortality is indicated in both males and females at 80 ppm. However the onset of early mortality begins much earlier into the study for male rats (Days 211-240) compared to females in which a significant increase in mortality was only seen at Days 361-390.
Bodyweights
A significant decrease in mean bodyweight was observed in rats exposed to 80 ppm acrylonitrile. Less significant, but similar weight decreases, were noted in the 20 ppm females after approximately 1 month exposure. A treatment-related effect on mean body weight was not observed in males at this dose level (20 ppm).
Water consumption
During the first 6 months of the study the exposed rats drank more water
Urinalysis
During the first 6 months of the study, exposed rats excreted lower specific gravity urine than control animals.
Organ weights
In the 80 ppm group of male rats a significantly increased relative organ to body weight ratio (p < 0.05) was observed for the brain, heart, and testes. However, since the body weight of fasted animals was significantly decreased (p < 0.05) in this group, the relative increase in these organs to body weight ratios were considered to be a reflection of the effect on body weight. In addition, the absolute kidney weight in the 80 ppm group of males was significantly decreased (p < 0.05). This observation was consistent with the decreased body weight and a decrease in the severity of chronic renal disease which was observed grossly and microscopically in these rats. The increased relative organ weights were a manifestation of the decreased bodyweight gain and do not indicate a specific target organ toxic effect. In the few surviving females there was a significantly increased (p < 0.05) liver to bodyweight ratio in the 20 ppm (45 mg/m3) group. The increased liver to body weight ratio and the slight increase in the absolute liver weight in these rats, as well as in the single surviving rat in the 80 ppm group, were interpreted to be the result of increased extramedullary haematopoiesis in the liver. This was a result of the greater number of bleeding tumours in these rats and was not interpreted to be indicative of a primary hepatoxic effect due to acrylonitrile exposure.
Clinical analyses
Haematology, urinalysis, and clinical chemistry determinations were performed at periodic intervals. The results showed that acrylonitrile exposure did not have a primary adverse effect on bone marrow, kidney, or liver function in either male or female rats. Occasional significant reduction of the packed cell volume (PVC), haemoglobin and in the RBC and WBC counts were noted. However these were interpreted as being secondary changes associated with decreased growth and tumour induction and haemorrhage, generalised stress, and inflammatory reactions resulting from exposure to acrylonitrile.
Pathology
Histopathological examination revealed increased pathological changes in the heart and lungs of male rats of both treatment groups. The authors indicated however that the changes seen were identical to effects seen in the control animals and were usually associated with chronic renal disease. Microscopic examination of the kidneys indicated a slight, non-statistically significant increase in the incidence of spontaneously occurring advanced chronic renal disease. However, this slight increase could have been due to increased demand on the kidneys, resulting from increased water consumption seen earlier in the study.
A treatment-related increase in extramedullary haemopoiesis in the liver and the spleen and an increase in focal liver cell necrosis was observed primarily at the 13-18 month and the 19-24 month intervals, with those in treated rats generally being observed at the earlier time intervals when compared with the controls. The finding of extramedullary haemopoiesis was considered to be secondary to the presence of large, benign mammary tumours in the animals, which occurred earlier in treated animals than controls. The presence of these tumours was frequently associated with haemorrhage and tissue damage or pressure necrosis due to contact with the wire mesh cage, the haemorrhage and blood loss in turn resulting in compensatory extramedullary haemopoiesis. The development of large, frequently ulcerated, necrotic and haemorrhagic ear canal (Zymbal gland) tumours in acrylonitrile treated rats contributed to this compensatory response. The presence of increased focal hepatic necrosis in these rats was also considered to be a secondary effect due to repeated episodes of blood loss and associated anaemia and hypoxia. It was concluded, therefore, that these findings were not indicative of a primary hepatotoxic effect of acrylonitrile. This is supported by the fact that the 6- and 12-month interim pathology data did not indicate any primary haemopoietic or liver toxicity attributable to acrylonitrile exposure, nor was any such effect demonstrated in other chronic toxicity studies in rats and dogs exposed to acrylonitrile by different routes.
Primary treatment-related effects were observed in the nasal turbinate mucosa of all rats examined in the 80 ppm group as well as in most of the rats in the 20 ppm group. The changes in both groups were qualitatively similar but much less severe in the 20 ppm group than in the 80 ppm group. These changes were confined to the turbinate region extending from the external nares into the region lined by respiratory epithelium. The inflammatory and degenerative changes present in the nasal turbinates were characterised by suppurative rhinitis, hyperplasia, focal erosions, and squamous metaplasia of the respiratory epithelium, with hyperplasia of the mucous secreting cells. These changes were interpreted to be a result of irritation due to exposure. In addition, in two of the 80 ppm female rats there was a microscopic metaplastic proliferation of the respiratory epithelium. Although the incidence of this lesion was not statistically significantly increased, it was considered treatment-related in view of its location in the same region of the nasal mucosa showing the degenerative and inflammatory changes and because of the historically low spontaneous incidence of this finding.
Focal perivascular cuffing and gliosis was reported in the brain. In males at 20 and 80 ppm, the incidence was 2/99 and 7/99 (p <0.05, one-sided), respectively, and, for females, the incidence was 2/100 and 8/100 (p <0.05, one-sided), respectively. A treatment-related increase in extramedullary haemopoiesis in the liver and the spleen and an increase in focal liver cell necrosis was observed primarily at the 13-18 month and the 19-24 month intervals, with findings in treated rats generally being observed at the earlier time intervals when compared with the controls. The finding of extramedullary haemopoiesis was considered (Quast, 2001 personal communication) to be secondary to the presence of large, benign mammary tumors and ear canal (Zymbal’s gland) tumors in the animals, which occurred earlier in treated animals than controls. It was concluded, therefore, that these findings were not indicative of a primary hepatotoxic effect of acrylonitrile.
An increased incidence of brain tumours was observed, although they were rarely the cause of death. Many of the tumours could not be detected by gross pathology, but were identified histopathologically as focal or multifocal glial cell tumours (astrocytomas). The incidence was significantly increased for both male and females at the 80 ppm exposure level compared to the controls. The incidence of proliferative glial cell lesions, suggestive of early tumours, was significantly increased in the 80 ppm males, but not in the females at any dose level. Collectively, proliferative changes in the glial cells (i.e. tumours and early proliferation suggestive of tumours) were significantly increased in the 20 ppm and 80 ppm females, but only in the 80 ppm males, compared to controls.
