Registration Dossier

Administrative data

Description of key information

Repeated exposure to acrylonitrile by the oral route results in damage to the gastrointestinal tract and neurotoxicity. The respiratory tract is affected following repeated exposure by inhalation; the critical effect of repeated inhalation exposure is identified as local irritation.  For repeated dose toxicity by the oral route, the key study is the F344 rat drinking water study of Johannsen & Levinskas (1980), from which a NOAEL of 3 ppm (equivalent to average daily dose levels of 0.25 mg/kg bw/d in males and 0.36 mg/kg bw/d in females) was derived. For repeated dose inhalation toxicity, the key study is the 2 -generation rat study of Nemec et al. (2008), from which a LOAEC of 5 ppm (11 mg/m3) was determined based on irritant effects on the nasal mucosa.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1977-1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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 21-23°C, 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 oral 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 used over the replacement period 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.
Dose / conc.:
0 ppm
Remarks:
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 randomly assigned to study groups 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 is not required for this study type.
Observations and examinations performed and frequency:
Animals were observed twice daily for overt signs of toxicity and mortality. Detailed cage-side examinations, including palpations to identify growths, were performed weekly. Ophthalmoscopic examinations were carried out pretest, and at 6, 12, 18, 23 (females) and 26 (males) months. Individual bodyweights were recorded weekly through the first 14 weeks of the study, bi-weekly between weeks 16 to 26, and monthly thereafter. Food and water consumption (over a 3 day period) was 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 bw/d.

Ten rats/sex/treatment group and 5 rats/sex from the control group were selected for periodic haematology, clinical chemistry and urinalysis pre-test, at 6, 12, 18 months and at study termination. Blood was drawn via the dorsal aorta from lightly anaesthetised 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 (ALT), 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, pituitary, 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:
Bodyweights, 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
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:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Mortality in both 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 controls. Due to low survival in the females at 100 ppm, all surviving females were sacrificed at 23 months. Mortality data are tabulated below.

Body weights for the males and females receiving 100 ppm were consistently lower (p<0.01) than the controls, while bodyweights 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 for the females at 100 ppm was consistently slightly lower than controls on a g/week basis, while this pattern was notable for the males of this group only following the first year of the study. On a g/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 bodyweights 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 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.

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. 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. Urine specific gravity was increased in males receiving 100 ppm at 18 months and at termination. Haematology data for the control and high dose groups are tabulated below.

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. 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. Organ weights and terminal body weights are tabultaed below.
Dose descriptor:
NOAEL
Effect level:
0.25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Equivalent to 3 ppm in the drinking water
Dose descriptor:
LOAEL
Effect level:
0.84 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Equivalent to 10 ppm in the drinking water
Dose descriptor:
NOAEL
Effect level:
0.36 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Equivalent to 3 ppm in the drinking water
Dose descriptor:
LOAEL
Effect level:
1.25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Equivalent to 10 ppm in the drinking water
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 ppm
System:
other: no specific target organ
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

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.

Haematology results

AN dose (ppm)

Study interval (month)

HGB (g%)

HCT (%)

RBC (106/mm³)

Retic. (% RBL)

PT (s)

WBC (10³/mm³)

Malesa

0

6

15.8±0.4

47±1

8.23±0.30

0.9±0.6

22.5±1.7

6.0±1.6

12

16.5±0.5

50±3

8.60±0.29

2.2±0.8

24.8±7.6

6.4±1.2

18

16.0±0.9

47±3

7.70±0.47

2.6±0.5

15.9±1.4

5.3±1.3

T

15.7±3.4

-b

6.55±1.55

2.7±2.7

13.2±0.6

6.9±1.7

100

6

15.7±0.4

48±2

8.41±0.22

1.0±0.6

22.1±2.5

5.6±1.5

12

16.1±0.4

49±1

8.59±0.30

2.2±0.8

22.5±1.8

6.1±0.8

18

15.7±2.8

47±8

7.69±1.53

2.3±1.0

15.8±1.0

5.8±1.8

T

14.3±3.7

-

6.46±1.52

1.9±0.8

13.4±0.6

6.3±2.1

Femalesa

0

6

16.4±0.9

49±4

8.25±0.3

1.0±0.5

18.9±1.8

7.6±2.0

12

16.8±0.4

50±2

8.16±0.27

2.2±0.9

22.4±2.1

5.8±0.9

18

16.2±0.4

47±1

7.53±0.14

2.7±0.7

15.8±1.2

4.7±1.7

T

15.9±0.8

47±3

7.28±0.59

1.2±0.7

13.4±1.8

3.6±0.9

100

6

16.4±0.7

49±3

8.06±0.22

1.0±0.5

20.0±1.8

7.4±1.7

12

16.1±0.8

48±2*

7.89±0.4

3.3±0.7**

24.5±3.2

5.1±1.6

18

14.1±2.8*

42±9

6.32±1.82

3.4±2.8

15.8±0.6

5.5±2.1

T

15.0±1.6

44±8

6.85±1.67

1.2±0.6

13.7±3.4

5.3±5.0

T = terminal sacrifice, * P< 0.05; ** P<0.01

aMean ± SD; n = 9 to 10 rats per group.

bData not recorded.

Selected mean abolsute organ weights at terminal sacrifice

AN dose (ppm)

Terminal body wt (g)

Brain (g)

Pituitary (g)

Adrenal (g)

Testis/ovary (g)

Heart (g)

Kidney (g)

Liver (g)

Malesa

0

318.9±44.8

1.95±0.07

0.0015±0.004

0.068±0.01

8.01±3.4

1.20±0.15

2.8±0.4

10.98±1.76

1

302.9±40.6

1.86±0.10

0.015±0.005

0.080±0.02*

9.80±7.63

1.21±0.16

3.53±1.40*

13.07±0.9*

3

316.8±26.4

1.45±0.07

0.015±0.003

0.069±0.01

8.46±3.13

1.15±0.07

2.87±0.21

11.58±1.31

10

320.2±26.6

1.95±0.07

0.019±0.011

0.069±0.01

7.32±2.65

1.20±0.01

3.09±0.39

13.53±3.63*

30

305.7±36.0

1.95±0.05

0.014±0.002

0.079±0.02*

5.93±4.06

1.16±0.12

2.76±0.26

10.47±1.69

100

283.3±33.4

1.98±0.08

0.016±0.006

0.067±0.01

11.14±0.0

1.18±0.10

2.93±0.65

11.73±1.65

Femalesa

0

254.0±20.4

1.80±0.05

0.011±0.003

0.071±0.008

0.068±0.017

0.95±0.07

1.96±0.14

7.59±1.0

1

264.5±16.2

1.81±0.03

0.012±0.002

0.069±0.007

0.069±0.012

0.91±0.04

1.89±0.13

7.17±0.7

3

263.5±11.1

1.81±0.05

0.010±0.001

0.074±0.008

0.070±0.009

0.93±0.03

2.03±0.32

7.63±1.6

10

250.2±24.6

1.79±0.06

0.010±0.002

0.073±0.010

0.078±0.022

0.93±0.06

1.91±0.16

7.86±1.8

30

251.5±18.1

1.79±0.05

0.010±0.001

0.080±0.016

0.068±0.020

1.28±0.09*

2.04±0.15

8.99±1.6*

100

222.3±38.8**

1.84±0.08

0.011±0.004

0.070±0.011

0.064±0.029

0.95±0.11

1.94±0.17

7.43±1.6

T = terminal sacrifice, * P< 0.05’ ** P<0.01

aMean ± SD; n = 7 to 10 rats per group.

Conclusions:
Due to the lack of a dose response relationship in the female mortality data, the NOAEL was considered to be 3 ppm for both males and females; equivalent to an average daily dose of 0.25 mg/kg bw/d in males and 0.36 mg/kg bw/d in females.
Executive summary:

Groups of F344 rats were exposed to acrylonitrile in the drinking water for approximately 2 years as part of a combined chronic toxicity/carcinogenicity study, at doses of 0, 1, 3, 10, 30 and 100 ppm. The study was terminated at 23 months in females because of low survival rates. The males were exposed for 26 months. A consistent decrease in survival, reduced bodyweight and reduced water intake, and small reductions in haematology parameters were observed in both sexes of the 100 ppm group. Mortality was significantly increased compared to controls in the 100 ppm group, while mortality in the males receiving 10 ppm and the females receiving 3 and 30 ppm was also significantly greater than controls. Organ to body weight ratios at various study intervals were consistently elevated in the high dose groups, and were thought to be related to the lower body weights seen in ths group. Due to the lack of a dose response relationship in the female mortality data, the NOAEL was considered to be 3 ppm for both males and females; equivalent to average daily dose levels of 0.25 mg/kg bw/d in males and 0.36 mg/kg bw/d in females.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
21st November 1995 to 21st February 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
other: NTP protocol
Principles of method if other than guideline:
The study was performed primarily as a range-finder for the subsequent carcinogenicity study
GLP compliance:
yes
Remarks:
: FDA
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
Male and female B6C3F1 mice were obtained from Taconic Laboratory Animals and Services (Germantown, NY). On receipt, the mice were 4 weeks old. The animals were quarantined for 12 (males) or 13 (females) days and were 6 weeks old on the first day of the study. Before the study began, five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease. At 4 weeks, serologic analyses were performed on five male and five female sentinel mice. Feed and water were provided ad libitum. Males were housed individually and females were housed five per cage. The animals were weighed initially, weekly, and at the end of the study. Individuals were identified by tail tattoo. Temperature of the animal room was 22±3°C, relative humidity: 50±15%, fluorescent light was provided 12 hours per day, and there were approximately 10 air changes per hour.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Acrylonitrile was administered in deionised water by gavage. The dosing volume was 10ml/kg bw.
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 deionized water. Stability studies of a 0.8145 mg/mL formulation were performed by the analytical chemistry laboratory 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 at the study laboratory using gas chromatography. During the 14-week study, the dose formulations were analyzed at the beginning, midpoint, and end of the study.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 days per week.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle (water) control
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 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.
Positive control:
Not relevant for this study type.
Observations and examinations performed and frequency:
Mice were observed twice daily; weighed initially, weekly, and at the end of the study; clinical findings were recorded on day 8 and weekly thereafter.
Blood was collected from the retroorbital sinus of all mice surviving to the end of the study (under anaesthesia). The following parameters were measured: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte and platelet morphology; mean cell volume; mean cell haemoglobin; mean cell haemoglobin concentration; total leucocyte and differential counts.
Sacrifice and pathology:
Mice were sacrificed at the end of the 14 week exposure period by carbon dioxide asphyxiation. Necropsies were performed on all animals. Organs weighed were heart, right kidney, liver, lung, spleen, right testis, and thymus. Complete histopathology was performed on 0, 20 (males), 40, and 60 mg/kg mice.
Other examinations:
At the end of the study, sperm samples were collected from up to 10 males in the 0, 5, 10, and 20 mg/kg groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from up to 10 females given 0, 10, 20, or 40 mg/kg for vaginal cytology evaluations. The percentage of time spent in the various oestrous cycle stages and estrous cycle length were evaluated.
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.
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:
effects observed, treatment-related
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
All male and nine female mice in the 60 mg/kg bw/d groups and eight male and three female mice in the 40 mg/kg bw/d groups died on the first day of the study. The mean body weight gain of 20 mg/kg bw/d males was less than that of the vehicle control group. Clinical findings included lethargy and abnormal breathing in the 40 mg/kg bw/d groups immediately after dosing. Surviving mice administered 40 mg/kg bw/d continued to show these signs of toxicity for several days until the mice appeared to develop a tolerance to acrylonitrile.

Leucocyte and lymphocyte counts were decreased in 20 mg/kg males and 40 mg/kg females, and a minimal haemolytic anaemia was observed in 40 mg/kg bw/d females. Heart weights of 20 mg/kg bw/d males were significantly greater than those of the vehicle controls, and left cauda epididymis weights of 10 and 20 mg/kg bw/d males were significantly increased. The incidences of chronic active inflammation and hyperplasia in the forestomach of 40 mg/kg bw/d females were significantly increased.
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Adverse effects were apparent at dose levels of 10 mg/kg bw/d and above.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (actual dose received)
System:
other: no specific system affected
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Collectively, the differences in the survival rates, mean body weight gains, and the increase in heart weights of males suggest that male mice may be more sensitive to acrylonitrile than female mice. Mild haematological effects were observed in mice surviving to the end of the study; however changes were generally minimal and were more prevalent in females.

Conclusions:
60 mg/kg bw/d acrylonitrile by gavage was lethal to all male mice within 24 hours, and to 9/10 female mice. No adverse effects were reported in mice that received 5mg/kg bw/d for 14 weeks.
Executive summary:

In a 14 -week range-finding study, acrylonitrile was administered by gavage to B6C3F1 male and female mice (10/sex/group), 5 days a week for 14 weeks at dose levels of 0, 5, 10, 20, 40 and 60 mg/kg bw/d. Administration of 60 mg/kg bw/d acrylonitrile by gavage was lethal to all male mice and to 9/10 female mice within 24 hours. Clinical findings included lethargy and abnormal breathing in the 40 mg/kg bw/d groups immediately after dosing. Surviving mice administered 40 mg/kg bw/d continued to show these signs of toxicity for several days until the mice appeared to develop a tolerance to acrylonitrile. No adverse effects were reported in mice that received 5 mg/kg bw/d for 14 weeks. Collectively, the differences in the survival rates, mean body weight gains, and the increase in heart weights of males suggest that male mice may be more sensitive to acrylonitrile than female mice. Leucocyte and lymphocyte counts were decreased in 20 mg/kg males and 40 mg/kg females, and a minimal haemolytic anaemia was observed in 40 mg/kg bw/d females. Heart weights of 20 mg/kg bw/d males were significantly greater than those of the vehicle controls, and left cauda epididymis weights of 10 and 20 mg/kg bw/d males were significantly increased. The incidences of chronic active inflammation and hyperplasia in the forestomach of 40 mg/kg bw/d females were significantly increased. A NOAEL of 5 mg/kg bw/d is proposed for this study.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
No further information available.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The vehicle control (distilled water) or acrylonitrile solution was administered via gavage for 5 days/week for 13 weeks.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information available.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily for 5 days per week (for a total of 13 weeks)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle (water) control

Dose / conc.:
1.2 mg/kg bw/day (actual dose received)
Dose / conc.:
2.4 mg/kg bw/day (actual dose received)
Dose / conc.:
4.8 mg/kg bw/day (actual dose received)
Dose / conc.:
9.6 mg/kg bw/day (actual dose received)
Dose / conc.:
12 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 male and 10 female mice per group. Additional male animals (41 to 71 per group) were included for satellite studies (see below).
Control animals:
yes, concurrent vehicle
Details on study design:
The study was a range-finding study conducted to determine dose levels for a subsequent carcinogenicity study. Additional male mice were included in each core study group for the collection of blood and tissue samples to examine acrylonitrile associated cellular proliferation, apoptosis, haemoglobin adduct formation and the production of the acrylonitrile metabolite cyanoethylene oxide (CEO).
Positive control:
A positive control was not included.
Observations and examinations performed and frequency:
Mice were observed for mortality and clinical signs of toxicity. Body weights were also recorded.
Sacrifice and pathology:
Clinical pathology, sperm morphology, vaginal cytology, gross patholoy and organ weights were determined.
Additional male mice were included in each core study group for the collection of blood and tissue samples to examine acrylonitrile associated ccellular proliferation, apoptosis, haemoglobin adduct formation, and production of cyanoethylene oxide.
Other examinations:
No other examinations reported.
Statistics:
Statistical analyses were carried out to evaluate the data.
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:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
All core animals survived, except a single mouse in the 4.8 mg/kg group which was sacrificed in a moribund condition. All mice in the satellite groups survived to scheduled sacrifice. There were no clinical signs of toxicity observed. Sporadic occurences of alopecia seen in several mice was a common background finding and not considered to be treatment related. Normal body weight gains were observed throughout the study, with the exception of one occasion due to a lack of supply of water overnight.

Biologically significant alterations were not detected in any of the haematological parameters. Statistically significant reductions in WBC values were seen in the 2.4 and 9.6 mg/kg males, but were not thought to be treatment-related as there was no dose-relationship. Statistically significant elevations in WBC values were seen in the 4.8 and 12.0 mg/kg females, but again there was no dose-relationship and values remained within the normal range for historical controls, so the effect was not thought to be biologically significant. A statistically significant increase in the mean HCT value in the 9.6 mg/kg females lay within the normal range. Statistically significant declines in lymphocyte counts present in the 1.2, 2.4 and 9.6 mg/kg males were not believed to be of biological significance because there was no dose-relationship and the values were within the normal range. Significant elevations in lymphocyte counts evident in the 4.8 and 12.0 mg/kg females, and an elevation in neutrophils in the 12.0 mg/kg females were believed to be within the normal biological variation range.

No treatment-related gross lesions were noted. There were isolated findings in males of preputial gland cysts and enlarged inguinal lymph nodes. There were isolated findings in females of ovarian cysts ad foci of ovarian tissue. These findings in males and females were considered to be unrelated to treatment, and are normal incidental findings in mice of this age and strain. A single tumour was observed in the study. The ovarian tumour was diagnosed as an ovarian choriocarcinoma, a germ cell tumour with trophoblastic differentiation. Although these tumours are rare, several have been reported in the strain of mouse in other studies. The tumour occurred in a control mouse, and therefore could not be compound related.