Recorded deaths were often attributable to severe ulceration of the Zymbal’s gland or mammary tissue tumours, and at the highest dose level (80 ppm) were also due to suppurative pneumonia due to the irritant effects of acrylonitrile on the lungs. The occurrence of Zymbal’s gland tumors was observed to be significantly increased in both male and female animals in the 80 ppm group (11/100 in both male and female, p < 0.05). For females the highest incidence occurred during the 13 to 18 month interval. An incidence of 3/100 was also seen in males exposed to 20 ppm, compared with 1/100 for control males, but no Zymbal’s gland tumors were seen in females at this exposure level. These tumors showed an increased incidence and a decreased latency period that was consistent with the life records of the palpable masses in this region. The type of tumour observed in both males and females was sebaceous squamous cell carcinoma of the external auditory canal gland, without metastasis. - Relevance of carcinogenic effects / potential:
- Clear evidence of carcinogenicity was seen in this study.
- Dose descriptor:
- LOAEC
- Effect level:
- 20 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Local nasal irritant effects
- Remarks on result:
- other: Effect type: toxicity
- Dose descriptor:
- NOAEC
- Effect level:
- 4 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Estimated value based on local irritancy at 20 ppm
- Remarks on result:
- other: Effect type: toxicity
- Dose descriptor:
- LOAEC
- Effect level:
- 20 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increased brain tumour incidence at 20 ppm
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- NOAEC
- Effect level:
- < 20 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increased brain tumour incidence at 20 ppm
- Remarks on result:
- other: Effect type: carcinogenicity
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 20 ppm
- System:
- central nervous system
- Organ:
- brain
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 80 ppm
- System:
- gastrointestinal tract
- Organ:
- duodenum
- tongue
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 80 ppm
- System:
- ear
- Organ:
- zymbal gland
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Clear evidence of carcinogenicity was seen in this study.The main tumours observed in rats exposed to acrylonitrile were microscopic brain tumours and Zymbal’s gland tumours.
- Executive summary:
Male and female Spartan Sprague-Dawley rats were exposed to acrylonitrile by inhalation at concentrations of 20 or 80 ppm, 6 hours/day, 5 days/week for 2 years. Controls were exposed to air only. Additional animals were included for interim sacrifice at 6 and 12 months. A statistically significant increase in mortality was observed within the first year in both male and female rats administered 80 ppm and in the females of the 20 ppm group during the last 10 weeks of the study. The apparent increase in the reported mortality for the 20 ppm females was principally due to early sacrifice of rats with large, benign, mammary gland tumours. These tumours are known to occur spontaneously in this strain at a high rate, but in this experiment the tumours were observed earlier and more frequently, and became larger in exposed animals. Primary treatment-related effects were observed in the nasal turbinate mucosa of all rats examined in the 80 ppm group as well as in most of the rats in the 20 ppm group. The changes in both groups were qualitatively similar but much less severe in the 20 ppm group than in the 80 ppm group. The main tumours observed in rats exposed to acrylonitrile were microscopic brain tumours and Zymbal’s gland tumours.
The EU RAR considered this study to be pivotal for risk assessment purposes. The key toxicological findings due to acrylonitrile exposure were local irritant effects in the nasal epithelium comprising suppurative rhinitis, hyperplasia, focal erosions, and squamous metaplasia of the respiratory epithelium, with hyperplasia of the mucous secreting cells. Effects were seen at the lowest effect level of 20 ppm used in the study, and this represents therefore a LOAEL. The EU RAR applied a safety factor of 5 to the level of 20 ppm to give a suggested NOAEL of 4 ppm. This approach was stated to be justified due to the nature of the effect (local irritancy) and the conclusion that other systemic, non-neoplastic findings in acrylonitrile-treated rats were secondary to the tumorigenic effects of acrylonitrile, rather than due to direct systemic toxicity.
- Endpoint:
- carcinogenicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- One year inhalation carcinogenicity study in rats
- GLP compliance:
- no
- Remarks:
- : older, non-standard published literature study
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- The animals were male and female Sprague-Dawley rats
- Route of administration:
- inhalation
- Type of inhalation exposure (if applicable):
- not specified
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- Rats were exposed to acrylonitrile 4 hours/day, 5 days/week, for 12 months.
- Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- No information available
- Duration of treatment / exposure:
- 12 months
- Frequency of treatment:
- 4 hours/day, 5 days/week
- Post exposure period:
- Following the 12 month exposure period, the rats were kept under observation until spontaneous death occured.
- Dose / conc.:
- 0 ppm
- Remarks:
- Untreated controls
- Dose / conc.:
- 5 ppm
- Remarks:
- Inhalation
- Dose / conc.:
- 10 ppm
- Remarks:
- Inhalation
- Dose / conc.:
- 20 ppm
- Remarks:
- Inhalation
- Dose / conc.:
- 40 ppm
- Remarks:
- Inhalation
- No. of animals per sex per dose:
- 30 rats/sex/group
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- Groups of male and female rats were exposed to acrylonitrile by inhalation for 12 months, and subsequently kept under observation until spontaneous death. A group of untreated rats acted as controls.
- Positive control:
- Not examined.
- Observations and examinations performed and frequency:
- Body weight, mortality.
- Sacrifice and pathology:
- Rats were necropsied for histopathologic evaluation.
- Other examinations:
- No further information available
- Statistics:
- No information available
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Details on results:
- Body weight was unaffected by exposure. There was statistically significant increase in the percentage of animals with benign and malignant tumours (P < 0.01) and malignant tumors alone (P < 0.01). The total malignant tumors per 100 animals was noted for several treated groups, but lacked a definitive dose-response relationship. There was no increase in Zymbal's gland tumors, extrahepatic angiosarcomas, or hepatomas. Encephalic glioma incidence was increased in rats exposed to 20 ppm (3.3%; 3/60) and 40 ppm (5%; 3/60). Although not statistically significant, this response was considered by the investigators to be of possible biological relevance because the brain was shown to be a target organ in the oral administration part of the study.
- Relevance of carcinogenic effects / potential:
- A statistically significant increase in malignant and total number of tumours occurred only in females at 5 ppm.