Histopathological findings in tissues also indicated that there were no effects of treatment. Epididymal sperm motility was significantly decreased, compared to controls, at the lowest and highest dose levels. As no other dose levels were affected, the finding was not thought to be bioloigcally significant. There were no other sperm morphology or vaginal cytology parameters affected at any dose level.
Dose descriptor:
NOAEL
Effect level:
12 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects of treatment were seen at the highest dose level of 12 mg/kg bw/d
Dose descriptor:
LOAEL
Effect level:
> 12 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects of treatment were seen at this (the highest) dose level
Key result
Critical effects observed:
no

No effects of treatment were seen in this study.

Conclusions:
A NOAEL of 12.0 mg/kg bw.d can be determined in the absence of any treatment-related effects.
Executive summary:

B6C3F1 male and female mice (10/sex/group) were administered acrylonitrile by gavage (in distilled water) on 5 days/week for 13 weeks at dose levels of 0, 1.2, 2.4, 4.8, 9.6, and 12.0 mg/kg bw/d for 13 weeks in order to determine dose levels for a subsequent carcinogenicity study.  Animals were assessed for survival, clinical observations, bodyweights, clinical pathology, sperm morphology and vaginal cytology, gross and microscopic pathology and organ weights. Additional male mice were included in each group for the collection of blood and tissue samples for special investigations to examine cellular proliferation, apoptosis, haemoglobin adduct formation, and the production of the metabolite CEO. No treatment-related effects on survival, clinical observations, bodyweights, clinical pathology, sperm morphology and vaginal cytology, gross or microscopic pathology or organ weights were observed.  Isolated (and statistically significant) effects on several parameters were noted but considered to be unrelated to treatment. All animals survived the 13 weeks of treatment. With the exception of a single male mouse in the group at 4.8 mg/kg bw/d (sacrificed in a moribund condition), all males assigned for special investigations all survived to scheduled sacrifice.  This single death in an intermediated dose group is not considered to be treatment-related.  No treatment-related signs of toxicity were reported. Alopecia was seen sporadically in several mice, but is a common background finding and was not considered to be treatment-related.  Normal bodyweight gains were achieved except on occasion once due to a lack of water supply overnight. Biologically significant alterations were not detected in any of the haematological parameters evaluated in mice of either sex. Statistically significant decreases in white blood cell values were seen males at 2.4 and 9.6 mg/kg bw/d but were not considered to be biologically significant in the absence of a dose-response relationship. Significantly elevated WBC counts were apparent in females at 4.8 and 12.0 mg/kg bw/d but were also not dose-related and were noted to be within the historical control range. A significantly increased mean haematocrit value seen in 9.6 mg/kg bw/d females was within the laboratory’s normal range. Statistically significant declines in lymphocyte counts present in males at 1.2, 2.4, and 9.6 mg/kg bw/d were not considered to be of biological significance in the absence of a dose-response relationship; values are reported to be within the normal range. Similarly, significant elevations in lymphocyte counts in 4.8 and 12.0 mg/kg bw/d females and elevated neutrophil count in 12.0 mg/kg bw/d females is within the normal biological variation range. Gross necropsy did not reveal any effects of treatment: isolated findings of preputial gland cysts and enlarged inguinal lymph nodes (males); ovarian cysts and foci of ovarian tissue in females were considered to be unrelated to the treatment since these lesions are normal findings in mice of this strain and age. A single ovarian tumour (choriocarcinoma, a germ cell tumour with trophoblastic differentiation) occurred in a control mouse in this study. Although these tumours are rare, several have been reported in B6C3F1 mice in studies conducted by the National Cancer Institute (NCI) and National Toxicology Program (NTP). Histopathology indicated no treatment-related effects. Investigations of sperm morphology and vaginal cytology, showed a significant decrease in epididymal sperm motility at the lowest dose level of 1.2 mg/kg bw/d and also at the highest dose levels of 12.0 mg/kg bw/d. Similar findings were not apparent at the intermediate dose levels of 2.4, 4.8, and 9.6 mg/kg bw/d; changes are not considered to be treatment-related in the absence of a clear dose-response relationship. No other parameters were significantly affected at any dose level. A NOAEL of 12.0 mg/kg bw/d can be determined for this study in the absence of any treatment-related effects.

Endpoint:
chronic toxicity: oral
Type of information:
other: expert revoew / secondary source
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
no guideline followed
Principles of method if other than guideline:
The EU RAR reports the results of a number of largely non-standard repeated dose oral toxicity studies in the rat and mouse.
GLP compliance:
no
Limit test:
no
Species:
other: various species were used in the reviewed studies
Route of administration:
other: gavage, drinking water
Details on oral exposure:
In a 90-day rat study (Humiston & Frauson, 1985), adult Sprague-Dawley rats received acrylonitrile in drinking water up to a dose equivalent of 42
mg/kg bw/d.

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days (Humiston & Frauson, 1975)
7 weeks (Barnes, 1970)
180 days (Quast et al, 1975)
Frequency of treatment:
Continous / daily (Humiston & Frauson, 1975)
Daily (Barnes, 1970)
Continous / daily (Quast et al, 1975)
Remarks:
Doses / Concentrations:
10, 17, 22, 38, 42 mg/kg bw/d
Basis:
other: approximate dose level based on water concentration (Humiston & Frauson, 1975)
Remarks:
Doses / Concentrations:
30, 50, 75 mg/kg bw/d
Basis:
other: actual gavage dose (Barnes, 1970)
Remarks:
Doses / Concentrations:
8-10, 16-17, 17-18 mg/kg bw/d
Basis:
other: approximate dose level based on water concentration (Quast et al, 1975)
No. of animals per sex per dose:
No details
Details on study design:
90-day drinking water (Humiston & Frauson, 1975)
7 week dose escalation (Barnes, 1970)
180 day drinking water (Quast et al, 1975)
Positive control:
Not required
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
: reduced weight gain at 22 mg/kg bw/d and above
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
: reduced food consumption at 17 mg/kg bw/d and above.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
: reduced water consumption at 10 mg/kg bw/d and above
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Description (incidence and severity):
: increased liver weights at 10 mg/kg bw/d and above
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Details on results:
90-day rat drinking wtaer study (Humiston & Frauson, 1975)

Treatment-related effects in Sprague-Dawley rats receiving acrylonitrile in drinking water up to a dose equivalent of 42 mg/kg bw/d weight for 90 days. Reduced water consumption was observed at dose levels above 10 mg/kg bw/d, while growth retardation occurred at levels of 22 mg/kg bw/d and higher in female rats and at 42 mg/kg bw/d in male rats. Food consumption was reduced for the first 7 weeks of the study at 38 mg/kg bw/d, while at a dose level of 17 mg/kg bw/d it was reduced in the first 2 weeks. Increased relative liver weight was observed at 10 mg/kg bw/d and above.

7-week dose escalation study (Barnes (1970)

No effects on body weight and no neurotoxic effects (i.e., gait, hindlimb activity, etc.) were observed in this study.

6-month dog study (Quast et al, 1975)

At 100 mg/l, a slight decrease in water and food intake and a slight increase in relative kidney weight was observed. Five dogs died, or were sacrificed because of debilitation, in each of the two higher dosage groups. In the dogs receiving 100 to 300 mg/l in the drinking water, early signs of toxicity included roughening of the coat and, later, retching, and vomiting. Terminal signs of lethargy, weakness, emaciation, and respiratory distress were noted.
Dose descriptor:
LOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased liver weights are reported at dose levels of 10 mg/kg bw/d and above; 90-day rat study (drinking water)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
The results of these studies confirm the repeated dose oral toxicity of acrylonitrile.
Executive summary:

A number of additional repeated dose oral toxicity studies are summarised in the EU RAR.

In a 7 -week dose escalation study (Barnes, 1970), groups of 6 rats were gavaged with acrylonitrile at a dose level of 30 mg/kg bw/d (15 doses), 50 mg/kg bw/d (7 doses) and 75 mg/kg bw/d (13 doses). No effects on body weight and no neurotoxic effects (i.e., gait, hindlimb activity, etc.) were observed in this study.

In a 90 -day rat drinking water study (Humiston & Frauson, 1975), adult Sprague-Dawley rats received acrylonitrile at dose levels equivalent to up to 42 mg/kg bw/d. Reduced water consumption was observed at dose levels above 10 mg/kg bw/d, while growth retardation occurred at levels of about 22 mg/kg bw/d and higher in female rats and at 42 mg/kg bw/d in males. Mean weekly food consumption was reduced for the first seven weeks of the study at a dose level of 38 mg/kg bw/d. At a dose level of 17 mg/kg bw/d, it was reduced in the first two weeks. Increased relative liver weight was observed at dose levels of 10 mg/kg bw/d and higher.

In a 6 -month dog study, Quast et al (1975), administered acrylonitrile in drinking water at concentrations of 100, 200, or 300 mg/L to groups of four male and four female beagle dogs. Average intakes for males were 10 mg/kg bw/d at 100 mg/L, 16 mg/kg bw/d at 200 mg/L and 17 mg/kg bw/d at 300 mg/L. For females, the corresponding average intakes were 8 mg/kg bw/d at 100 mg/L, 17 mg/kg bw/d at 200 mg/L and 18 mg/kg bw/d at 300 mg/L. At 100 mg/L, a slight decrease in water and food intake and a slight increase in relative kidney weight was observed. Five dogs died, or were sacrificed because of debilitation, in each of the two higher dosage groups. In the dogs receiving 100 to 300 mg/L in the drinking water, early signs of toxicity included roughening of the coat and, later, retching, and vomiting. Terminal signs of lethargy, weakness, emaciation, and respiratory distress were noted.

Working et al (1987) administered acrylonitrile by gavage to groups of 10 male rats at levels of 45, 60, 68, 75, and 90 mg/kg bw/d for five days. Some treated animals died during or immediately after dosing at all dose levels above 60 mg/kg bw/d. All rats treated with 60 mg/kg bw/d survived the entire 42-day observation period. While no deaths were observed at the 60 mg/kg bw/d dose level, decreases in body weight were present for three to four weeks after dosing. Four animals receiving 68 mg/kg bw/d died on the first day after dosing.

Endpoint:
sub-chronic toxicity: 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
Principles of method if other than guideline:
The authors carried out a number of studies in order to elucidate the sub-acute and chronic actions of acrylonitrile on the adrenals, stomach, and duodenum of experimental animals by correlation of biochemical, functional, and morphological findings, and to gain insight into mechanisms of action.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
female
Route of administration:
other: drinking water and gavage
Vehicle:
water
Details on oral exposure:
Rats were exposed to 0, 0.0001% (1 ppm), 0.002%, 0.01%, 0.05% or 0.2% acrylonitrile in drinking water, or to the same amount of the chemical given through daily gavage, for 7, 21 or 60 days.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
7, 21 or 60 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 0.0001%, 0.002%, 0.01%, 0.05%, 0.2%
Basis:
nominal in water
No. of animals per sex per dose:
3-5 (females)
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Treatment-related effects occurred consistently at the 100 ppm level via drinking water, with 20 ppm representing a NOAEL (equivalent to an intake of 4 mg/kg bw/d).
Critical effects observed:
not specified

In general, no overt signs of intoxication was noted and mortality only occurred in the 2000 ppm dose group, in which 2/18 rats died from severe bilateral adrenal hemorrhage and necrosis. Decreased water and food intake was observed in both the 2000 and the 500 ppm drinking water groups and following 100 mg/kg bw twice daily by gavage. Adrenal weights were decreased in 7, 14, and 21 day studies in rats receiving 500 and 2000 ppm in drinking water, accompanied in the 2000 ppm group by polyuria. Pair-fed controls to the 2000 ppm group also showed a decreased relative adrenal weight, but urinary output was normal. However, animals given the equivalent of 2000 ppm (100 mg/kg bw twice daily) by gavage showed an enlargement of the adrenals, again accompanied by polyuria. Following 60 days administration in drinking water, there was also a significant increase in adrenal weight that was particularly prominent in the group given 60 mg/kg bw (equivalent to 500 ppm) daily. Histological examination of the adrenals from rats administered 500 and 2000 ppm in drinking water for 7, 14, or 21 days revealed atrophy in the adrenal cortex (especially the zona fasciculata). In contrast, cellular hyperplasia with normal size or slightly shrunken cells was seen in the adrenals from rats given equivalent amounts by gavage and in animals administered 500 ppm in drinking water for 60 days.

Plasma levels of corticosterone showed a dose-dependent decrease in rats administered 100, 500 or 2000 ppm in drinking water, with larger decreases being seen when acrylontrile was administered by gavage. The decrease noted in the 2000 ppm group (14 days administration) was even more marked in pair-fed controls. Plasma aldosterone levels were less affected by administration of acrylonitrile Effects were only seen at high levels and after prolonged exposure. A significant decrease was observed only after administration by gavage of 60 mg/kg bw for 60 days.

Other effects reported in these studies were increased liver weights following a 21-day administration period, with a decrease being reported after 60 days. Kidneys were enlarged in the 100 ppm group after 60 days of administration and in the 500 ppm group after 21 days. Hyperplasia was observed in regions of the gastric mucosa of rats receiving 100 and 500 ppm in drinking water for at least 21 days. Treatment-related effects occurred consistently at the 100 ppm level via drinking water, with 20 ppm representing a NOAEL (equivalent to an intake of 4 mg/kg bw/d).

Conclusions:
The results suggest that the effects of acrylonitrile on the adrenals were in part attributable to its inherent toxicity and the consequences of decreased food and especially water intake (probably due to its unpalatability in drinking water even at 20 ppm).
Executive summary:

The stated aim of this study was to investigate the sub-acute and sub-chronic toxicity of acrylonitrile to the adrenals, stomach and duodenum by correlating biochemical, functional and morphologic investigations, and to elucidate the mechanism of toxicity of acrylonitrile. Rats were exposed to 0, 0.0001% (1 ppm), 0.002%, 0.01%, 0.05% or 0.2% acrylonitrile in drinking water, or to the same dose level administered by gavage, for 7, 21 or 60 days. Acrylonitrile caused a time- and dose-dependent decrease in plasma corticosterone levels; aldosterone was affected only by the high dose level and prolonged time of exposure. Young rats were noted to be more susceptible than adults to this action of acrylonitrile. The adrenal cortex, especially the zona fasciculata, was atrophic in rats exposed through drinking water. At dose levels of 0.05% and 0.2%, administration also caused decreased food intake and body weight gain. The adrenals were enlarged with a hyperplastic zona fasciculata after daily gavage doses of acrylonitrile. Ingestion of the chemical did not interfere with compensatory enlargement of the adrenal gland following unilateral adrenalectomy. On the other hand, the ACTH-induced elevation of corticosterone plasma concentration was significantly attenuated by acrylonitrile in drinking water. Electron microscopy of the adrenal glands revealed no consistent changes in the steroid-producing cells. The authors postulate that accelerated turnover of circulating corticoids and/or interference with the secretion or action of ACTH may primarily be responsible for the decreased plasma levels of corticosterone and aldosterone in rats that ingest acrylonitrile. The mucosa in the stomach at the junction of the forestomach and glandular region of animals that had ingested acrylonitrile was hyperplastic. The corpus also showed regional mucosal hyperplasia. Changes were associated with an elevated concentration of non-protein sulphydryls mostly in the mucosa of the glandular stomach. A similar, less prominent elevation also occurred in the proximal duodenum. The results of the study suggest that the effects of acrylonitrile on the adrenals were in part attributable to its inherent toxicity and the consequences of decreased food and especially water intake (probably due to its unpalatability in drinking water even at 20 ppm)

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
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:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Principles of method if other than guideline:
2 year oral (drinking water) combined chronic toxicity and oncogenicity study
GLP compliance:
yes
Remarks:
the original study was performed to GLP, the study is also reported in a published paper
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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 21-23°C, 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 oral exposure:
Rats were exposed to acrylonitrile in their drinking water continuously for up to 2 years. The dose levels 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
Dose / conc.:
0 ppm
Remarks:
Control: untreated drinking water
Dose / conc.:
35 ppm
Remarks:
In drinking water: throughout the study period
Dose / conc.:
100 ppm
Remarks:
In drinking water: increased from 85 ppm on Day 22
Dose / conc.:
300 ppm
Remarks:
In drinking water: increased from 210 ppm on Day 22
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 sub-chronic 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 required for this study type
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:
Mortality

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 tabulated below.

Clinical investigations

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 (nutritional anaemia.).

Bodyweights

Mean bodyweights of males and females were adversely affected early in the study with the magnitude of the difference increasing in severity throughout the study. The decreased weights were dose related and associated with increasing dose level. Mean bodyweights in the 300 ppm group were significantly decreased within the first 5 days and remained so throughout the study. Within the first month, bodyweights of 100 ppm male and female rats were also significantly decreased. Bodyweights of 35 ppm rats were generally decreased throughout the study with fewer time intervals identified as statistically significant when compared with the 100 and 300 ppm groups. Near the end of the study individual and mean group body weights were more variable due to age-related changes, fewer surviving rats, and a greater number of rats bearing different sized single or multiple tumours. Body weight data are tabulated below.

Food and water consumption

In general, food consumption was decreased in a dose-related manner. The effect on food consumption was immediately evident and was significantly decreased in the 100 and 300 ppm groups within the first week and remained so throughout the study. In 300 ppm rats their feed intake (g/rat/day) was decreased compared to controls at 23/24 measurements, and was identified as statistically significantly decreased 12/24 times in males and 16/24 times in females. The 35 ppm rats usually ate less than controls, however it was only occasionally identified as statistically significant in females. In general, the decreased feed intake was more apparent in all female treated groups than males when compared with their respective controls.