- Dose descriptor:
- LOAEC
- Effect level:
- 5 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Borderline increase in tumours at 5 ppm
- Remarks on result:
- other: Effect type: carcinogenicity
- Dose descriptor:
- NOAEC
- Effect level:
- < 5 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Borderline increase in tumours at 5 ppm
- Remarks on result:
- other: Effect type: carcinogenicity
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 5 ppm
- System:
- central nervous system
- Organ:
- brain
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- A slight increase in brain tumours was seen at the two highest exposure concentrations. The results were considered by the authors to indicate a borderline carcinogenic effect, but are consistent with other studies.
- Executive summary:
The effects of inhalation exposure to 5, 10, 20 and 40 ppm acrylonitrile, 4 hours/day, 5 days/week for 12 months were investigated in groups of male and female Sprague-Dawley rats. One group of untreated rats acted as controls. After the 12 month exposure period, rats were kept under observation until spontaneous death. There were no apparent effects on mortality or body weights throughout the study. A statistically significant increase in the percentage of animals bearing benign and malignant tumours (p < 0.01), malignant tumours alone (p < 0.01) and in the number of total malignant tumours per 100 animals was found in several treated groups, although a strong dose reponse relationship was not established. Slight to moderate increases in tumour incidence were observed in the mammary gland, forestomach and CNS, but none of these were statistically significant. No increase in Zymbal gland tumours, extrahepatic angiosarcomas and hepatomas was observed, 3/60 and 2/60 encephalic gliomas were observed in animals exposed to the two highest concentrations of acrylonitrile. Whilst this finding did not achieve statistical significance, it is biologically significant given that the brain was clearly shown to be the target organ in rats following oral adminsitration. The authors suggested that the carcinogenicity of acrylonitrile was influenced by the age of the animals at the start of treatment, and was dependent on the concentration administered and duration of treatment. The results were considered by the authors to indicate a borderline carcinogenic effect, but are consistent with other studies.
Referenceopen allclose all
Cumulative mortality data
Days on test |
Sex |
0 ppm |
20 ppm |
80 ppm |
Sex |
0 ppm |
20 ppm |
80 ppm |
0-30 |
M |
0 |
1 |
0 |
F |
1 |
0 |
0 |
31-60 |
M |
0 |
1 |
0 |
F |
1 |
0 |
0 |
61-90 |
M |
0 |
1 |
1 |
F |
1 |
0 |
0 |
91-120 |
M |
0 |
2 |
1 |
F |
1 |
0 |
0 |
121-150 |
M |
1 |
2 |
1 |
F |
1 |
0 |
0 |
151-180 |
M |
2 |
3 |
2 |
F |
1 |
0 |
0 |
181-210 |
M |
2 |
3 |
6 |
F |
1 |
1 |
1 |
211-240 |
M |
2 |
4 |
12a |
F |
1 |
1 |
2 |
241-270 |
M |
2 |
5 |
13a |
F |
3 |
1 |
4 |
271-300 |
M |
2 |
5 |
14a |
F |
5 |
1 |
6 |
301-330 |
M |
3 |
6 |
16a |
F |
5 |
2 |
9 |
331-360 |
M |
3 |
6 |
18a |
F |
7 |
2 |
11 |
361-390 |
M |
4 |
8 |
19a |
F |
9 |
3 |
19a |
391-420 |
M |
6 |
9 |
22a |
F |
11 |
5 |
27a |
421-450 |
M |
11 |
12 |
24a |
F |
14 |
10 |
33a |
451-480 |
M |
14 |
15 |
28a |
F |
14 |
14 |
41a |
481-510 |
M |
19 |
26 |
39a |
F |
19 |
22 |
57a |
511-540 |
M |
23 |
34 |
47a |
F |
26 |
31 |
71a |
541-570 |
M |
27 |
38 |
56a |
F |
34 |
36 |
80a |
571-600 |
M |
35 |
47 |
63a |
F |
36 |
43 |
88a |
601-630 |
M |
43 |
59a |
76a |
F |
50 |
54 |
94a |
631-660 |
M |
62 |
68 |
83a |
F |
63 |
70 |
98a |
661-690 |
M |
71 |
72 |
85a |
F |
66 |
81a |
98a |
691-720 |
M |
78 |
81 |
94a |
F |
71 |
88a |
99a |
721-735 |
M |
82 |
86 |
96a |
F |
78 |
91a |
99a |
Total No. Rats |
|
100 |
100 |
100 |
|
100 |
100 |
100 |
asignificantly different from control
Note: Data listed as number dead which is equal to percent dead
Summary of tumours.
Sex |
Concentration (ppm) |
Zymbals Gland carcinoma |
Tongue papilloma, carcinoma |
Mammary Gland fibroadenoma |
Small Intestines cystadenocarcinoma |
Brain astrocytoma |
Male |
0 |
1/100 |
1/96 |
- |
2/99 |
0/100 |
20 |
3/100 |
0/14 |
- |
2/20 |
4/99 |
|
80 |
11/100* |
7/89* |
- |
14/98* |
15/99* |
|
Female |
0 |
0/100 |
- |
79/100 |
- |
0/100 |
20 |
0/100 |
- |
95/100* |
- |
4/100* |
|
80 |
10/100* |
- |
75/100 |
- |
17/100* |
* significantly different from control incidence
Nasal findings
Histopathological findings |
Exposure concentration |
|||||
Control |
20 ppm |
80 ppm |
||||
M |
F |
M |
F |
M |
F |
|
Hyperplasia of respiratory epithelium in the nasal turbinate mucosa |
0/11 |
0/11 |
4/12 |
2/10 |
10/10 |
5/10 |
Squamous metaplasia of respiratory epithelium in the nasal turbinate mucosa |
0/11 |
0/11 |
1/12 |
2/10 |
7/12 |
5/10 |
Hyperplasia of mucous secreting cells |
0/11 |
0/11 |
7/12 |
8/10 |
2/10 |
8/10 |
Figures indicate no. showing effect/no. of animals in which the nasal passages were examined microscopically
A statistically significant increase in the percentage of animals bearing benign and malignant tumours (p<0.01), malignant tumours alone (p<0.01) and in the number of total malignant tumours per 100 animals was found in several treated groups, although a strong dose reponse relationship was not established. Slight to moderate increases in tumour incidence were observed in the mammary gland, forestomach and CNS, but none of these were statistically significant. No increase in Zymbal gland tumours, extrahepatic angiosarcomas and hepatomas was observed, 3/60 and 2/60 gliomas were observed in animals exposed to the two highest concentrations of acrylonitrile. Whilst this finding did not achieve statistical significance, it is biologically significant given that the brain was clearly shown to be the target organ in rats following oral adminsitration.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- LOAEC
- 20 mg/m³
- Study duration:
- chronic
- Species:
- rat
- Quality of whole database:
- A proprietary study is supported by published data.