Both male and female rats restricted their water intake in a dose-related fashion at all treatment levels. Within 3 test days, water consumption was significantly decreased in all dose groups except the 35 ppm males. During the first 10 days males and females in the 300 ppm group decreased their water intake by 36 and 38%, respectively. Water consumption was measured 26 times during the study and it was significantly decreased in males at 35 ppm (13/26), 100 ppm (24/26), and 300 ppm (26/26); and in females at 35 ppm (21/26), 100 ppm (23/26) and 300 ppm (26/26). Water consumption is tabulated below.

Clinical signs

The clinical appearance and behaviour of the rats was normal early in the study, however some rats in the two higher dose groups developed an 'unthrifty' appearance later in the study due to reduced weight gain. It was thought likely that the marked stress associated with the significantly decreased water consumption, food consumption, body weights, moribundity, and complications due to multiple tumours may have significantly influenced the incidence of clinical signs of the rats. During the study, control and treated rats exhibited clinical signs of erratic movements, trembliing, circling, and limb weakness suggestive of nervous system dysfunction. These observations were more frequently noted in males than in females, despite the higher intake in females. Generally in control rats, the clinical signs were correlated with necropsy observations of end stage kidney disease or with a pituitary tumour. However, an occasional male rat of the higher dose groups had the same clinical signs in the absence of severe kidney disease or a pituitary tumour.

Organ weights

The mean fasted bodyw eights, organ weights and organ to bodyweight ratios were unaffected in the few surviving male and female rats in the 35 and 100 ppm groups. No rats in the 300 ppm group survived to study termination. The fasted body weights of the treated rats were slightly lower than controls and were consistent with their lower in-life body weights.

Gross pathology

The gross pathologic observations on the ten rats per sex per dose necropsied for the 1 year interim study identified treatment-related effects in the forestomach of males and females in the 100 and 300 ppm groups. The forestomach of males in the 100 and 300 ppm groups was pale and thickened in 1/10 (100 ppm) and in 3/10 (300 ppm), while in females it was observed in 2/10 (100 ppm) and 3/10 (300 ppm). Gastric erosion or ulcer was also observed in the forestomach of 1 male in each of the 100 and 300 ppm groups. At study termination (2 years), the primary adverse treatment related effect in both sexes involved the stomach. The changes were located in the non-glandular forestomach, and were characterised by hyperplasia and/or hyperkeratosis suggestive of chronic irritation. Other observations in the stomach (gastric erosions/ulcers, focal white plaque formation, decreased feed, and blood or bile present) may have been affected by treatment, however, they were reported to generally reflect changes associated with other organ system effects, tumours, or general moribundity.

The results of microscopic findings in the ten rats per sex per dose from the 1-year interim study identified the forestomach as the only tissue with non-tumorous changes. There was squamous cell hyperplasia of the forestomach of males in 4/10 (100 ppm) and 10/10 (300 ppm), while in females 7/10 (100 ppm) and 9/10 (300 ppm) were affected. At study termination (2 years), numerous microscopic diagnoses were made in various tissues. Squamous cell epithelial hyperplasia and/or hyperkeratosis of the forestomach in all dose groups of both sexes was significantly increase, although not identified statistically so in the 35 ppm males. The epithelial changes in the forestomach of some rats were also associated with inflammation and focal ulceration due to acrylonitrile irritation. In the brain, gliosis with or without perviascular cuffing was significantly increased in the low and middle dose females, however, even though the incidence in 300 ppm females was not statistically significant it was interpreted as of toxicological significance. The incidence for the observation in males was not statistically significant, but did show a dose response relationship so again was considered to be of toxicological relevance. Other non-tumorous histopathological changes were observed, but were not thought to be treatment related.

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.

Neoplastic pathology, histpathology and tumour data are presented in detail in IUCLID section 7.7
Dose descriptor:
NOAEL
Effect level:
< 3.4 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Equivalent to 35 ppm in the drinking water
Dose descriptor:
NOAEL
Effect level:
< 4.4 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Equivalent to 35 ppm in the drinking water
Dose descriptor:
LOAEL
Effect level:
3.4 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Equivalent to 35 ppm in the drinking water
Dose descriptor:
LOAEL
Effect level:
4.4 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Equivalent to 35 ppm in the drinking water
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
35 ppm
System:
other: no specific effects
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Mortality data

Dose level (ppm)

Sex of animal

No. animals

No. 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)

Mean body weights (g)

Days on test

Dose level (ppm)

Males

Females

0

35

100

300

0

35

100

300

0

312±21

306±26

309±17

309±20

212±13

206±10*

207±10*

209±11

5

340±18

331±13

329±14

314±20*

224±17

225±11

217±8

211±8*

12

366±20

355±20

349±16

339±20*

237±19

236±12

227±9

223±11*

19

363±30

334±32*

345±20

325±35*

239±22

237±16

231±15

227±9

28

397±27

377±40*

376±27*

350±26*

252±15

245±12*

242±13*

236±14*

33

421±26

401±17

395±21*

374±30*

261±18

259±17

256±10

240±14*

40

442±29

427±19

410±23*

390±32*

273±19

270±13

261±11

247±13*

47

453±33

438±43

427±24*

391±28*

274±17

272±13

265±13

244±16*

56

459±33

447±37

434±33*

400±28*

274±15

268±14

263±15*

249±17*

61

473±30

458±25

438±27*

412±35*

283±20

278±16

269±12

255±14*

68

480±30

469±24

444±34*

421±31*

284±20

274±18

270±13

256±15*

75

500±30

488±27

461±40*

437±34*

292±21

288±18

282±15

264±13*

82

504±32

492±26

467±35*

433±40*

297±22

289±16

284±14

261±12*

91

510±38

497±44

479±37*

438±36*

299±18

289±16*

281±16*

267±16*

105

524±41

511±45

493±41

449±38*

305±17

298±15*

289±16*

273±17*

110

532±36

522±29

482±39*

448±47*

312±24

303±19

292±12*

272±16*

117

538±44

524±48

497±45*

454±37*

312±19

301±17

289±20*

273±17*

124

555±41

530±48

485±47*

458±36*

315±24

306±16

292±19*

273±17*

145

564±48

543±52

509±47*

469±38*

320±20

310±17

297±17*

279±17*

174

584±50

561±55*

523±48*

475±36*

322±22

313±19

301±19*

282±18*

208

597±53

571±57*

536±44*

487±39*

337±23

321±22

311±24*

292±22*

238

617±56

592±58*

557±48*

502±40*

341±24

331±22

317±24*

299±22*

272

636±60

612±62

568±48*

509±40*

349±25

338±23

322±23*

301±28*

301

649±55

621±63*

571±60*

516±43*

357±34

347±31

326±29*

308±39*

329

647±53

626±61

583±50*

518±40*

367±31

353±28*

333±34*

302±28*

363

652±50

615±71*

579±51*

511±45*

368±37

356±35

334±44*

300±30*

392

654±52

633±63

581±61*

513±55*

379±46

357±33*

339±34*

320±40*

427

670±52

629±71*

592±54*

507±63*

384±42

368±49

351±46*

330±64*

455

651±56

608±62*

582±60*

497±63*

384±51

365±49

361±80

318±56*

482

636±58

599±62*

568±67*

482±70*

384±45

379±59

350±44*

337±73*

509

633±59

591±70*

570±62*

495±60*

387±44

391±74

364±60

318±53*

549

632±59

564±86*

582±67*

491±69*

404±53

395±88

372±48

352±87*

573

632±56

590±53*

581±75*

483±73*

413±60

377±57

377±45

354±52*

601

617±61

557±65*

584±81

492±93*

422±82

397±75

423±79

352±49*

634

593±66

538±69*

540±60*

443±89*

422±63

425±105

433±99

346±44*

665

572±93

552±80

528±60

444±90*

415±72

397±93

431±85

354±107

692

571±49

523±57

515±72

-

419±92

418±97

535±109

-

738

532±65

465±66

511±72

-

370±64

357±96

389±-

-

746

502±66

458±45

469±81

-

360±61

340±88

341±-

-

Each value represents mean ± SD for a minimum of 15 animals during the first 110 days, 30 animals on day 124, and all animals thereafter. – Indicates no data available.

* Significantly different from control mean by Dunnett’s test, p<0.05.

Mean daily water consumption (g per rat per day)

Days on test

Dose level (ppm)

Males

Females

0

35

100

300

0

35

100

300

0-3

47±4

44±6

40±3*

29±4*

35±3

33±2*

31±2*

22±2*

4-6

52±5

46±4*

44±6*

36±4*

41±3

37±3*

32±2*

24±1*

7-10

50±4

43±4*

37±2*

31±2*

38±6

33±3*

23±2*

25±4*

11-13

53±5

47±4*

39±4*

32±4*

43±5

36±3*

29±2*

23±3*

18-20

50±6

36±5*

42±3*

30±5*

45±5

33±3*

32±4*

23±2*

25-27

50±5

47±4

40±5*

33±3*

41±10

38±5

30±5*

22±5*

32-34

52±4

42±5*

38±3*

27±8*

40±9

33±3

23±9*

22±2*

39-41

48±5

44±6*

37±2*

31±2*

41±3

34±2*

28±2*

23±3*

46-48

51±8

43±5*

39±4*

32±3*

47±5

35±2*

30±2*

24±1*

53-55

47±5

42±6

37±5*

30±4*

43±6

35±3*

28±2*

23±3*

60-62

47±6

42±4

39±6*

32±4*

40±5

36±4

29±2*

25±8*

67-69

46±7

41±4

36±8*

31±6*

44±6

34±5*

40±3*

21±3*

74-76

48±6

40±7*

37±10*

32±3*

42±3

36±5*

29±3*

23±3*

81-83

48±5

43±5*

40±5*

32±4*

45±5

38±3*

31±3*

23±3*

88-91

47±6

43±4*

37±3*

30±2*

42±3

35±4*

28±5*

24±2*

119-122

48±7

39±4*

38±6*

30±4*

41±9

33±3*

28±4*

22±3*

147-150

45±5

40±5

36±5*

28±4*

41±5

33±4*

28±3*

22±1*

175-178

45±6

41±5

35±4*

28±2*

38±11

33±5*

27±3*

21±3*

210-213

50±6

45±5

38±3*

33±4*

45±8

38±9*

32±5*

30±4*

273-276

46±5

41±7*

37±5*

29±3*

45±6

35±3*

30±4*

23±3*

340-342

55±8

48±9*

40±6*

32±5*

50±6

42±5*

35±3*

26±3*

364-367

53±6

50±7

41±6*

36±5*

50±4

42±6*

34±7*

26±4*

455-458

51±19

39±7

37±14*

33±11*

42±8

33±7*

31±5*

25±8*

543-545

60±19

66±19

49±14

39±13*

60±12

48±11

41±11

27±8*

634-637

48±18

46±12

34±10*

27±11*

44±13

32±15

33±15

16±4*

739-741

72±13

78±8

65±23

-

61±14

47±10*

57-

-

Each value represents mean ± SD for 15 cages (initially 2 rats per cage). – Indicates no data available.

* Significantly different from control mean by Dunnett’s test, p<0.05.

Conclusions:
Exposure to acrylonitrile in the drinking water for 2 years resulted in higher mortality and numerous toxic effects. 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 in a combined chronic toxicity/carcinogenicity study. 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 imitation and persisted throughout the study in all treatment groups, with females being more severely affected than males. Clinical observations showed that treated animals were unthrifty (lack of normal growth), 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 initially noted in the highest dose level rats, however the same observations occurred at the lower doses as the study progressed. Haematology, clinical chemistry, urinalysis and organ weights at study termination were not significantly affected. Non-neoplastic and neoplastic lesions were found at an increased incidence in a number of tissues of both sexes at all treatment levels. The primary non-neoplastic effects of exposure occurred in the forestomach (hyperplasia and/or hyperkeratosis suggestive of chronic irritation) and the central nervous system (gliosis with or without perivascular cuffing) of rats of both sexes and involved all treatment groups. Due to treatment-related effects in all exposed groups, A NOAEL could not be identified for this study. The LOAEL for this study is therefore a drinking water concentration of 35 ppm, calculated to be equivalent to dose levels of 3.4 and 4.4 mg/kg bw/d acrylonitrile in males and females respectively.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
0.25 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Reliable data are available in the rat and mouse, using gavage or drinking water administration and study durations of up to 2 years.
System:
gastrointestinal tract
Organ:
stomach

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
Rats were exposed to acrylonitrile by inhalation as part of a 2-generation reproductive toxicity study
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Crl:CD(SD) Sprague-Dawley albino virgin male and female rats, obtained from Charles River Laboratories, NC. Rats were acclimated for 14 days, during which they were observed twice daily for mortality and moribundity. Rats were groups housed by sex for 3 days, then housed individually (except during mating) in suspended wire mesh cages. Following mating females were transferred to plastic maternity cages with nesting material. Basal diet (PMI Nutrition International, Certified Rodent LabDiet 5002) and reverse osmosis treated water were available ad libitum, except during exposure. Animals were maintained on a 12 hour photoperiod, at 71◦F ± 5◦F and 30% to 70% humidity. The F0 generation was approximately 8 weeks old at initiation of exposure, the F1 generation approximately 4 weeks old. Animals in the study were maintained in accordance with the Animal Welfare Act (1966) and the Guide for the Care and Use of Laboratory Animals.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No information available
Details on inhalation exposure:
Each group of animals was exposed to acrylonitrile vapour in a 2-cubic-metre stainless steel and glass whole-body inhalation chamber operated under dynamic conditions. Chamber temperature (20-25°C), relative humidity (30-70%), ventilation (12-15 air changes per hour), and negative pressure within the chambers were monitored. Cages were sequentially rotated around the available rack positions within the chamber on a daily basis, to minimise any potential variation due to positioning. The control group was exposed to clean filtered air under identical conditions to those used for the acrylonitrile exposure groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure concentrations of the vapour were measured approximately every 35 minutes (i.e. 9 to 10 times) during each daily exposure period via gas chromatography, using sensors placed approximately in the centre of the chamber, within the general breathing zone of the animals.
Duration of treatment / exposure:
Rats were exposed for 6 hours a day, 7 days a week, for 10 weeks prior to mating, during mating, gestation and lactation/.
Frequency of treatment:
Daily.
Dose / conc.:
0 ppm
Dose / conc.:
5 ppm
Dose / conc.:
15 ppm
Dose / conc.:
45 ppm
Dose / conc.:
90 ppm
No. of animals per sex per dose:
25 rats per sex per concentration
Control animals:
yes
Details on study design:
The study was perfomed as a two-generation reproductive toxicity study. The general toxicity data presented here are from the parental generation.

25 males and 25 females (F0 generation) in each of 5 groups were exposed to acrylonitrile (0, 5, 15, 45 or 90 ppm) 6 hours a day, 7 days a week for 10 weeks. The males were exposed for 10 weeks prior to mating and throughout mating until 1 day prior to euthanasia. The females were exposed for 10 weeks prior to mating and throughout mating, gestation, and lactation until 1 day prior to euthanasia. Exposure of the dams was suspended for 5 days following parturition (lactation days (LDs) 0 to 4) to avoid confounding nesting and nursing behaviour and neonatal survival. Exposure of the dams resumed on LD5, they were removed from the litters for 6 hours exposure at about the same time each day.

Animals found to be in good general health were allocated to groups based on body weight stratification and randomised in a block design by a computer generated program.
Positive control:
Not required for this study type
Observations and examinations performed and frequency:
Detailed physical examinations were recorded weekly for all parental animals (F0). All animals were observed twice daily for appearance, behaviour, moribundity, mortality and pharmacotoxic signs prior to exposure and within 1 hour after exposure. Females were also observed twice daily during the period of expected parturition for dystocia or other difficulties.

Individual body weights were recorded weekly throughout the study and prior to scheduled necropsy. Individual female bodyw eights were recorded weekly until evidence of copulation was observed and on GDs 0, 4, 7, 11, 14 and 20, and on LDs 1, 4, 7, 14, 21 and 28.

Parental food consumption was determined on the same days as the body weight measurements, except during the mating period when measurement of food consumption was suspended due to cohabitation.

Plasma and red blood cell (RBC) cholinesterase determinations were conducted on 10 rats/sex of the F0 parental generation from the control and 90 ppm groups, and from 10 rats/sex of the F1 parental generation from the control, 5, 15 and 45 ppm groups. Blood samples were collected from the tail vein following the daily 6 hour exposure 2 days prior to scheduled euthanasia. EDTA was used as the anticoagulant.
Sacrifice and pathology:
Surviving F0 adults were euthanised and necropsied following completion of weaning of their offspring (F1 and F2 pups respectively). Selected F0 and F1 parental tissues and organs were fixed by immersion in 10% neutral-buffered formalin for possible histopathological examination. Microscopic evaluations were performed on the following tissues for 10 randomly selected parental animals per sex (with confirmed sire or pregnancy) from the control and high-exposure groups: adrenal glands, prostate, brain, pituitary, seminal vesicles, right epididymis (caput, corpus and cauda), right testis, vagina, cervix, coagulating gland, uterus, oviducts, and ovaries (one section from each ovary was examined). Nasal cavities, lungs and gross lesions from all control, 5, 15 and 45 ppm groups were examined microscopically. Periodic acid-Schiff (PAS) and haematoxylin staining were used for the right tests and epididymis and haematoxylin-eosin staining was used for all other tissues. Organs weighed from all parental animals included adrenals, brain, total and cauda epididymis (weighed separately), kidneys, liver, lungs (prior to inflation with 10% neutral-buffered formalin), ovaries, pituitary, prostate, seminal vesicles with coagulating glands and accessory fluids, spleen, testes (weighed separately), thryroid, and uterus with oviducts and cervix.
Other examinations:
Plasma and red blood cell activities were assessed using a modified Ellman method.
Statistics:
All statistical analyses were conducted using two-tailed tests unless otherwise specified, comparing each exposure group to the control group. Data obtained from nongravid animals were excluded from analyses following the mating period. Parental body weight and food consumption data, absolute and relative organ weights, and RBC and plasma cholinesterase data were subjected to a one-way ANOVA among all groups. If the ANOVA was significant, Dunnett's test was used for the pairwise comparisons to the control group. Histopathologic findings in protocol-specified tissues were evaluated using a two-tailed Fisher's Exact test. Significance was accepted at the 5% and 1% 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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
There were no treatment-related mortalities at any exposure level evaluated. Spontaneous deaths occurred in parental animals of F0 and F1 generations; 1 F0 female each in the 5 and 45ppm groups were found dead. There were no signs of toxicity although there was evidence of dystocia in the 45 ppm female and the 5ppm female failed to initiate parturition before death.

There were no effects on bodyweights, weight gains, or food consumption at exposure levels of 5 and 15 ppm. Bodyweight gains for the 45 and 90 ppm males were statistically reduced relative to controls during the first 3 weeks of exposure, resulting in persistant and generally statistically significant body weight depression (up to 11.8%). Food consumption was also decreased for these males, generally in parallel with the bodyweight effects. Decreased food consumption and body weight gains were also noted for the females exposed to 45 and 90 ppm during the first 2 weeks of treatment and throughout gestation, resulting in decreased bodyweights (generally statistically significant) for 45 ppm females at study week 2 (-4.5%) and 90 ppm females throughout the 10 week premating period and gestation (7.5-9.1%). Body weights in the 90 ppm females were also depressed during lactation (5.8-11.5%) but did not achieve statistical significance, and were not accompanied by food consumption deficits.

Clincal findings consistent with the irritant properties of acrylonitrile (clear/red material around the nose, eyes and mouth and on the forelimbs) were observed for the rats exposed to 90 ppm throughout the exposure period within 1 hour following completion of daily exposure, but did not generally persist to the following day. Wet, cool tails were also noted for these animals, to a greater extent in the males, within 1 hour following exposure.

Increased absolute and/or relative (to final body weight) liver weights were noted for the 90 ppm group F0 males and females.

RBC cholinesterase activity was unaffected in males and females at exposure levels of 90 ppm. Plasma cholinesterase activity in the females exposed to 90 ppm was 40% lower than controls and was also lower than the mean value in the performing laboratory’s historical control database for approximately age-matched animals. However, there were no corresponding clinically observed functional deficits or inhibition of RBC cholinesterase activity in these females, and no effects on plasma or RBC cholinesterase activity were noted for the males. The reason for this could not be conclusively determined, but was not considered to be of toxicological significance in the absence of corresponding changes in RBC cholinesterase levels or associated clinical observations.

There were no treatment-related findings at histopathological examination.
Dose descriptor:
LOAEC
Effect level:
5 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathology of the nasal cavity (local irritant effects) at this (lowest) dose level
Dose descriptor:
NOAEC
Effect level:
< 5 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathology of the nasal cavity (local irritant effects) at all dose levels
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 ppm
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

The analyses of the chamber atmospheres indicated that the mean analytical values of acrylonitrile ± SD for the 5, 15, 45 and 90 ppm groups were; 5.0±0.30, 15.1±0.69, 45.3±1.51, and 89.4±3.58 ppm, respectively. No test chemical was detected in the control atmospheres.

Conclusions:
Sub-chronic inhalation exposure of rats to acrylonitrile vapour resulted in reduced weight gain and food consumption and clinical signs consistent with respiratory irritation. Histopathological investigation revealed findings in the nasal cavity consistent with local irritation in all exposure groups. A NOAEC cannot be determined for this study.
Executive summary:

A two-generation reproductive toxicity study was conducted in Sprague-Dawley rats; the data presented here relate to the repeated dose inhalation toxicity effects on the parental animals (F0 generation). Twenty-five rats/sex/group were exposed to vapour atmospheres of acrylonitrile via whole-body inhalation at concentrations of 0, 5, 15, 45 and 90 ppm, 6 hours daily, on 7 days a week for 10 weeks. Males were exposed for 10 weeks prior to mating and throughout mating until 1 day prior to termination. Females were exposed for 10 weeks prior to mating and throughout mating, gestation, and lactation until 1 day prior to termination. Exposure of the dams was suspended for 5 days following parturition (lactation days 0 -4) to avoid confounding nesting and nursing behaviour and neonatal survival. Exposure of the dams resumed on Day 5; rats were removed from the litters for 6 hours exposure at about the same time each day. There were no exposure-related mortalities. Bodyweight gain was significantly reduced at 45 and 90 ppm. Food consumption was also reduced at these dose levels, but the difference was only significant at 90 ppm. Clinical signs indicative of the irritant properties of acrylonitrile were observed in rats exposed to 90 ppm throughout the exposure period and within 1 hour of cessation of exposure; the irritant effects of the test material did not generally persist to the following day. Acrylonitrile-related microscopic alterations were limited to morphologically similar nasal lesions in the F0 males and females at 45 ppm, F1 males at 5, 15, and 45 ppm, and the F1 females at 15 and 45 ppm. Four levels of the nasal cavity were examined microscopically for the 5, 15, and 45 ppm groups. Lesions showed a clear exposure-response relationship in incidence and included respiratory/transitional epithelial hyperplasia, sub-acute inflammation, squamous metaplasia, and/or degeneration of the olfactory epithelium. The majority of the lesions were present in the most rostral section (level I) of the nasal tissues examined and are consistent with site-of-contact irritation resulting from exposure to irritant chemicals as reported in the literature by a number of authors. All of the nasal lesions noted in this study are common findings in the nasal epithelium of the rat following sub-chronic to chronic inhalation exposure with an irritating compound and represent the effects of local irritation, rather than a systemic effect. No other treatment-related histopathological findings were noted at any exposure level. Based on the incidence of local irritant effects in the nasal cavity at all exposure levels, a NOAEC cannot be determined for this study. A LOAEC of 5 ppm is determined.

Endpoint:
chronic toxicity: 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:
Inhalation exposure for 12 months; assessment of chronic toxicity and carcinogenicity
GLP compliance:
not specified
Remarks:
: older study, pre-dates GLP
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
No further information.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No information
Details on inhalation exposure:
No further information
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 per day, 5 days per week
Dose / conc.:
0 ppm
Remarks:
Untreated control
Dose / conc.:
5 ppm
Dose / conc.:
10 ppm
Dose / conc.:
20 ppm
Dose / conc.:
40 ppm
No. of animals per sex per dose:
30 rats per sex per dose
Control animals:
yes, concurrent no treatment
Details on study design:
Following the 12 month exposure rats were kept under observation without acrylonitrile exposure until spontaneous death.
Positive control:
No positive control.
Observations and examinations performed and frequency:
Observations included mortality, body weight, and neoplastic examinations.
Sacrifice and pathology:
Rats were not sacrificed, and instead kept until spontaneous death.
Other examinations:
No further examinations
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:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Slight increases in tumour incidences were reported in the mammary glands (males and females), forestomach (males) and skin (females), but none of these were statistically significant. No increase in mortality related to acrylonitrile treatment was observed. A significant increase in malignant and total number of tumours occurred in the 5ppm females. No effects on body weight were observed.
Dose descriptor:
NOAEC
Effect level:
5 ppm
Sex:
male
Dose descriptor:
LOAEC
Effect level:
5 ppm
Sex:
female
Dose descriptor:
NOAEC
Effect level:
< 5 ppm
Sex:
female
Dose descriptor:
NOAEC
Effect level:
>= 5 - <= 10 ppm
Sex:
male/female
Basis for effect level:
other: Borderline effects in females at 5 ppm indicate a NOAEC of between 5 and 10 ppm
Dose descriptor:
LOAEC
Effect level:
10 ppm
Sex:
male
Critical effects observed:
not specified

The increase in tumour incidences was considered by the authors to indicate a borderline carcinogenic effect.

Conclusions:
The authors concluded that the results indicated a borderline carcinogenic effect in females.
Executive summary:

Male and female rats were exposed to acrylonitrile vapour at concentrations of 0, 5, 10, 20 or 40 ppm for 4 hours per day, 5 days per week for 12 months. Rats were not sacrificed and instead kept under observation without acrylonitrile exposure, following the 12 month period until spontaneous death. Slight increases in tumour incidences were observed, but none were statistically significant. A significant increase in malignant and total number of tumours was seen in the 5 ppm females. There was no increase in mortality in the treated rats or bodyweight effects and borderline effects on carcinogenicity were seen in females only at 5 ppm. The study focused on neoplastic changes and provided little useful information for the assessment of chronic toxicity of acrylonitrile. A NOAEC of between 5 -10 ppm can be estimated

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
no guideline followed
Principles of method if other than guideline:
Gut et al (1985) studied the effect on intermediary metabolism in rats following repeated exposure to acrylonitrile via inhalation.
Bhooma et al (1992) studied the effect of acrylonitrile on the procoagulant activity (PCA) of rat lung.
Brewer (1976) investigated the repeated dose inhalation toxicity in a number of species
GLP compliance:
no
Limit test:
no
Species:
other: rat, dog, mouse
Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
air
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
5 days: Gut et al (1985)
5 days: Bhooma et al (1992)
90 days: Brewer (1976)
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
130 ppm (Gut et al)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 ppm (Bhooma et al)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 24, 54 ppm (dog), 0, 24, 54, 108 ppm (rat, mouse) (Brewer, 1976)
Basis:
nominal conc.
Control animals:
yes
Critical effects observed:
not specified

Gut et al (1985)

The body weight gradually decreased over the 5 days of exposure, and inspection of the abdominal cavity revealed a marked decrease of intra-abdominal fat. The weight of the liver decreased, while the weight of the brain did not. There were no changes in the absolute nor relative weights of the kidneys, lungs and adrenals. The relative weight of the liver significantly decreased (P <0.05), but that of the brain increased by (P <0.05) due to the body weight decrease. Clinical chemistry and biochemical measurements showed a significantly decreased (P <0.05) serum concentration of cholesterol and triglycerides, but the liver concentrations of phospholipids and esterified fatty acids were unchanged. The liver microsomal protein and cytochrome P-450 content decreased significantly (P <0.001), while the levels of glucose, lactate and pyruvate in the blood and brain increased significantly (up to 250% compared to controls). Microscopic examination of the lungs, liver, kidneys and adrenals did not show histopathological changes and the numbers and enzyme activities of alveolar macrophages were also unaffected.

Bhooma et al (1992), Hopper et al, 1981

An increased coagulation capability of alveolar macrophages of the lung detected in this study was indicative of a lung-damaging effect. The elevation of alveolar macrophage procoagulant activity occurred from day 1 to 14 post-exposure. On the 28th day the levels returned to normal. BAL-PCA increased at this time, possibly due to the release of macrophagic PCA into BAL facilitated by fibrin degradation products. Hopper et al (1981) observed that the formation of fibrin networks on the surfaces of stimulated peritoneal macrophages impaired their mobility and that macrophage-associated PCA appeared to promote the formation of these fibrin networks. The Bhooma study also demonstrated fibrin network formation in the lung following exposure to acrylonitrile. The elevated macrophage PCA level up to day 14 and the decrease on day 28 illustrates the dynamic interplay between procoagulant activity and fibrinolytic factors. However it should be noted that the exposure level of acrylonitrile in the Bhooma experiment high (100 ppm) and the exposure regime was 5 hours/day for 5 continuous days.

Brewer (1976)

In the dog study at 54 ppm (121.5 mg/m3), 1/3 males and 2/3 females died. All animals showed some decrease in weight gain. Clinical symptoms included rhinitis, ataxia and increased diuresis. Organ weights of the liver, kidney, spleen, adrenal gland, lungs, gonads, thyroid gland, heart and brain were similar to those of the control animals. The haematological and clinical chemistry findings were also similar to those in controls, other than a slight increase in serum alkaline phosphatase. Histopathological examination revealed focal macrophage infiltration, focal fibrosis and multifocal bronchopneumonia in 1/3 males and 3/3 females. Dogs exposed to 24 ppm (54 mg/m3) acrylonitrile showed no mortality but signs of lung irritation were observed even at this low-dose level, comprising focal alveolar macrophage infiltration and multifocal bronchopneumonia (2/3) in female dogs only. Serum alkaline phosphatase was also slightly increased. The NO(A)EL for in dogs is thus below 24 ppm (54 mg/m3).

In the rodent study, 5/40 control rats, 5/40 at 24 ppm (54 mg/m3), 5/40 at 54 ppm (121.5 mg/m3) and 18/40 at 108 ppm (243 mg/m3) died during the study, while the comparable figures in mice were 23/30 controls, 21/30 at 24 ppm (54 mg/m3), 15/30 at 54 ppm (121.5 mg/m3) and 27/30 at 108 ppm (243 mg/m3). Mortality in mice and rats was therefore unaffected by exposure to 24 and 54 ppm (54 mg/m3 and 121.5 mg/m3) acrylonitrile. However an increased lethality was seen in rats exposed to a level of 108 ppm (243 mg/m3), with some increase in deaths also being seen in mice at this level. As in dogs, clinical symptoms included body weight retardation, rhinitis, ataxia and increased diuresis. Organ weights, haematological, clinical chemistry findings were similar to those in control animals. Histopathological examination of tissues in dogs revealed treatment-related changes in the lung of some dogs at dose levels of 24 and 54 ppm (54 mg/m3 and 121.5 mg/m3). These changes were exposure-related with regard to incidence and relative severity. The changes consisted of focal to multifocal suppurative bronchopneumonia, and focal aggregates of alveolar macrophages in the alveolar lumina. These lesions were indicative of mild irritation. There were three mortalities at the 54 ppm (121.5 mg/m3) dose level. The focal haemorrhages described in the lung of sacrificed animals were agonal lesions related to the method of sacrifice. Any other changes seen in other tissues were lesions of naturally occurring diseases and they were present among both control and test animals. The histopathological examination of the tissues in rats resulted in findings confined to the lung, only in rats exposed to 108 ppm (243 mg/m3)), which were indicative of irritation. These changes consisted of a slight to mild increase in number of alveolar macrophages in the lumina of alveoli and a suppurative bronchopneumonia. No histopathological alterations were noted among the test mice. Asphyxiation secondary to the sub-acute bronchopneumonia in affected animals was the major cause of death. Chronic respiratory disease was present in the lung and trachea of all the control and most of the test animals.

Executive summary:

The EU RAR summarises a number of additional studies investigating the repeated exposure inhalation toxicity of acrylonitrile. The studies are not of standard design or are considered to be of questionable quality, and therefore are not considered to be of critical relevance to the risk assessment. Gut et al. (1985) studied the effect on intermediary metabolism in rats following repeated exposure to acrylonitrile via inhalation. Male Wistar rats were exposed to acrylonitrile at a concentration of 130 ppm (280 mg/m3) for 8 hours/day for 5 days. Bhooma et al. (1992) studied the effect of acrylonitrile on the procoagulant activity (PCA) of rat lung. In this study 6 rats were exposed to acrylonitrile levels of 100 ppm (225 mg/m3) for 5 hours/day for 5 days. The lungs (together with other organs) were removed. The lungs were lavaged 6 times with 5 ml cold, sterile, isotonic saline, with collection of the lavage effluent. Cells were counted in a haemocytometer chamber and viability was determined. Levels of PCA in macrophages and bronchoalveolar lavage (BAL) fluid were measured on day 1, 3, 5, 7, 14 and 28 days after acrylonitrile exposure. 90-day subacute inhalation studies (Brewer, 1976) were carried out in groups of 6 beagle dogs (3 male and 3 female) exposed to mean atmospheric concentrations of 0, 24 (54 mg/m3) and 54 ppm (121.5 mg/m3) acrylonitrile, and in 40 albino rats and 30 albino CD-1 mice exposed to 0, 24 (54 mg/m3), 54 ppm (121.5 mg/m3 and 108 (243 mg/m3). The exposure regime was 6 hours per day, 5 days per week for a total of 57 exposures. Microscopic examinations of brain (cerebrum, cerebellum and pons), bronchi, small intestine, gonads, gall bladder (dogs only), heart, kidney, liver, lungs, lymph nodes, spleen, trachea and thyroid were carried out in untreated controls (dog) and dogs at 24 and 54 ppm (54 mg/m3 and 121.5 mg/m3), untreated controls (rats and mice) and rats and mice at 108 ppm (243 mg/m3). The EU RAR concludes that the quality of the Brewer studies is questionable due to the presence of chronic respiratory disease in the rodents and the high mortality in all exposure groups. The value of this study for risk assessment purposes was therefore concluded to be limited. The results suggest that the main effect of acrylonitrile inhalation in dogs was irritation of the lungs, to which the females seemed to be more sensitive than males. As with acute exposure, dogs were more sensitive to acrylonitrile than rats or mice, while the two rodent species seemed to be equally sensitive. The results indicate that the sub-chronic NOAEC is less than 24 ppm (54 mg/m3) in the most sensitive species, the dog, as lung irritation was still seen at this low-dose level. For the purposes of risk assessment (while recognising the limitations of the studies) this exposure level may be considered to be a LOAEC.