Carcinogenicity: via dermal route
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
Overview
There is an unusually large experimental carcinogenicity dataset for acrylonitrile in rodents, particularly in the rat. Under experimental conditions, acrylonitrile is clearly a multisite carcinogen in rodents. It is notable, however, that a number of high quality human epidemiological studies are available for acrylonitrile and these have not found any consistent evidence of increased cancer risk across studies. The most informative human epidemiology studies do not support a causal relationship between acrylonitrile and increased cancer risk.
Summary of findings in animal studies
A total of fourteen rat cancer bioassays are reported for acrylonitrile reported, using exposure in the drinking water (seven studies), by oral gavage (two studies) and by inhalation exposure (five studies). A single mouse cancer bioassay has been reported, and uses gavage exposure. Findings show irritation and neoplasia at the site of contact (i.e. the forestomach in studies using the oral route; the nasal passages in studies using inhalation). In addition, increases in brain and Zymbal’s gland tumours are seen relatively consistently in studies in the rat.
Critical carcinogenic effect
The rat brain tumour response has been identified as the predominant cancer effect following acrylonitrile exposure. Other tumour sites are also reported in some studies but are not seen consistently across studies; these tissues include the mammary gland and liver. Harderian gland and forestomach tumors were seen in the single mouse study. The cellular aetiology of the CNS tumours observed in the chronic rat studies has been investigated further. These tumours were identified as glial tumours in some studies and as astrocytomas in other studies. Based on a reevaluation of tissues selected from the Quast (2002) drinking water study and from the Dow (1980) inhalation study using a panel of immunohistochemical antibodies, these tumours are now considered to be malignant microgliomas (Kolenda-Roberts et al., 2012; EPL, 2014).
Mode of action
The mode of action by which acrylonitrile induces rodent tumors, particularly in the brain of rats, has been the subject of intensive research.
Evidence for mutagenicity
The mutagenicity of acrylonitrile and its metabolite CNEO has been investigated extensively in studies in vitro and in vivo. Critical aspects are discussed below.
In vitro studies
Several studies have reported DNA strand breakage following exposures to acrylonitrile or CNEO using various methods including standard comet assays. As almost all of these studies used alkaline treatment based methods, it is not possible to differentiate between the presence of alkali labile sites and true strand breakage. The early positive report using the B. subtilis rec assay is free of this complication (Kawachi et al., 1980). Three studies that employed the standard alkaline comet assay to measure the induction of DNA strand breaks or alkali labile sites in mammalian cells were negative. Aacrylonitrile failed to elicit a SOS response in either E. coli or S. typhimurium. Several studies have reported on the induction of UDS as a manifestation of acrylonitrile or CNEO stimulated DNA repair. In general the positive reports have employed LSC and are therefore of little relevance. One study, however, reports the induction of UDS (detected using autoradiography) in human mammary epithelial cells exposed to CNEO. It is notable, however, the concentrations of CNEO used in this study were massive compared to thos likley to be encountered in target tissues in vivo. Variable results have been reported for the induction of SCE following exposure to acrylonitrile. Metabolic activation was not always required for a positive response, even in the same cell type (e.g. CHO cells), while other cells may have been metabolically competent (e.g. liver cells). Results were also conflicting when human PBLs were the target cells. As with all in vitro studies, the concentrations used were large compared to those likely to be attained in the target tissue in vivo. One study measured the production of p53 and p21 proteins in the culture medium by human embryonic fibroblasts following in vitro exposure to acrylonitrle. Significant increases over baseline suggest DNA damag; however the significance of this observation is unknown.
The results of studies investigating gene level mutagenicity in prokaryotic microorganisms have been variable, but certain patterns emerge. In Salmonella tester strains requiring a reverse mutation to occur by a frame shift change, results have been mostly negative. In contrast, most studies in strains carrying the his G46 allele have been positive, usually requiring metabolic activation. Reversion of G46 requires a base substitution mutation at G or C. The only Salmonella strain used in studies with acrylonitrile that scored for reverse base substitution mutations involving AT base pairs gave mostly negative results. However, studies in E. coli, where the critical target site for reverse base substitution mutations also involves AT base pairs, gave both negative and positive results for acrylonitrile. Both gene and chromosome level mutations induced by acrylonitrile have been assessed in eukaryotic microorganisms. Variable results have been reported for gene mutations, employing a variety of loci. By contrast, most studies of acrylonitrile-induced chromosome level changes have been reported as positive. Many of these chromosome level mutations involve intra-chromosome recombinations and/or gene conversions. An assay in S. cerevisiae specifically designed to sensitively detect oxidative damage mutagenesis (DEL assay) reports positive results for acrylonitrile. Many studies have demonstrated acrylonitrile-induced gene level mutations in cultured mammalian cells. Most studies have used the L5178Y mouse lymphoma cell line and have reported positive results, at least for Tk-/- mutations. Large and small colony analyses were not conducted for any of the L5178Y assays used in studies with acrylonitrile; therefore the relative contributions of point mutations versus deletion mutations cannot be inferred. L5178Y cells are particularly sensitive to all mutations, in part because they have a mutation in the p53 tumour suppressor gene and in part because they may be especially sensitive to oxidative damage. The Tk locus also allows a wide variety of mutational events to be scored because of the presence on the homologous chromosome. Studies of in vitro gene mutations in other cell lines have given variable results. Molecular analyses of both Tk-/- and Hprt mutants isolated from acrylonitrile-treated human lymphoblastoid cells suggest that point mutations may predominate over deletion mutations. The conditions of this experiment, however, may have biased results towards the recovery of mutants resulting from point mutations. Studies of the hprt mutations induced by CNEO indicated that base-substitution mutations involving both AT and GC base pairs occur. Many studies of acrylonitrile-induced chromosome level mutations in mammalian cells have been reported as positive, with most requiring metabolic activation. One negative study was the only study to use human PBLs, where the cell characteristics mimic those of normal human cells in vivo. The positive studies of acrylonitrile-induced chromosome level mutations have been in exponentially growing cell lines adapted to in vitro culture conditions. As for the in vitro studies of DNA damage events, all studies of in vitro mutations have also used acrylonitrile and/or CNEO concentrations much greater than would be achieved systemically in vivo.