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
not specified
Remarks:
: older study, pre-dates mandatory GLP
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Sprague-Dawley (Spartan sub-strain) rats.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No information
Details on inhalation exposure:
No further information
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information available
Duration of treatment / exposure:
Rats were exposed for 6 hours per day, 5 days per week, for 2 years.
Frequency of treatment:
6 hours per day, 5 days per week.
Dose / conc.:
0 ppm
Remarks:
Control (untreated)
Dose / conc.:
20 ppm
Remarks:
45 mg/m3
Dose / conc.:
80 ppm
Remarks:
180 mg/m3
No. of animals per sex per dose:
100 rats per sex per concentration, plus an additional 7 rats per sex per concentration for sacrifice at 6 months, and 13 rats per sex per concentration for sacrifice at 12 months.
Control animals:
yes, concurrent no treatment
Details on study design:
Control rats were exposed to air only. 100 rats per sex were exposed to one of two acrylonitrile concentrations . An additional 7 rats per sex per concentration for sacrifice at 6 months, and 13 rats per sex per concentration for sacrifice at 12 months.
Positive control:
No positive control; not relevant to this study type
Observations and examinations performed and frequency:
Examinations performed included: clinical observations, body weights, organ weights, water consumption, haematology, urinalysis, clinical chemistry and mortality.
Sacrifice and pathology:
Gross pathology was carried out on all rats. Complete histopathology examinations were carried out on 40 organs of rats in the control and high dose group.
Other examinations:
No further examinations were made.
Statistics:
No information available.
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):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Clinical observations detected various toxic effects; decreases in body weight, early mortality, 'unthrifty' clinical appearance (retarded weight gain), earlier onset of tumours, and more frequently observed palpable tumours. They were most apparent and occurred earliest in the high dose group (80ppm). A significant decrease in mean body weight was observed in rats exposed to 80 ppm. Similar weight decreases were noted in the 20 ppm females after approximately 1 month exposure. During the first 6 months the exposed rats drank more water and appeared to excrete lower specific gravity urine than the controls.

In the 80 ppm group of male rats a significantly increased relative organ to body weight ratio was observed for the brain, heart and testes. The absolute kidney weight in the 80 ppm group of males was significantly decreased. In the few surviving females there was a significantly increased liver to body weight ratio and there was a slight increase in the absolute liver weight in these rats, as well as in the single surviving rat in the 80 ppm group.

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.

A statistically significant increase in mortality was observed within the first year in both male and female rats in the 80 ppm group, and in the females in the 20 ppm during the last 10 weeks of the study. The increase in the 20 ppm females was due to early sacrifice of rats with large benign mammary gland tumours. The onset of early mortality began much earlier into the study for male rats (days 211-240), compared to the females (days 361-390).

Histopathology revealed increased pathological changes in the heart and lungs of male rats of both treatment groups. However the changes seen were reportedly identical to effects seen in the control animals (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.

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 presence of large benign mammary tumours (occurring earlier in exposed rats compared to controls) was frequently associated with haemorrhage and tissue damage or pressure necrosis, resulting in compensatory extramedullary haemopoiesis. The development of large and frequently ulcerated, necrotic and haemorrhagic ear canal (Zymbal gland) tumours in acrylonitrile treated rats contributed to this compensatory response.

A treatment-related effect was observed in the nasal turbinate mucosa of all rats examined in the 80 ppm group as well as in some of the rats in the 20 ppm group. The changes in the exposure groups were similar but much less severe in the 20 ppm group than in the 80 ppm group. More pronounced changes were observed in males exposed to 80ppm between months 19 and 24. Changes observed included: suppurative rhinitis, hyperplasia in the region of the nasal turbinate mucosa lined by the respiratory epithelium, focal erosion of mucosa lining the respiratory epithelium, squamous
metaplasia of the respiratory epithelium, etc. Similar occurrences (though fewer in number) were noted for the 20 ppm males only at the terminal kill.
In the females effects on the nasal turbinate mucosa were first observed at month 19 for the 80ppm group, with similar though less frequent effects only being observed at terminal kill in the 20ppm group. Some of these changes were noted in the female control animals either at 19 months or at the terminal kill.

In 2 of the 80 ppm female rats there was a microscopic metaplastic proliferation of the respiratory epithelium.

Treatment-related non-neoplastic lesions were also detected in the brain, characterised by focal perivascular cuffing and gliosis. In males at 20 and 80ppm the incidence was 2/997 and 7/995 (p < 0.05, onesided), respectively and for females the incidence was 2/100 and 8/100 (p < 0.05, one-sided), respectively.
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
20 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Local irritant effects on the nasal turbinates (M, F), bodyweight effects (F).
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
20 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Bodyweight effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 ppm
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

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 mortality for 20 ppm females was principally due to early sacrifice of rats with large, benign mammary gland tumours which occur spontaneously at a high rate in this rat strain, but in this study were observed earlier and more frequently than in controls, and became larger in exposed animals. Statistically significant early mortality is indicated in both sexes at 80 ppm. The onset of mortality was apparent much earlier for males (Day 211 - 240) than for females in which a significant increase in mortality was only seen at days 361 -390.

Clinical observations detected a variety of adverse effects including bodyweight decreases, early mortality, 'unthrifty' appearance (retarded weight gain), earlier onset of tumors and more frequently observed palpable tumors. These observations were most apparent and occurred earliest at 80 ppm. A significant decrease in mean bodyweight was observed in rats exposed to 80 ppm; less significant (but similar) weight decreases were also noted in females at 20 ppm after approximately one month. A treatment-related effect on mean bodyweight was not observed in males at 20 ppm. During the first six months, exposed rats drank more water and appeared to excrete lower specific gravity urine than controls.

Increased organ to body weight ratios were seen in rats exhibiting significantly reduced body weights, therefore the relative increase in the organ weight ratio was considered to be a reflection of the effect on bodyweight. A reduction in kidney weight in the 80 ppm males was consistent with the decreased bodyweight and a decrease in the severity of chronic renal disease which was observed grossly and microscopically. The increased liver to body weight ratio seen in the females was interpreted to be the result of extramedullary haematopoiesis in the liver. This was a result of the greater number of bleeding tumours in these rats, and was thought to be a secondary effect due to repeated episodes of blood loss and associated anaemia and hypoxia. It was concluded that these findings were not indicative of a primary hepatotoxic effect of acrylonitrile. This was 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.

Ocassional reductions in PCV, haemoglobin concentration and WBC counts 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.

The increase in the incidence of spontaneous advanced chronic renal disease could have been due to increased demand on the kidneys due to increased water consumption seen in the first 6 months of the study.

The incidence of microscopic metaplastic proliferation in the respiratory epithelium was not statistically significantly increased, however 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 change.

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

Conclusions:
The LOAEC was 20 ppm under the conditions of this study; a NOAEC of 5 ppm can be estimated.
Executive summary:

The study demonstrates treatment-related non-neoplastic changes in Sprague- Dawley (Spartan) rats exposed to 20 or 80 ppm acrylonitrile for six hours/day, five days/week for 104 weeks. Findings consisted of effects on bodyweight and early mortality in both sexes at 80 ppm and in females at 20 ppm. As a result of irritation, inflammatory and degenerative changes (hyperplasia and metaplasia of the respiratory epithelium) were present in the nasal turbinates of both exposure groups. A significantly increased number of rats 80 ppm also showed focal gliosis and perivascular cuffing in the brain. The key toxicological findings in this study were considered to be local irritant effects in the nasal epithelium comprising suppurative rhinitis, hyperplasia, focal erosions, and squamous metaplasia of the respiratory epithelium, with hyperplasia of the mucus-secreting cells. Effects were seen at the lowest exposure level of 20 ppm. The EU RAR suggests that, as effects were due to local irritancy and the other systemic, non-neoplastic findings in treated rats were secondary to carcinogenicity rather than direct systemic toxicity, the application of an uncertainty factor of five to the LOAEC of 20 ppm could be used to derive a suggested NOAEC of 4 ppm (9 mg/m3).

Endpoint:
short-term repeated dose toxicity: 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:
The study predates guidelines. Investigation of the subacute repeated inhalation toxicity study of acrylonitrile in various species.
GLP compliance:
no
Remarks:
: older published study, predates GLP
Limit test:
no
Species:
other: rats, rabbits, guinea pigs, dogs, cats and monkeys
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
The animals were dogs, rhesus monkeys, rats, guinea pigs, rabbits and cats. In study 3 the 16 rats were described as comprising 8 adult animals and 8 young animals.
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No information available.
Details on inhalation exposure:
The animals were exposed by inhalation to acrylonitrile vapour
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Study 1: 4 weeks (dogs and monkeys)
Study 2: 8 weeks (rats, guinea pigs, rabbits and cats)
Study 3: 8 weeks (rats, guinea pigs, rabbits, cats and monkeys)
Frequency of treatment:
4 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
56 ppm (126 mg/m³)
Basis:
other: average concentration, Study 1 (dogs and monkeys)
Remarks:
Doses / Concentrations:
100 ppm (225 mg/m³)
Basis:
other: average concentration, Study 2 (rats, guinea pigs, rabbits and cats)
Remarks:
Doses / Concentrations:
153 ppm (344 mg/m³)
Basis:
other: average concentration, Study 3 (rats, guinea pigs, rabbits, cats and monkeys)
No. of animals per sex per dose:
The sex of the animals was not specified.
Study 1: 2 dogs and 4 monkeys.
Study 2: 16 rats, 16 guinea pigs, 3 rabbits and 4 cats.
Study 3: 16 rats (8 adult and 8 young), 16 guinea pigs, 4 rabbits, 4 cats and 2 monkeys.
Control animals:
no
Details on study design:
In study 2, three female rats were allowed to give birth. It is not clear whether the rats were pregnant during the exposures or if mating occurred after completion of the 8 week exposure period.
Positive control:
A positive control was not included.
Observations and examinations performed and frequency:
Animals were observed for mortality and signs of toxicity during the exposure periods. Body weights were recorded for some of the species. Weekly blood counts (RBC, WBC, haemoglobin and differential counts) were made on four rats and four rabbits during the 8 week exposure to 153 ppm.
Sacrifice and pathology:
Histopathological examination was carried out at the end of the study. Paraffin sections were made from the spleen, kidneys, liver, lung, heart, pancreas, lymph nodes, stomach, duodenum, jejunum, ileum, and large intestine, from a representative number of animals. Sections from all of the spleens and a representative number livers were treated with acidulated ferro-cyanide to demonstrate the presence or absence of iron-bearing pigment. A total of 680 sections from 18 rats, 6 rabbits, 6 cats, 16 guinea pigs and 1 monkey were examined.
Other examinations:
No other examinations reported.
Statistics:
Formal statistical analyses were not conducted.
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:
effects observed, treatment-related
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:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Study 1: All 4 monkeys survived the exposures, and showed no evidence of toxicity during the 4 week exposure. There were no signs of cumulative action in monkeys. After the first 4 hours exposure, one dog died in convlusions while the second dog developed a transitory weakness, with a paralysis of the hind legs after the 5th, 13th and 14th exposure. Subsequent exposures were reported to be well tolerated in the surviving dog.

Study 2: Exposure to 100 ppm for 8 weeks (4 hours/day, 5 days/week) was well tolerated by the rats, the only reported sign of toxicity was slight lethargy during exposure. Three of the female rats gave birth to and raised normal litters. Guinea pigs gained weight moderately and showed slight lethargy during the exposure, but otherwise no toxic effects were reported. Signs of toxicity reported in the rabbits were drowsy and listless behaviour during exposure, and no weight gain during the exposure period. Signs of toxicity reported in the cats were occasional vomiting, lethargy and weight loss. One cat developed transitory weakness of the hind legs after the 3rd exposure, with death occuring after the 11th exposure. The remaining three cats survived the exposure period with minimal toxic effects. There was no evidence of cumulative action in any species.

Study 3: Rats lost weight, developed rough coats and poor general physical condition. 50% of the adult rats died during the third and fourth week of exposure. The 8 young rats showed definite impairment of growth, and marked irritaion of the eyes and nose. The adult rats also showed irritation of the eyes and nose. One of the young rats died during the third week of exposure, and another 4 died by the end of the fifth week. Guinea pigs showed irritation of the eyes and nose, and salivation during the first week of exposure. Three of the 16 guinea pigs died during the fifth week of exposure. The remaining animals gained weight slightly and were in fair condition by the end of the study. Rabbits showed moderate irritation of the eyes and nose. One of the 4 rabbits died during the first week of exposure. Cats were severely affected. All showed severe signs of distress with each exposure and were frequently in collapse at the end of the exposure period. The cats suffered from marked nasal and conjunctival irritation, and they all developed transitory weakness of the hind legs. One cat died after the second exposure. Clinical signs seen in monkeys were sleepiness and weakness, loss of appetitie, and frequent salivation and vomiting. One monkey died after 6 weeks of exposure, and the second animal was in complete collapse after each exposure during the last 2 weeks of the study. There was no definite evidence of cumulative action in any species.

Histopathology: A slight amount of hemosiderosis indicative of blood destruction was seen in the spleen of rats. This increased in degree and occurred in a greater number of animals with the higher concentrations. Negligible amounts were noted in the spleens of cats, guinea pigs and rabbits.
Evidence of renal irritation was noted in most animals. Hyaline casts were present in the straight collecting tubules of all of the animals exposed in Studies 2 and 3, with the exception of 1 cat and 1 rabbit receiving 100 ppm, and the monkey exposed to 153 ppm. Subacute interstitial nephritis, characterised by focal collections of lymphocytes, a few polymorphonuclear leukocytes and sometimes accompanied by small areas of fibrosis with occasional distension of the tubules, was found in a significant number of the animals, although it was never extensive. The monkey and all the rats exposed to 100 ppm did not show these changes. The species difference in relation to kidney involvement was otherwise not significant, although the guinea pigs and rabbits appeared to be the most affected. As these animals were symptomatically the least susceptible, it may be assumed that the greater renal damage could indicate either more active excretion or a difference in species metabolism.
Subacute bronchopneumonia, characterised by congestion and oedema of the alveolar walls, extravasation of red cells and serum into the alveoli, and focal collections of lymphocytes and polymorphonuclear leukocytes, was present in all but one guinea pig and rabbit respectively, in the mokney, and about one-third of the rats. No pneumonic changes occurred in the cats, whereas liver damage was only observed in the cats.

Haematology (4 rats and 4 rabbits, 153 ppm exposure): The red and white blood cell counts and haemoglobin determinations remained within normal limits. The differential counts revealed an increase in eosinophils in both rats and rabbits, ranging from no eosinophils at the end of the first week to a maximum of 35, 42, 36 and 25% in the rabbits, and from 1% to a maximum of 21% in the rats.
Dose descriptor:
NOEC
Effect level:
56 ppm
Sex:
not specified
Basis for effect level:
other: There were no effects of 4 weeks exposure (4 hours/day, 5 days/week) to 56 ppm acrylonitrile in 4 rhesus monkeys

The experiments demonstrated that dogs are more susceptible to acrylonitrile than monkeys. Cats were shown to be more sensitive to acrylonitrile than rodents and rabbits. Repeated exposures to 153 ppm were toxic to guinea pigs, rats, rabbits, and were more strongly toxic to monkeys and cats. Exposure to 153 ppm resulted in irritation of the eyes and nose, loss of appetite, gastro-intestinal disturbances, and an incapacitating weakness of the hind legs from which the animals recovered relatively rapidly. No cause for the increase in eosinophils in rats and rabbits of the 153 ppm could be determined.

Conclusions:
This non-standard investigative study of the repeated exposure inhalation toxicity of acrylonitrile in various species identified target organs as the nervous system, kidney and resipratory tract.
Executive summary:

The effects of repeated inhalation exposure to acrylonitrile were evaluated in rats, rabbits, guinea pigs, dogs, cats and rhesus monkeys. The experiment was split into three studies: Study 1: 4 weeks exposure (4 hours/day, 5 days/week) to 56 ppm acrylonitrile in air, dogs and monkeys; Study 2: 8 weeks exposure (4 hours/day 5 days/week) to 100 ppm acrylonitrile in air, rats, guinea pigs, rabbits and cats; Study 3: 8 weeks exposure (4 hours/day 5 days/week) to 153 ppm acrylonitrile in air, rats, guinea pigs, rabbits, cats and monkeys. One dog (n=2) died following the fourth exposure to 56 ppm. One cat (n=4) died following the eleventh exposure to 100 ppm. Four adult rats (n=8) died during the third and fourth week of exposure to 153 ppm, and five young rats (n=8) died during the third and fifth exposure weeks. Three guinea pigs (n=16) died during the fifth week of exposure to 153 ppm. One rabbit (n=4) died during the first week of exposure to 153 ppm. One cat (n=4) died after the second exposure, and one monkey (n=2) died during week 6 of exposure. The experiments demonstrated that dogs are more susceptible to acrylonitrile than monkeys. Cats were shown to be more sensitive to acrylonitrile than rodents and rabbits. Repeated exposures to 153 ppm were toxic to guinea pigs, rats, rabbits, and were more strongly toxic to monkeys and cats. Exposure to 153 ppm resulted in irritation of the eyes and nose, loss of appetite, gastro-intestinal disturbances, and an incapacitating weakness of the hind legs from which the animals recovered relatively rapidly. Target organs identified were the nervous system (transitory limb weakness and/or paralysis in dogs and cats), the kidney (histopathological changes in rats and rabbits), the upper respiratory tract (nasal irritation in all species studied) and the lung (bronchopneumonia in all species except cats).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
11 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
High quality data are available for the rat, with study durations of up to 2 years.
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
Rats were exposed to acrylonitrile by inhalation as part of a 2-generation reproductive toxicity study
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Crl:CD(SD) Sprague-Dawley albino virgin male and female rats, obtained from Charles River Laboratories, NC. Rats were acclimated for 14 days, during which they were observed twice daily for mortality and moribundity. Rats were groups housed by sex for 3 days, then housed individually (except during mating) in suspended wire mesh cages. Following mating females were transferred to plastic maternity cages with nesting material. Basal diet (PMI Nutrition International, Certified Rodent LabDiet 5002) and reverse osmosis treated water were available ad libitum, except during exposure. Animals were maintained on a 12 hour photoperiod, at 71◦F ± 5◦F and 30% to 70% humidity. The F0 generation was approximately 8 weeks old at initiation of exposure, the F1 generation approximately 4 weeks old. Animals in the study were maintained in accordance with the Animal Welfare Act (1966) and the Guide for the Care and Use of Laboratory Animals.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No information available
Details on inhalation exposure:
Each group of animals was exposed to acrylonitrile vapour in a 2-cubic-metre stainless steel and glass whole-body inhalation chamber operated under dynamic conditions. Chamber temperature (20-25°C), relative humidity (30-70%), ventilation (12-15 air changes per hour), and negative pressure within the chambers were monitored. Cages were sequentially rotated around the available rack positions within the chamber on a daily basis, to minimise any potential variation due to positioning. The control group was exposed to clean filtered air under identical conditions to those used for the acrylonitrile exposure groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure concentrations of the vapour were measured approximately every 35 minutes (i.e. 9 to 10 times) during each daily exposure period via gas chromatography, using sensors placed approximately in the centre of the chamber, within the general breathing zone of the animals.
Duration of treatment / exposure:
Rats were exposed for 6 hours a day, 7 days a week, for 10 weeks prior to mating, during mating, gestation and lactation/.
Frequency of treatment:
Daily.
Dose / conc.:
0 ppm
Dose / conc.:
5 ppm
Dose / conc.:
15 ppm
Dose / conc.:
45 ppm
Dose / conc.:
90 ppm
No. of animals per sex per dose:
25 rats per sex per concentration
Control animals:
yes
Details on study design:
The study was perfomed as a two-generation reproductive toxicity study. The general toxicity data presented here are from the parental generation.

25 males and 25 females (F0 generation) in each of 5 groups were exposed to acrylonitrile (0, 5, 15, 45 or 90 ppm) 6 hours a day, 7 days a week for 10 weeks. The males were exposed for 10 weeks prior to mating and throughout mating until 1 day prior to euthanasia. The females were exposed for 10 weeks prior to mating and throughout mating, gestation, and lactation until 1 day prior to euthanasia. Exposure of the dams was suspended for 5 days following parturition (lactation days (LDs) 0 to 4) to avoid confounding nesting and nursing behaviour and neonatal survival. Exposure of the dams resumed on LD5, they were removed from the litters for 6 hours exposure at about the same time each day.

Animals found to be in good general health were allocated to groups based on body weight stratification and randomised in a block design by a computer generated program.
Positive control:
Not required for this study type
Observations and examinations performed and frequency:
Detailed physical examinations were recorded weekly for all parental animals (F0). All animals were observed twice daily for appearance, behaviour, moribundity, mortality and pharmacotoxic signs prior to exposure and within 1 hour after exposure. Females were also observed twice daily during the period of expected parturition for dystocia or other difficulties.

Individual body weights were recorded weekly throughout the study and prior to scheduled necropsy. Individual female bodyw eights were recorded weekly until evidence of copulation was observed and on GDs 0, 4, 7, 11, 14 and 20, and on LDs 1, 4, 7, 14, 21 and 28.

Parental food consumption was determined on the same days as the body weight measurements, except during the mating period when measurement of food consumption was suspended due to cohabitation.

Plasma and red blood cell (RBC) cholinesterase determinations were conducted on 10 rats/sex of the F0 parental generation from the control and 90 ppm groups, and from 10 rats/sex of the F1 parental generation from the control, 5, 15 and 45 ppm groups. Blood samples were collected from the tail vein following the daily 6 hour exposure 2 days prior to scheduled euthanasia. EDTA was used as the anticoagulant.
Sacrifice and pathology:
Surviving F0 adults were euthanised and necropsied following completion of weaning of their offspring (F1 and F2 pups respectively). Selected F0 and F1 parental tissues and organs were fixed by immersion in 10% neutral-buffered formalin for possible histopathological examination. Microscopic evaluations were performed on the following tissues for 10 randomly selected parental animals per sex (with confirmed sire or pregnancy) from the control and high-exposure groups: adrenal glands, prostate, brain, pituitary, seminal vesicles, right epididymis (caput, corpus and cauda), right testis, vagina, cervix, coagulating gland, uterus, oviducts, and ovaries (one section from each ovary was examined). Nasal cavities, lungs and gross lesions from all control, 5, 15 and 45 ppm groups were examined microscopically. Periodic acid-Schiff (PAS) and haematoxylin staining were used for the right tests and epididymis and haematoxylin-eosin staining was used for all other tissues. Organs weighed from all parental animals included adrenals, brain, total and cauda epididymis (weighed separately), kidneys, liver, lungs (prior to inflation with 10% neutral-buffered formalin), ovaries, pituitary, prostate, seminal vesicles with coagulating glands and accessory fluids, spleen, testes (weighed separately), thryroid, and uterus with oviducts and cervix.
Other examinations:
Plasma and red blood cell activities were assessed using a modified Ellman method.
Statistics:
All statistical analyses were conducted using two-tailed tests unless otherwise specified, comparing each exposure group to the control group. Data obtained from nongravid animals were excluded from analyses following the mating period. Parental body weight and food consumption data, absolute and relative organ weights, and RBC and plasma cholinesterase data were subjected to a one-way ANOVA among all groups. If the ANOVA was significant, Dunnett's test was used for the pairwise comparisons to the control group. Histopathologic findings in protocol-specified tissues were evaluated using a two-tailed Fisher's Exact test. Significance was accepted at the 5% and 1% 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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
There were no treatment-related mortalities at any exposure level evaluated. Spontaneous deaths occurred in parental animals of F0 and F1 generations; 1 F0 female each in the 5 and 45ppm groups were found dead. There were no signs of toxicity although there was evidence of dystocia in the 45 ppm female and the 5ppm female failed to initiate parturition before death.

There were no effects on bodyweights, weight gains, or food consumption at exposure levels of 5 and 15 ppm. Bodyweight gains for the 45 and 90 ppm males were statistically reduced relative to controls during the first 3 weeks of exposure, resulting in persistant and generally statistically significant body weight depression (up to 11.8%). Food consumption was also decreased for these males, generally in parallel with the bodyweight effects. Decreased food consumption and body weight gains were also noted for the females exposed to 45 and 90 ppm during the first 2 weeks of treatment and throughout gestation, resulting in decreased bodyweights (generally statistically significant) for 45 ppm females at study week 2 (-4.5%) and 90 ppm females throughout the 10 week premating period and gestation (7.5-9.1%). Body weights in the 90 ppm females were also depressed during lactation (5.8-11.5%) but did not achieve statistical significance, and were not accompanied by food consumption deficits.

Clincal findings consistent with the irritant properties of acrylonitrile (clear/red material around the nose, eyes and mouth and on the forelimbs) were observed for the rats exposed to 90 ppm throughout the exposure period within 1 hour following completion of daily exposure, but did not generally persist to the following day. Wet, cool tails were also noted for these animals, to a greater extent in the males, within 1 hour following exposure.

Increased absolute and/or relative (to final body weight) liver weights were noted for the 90 ppm group F0 males and females.

RBC cholinesterase activity was unaffected in males and females at exposure levels of 90 ppm. Plasma cholinesterase activity in the females exposed to 90 ppm was 40% lower than controls and was also lower than the mean value in the performing laboratory’s historical control database for approximately age-matched animals. However, there were no corresponding clinically observed functional deficits or inhibition of RBC cholinesterase activity in these females, and no effects on plasma or RBC cholinesterase activity were noted for the males. The reason for this could not be conclusively determined, but was not considered to be of toxicological significance in the absence of corresponding changes in RBC cholinesterase levels or associated clinical observations.

There were no treatment-related findings at histopathological examination.
Dose descriptor:
LOAEC
Effect level:
5 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathology of the nasal cavity (local irritant effects) at this (lowest) dose level
Dose descriptor:
NOAEC
Effect level:
< 5 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathology of the nasal cavity (local irritant effects) at all dose levels
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 ppm
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

The analyses of the chamber atmospheres indicated that the mean analytical values of acrylonitrile ± SD for the 5, 15, 45 and 90 ppm groups were; 5.0±0.30, 15.1±0.69, 45.3±1.51, and 89.4±3.58 ppm, respectively. No test chemical was detected in the control atmospheres.

Conclusions:
Sub-chronic inhalation exposure of rats to acrylonitrile vapour resulted in reduced weight gain and food consumption and clinical signs consistent with respiratory irritation. Histopathological investigation revealed findings in the nasal cavity consistent with local irritation in all exposure groups. A NOAEC cannot be determined for this study.
Executive summary:

A two-generation reproductive toxicity study was conducted in Sprague-Dawley rats; the data presented here relate to the repeated dose inhalation toxicity effects on the parental animals (F0 generation). Twenty-five rats/sex/group were exposed to vapour atmospheres of acrylonitrile via whole-body inhalation at concentrations of 0, 5, 15, 45 and 90 ppm, 6 hours daily, on 7 days a week for 10 weeks. Males were exposed for 10 weeks prior to mating and throughout mating until 1 day prior to termination. Females were exposed for 10 weeks prior to mating and throughout mating, gestation, and lactation until 1 day prior to termination. Exposure of the dams was suspended for 5 days following parturition (lactation days 0 -4) to avoid confounding nesting and nursing behaviour and neonatal survival. Exposure of the dams resumed on Day 5; rats were removed from the litters for 6 hours exposure at about the same time each day. There were no exposure-related mortalities. Bodyweight gain was significantly reduced at 45 and 90 ppm. Food consumption was also reduced at these dose levels, but the difference was only significant at 90 ppm. Clinical signs indicative of the irritant properties of acrylonitrile were observed in rats exposed to 90 ppm throughout the exposure period and within 1 hour of cessation of exposure; the irritant effects of the test material did not generally persist to the following day. Acrylonitrile-related microscopic alterations were limited to morphologically similar nasal lesions in the F0 males and females at 45 ppm, F1 males at 5, 15, and 45 ppm, and the F1 females at 15 and 45 ppm. Four levels of the nasal cavity were examined microscopically for the 5, 15, and 45 ppm groups. Lesions showed a clear exposure-response relationship in incidence and included respiratory/transitional epithelial hyperplasia, sub-acute inflammation, squamous metaplasia, and/or degeneration of the olfactory epithelium. The majority of the lesions were present in the most rostral section (level I) of the nasal tissues examined and are consistent with site-of-contact irritation resulting from exposure to irritant chemicals as reported in the literature by a number of authors. All of the nasal lesions noted in this study are common findings in the nasal epithelium of the rat following sub-chronic to chronic inhalation exposure with an irritating compound and represent the effects of local irritation, rather than a systemic effect. No other treatment-related histopathological findings were noted at any exposure level. Based on the incidence of local irritant effects in the nasal cavity at all exposure levels, a NOAEC cannot be determined for this study. A LOAEC of 5 ppm is determined.

Endpoint:
chronic toxicity: 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:
Inhalation exposure for 12 months; assessment of chronic toxicity and carcinogenicity
GLP compliance:
not specified
Remarks:
: older study, pre-dates GLP
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
No further information.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No information
Details on inhalation exposure:
No further information
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 per day, 5 days per week
Dose / conc.:
0 ppm
Remarks:
Untreated control
Dose / conc.:
5 ppm
Dose / conc.:
10 ppm
Dose / conc.:
20 ppm
Dose / conc.:
40 ppm
No. of animals per sex per dose:
30 rats per sex per dose
Control animals:
yes, concurrent no treatment
Details on study design:
Following the 12 month exposure rats were kept under observation without acrylonitrile exposure until spontaneous death.
Positive control:
No positive control.
Observations and examinations performed and frequency:
Observations included mortality, body weight, and neoplastic examinations.
Sacrifice and pathology:
Rats were not sacrificed, and instead kept until spontaneous death.
Other examinations:
No further examinations
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:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Slight increases in tumour incidences were reported in the mammary glands (males and females), forestomach (males) and skin (females), but none of these were statistically significant. No increase in mortality related to acrylonitrile treatment was observed. A significant increase in malignant and total number of tumours occurred in the 5ppm females. No effects on body weight were observed.
Dose descriptor:
NOAEC
Effect level:
5 ppm
Sex:
male
Dose descriptor:
LOAEC
Effect level:
5 ppm
Sex:
female
Dose descriptor:
NOAEC
Effect level:
< 5 ppm
Sex:
female
Dose descriptor:
NOAEC
Effect level:
>= 5 - <= 10 ppm
Sex:
male/female
Basis for effect level:
other: Borderline effects in females at 5 ppm indicate a NOAEC of between 5 and 10 ppm
Dose descriptor:
LOAEC
Effect level:
10 ppm
Sex:
male
Critical effects observed:
not specified

The increase in tumour incidences was considered by the authors to indicate a borderline carcinogenic effect.

Conclusions:
The authors concluded that the results indicated a borderline carcinogenic effect in females.
Executive summary:

Male and female rats were exposed to acrylonitrile vapour at concentrations of 0, 5, 10, 20 or 40 ppm for 4 hours per day, 5 days per week for 12 months. Rats were not sacrificed and instead kept under observation without acrylonitrile exposure, following the 12 month period until spontaneous death. Slight increases in tumour incidences were observed, but none were statistically significant. A significant increase in malignant and total number of tumours was seen in the 5 ppm females. There was no increase in mortality in the treated rats or bodyweight effects and borderline effects on carcinogenicity were seen in females only at 5 ppm. The study focused on neoplastic changes and provided little useful information for the assessment of chronic toxicity of acrylonitrile. A NOAEC of between 5 -10 ppm can be estimated

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
no guideline followed
Principles of method if other than guideline:
Gut et al (1985) studied the effect on intermediary metabolism in rats following repeated exposure to acrylonitrile via inhalation.
Bhooma et al (1992) studied the effect of acrylonitrile on the procoagulant activity (PCA) of rat lung.
Brewer (1976) investigated the repeated dose inhalation toxicity in a number of species
GLP compliance:
no
Limit test:
no
Species:
other: rat, dog, mouse
Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
air
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
5 days: Gut et al (1985)
5 days: Bhooma et al (1992)
90 days: Brewer (1976)
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
130 ppm (Gut et al)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 ppm (Bhooma et al)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 24, 54 ppm (dog), 0, 24, 54, 108 ppm (rat, mouse) (Brewer, 1976)
Basis:
nominal conc.
Control animals:
yes
Critical effects observed:
not specified

Gut et al (1985)

The body weight gradually decreased over the 5 days of exposure, and inspection of the abdominal cavity revealed a marked decrease of intra-abdominal fat. The weight of the liver decreased, while the weight of the brain did not. There were no changes in the absolute nor relative weights of the kidneys, lungs and adrenals. The relative weight of the liver significantly decreased (P <0.05), but that of the brain increased by (P <0.05) due to the body weight decrease. Clinical chemistry and biochemical measurements showed a significantly decreased (P <0.05) serum concentration of cholesterol and triglycerides, but the liver concentrations of phospholipids and esterified fatty acids were unchanged. The liver microsomal protein and cytochrome P-450 content decreased significantly (P <0.001), while the levels of glucose, lactate and pyruvate in the blood and brain increased significantly (up to 250% compared to controls). Microscopic examination of the lungs, liver, kidneys and adrenals did not show histopathological changes and the numbers and enzyme activities of alveolar macrophages were also unaffected.

Bhooma et al (1992), Hopper et al, 1981

An increased coagulation capability of alveolar macrophages of the lung detected in this study was indicative of a lung-damaging effect. The elevation of alveolar macrophage procoagulant activity occurred from day 1 to 14 post-exposure. On the 28th day the levels returned to normal. BAL-PCA increased at this time, possibly due to the release of macrophagic PCA into BAL facilitated by fibrin degradation products. Hopper et al (1981) observed that the formation of fibrin networks on the surfaces of stimulated peritoneal macrophages impaired their mobility and that macrophage-associated PCA appeared to promote the formation of these fibrin networks. The Bhooma study also demonstrated fibrin network formation in the lung following exposure to acrylonitrile. The elevated macrophage PCA level up to day 14 and the decrease on day 28 illustrates the dynamic interplay between procoagulant activity and fibrinolytic factors. However it should be noted that the exposure level of acrylonitrile in the Bhooma experiment high (100 ppm) and the exposure regime was 5 hours/day for 5 continuous days.