In vivo studies
Positive results of comet assays in multiple tissues following intraperitoneal exposure to acrylonitrile in mice and rats has been reported by one group with a notable paradoxical observation between the two species as to findings in brain; positive results are reported for in mice (a species in which brain tumours are not reported) whereas negative results are reported in rats (in which species brain tumours are reported with some consistency). Three subsequent standard alkaline comet assay studies in rats administered acrylonitrile in drinking water were negative in lymphocytes, brain and Zymbal’s gland. DNA damage (in the form of fragmentation of brain DNA) has also been reported following the oral administration of acrylonitrile in drinking water to Wistar rats. This effect appeared to be due to oxidative damage as it was associated with markers of oxidative stress and was inhibited by concomitant antioxidant administration. However, a later study failed to find markers of oxidative stress in this same strain of rats following treatment with acrylonitrile. Several studies have reported positive UDS responses in rats administered acrylonitrile either orally or by injection. All positive studies used LSC to quantify the UDS response. One study found that the UDS response was associated with GSH depletion, was made greater by depleting GSH before acrylonitrile treatment and was inhibited by the prior administration of sulphydryl compounds. A single study that used autoradiography failed to find a positive UDS response in the testis or liver DNA following oral administration of acrylonitrile. There have been two in vivo studies of SCE in mice; one study reported a weak positive response while the other reported a clear positive result in bone marrow following intraperitoneal administration. Hprt gene mutations have been induced in acrylonitrile-treated mice and rats, including in CYP2E1 knock-out mice that should be incapable of metabolising acrylonitrile to CNEO. The positive mutagenicity responses reported in rats were in cells in which specific DNA adducts did not increase over background levels, thereby suggesting mutagenic mechanisms other than direct DNA reactivity. In vivo gene mutations of the lacZ gene were not found in any tissues of transgenic mice following exposure to acrylonitrile. In contrast to the positive studies of gene mutations, several studies assessing chromosome level mutations arising in either somatic or germ cells in mice or rats administered acrylonitrile by a variety of routes have given negative results. There is therefore a clear disparity between the absence of clastogenicity of acrylonitrile in vivo and the more numerous positive in vitro studies.
Positive results have been reported for acrylonitrile-induced mutations in the rapidly dividing somatic cells of Drosophila in studies conducted as part of the IPCS CCSST 1985 series. The assays employed were then novel, reflecting both gene and chromosome level changes, with the latter resulting from somatic recombination. An additional somatic mutation assay, reported as positive, detected mutational events at an unstable genetic locus, with the mutations thought to have been mainly deletions. Two kinds of germinal/heritable mutations have been assayed in Drosophila studies. One detects sex-chromosome aneuploidy and gave a positive result. It is of interest that mechanistic studies of this system have revealed that other nitriles, while not rodent carcinogens, are important agents in the induction of this aneuploidy due to effects on spindle formation (Sehgal et al., 1990; Osgood & Cyr, 1998). Two studies of heritable SLRL gene mutations with acrylonitrile are negative. Acrylonitrile exposure concentrations used in the Drosophila studies were high compared to those used in mammalian studies.
Human data
Studies in acrylonitrile-exposed workers have assessed chromosome aberration and HPRT gene mutations as biomarkers of effect. In addition DNA strand breaks (or alkali labile sites), sex chromosome aneuploidy and various measures of sperm quality have been assessed in exposed workers. The results of studies of chromosome level mutations are mixed, with some being ambiguous, including a study in Portugal that was reported as positive but in which the pattern of aberration frequency elevations between exposed worker groups calls into question acrylonitrile as the causative agent. A study of Hungarian workers exposed to at least two chemicals, with the acrylonitrile exposure levels being high, is more difficult to interpret. Similarly, studies in Czech workers show results of uncertain significance, i.e. different patterns of chromosome aberration seen by FISH analysis without an overall increase in aberration frequency and increases in the second (but not first) study of chromosome aberration determined by standard methods but not by FISH. Limitations in Chinese studies that influence interpretation are also identified. A study of HPRT mutation in acrylonitrile and DMF-exposed workers used an assay that is known to produce artifacts. A study of exposed workers in the Czech Republic reported failure of the exposures to induce expression of two well known tumor suppressor genes as manifest by increased serum levels of the respective proteins. Mitochondrial deletions have been reported in exposed workers similar to those found in elderly individuals. Finally, modest exposures to acrylonitrile have been associated with DNA damage and aneuploidy in sperm; other parameters of sperm quality were also affected. A mechanism underlying these finding is unknown but the authors list oxidative stress as a possibility.
Evidence for direct and indirect mutagenicity
There is ample evidence that both acrylonitrile and its metabolite CNEO reach critical cancer target tissues in vivo. Administered radiolabelled material is widely distributed among tissues and organs. However, the radioactivity is shown to be bound primarily to proteins. In terms of critical specific DNA adducts, only N7OEG has been found, and only in the liver (a non-target tissue) and by a single laboratory. The available evidence therefore indicates that such adducts are either not efficiently produced or are rapidly repaired, at least in the brain. It is notable, however, that N7OEG adducts induced by ethylene oxide are readily detected, thereby arguing against rapid repair as a reason for the failure to detect adducts. The failure to find acrylonitrile or CNEO-specific DNA adducts in the brain challenges the assumption that mutations resulting from direct DNA reactivity result in tumours in this target tissue. In contrast to acrylonitrile-specific adducts, however, several studies have demonstrated that exposure to acrylonitrile does induce the formation of 8oxoG DNA adducts in brain at tumourigenic doses that may result in mutations. The implication of these fidnings is that, if mutation is a critical early event in the production of brain tumours, this is most likely to be a secondary effect; i.e. indirect mutagenicity. This finding is consistent with those of Klaunig & Forney (2009) that acrylonitrile (which causes brain tumours in rat studies) induces 8oxoG adducts in astrocytes in vitro, while methacrylonitrile (which does not cause brain tumours) does not induce the formation of 8oxoG adducts. It is also important to note that several tissues have been evaluated for the induction of somatic mutations by acrylonitrile in target tissues in mice, and none have been found (Lambert et al., 2005). These findings also argue against direct mutagenicity as a causative factor of carcinogenicity in these tissues.
The mode of action (MoA) for the rodent carcinogenicity of acrylonitrile is likely to be complex and is, in part, through mutagenicity. The mutagenicity, however, has two underlying mechanisms – direct and indirect, with current evidence indicating that the latter may be more prominent than the former. Furthermore, both the direct and indirect mutagenicity of acrylonitrile may be enhanced or even manifested by associated tissue effects such as increased cell proliferation and the generation of oxidative stress. Acrylonitrile also induces cellular effects such as cell transformation and gap junction inhibition that are not genotoxic, but which may serve to enhance carcinogenicity. These two non-genotoxic effects may modulate the rodent carcinogenicity of acrylonitrile in a tissue-specific manner; those tissues with a high spontaneous tumour incidence may have a correspondingly high number of spontaneously initiated cells, needing only promotion to evolve a cancer. The rodent brain may be such a tissue.
Justification for classification or non-classification
Acrylonitrile is listed on Annex VI of the CLP Regulation and has a harmonised classification for carcinogenicity in Category 1B. Given the consistent and clear observations of a lack of effect in occupationally exposed humans, it would seem more appropriate to reinterpret the hazard as a 'suspected human carcinogen' (GHS Category 2). The reason for the difference between occupationally exposed humans and the results of the animal studies is unclear, however this could be due to either the MoA in rats not being relevant to humans, or that acrylonitrile carcinogenicity is a threshold effect and human exposure does not exceed this threshold.
Although research is presently unable to fully define a mode of action for acrylonitrile carcinogenicity, the existence of a threshold principle is entirely plausible based on the existing data. Kirman et al (2005) were able to show the link between occupational human exposure and the results of the rodent cancer assays by modelling the exposure concentrations based on internal CEO levels. This demonstrated that even the highest occupationally relevant exposure concentrations in humans (which are now no longer permitted since changes in legislation), gave rise to an internal concentration at the very lowest animal exposure levels where significant cancer risk was not apparent in the animals. Whilst a cancer risk in humans at high concentrations cannot presently be entirely ruled out, the occupational exposures presently imposed are clearly below a threshold for cancer. At the time of the EU RAR (2004), there was little mechanistic evidence to support the threshold carcinogen hypothesis, therefore acrylonitrile was conservatively classed as a non-threshold (direct DNA acting) carcinogen. As well as recent updates to the epidemiology dataset, numerous recent mechanistic studies have highlighted the link between effects in rat tissue and oxidative stress and the cell transformation capacity of acrylonitrile. Mechanistic data therefore strongly indicate that the carcinogenicity of acrylonitrile occurs through an indirect DNA-reactive mechanism secondary to the induction of oxidative stress in the target tissue, through the (non-genotoxic) transformation of initiated cells, or through a combination of these threshold mechanisms.
IARC consideration of acrylonitrile
It is notable that the IARC (Monograph 71; 1999) downgraded their carcinogenicity classification of acrylonitrile to Group 2B (possibly carcinogenic to humans). This assessment was based on a consideration of the genotoxicity data, animal carcinogenicity and human epidemiological data. It was concluded that, while acrylonitrile was mutagenic in vitro, the results of studies in vivo were largely negative. The clear evidence of carcinogenicity in studies in experimental animals was not considered to be reflected in the epidemiology. The IARC concluded that, on balance, and given the largely unsupportive findings from the other epidemiology studies, the evidence of an increased incidence of lung cancer reported in exposed workers in one early study was not considered to be sufficiently strong to conclude that there was a credible association between acrylonitrile exposure and lung cancer. The earlier indications of an increased cancer risk in workers exposed to acrylonitrile were therefore not confirmed by the more recent studies, which were also considered to be more informative. The IARC Group 2B classification is comparable to the GHS/CLP Category 2 classification for carcinogenicity.
Additional information
The carcinogenicity of acrylonitrile has been investigated in a relatively large number of animal studies using administration in the drinking water, by gavage and by inhalation. While the studies are of variable quality, the results of the studies are consistently positive. Numerous epidemiology studies investigating cancer incidence in exposed workers are also available and are discussed in detail elsewhere in this dossier.
Mouse gavage study: NTP (2001)
Clear evidence of the carcinogenicity was seen in this study in male and female B6C3F1 mice administered acrylonitrile by gavage at dose levels of 0, 2.5, 10 or 20 mg/kg bw/d for 2 years. Reduced survival was seen at 20 mg/kg bw/d. Evidence of local gastric irritation was seen at 20 mg/kg bw/d. Increased incidences of forestomach tumours were seen in both sexes at 10 and 20 mg/kg bw/d. Harderian gland hyperplasia was increased in males at 10 mg/kg bw/d; increased incidences of Harderian gland tumours were seen in all treated groups of males and in females at 10 and 20 mg/kg bw/d. Ovarian and bronchio-alveolar tumour incidences were increased in females at 10 mg/kg bw/d.
Rat drinking-water study: Quast (1980, 2002)
Male and female Sprague-Dawley rats were administered acrylonitrile in drinking water at concentrations of 0, 35, 100 or 300 ppm. Early mortality was noted in both sexes at 300 ppm. Reduced weight gain and food consumption were seen in all treatment groups, with evidence of local gastric irritation. Increased tumour incidences were seen in one or more dose levels in the brain, Zymbal’s gland, forestomach, tongue, small intestine and mammary gland.
Rat drinking-water study: Bigner et al. (1986)
In a study specifically designed to investigate the incidence and origin of brain tumours, male and female F344 rats were administered acrylonitrile in the drinking water at concentrations of 100 and 500 ppm. Treated groups showed effects on mortality and weight gain, with increased incidences of clinical signs consistent with neurotoxicity. Increased incidences of brain tumours were seen in both treated groups, with increased incidences of tumours in other organs (skin, stomach, Zymbal’s gland) also noted. Although the brain tumours noted in this study morphologically closely resembled astrocytic tumours commonly seen in this rat strain, specific staining did not reveal the presence of GFAP thus indicating a different cellular origin.
Rat drinking-water study: Gallagher et al. (1988)
Male CD rats (20/group) were administered acrylonitrile in the drinking water at concentrations of 0, 20, 100 or 500 ppm for two years. Increased mortality was seen at 500 ppm, with bodyweight effects seen at 100 and 500 ppm. Increased incidences of Zymbal’s gland tumours were seen at 100 and 500 ppm, with forestomach papillomatous changes also noted at 500 ppm and considered likely to be secondary to local irritation. The incidences of tumours in other organs and tissues were not affected by treatment, however the small group size may have limited the power of the study to detect carcinogenicity.