Brewer (1976)

In the dog study at 54 ppm (121.5 mg/m3), 1/3 males and 2/3 females died. All animals showed some decrease in weight gain. Clinical symptoms included rhinitis, ataxia and increased diuresis. Organ weights of the liver, kidney, spleen, adrenal gland, lungs, gonads, thyroid gland, heart and brain were similar to those of the control animals. The haematological and clinical chemistry findings were also similar to those in controls, other than a slight increase in serum alkaline phosphatase. Histopathological examination revealed focal macrophage infiltration, focal fibrosis and multifocal bronchopneumonia in 1/3 males and 3/3 females. Dogs exposed to 24 ppm (54 mg/m3) acrylonitrile showed no mortality but signs of lung irritation were observed even at this low-dose level, comprising focal alveolar macrophage infiltration and multifocal bronchopneumonia (2/3) in female dogs only. Serum alkaline phosphatase was also slightly increased. The NO(A)EL for in dogs is thus below 24 ppm (54 mg/m3).

In the rodent study, 5/40 control rats, 5/40 at 24 ppm (54 mg/m3), 5/40 at 54 ppm (121.5 mg/m3) and 18/40 at 108 ppm (243 mg/m3) died during the study, while the comparable figures in mice were 23/30 controls, 21/30 at 24 ppm (54 mg/m3), 15/30 at 54 ppm (121.5 mg/m3) and 27/30 at 108 ppm (243 mg/m3). Mortality in mice and rats was therefore unaffected by exposure to 24 and 54 ppm (54 mg/m3 and 121.5 mg/m3) acrylonitrile. However an increased lethality was seen in rats exposed to a level of 108 ppm (243 mg/m3), with some increase in deaths also being seen in mice at this level. As in dogs, clinical symptoms included body weight retardation, rhinitis, ataxia and increased diuresis. Organ weights, haematological, clinical chemistry findings were similar to those in control animals. Histopathological examination of tissues in dogs revealed treatment-related changes in the lung of some dogs at dose levels of 24 and 54 ppm (54 mg/m3 and 121.5 mg/m3). These changes were exposure-related with regard to incidence and relative severity. The changes consisted of focal to multifocal suppurative bronchopneumonia, and focal aggregates of alveolar macrophages in the alveolar lumina. These lesions were indicative of mild irritation. There were three mortalities at the 54 ppm (121.5 mg/m3) dose level. The focal haemorrhages described in the lung of sacrificed animals were agonal lesions related to the method of sacrifice. Any other changes seen in other tissues were lesions of naturally occurring diseases and they were present among both control and test animals. The histopathological examination of the tissues in rats resulted in findings confined to the lung, only in rats exposed to 108 ppm (243 mg/m3)), which were indicative of irritation. These changes consisted of a slight to mild increase in number of alveolar macrophages in the lumina of alveoli and a suppurative bronchopneumonia. No histopathological alterations were noted among the test mice. Asphyxiation secondary to the sub-acute bronchopneumonia in affected animals was the major cause of death. Chronic respiratory disease was present in the lung and trachea of all the control and most of the test animals.

Executive summary:

The EU RAR summarises a number of additional studies investigating the repeated exposure inhalation toxicity of acrylonitrile. The studies are not of standard design or are considered to be of questionable quality, and therefore are not considered to be of critical relevance to the risk assessment. Gut et al. (1985) studied the effect on intermediary metabolism in rats following repeated exposure to acrylonitrile via inhalation. Male Wistar rats were exposed to acrylonitrile at a concentration of 130 ppm (280 mg/m3) for 8 hours/day for 5 days. Bhooma et al. (1992) studied the effect of acrylonitrile on the procoagulant activity (PCA) of rat lung. In this study 6 rats were exposed to acrylonitrile levels of 100 ppm (225 mg/m3) for 5 hours/day for 5 days. The lungs (together with other organs) were removed. The lungs were lavaged 6 times with 5 ml cold, sterile, isotonic saline, with collection of the lavage effluent. Cells were counted in a haemocytometer chamber and viability was determined. Levels of PCA in macrophages and bronchoalveolar lavage (BAL) fluid were measured on day 1, 3, 5, 7, 14 and 28 days after acrylonitrile exposure. 90-day subacute inhalation studies (Brewer, 1976) were carried out in groups of 6 beagle dogs (3 male and 3 female) exposed to mean atmospheric concentrations of 0, 24 (54 mg/m3) and 54 ppm (121.5 mg/m3) acrylonitrile, and in 40 albino rats and 30 albino CD-1 mice exposed to 0, 24 (54 mg/m3), 54 ppm (121.5 mg/m3 and 108 (243 mg/m3). The exposure regime was 6 hours per day, 5 days per week for a total of 57 exposures. Microscopic examinations of brain (cerebrum, cerebellum and pons), bronchi, small intestine, gonads, gall bladder (dogs only), heart, kidney, liver, lungs, lymph nodes, spleen, trachea and thyroid were carried out in untreated controls (dog) and dogs at 24 and 54 ppm (54 mg/m3 and 121.5 mg/m3), untreated controls (rats and mice) and rats and mice at 108 ppm (243 mg/m3). The EU RAR concludes that the quality of the Brewer studies is questionable due to the presence of chronic respiratory disease in the rodents and the high mortality in all exposure groups. The value of this study for risk assessment purposes was therefore concluded to be limited. The results suggest that the main effect of acrylonitrile inhalation in dogs was irritation of the lungs, to which the females seemed to be more sensitive than males. As with acute exposure, dogs were more sensitive to acrylonitrile than rats or mice, while the two rodent species seemed to be equally sensitive. The results indicate that the sub-chronic NOAEC is less than 24 ppm (54 mg/m3) in the most sensitive species, the dog, as lung irritation was still seen at this low-dose level. For the purposes of risk assessment (while recognising the limitations of the studies) this exposure level may be considered to be a LOAEC.

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
not specified
Remarks:
: older study, pre-dates mandatory GLP
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Sprague-Dawley (Spartan sub-strain) rats.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No information
Details on inhalation exposure:
No further information
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information available
Duration of treatment / exposure:
Rats were exposed for 6 hours per day, 5 days per week, for 2 years.
Frequency of treatment:
6 hours per day, 5 days per week.
Dose / conc.:
0 ppm
Remarks:
Control (untreated)
Dose / conc.:
20 ppm
Remarks:
45 mg/m3
Dose / conc.:
80 ppm
Remarks:
180 mg/m3
No. of animals per sex per dose:
100 rats per sex per concentration, plus an additional 7 rats per sex per concentration for sacrifice at 6 months, and 13 rats per sex per concentration for sacrifice at 12 months.
Control animals:
yes, concurrent no treatment
Details on study design:
Control rats were exposed to air only. 100 rats per sex were exposed to one of two acrylonitrile concentrations . An additional 7 rats per sex per concentration for sacrifice at 6 months, and 13 rats per sex per concentration for sacrifice at 12 months.
Positive control:
No positive control; not relevant to this study type
Observations and examinations performed and frequency:
Examinations performed included: clinical observations, body weights, organ weights, water consumption, haematology, urinalysis, clinical chemistry and mortality.
Sacrifice and pathology:
Gross pathology was carried out on all rats. Complete histopathology examinations were carried out on 40 organs of rats in the control and high dose group.
Other examinations:
No further examinations were made.
Statistics:
No information available.
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):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Clinical observations detected various toxic effects; decreases in body weight, early mortality, 'unthrifty' clinical appearance (retarded weight gain), earlier onset of tumours, and more frequently observed palpable tumours. They were most apparent and occurred earliest in the high dose group (80ppm). A significant decrease in mean body weight was observed in rats exposed to 80 ppm. Similar weight decreases were noted in the 20 ppm females after approximately 1 month exposure. During the first 6 months the exposed rats drank more water and appeared to excrete lower specific gravity urine than the controls.

In the 80 ppm group of male rats a significantly increased relative organ to body weight ratio was observed for the brain, heart and testes. The absolute kidney weight in the 80 ppm group of males was significantly decreased. In the few surviving females there was a significantly increased liver to body weight ratio and there was a slight increase in the absolute liver weight in these rats, as well as in the single surviving rat in the 80 ppm group.

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.

A statistically significant increase in mortality was observed within the first year in both male and female rats in the 80 ppm group, and in the females in the 20 ppm during the last 10 weeks of the study. The increase in the 20 ppm females was due to early sacrifice of rats with large benign mammary gland tumours. The onset of early mortality began much earlier into the study for male rats (days 211-240), compared to the females (days 361-390).

Histopathology revealed increased pathological changes in the heart and lungs of male rats of both treatment groups. However the changes seen were reportedly identical to effects seen in the control animals (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.

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 presence of large benign mammary tumours (occurring earlier in exposed rats compared to controls) was frequently associated with haemorrhage and tissue damage or pressure necrosis, resulting in compensatory extramedullary haemopoiesis. The development of large and frequently ulcerated, necrotic and haemorrhagic ear canal (Zymbal gland) tumours in acrylonitrile treated rats contributed to this compensatory response.

A treatment-related effect was observed in the nasal turbinate mucosa of all rats examined in the 80 ppm group as well as in some of the rats in the 20 ppm group. The changes in the exposure groups were similar but much less severe in the 20 ppm group than in the 80 ppm group. More pronounced changes were observed in males exposed to 80ppm between months 19 and 24. Changes observed included: suppurative rhinitis, hyperplasia in the region of the nasal turbinate mucosa lined by the respiratory epithelium, focal erosion of mucosa lining the respiratory epithelium, squamous
metaplasia of the respiratory epithelium, etc. Similar occurrences (though fewer in number) were noted for the 20 ppm males only at the terminal kill.
In the females effects on the nasal turbinate mucosa were first observed at month 19 for the 80ppm group, with similar though less frequent effects only being observed at terminal kill in the 20ppm group. Some of these changes were noted in the female control animals either at 19 months or at the terminal kill.

In 2 of the 80 ppm female rats there was a microscopic metaplastic proliferation of the respiratory epithelium.

Treatment-related non-neoplastic lesions were also detected in the brain, characterised by focal perivascular cuffing and gliosis. In males at 20 and 80ppm the incidence was 2/997 and 7/995 (p < 0.05, onesided), respectively and for females the incidence was 2/100 and 8/100 (p < 0.05, one-sided), respectively.
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
20 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Local irritant effects on the nasal turbinates (M, F), bodyweight effects (F).
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
20 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Bodyweight effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 ppm
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

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 mortality for 20 ppm females was principally due to early sacrifice of rats with large, benign mammary gland tumours which occur spontaneously at a high rate in this rat strain, but in this study were observed earlier and more frequently than in controls, and became larger in exposed animals. Statistically significant early mortality is indicated in both sexes at 80 ppm. The onset of mortality was apparent much earlier for males (Day 211 - 240) than for females in which a significant increase in mortality was only seen at days 361 -390.

Clinical observations detected a variety of adverse effects including bodyweight decreases, early mortality, 'unthrifty' appearance (retarded weight gain), earlier onset of tumors and more frequently observed palpable tumors. These observations were most apparent and occurred earliest at 80 ppm. A significant decrease in mean bodyweight was observed in rats exposed to 80 ppm; less significant (but similar) weight decreases were also noted in females at 20 ppm after approximately one month. A treatment-related effect on mean bodyweight was not observed in males at 20 ppm. During the first six months, exposed rats drank more water and appeared to excrete lower specific gravity urine than controls.

Increased organ to body weight ratios were seen in rats exhibiting significantly reduced body weights, therefore the relative increase in the organ weight ratio was considered to be a reflection of the effect on bodyweight. A reduction in kidney weight in the 80 ppm males was consistent with the decreased bodyweight and a decrease in the severity of chronic renal disease which was observed grossly and microscopically. The increased liver to body weight ratio seen in the females was interpreted to be the result of extramedullary haematopoiesis in the liver. This was a result of the greater number of bleeding tumours in these rats, and was thought to be a secondary effect due to repeated episodes of blood loss and associated anaemia and hypoxia. It was concluded that these findings were not indicative of a primary hepatotoxic effect of acrylonitrile. This was 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.

Ocassional reductions in PCV, haemoglobin concentration and WBC counts 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.

The increase in the incidence of spontaneous advanced chronic renal disease could have been due to increased demand on the kidneys due to increased water consumption seen in the first 6 months of the study.

The incidence of microscopic metaplastic proliferation in the respiratory epithelium was not statistically significantly increased, however 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 change.

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

Conclusions:
The LOAEC was 20 ppm under the conditions of this study; a NOAEC of 5 ppm can be estimated.
Executive summary:

The study demonstrates treatment-related non-neoplastic changes in Sprague- Dawley (Spartan) rats exposed to 20 or 80 ppm acrylonitrile for six hours/day, five days/week for 104 weeks. Findings consisted of effects on bodyweight and early mortality in both sexes at 80 ppm and in females at 20 ppm. As a result of irritation, inflammatory and degenerative changes (hyperplasia and metaplasia of the respiratory epithelium) were present in the nasal turbinates of both exposure groups. A significantly increased number of rats 80 ppm also showed focal gliosis and perivascular cuffing in the brain. The key toxicological findings in this study were considered to be local irritant effects in the nasal epithelium comprising suppurative rhinitis, hyperplasia, focal erosions, and squamous metaplasia of the respiratory epithelium, with hyperplasia of the mucus-secreting cells. Effects were seen at the lowest exposure level of 20 ppm. The EU RAR suggests that, as effects were due to local irritancy and the other systemic, non-neoplastic findings in treated rats were secondary to carcinogenicity rather than direct systemic toxicity, the application of an uncertainty factor of five to the LOAEC of 20 ppm could be used to derive a suggested NOAEC of 4 ppm (9 mg/m3).

Endpoint:
short-term repeated dose toxicity: 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:
The study predates guidelines. Investigation of the subacute repeated inhalation toxicity study of acrylonitrile in various species.
GLP compliance:
no
Remarks:
: older published study, predates GLP
Limit test:
no
Species:
other: rats, rabbits, guinea pigs, dogs, cats and monkeys
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
The animals were dogs, rhesus monkeys, rats, guinea pigs, rabbits and cats. In study 3 the 16 rats were described as comprising 8 adult animals and 8 young animals.
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No information available.
Details on inhalation exposure:
The animals were exposed by inhalation to acrylonitrile vapour
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Study 1: 4 weeks (dogs and monkeys)
Study 2: 8 weeks (rats, guinea pigs, rabbits and cats)
Study 3: 8 weeks (rats, guinea pigs, rabbits, cats and monkeys)
Frequency of treatment:
4 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
56 ppm (126 mg/m³)
Basis:
other: average concentration, Study 1 (dogs and monkeys)
Remarks:
Doses / Concentrations:
100 ppm (225 mg/m³)
Basis:
other: average concentration, Study 2 (rats, guinea pigs, rabbits and cats)
Remarks:
Doses / Concentrations:
153 ppm (344 mg/m³)
Basis:
other: average concentration, Study 3 (rats, guinea pigs, rabbits, cats and monkeys)
No. of animals per sex per dose:
The sex of the animals was not specified.
Study 1: 2 dogs and 4 monkeys.
Study 2: 16 rats, 16 guinea pigs, 3 rabbits and 4 cats.
Study 3: 16 rats (8 adult and 8 young), 16 guinea pigs, 4 rabbits, 4 cats and 2 monkeys.
Control animals:
no
Details on study design:
In study 2, three female rats were allowed to give birth. It is not clear whether the rats were pregnant during the exposures or if mating occurred after completion of the 8 week exposure period.
Positive control:
A positive control was not included.
Observations and examinations performed and frequency:
Animals were observed for mortality and signs of toxicity during the exposure periods. Body weights were recorded for some of the species. Weekly blood counts (RBC, WBC, haemoglobin and differential counts) were made on four rats and four rabbits during the 8 week exposure to 153 ppm.
Sacrifice and pathology:
Histopathological examination was carried out at the end of the study. Paraffin sections were made from the spleen, kidneys, liver, lung, heart, pancreas, lymph nodes, stomach, duodenum, jejunum, ileum, and large intestine, from a representative number of animals. Sections from all of the spleens and a representative number livers were treated with acidulated ferro-cyanide to demonstrate the presence or absence of iron-bearing pigment. A total of 680 sections from 18 rats, 6 rabbits, 6 cats, 16 guinea pigs and 1 monkey were examined.
Other examinations:
No other examinations reported.
Statistics:
Formal statistical analyses were not conducted.
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:
effects observed, treatment-related
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:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Study 1: All 4 monkeys survived the exposures, and showed no evidence of toxicity during the 4 week exposure. There were no signs of cumulative action in monkeys. After the first 4 hours exposure, one dog died in convlusions while the second dog developed a transitory weakness, with a paralysis of the hind legs after the 5th, 13th and 14th exposure. Subsequent exposures were reported to be well tolerated in the surviving dog.

Study 2: Exposure to 100 ppm for 8 weeks (4 hours/day, 5 days/week) was well tolerated by the rats, the only reported sign of toxicity was slight lethargy during exposure. Three of the female rats gave birth to and raised normal litters. Guinea pigs gained weight moderately and showed slight lethargy during the exposure, but otherwise no toxic effects were reported. Signs of toxicity reported in the rabbits were drowsy and listless behaviour during exposure, and no weight gain during the exposure period. Signs of toxicity reported in the cats were occasional vomiting, lethargy and weight loss. One cat developed transitory weakness of the hind legs after the 3rd exposure, with death occuring after the 11th exposure. The remaining three cats survived the exposure period with minimal toxic effects. There was no evidence of cumulative action in any species.