F344 rat drinking-water study: Johannsen & Levinskas (2002)
In this study, acrylonitrile was administered in the drinking water for approximately 2 years to groups of 100 male and 100 female F344 rats at nominal concentrations of 1, 3, 10, 30 and 100 ppm. Two additional groups, each of 100 males and 100 females, were used as untreated controls. The average daily intake was 0, 0.1, 0.3, 0.8, 2.5 or 8.4 mg/kg bw/d respectively, for males and 0, 0.1, 0.4, 1.3, 3.7 or 10.9 mg/kg bw/d respectively for females. Clinical biochemistry, interim necropsies, organ weights and microscopic evaluation of tissues and organs were performed on 10 rats/sex/group following treatment for 6, 12 and 18 months and at study termination. Females were sacrificed after treatment for 24 months; males after treatment for 26 months. A consistent decrease in survival, lower body weight and reduced water intake and small reductions in haematological parameters were observed in both sexes at 100 ppm. Increased numbers of early deaths were observed in males at 10 ppm and females at 30 ppm. Relative organ weights at various study intervals were consistently elevated in the high dose group; findings are attributable to lower body weights. At the same intervals, mean absolute weights were either comparable to controls or only slightly elevated and few changes in weight ratios were seen when organ weights were compared with brain weights. No biochemical changes suggested a treatment-related effect. An increase in urine specific gravity in 100 ppm male rats reflected the reduced water consumption by this group. The only significant non-neoplastic finding observed histologically was a dose-related increase in hyperplasia/hyperkeratosis in squamous cells of the forestomach in male and female rats at concentrations of 3 ppm and higher. This observation correlated with the induction of treatment-related squamous cell tumours (papillomas and carcinomas) of the forestomach seen primarily in rats in these groups. Mammary gland carcinomas were observed only in female groups. Both sexes given 10 ppm acrylonitrile or more had astrocytomas of the brain/spinal cord and adenomas/carcinomas of the Zymbal's gland.
Spartan rat drinking-water study: Johannsen & Levinskas (2002)
Spartan Sprague-Dawley rats (100/sex/group) were administered acrylonitrile continually at concentrations of 0 (controls), 1 or 100 ppm in the drinking water. The equivalent mean doses of acrylonitrile were 0, 0.09 and 8.0 mg/kg bw/d in males; 0, 0.15 and 10.7 mg/kg bw/d in females. Groups of ten rats/sex were sacrificed at 6, 12 and 18 months and at study termination. Ophthalmoscopic, haematological, clinical biochemistry, urinalysis and full histopathological exams were performed on control and high dose groups and in lower dose groups, as required, to define dose-responses of observed effects. All animals were necropsied and underwent microscopic examination of target tissues, including brain, ear canal, stomach, spinal cord and any grossly observable tissue masses. High dose male and female rats exhibited statistically decreased bodyweights. Food consumption and water intake were also reduced. Due to increased deaths in groups of high dose rats, surviving males and females were terminated after 22 and 19 months, respectively. Small, sometimes statistically significant, reductions in haemoglobin, haematocrit and erythrocyte count were observed in male and female rats at 100 ppm drinking water. Organ weight findings were not observed and there were no changes in clinical biochemistry. Absolute kidney weights were increased in high dose female rats only. Male and female rats from high dose groups had a higher incidence of palpable masses of the head and the non-glandular stomach and, in females only, the mammary region. In both sexes, treatment-related tumours of the central nervous system (brain, spinal cord), ear canal, and gastrointestinal tract were observed in rats administered 100 ppm.
Spartan rat gavage study: Johannsen & Levinskas (2002)
In this study, Spartan Sprague-Dawley rats (100/sex/group) were administered lifetime oral doses of acrylonitrile by gavage at 0, 0.1 or 10 mg/kg bw/d, 7 days per week. The doses selected were designed to approximate the same daily intake of acrylonitrile in the drinking water study by the same authors. Groups of ten rats/sex were sacrificed at 6, 12 and 18 months and at study term. Ophthalmoscopic, haematological, clinical biochemistry, urinalysis and full histopathological exams were performed on control and high dose groups. Similar tests were done in lower dose groups, as required, to define dose-responses of observed effects. All animals were necropsied and underwent microscopic examination of target tissues, including brain, ear canal, stomach, spinal cord and any observable tissue masses. High dose male and female rats exhibited statistically decreased body weights. Food consumption and water intake were unaffected. Due to increased deaths in groups of high dose rats, all test groups were terminated after 20 months of treatment. Small, sometimes statistically significant, reductions in haemoglobin, haematocrit and erythrocyte count were observed in male and female rats in the high dose group. There were increases in absolute or relative organ weight ratios for liver and adrenal in the high dose groups, but could not be correlated with acrylonitrile toxicity in the absence of adverse clinical biochemistry or microscopic findings. Absolute kidney weights were increased in high dose male and female rats. Male and female rats from the high dose group had a higher incidence of palpable masses of the head and the non-glandular stomach and, in females only, the mammary region. In both sexes, treatment-related tumours of the central nervous system (brain, spinal cord), ear canal and gastrointestinal tract, and in females only, the mammary gland were observed in rats administered 10 mg/kg bw/d.
Comparison of results with the drinking water study
Spartan rats from the gavage study had a substantially higher incidence of acrylonitrile-related site-specific tumours than rats of the same strain administered similar dose levels of acrylonitrile via their drinking water study counterparts. While a similar spectrum of tumours was produced by both dosing regimens, there were some notable differences in organ-specific incidence of tumours. Astrocytomas of the brain and spinal cord were found at a higher incidence in those rats exposed continuously to acrylonitrile administered in the drinking water compared to bolus dosing by gavage. Conversely, a higher incidence of squamous cell carcinomas/papillomas of the forestomach and adenocarcinomas of the intestine and, in females only, carcinomas of the mammary gland were observed in high dose rats receiving acrylonitrile by gavage. An increase in the degree of severity of forestomach hyperplasia was observed in all high dose groups of animals, irrespective of mode of administration. These effects were more pronounced, were correlated with a much higher incidence of forestomach tumours, and were identified earlier (12 months) in the gavage study in which there was direct tissue contact with a more concentrated acrylonitrile solution.
Rat gavage study (Maltoni et al., 1977; 1988)
Acrylonitrile was administered orally to male and female Sprague-Dawley rats by gavage in olive oil, at a single daily dose of 5 mg/kg bw 3 times weekly for 52 weeks. The rats were then observed until spontaneous death occurred. There were no effects on survival or body weight of the test animals. No treatment-related histological changes were observed in liver, kidneys and lung. Acrylonitrile administration did not affect the percentage of animals bearing benign and malignant tumours, the number of animals bearing malignant tumours only, the number of total malignant tumours per 100 animals or the incidence of Zymbal gland carcinomas, extrahepatic angiosarcomas, hepatomas and encephalic gliomas. The only increase in incidence of tumours was in the mammary gland and forestomach of female rats.
Rat inhalation study (Quast et al., 1980a)
Male and female Spartan Sprague-Dawley rats were exposed to acrylonitrile by inhalation at concentrations of 20 or 80 ppm, 6 hours/day, 5 days/week for 2 years. Controls were exposed to air only. Additional animals were included for interim sacrifice at 6 and 12 months. A statistically significant increase in mortality was observed within the first year in both male and female rats administered 80 ppm and in the females of the 20 ppm group during the last 10 weeks of the study. The apparent increase in the reported mortality for the 20 ppm females was principally due to early sacrifice of rats with large, benign, mammary gland tumours. These tumours are known to occur spontaneously in this strain at a high rate, but in this experiment the tumours were observed earlier and more frequently, and became larger in exposed animals. Primary treatment-related effects were observed in the nasal turbinate mucosa of all rats examined in the 80 ppm group as well as in most of the rats in the 20 ppm group. The changes in both groups were qualitatively similar but much less severe in the 20 ppm group than in the 80 ppm group. The main tumours observed in rats exposed to acrylonitrile were microscopic brain tumours and Zymbal’s gland tumours.
Rat inhalation study: Maltoni et al. (1977, 1988)
The effects of inhalation exposure to 5, 10, 20 and 40 ppm acrylonitrile, 4 hours/day, 5 days/week for 12 months were investigated in groups of male and female Sprague-Dawley rats. One group of untreated rats acted as controls. After the 12 month exposure period, rats were kept under observation until spontaneous death. There were no apparent effects on mortality or body weights throughout the study. A statistically significant increase in the percentage of animals bearing benign and malignant tumours (p < 0.01), malignant tumours alone (p < 0.01) and in the number of total malignant tumours per 100 animals was found in several treated groups, although a strong dose-response relationship was not established. Slight to moderate increases in tumour incidence were observed in the mammary gland, forestomach and CNS, but none of these were statistically significant. No increase in Zymbal’s gland tumours, extrahepatic angiosarcomas and hepatomas was observed, 3/60 and 2/60 ecephalic gliomas were observed in animals exposed to the two highest concentrations of acrylonitrile. Whilst this finding did not achieve statistical significance, it is biologically significant given that the brain was clearly shown to be the target organ in rats following oral administration . The authors suggested that the carcinogenicity of acrylonitrile was influenced by the age of the animals at the start of treatment, and was dependent on the concentration administered and duration of treatment. The results were considered by the authors to indicate a borderline carcinogenic effect.
Three-generation study: Friedman & Beliles (2002)
Although not primarily intended to assess carcinogenicity, this three-generation toxicity drinking water study in Sprague-Dawley rats identifies increased tumour incidences, The study evaluated tumour pathology for selected tissues in F0, F1, and F2 adult females exposed for 20 weeks after weaning of the second littering of pups (giving a total exposure period of approximately 1 year). This evaluation was conducted to assess whether there was any increased susceptibility to specific tumour types resulting from perinatal exposure to acrylonitrile. The brain was examined in all adult females surviving to scheduled necropsy; the majority of other tissues were evaluated histopathologically only if they demonstrated gross lesions or masses. A low incidence of astrocytomas and Zymbal’s gland tumors was found in all generations. Tumour incidence was slightly greater in the F1 animals that had perinatal exposure compared to the F0 animals that did not, but was very similar in the F0 animals that did not have exposure in utero or during lactation and the F2 animals that did. It should be noted that the number of animals evaluated was low compared to a standard carcinogenicity evaluation. The findings of this study suggest that in utero or perinatal exposure to acrylonitrile does not lead to an increased incidence of astrocytomas compared to that seen after adult exposure alone.
Human epidemiological studies
The numerous epidemiology studies investigating cancer incidence in exposed workers are individually summarised elsewhere. Individual studies have reported associations between acrylonitrile exposure and increased cancer incidence, however the studies have limitations and more recent updates of individual cohorts have not identified any increase in cancer risk. Recent meta-analyses and reviews have concluded that the existing epidemiology data do not support and increased risk of cancer resulting from acrylonitrile exposure.
Reviews
The peer review of acrylonitrile performed by TERA (2004), noted that the acrylonitrile database contains unusually extensive epidemiology data. It was concluded that no increased cancer risk has been consistently observed in several different large, well-conducted epidemiology studies using several different occupational cohorts in several different countries. Overall, the epidemiological data was noted by the TERA review to have three striking features: (1) the size and completeness of the database; (2) the lack of consistently positive findings across studies; and (3) the lack of a clear dose-response relationship for human cancer. Overall, the panel concluded that the epidemiology data do not support an association between acrylonitrile and increased cancer risk in humans, but that such an association could not be ruled out completely. It was concluded that linear extrapolation from the animal data was not supported by the available epidemiology data and that the overall weight of the evidence suggests that acrylonitrile may be carcinogenic to humans at high doses based on extrapolation from rat studies, but the cancer risk associated with the low levels to which humans have been exposed in occupational settings is negligible. In an assessment of the mechanism and dose-response relationship for carcinogenicity, Kirman et al (2005) also concluded, based on a weight of evidence approach, that the data support a non-linear extrapolation.
Summary of carcinogenicity
It is clear that acrylonitrile is carcinogenic at numerous sites in rodent bioassays: all rodent studies demonstrate a positive response. In contrast, the weight of evidence from numerous epidemiological studies does not support an association between worker exposure to acrylonitrile and increased cancer risk. The reason for the marked difference between occupationally exposed humans and the results of the animal studies is unclear, however this could be due to either the MoA in rats not being relevant to humans, or that acrylonitrile carcinogenicity is a threshold effect and human exposure does not exceed this threshold.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