Study 3: Rats lost weight, developed rough coats and poor general physical condition. 50% of the adult rats died during the third and fourth week of exposure. The 8 young rats showed definite impairment of growth, and marked irritaion of the eyes and nose. The adult rats also showed irritation of the eyes and nose. One of the young rats died during the third week of exposure, and another 4 died by the end of the fifth week. Guinea pigs showed irritation of the eyes and nose, and salivation during the first week of exposure. Three of the 16 guinea pigs died during the fifth week of exposure. The remaining animals gained weight slightly and were in fair condition by the end of the study. Rabbits showed moderate irritation of the eyes and nose. One of the 4 rabbits died during the first week of exposure. Cats were severely affected. All showed severe signs of distress with each exposure and were frequently in collapse at the end of the exposure period. The cats suffered from marked nasal and conjunctival irritation, and they all developed transitory weakness of the hind legs. One cat died after the second exposure. Clinical signs seen in monkeys were sleepiness and weakness, loss of appetitie, and frequent salivation and vomiting. One monkey died after 6 weeks of exposure, and the second animal was in complete collapse after each exposure during the last 2 weeks of the study. There was no definite evidence of cumulative action in any species.

Histopathology: A slight amount of hemosiderosis indicative of blood destruction was seen in the spleen of rats. This increased in degree and occurred in a greater number of animals with the higher concentrations. Negligible amounts were noted in the spleens of cats, guinea pigs and rabbits.
Evidence of renal irritation was noted in most animals. Hyaline casts were present in the straight collecting tubules of all of the animals exposed in Studies 2 and 3, with the exception of 1 cat and 1 rabbit receiving 100 ppm, and the monkey exposed to 153 ppm. Subacute interstitial nephritis, characterised by focal collections of lymphocytes, a few polymorphonuclear leukocytes and sometimes accompanied by small areas of fibrosis with occasional distension of the tubules, was found in a significant number of the animals, although it was never extensive. The monkey and all the rats exposed to 100 ppm did not show these changes. The species difference in relation to kidney involvement was otherwise not significant, although the guinea pigs and rabbits appeared to be the most affected. As these animals were symptomatically the least susceptible, it may be assumed that the greater renal damage could indicate either more active excretion or a difference in species metabolism.
Subacute bronchopneumonia, characterised by congestion and oedema of the alveolar walls, extravasation of red cells and serum into the alveoli, and focal collections of lymphocytes and polymorphonuclear leukocytes, was present in all but one guinea pig and rabbit respectively, in the mokney, and about one-third of the rats. No pneumonic changes occurred in the cats, whereas liver damage was only observed in the cats.

Haematology (4 rats and 4 rabbits, 153 ppm exposure): The red and white blood cell counts and haemoglobin determinations remained within normal limits. The differential counts revealed an increase in eosinophils in both rats and rabbits, ranging from no eosinophils at the end of the first week to a maximum of 35, 42, 36 and 25% in the rabbits, and from 1% to a maximum of 21% in the rats.
Dose descriptor:
NOEC
Effect level:
56 ppm
Sex:
not specified
Basis for effect level:
other: There were no effects of 4 weeks exposure (4 hours/day, 5 days/week) to 56 ppm acrylonitrile in 4 rhesus monkeys

The experiments demonstrated that dogs are more susceptible to acrylonitrile than monkeys. Cats were shown to be more sensitive to acrylonitrile than rodents and rabbits. Repeated exposures to 153 ppm were toxic to guinea pigs, rats, rabbits, and were more strongly toxic to monkeys and cats. Exposure to 153 ppm resulted in irritation of the eyes and nose, loss of appetite, gastro-intestinal disturbances, and an incapacitating weakness of the hind legs from which the animals recovered relatively rapidly. No cause for the increase in eosinophils in rats and rabbits of the 153 ppm could be determined.

Conclusions:
This non-standard investigative study of the repeated exposure inhalation toxicity of acrylonitrile in various species identified target organs as the nervous system, kidney and resipratory tract.
Executive summary:

The effects of repeated inhalation exposure to acrylonitrile were evaluated in rats, rabbits, guinea pigs, dogs, cats and rhesus monkeys. The experiment was split into three studies: Study 1: 4 weeks exposure (4 hours/day, 5 days/week) to 56 ppm acrylonitrile in air, dogs and monkeys; Study 2: 8 weeks exposure (4 hours/day 5 days/week) to 100 ppm acrylonitrile in air, rats, guinea pigs, rabbits and cats; Study 3: 8 weeks exposure (4 hours/day 5 days/week) to 153 ppm acrylonitrile in air, rats, guinea pigs, rabbits, cats and monkeys. One dog (n=2) died following the fourth exposure to 56 ppm. One cat (n=4) died following the eleventh exposure to 100 ppm. Four adult rats (n=8) died during the third and fourth week of exposure to 153 ppm, and five young rats (n=8) died during the third and fifth exposure weeks. Three guinea pigs (n=16) died during the fifth week of exposure to 153 ppm. One rabbit (n=4) died during the first week of exposure to 153 ppm. One cat (n=4) died after the second exposure, and one monkey (n=2) died during week 6 of exposure. The experiments demonstrated that dogs are more susceptible to acrylonitrile than monkeys. Cats were shown to be more sensitive to acrylonitrile than rodents and rabbits. Repeated exposures to 153 ppm were toxic to guinea pigs, rats, rabbits, and were more strongly toxic to monkeys and cats. Exposure to 153 ppm resulted in irritation of the eyes and nose, loss of appetite, gastro-intestinal disturbances, and an incapacitating weakness of the hind legs from which the animals recovered relatively rapidly. Target organs identified were the nervous system (transitory limb weakness and/or paralysis in dogs and cats), the kidney (histopathological changes in rats and rabbits), the upper respiratory tract (nasal irritation in all species studied) and the lung (bronchopneumonia in all species except cats).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
11 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
High quality data are available for the rat, with study durations of up to 2 years.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The repeated dose toxicity of acrylonitrile has been investigated in high quality studies using oral (gavage or drinking water) administration to rats and mice and has been extensively investigated in rats using inhalation exposure, with limited non-standard investigation in a number of other species.

 

Repeated dose oral toxicity

Studies in the mouse

Two sub-chronic range-finding studies are available, using gavage administration. In a 14 -week range-finding study (NTP, 2001), acrylonitrile was administered by gavage to B6C3F1 male and female mice (10/sex/group), 5 days a week for 14 weeks at dose levels of 0, 5, 10, 20, 40 and 60 mg/kg bw/d. Administration of 60 mg/kg bw/d acrylonitrile by gavage was lethal to all male mice and to 9/10 female mice within 24 hours. Clinical findings included lethargy and abnormal breathing in the 40 mg/kg bw/d groups immediately after dosing. Surviving mice administered 40 mg/kg bw/d continued to show these signs of toxicity for several days until the mice appeared to develop a tolerance to acrylonitrile. No adverse effects were reported in mice that received 5 mg/kg bw/d for 14 weeks. Collectively, the differences in the survival rates, mean body weight gains, and the increase in heart weights of males suggest that male mice may be more sensitive to acrylonitrile than female mice. Leucocyte and lymphocyte counts were decreased in 20 mg/kg males and 40 mg/kg females, and a minimal haemolytic anaemia was observed in 40 mg/kg bw/d females. Heart weights of 20 mg/kg bw/d males were significantly greater than those of the vehicle controls, and left cauda epididymis weights of 10 and 20 mg/kg bw/d males were significantly increased. The incidences of chronic active inflammation and hyperplasia in the forestomach of 40 mg/kg bw/d females were significantly increased. A NOAEL of 5 mg/kg bw/d is proposed for this study.

 

In a further study (Serota et al., 1996), B6C3F1 male and female mice (10/sex/group) were administered acrylonitrile by gavage (in distilled water) on 5 days/week for 13 weeks at dose levels of 0, 1.2, 2.4, 4.8, 9.6, and 12.0 mg/kg bw/d for 13 weeks in order to determine dose levels for a subsequent carcinogenicity study. Animals were assessed for survival, clinical observations, bodyweights, clinical pathology, sperm morphology and vaginal cytology, gross and microscopic pathology and organ weights. Additional male mice were included in each group for the collection of blood and tissue samples for special investigations to examine cellular proliferation, apoptosis, haemoglobin adduct formation, and the production of the metabolite CEO. No treatment-related effects on survival, clinical observations, bodyweights, clinical pathology, sperm morphology and vaginal cytology, gross or microscopic pathology or organ weights were observed.  Isolated (and statistically significant) effects on several parameters were noted but considered to be unrelated to treatment. All animals survived the 13 weeks of treatment. With the exception of a single male mouse in the group at 4.8 mg/kg bw/d (sacrificed in a moribund condition), all males assigned for special investigations all survived to scheduled sacrifice.  This single death in an intermediated dose group is not considered to be treatment-related.  No treatment-related signs of toxicity were reported. Alopecia was seen sporadically in several mice, but is a common background finding and was not considered to be treatment-related.  Normal bodyweight gains were achieved except on occasion once due to a lack of water supply overnight. Biologically significant alterations were not detected in any of the haematological parameters evaluated in mice of either sex. Statistically significant decreases in white blood cell values were seen males at 2.4 and 9.6 mg/kg bw/d but were not considered to be biologically significant in the absence of a dose-response relationship. Significantly elevated WBC counts were apparent in females at 4.8 and 12.0 mg/kg bw/d but were also not dose-related and were noted to be within the historical control range. A significantly increased mean haematocrit value seen in 9.6 mg/kg bw/d females was within the laboratory’s normal range. Statistically significant declines in lymphocyte counts present in males at 1.2, 2.4, and 9.6 mg/kg bw/d were not considered to be of biological significance in the absence of a dose-response relationship; values are reported to be within the normal range. Similarly, significant elevations in lymphocyte counts in 4.8 and 12.0 mg/kg bw/d females and elevated neutrophil count in 12.0 mg/kg bw/d females is within the normal biological variation range. Gross necropsy did not reveal any effects of treatment: isolated findings of preputial gland cysts and enlarged inguinal lymph nodes (males); ovarian cysts and foci of ovarian tissue in females were considered to be unrelated to the treatment since these lesions are normal findings in mice of this strain and age. A single ovarian tumour (choriocarcinoma, a germ cell tumour with trophoblastic differentiation) occurred in a control mouse in this study. Although these tumours are rare, several have been reported in B6C3F1 mice in studies conducted by the National Cancer Institute (NCI) and National Toxicology Program (NTP). Histopathology indicated no treatment-related effects. Investigations of sperm morphology and vaginal cytology, showed a significant decrease in epididymal sperm motility at the lowest dose level of 1.2 mg/kg bw/d and also at the highest dose levels of 12.0 mg/kg bw/d. Similar findings were not apparent at the intermediate dose levels of 2.4, 4.8, and 9.6 mg/kg bw/d; changes are not considered to be treatment-related in the absence of a clear dose-response relationship. No other parameters were significantly affected at any dose level. A NOAEL of 12.0 mg/kg bw/d can be determined for this study in the absence of any treatment-related effects.

 

Studies in the rat

The repeated toxicity of acrylonitrile has been fully investigated as part of comprehensive and high quality combined studies of chronic toxicity and carcinogenicity in the rat. The studies also incorporate investigations made at interim sacrifices. The results of the carcinogenicity assessments from these studies are presented in IUCLID Section 7.7.

 

In one study (Quast et al. (1980, 2002)), male and female Sprague-Dawley rats were exposed to acrylonitrile in drinking water for 2 years in a combined chronic toxicity/carcinogenicity study. 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 initation and persisted throughout the study in all treatment groups, with females being more severely affected than males. Clinical observations showed that treated animals were 'unthrifty' (retarded growth), 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. Haematology, clinical chemistry, urinalysis and organ weights at study termination were not significantly affected. Non-neoplastic and neoplastic lesions were found at an increased incidence in a number of tissues of both sexes at all treatment levels. The primary non-neoplastic effects of exposure occurred in the forestomach (hyperplasia and/or hyperkeratosis suggestive of chronic irritation) and the central nervous system (gliosis with or without perviascular cuffing) of rats of both sexes and involved all treatment groups. Due to treatment-related effects in all exposed groups, A NOAEL could not be identified for this study. The LOAEL for this study is therefore a drinking water concentration of 35 ppm, calculated to be equivalent to dose levels of 3.4 and 4.4 mg/kg bw/d acrylonitrile in males and females respectively.

In a second study (Johannsen & Levinskas, 2002), Groups of F344 rats were exposed to acrylonitrile in the drinking water for approximately 2 years as part of a combined chronic toxicity/carcinogenicity study, at doses of 0, 1, 3, 10, 30 and 100 ppm. The study was terminated at 23 months in females because of low survival rates. The males were exposed for 26 months. A consistent decrease in survival, reduced bodyweight and reduced water intake, and small reductions in haematology parameters were observed in both sexes of the 100 ppm group. Mortality was significantly increased compared to controls in the 100 ppm group, while mortality in the males receiving 10 ppm and the females receiving 3 and 30 ppm was also significantly greater than controls. Organ to body weight ratios at various study intervals were consistently elevated in the high dose groups, and were thought to be related to the lower body weights seen in this group. Due to the lack of a dose response relationship in the female mortality data, the NOAEL was considered to be 3 ppm for both males and females; equivalent to average daily dose levels of 0.25 mg/kg bw/d in males and 0.36 mg/kg bw/d in females.

  

In a non-standard published study, Szabo et al. (1984) investigated the sub-acute and sub-chronic toxicity of acrylonitrile to the adrenals, stomach and duodenum by correlating biochemical, functional and morphologic investigations, and to elucidate the mechanism of toxicity of acrylonitrile. Rats were exposed to 0, 0.0001% (1 ppm), 0.002%, 0.01%, 0.05% or 0.2% acrylonitrile in drinking water, or to the same dose level administered by gavage, for 7, 21 or 60 days. Acrylonitrile caused a time- and dose-dependent decrease in plasma corticosterone levels; aldosterone was affected only by the high dose level and prolonged time of exposure. Young rats were noted to be more susceptible than adults to this action of acrylonitrile. The adrenal cortex, especially the zona fasciculata, was atrophic in rats exposed through drinking water. At dose levels of 0.05% and 0.2%, administration also caused decreased food intake and body weight gain. The adrenals were enlarged with a hyperplastic zona fasciculata after daily gavage doses of acrylonitrile. Ingestion of the chemical did not interfere with compensatory enlargement of the adrenal gland following unilateral adrenalectomy. On the other hand, the ACTH-induced elevation of corticosterone plasma concentration was significantly attenuated by acrylonitrile in drinking water. Electron microscopy of the adrenal glands revealed no consistent changes in the steroid-producing cells. The authors postulate that accelerated turnover of circulating corticoids and/or interference with the secretion or action of ACTH may primarily be responsible for the decreased plasma levels of corticosterone and aldosterone in rats that ingest acrylonitrile. The mucosa in the stomach at the junction of the forestomach and glandular region of animals that had ingested acrylonitrile was hyperplastic. The corpus also showed regional mucosal hyperplasia. Changes were associated with an elevated concentration of non-protein sulphydryls mostly in the mucosa of the glandular stomach. A similar, less prominent elevation also occurred in the proximal duodenum. The results of the study suggest that the effects of acrylonitrile on the adrenals were in part attributable to its inherent toxicity and the consequences of decreased food and especially water intake (probably due to its unpalatability in drinking water even at 20 ppm)

 

A number of additional repeated dose oral toxicity studies are summarised in the EU RAR. In a 7 -week dose escalation study (Barnes, 1970), groups of 6 rats were gavaged with acrylonitrile at a dose level of 30 mg/kg bw/d (15 doses), 50 mg/kg bw/d (7 doses) and 75 mg/kg bw/d (13 doses). No effects on body weight and no neurotoxic effects (i.e., gait, hindlimb activity, etc.) were observed in this study. In a 90 -day rat drinking water study (Humiston & Frauson, 1975), adult Sprague-Dawley rats received acrylonitrile at dose levels equivalent to up to 42 mg/kg bw/d. Reduced water consumption was observed at dose levels above 10 mg/kg bw/d, while growth retardation occurred at levels of about 22 mg/kg bw/d and higher in female rats and at 42 mg/kg bw/d in males. Mean weekly food consumption was reduced for the first seven weeks of the study at a dose level of 38 mg/kg bw/d. At a dose level of 17 mg/kg bw/d, it was reduced in the first two weeks. Increased relative liver weight was observed at dose levels of 10 mg/kg bw/d and higher. Working et al. (1987) administered acrylonitrile by gavage to groups of 10 male rats at levels of 45, 60, 68, 75, and 90 mg/kg bw/d for five days. Some treated animals died during or immediately after dosing at all dose levels above 60 mg/kg bw/d. All rats treated with 60 mg/kg bw/d survived the entire 42-day observation period. While no deaths were observed at the 60 mg/kg bw/d dose level, decreases in body weight were present for three to four weeks after dosing. Four animals receiving 68 mg/kg bw/d died on the first day after dosing

 

Study in the dog

In a 6 -month dog study, Quast et al. (1975) (reported in ECB, 2004), administered acrylonitrile in drinking water at concentrations of 100, 200, or 300 mg/L to groups of four male and four female beagle dogs. Average intakes for males were 10 mg/kg bw/d at 100 mg/L, 16 mg/kg bw/d at 200 mg/L and 17 mg/kg bw/d at 300 mg/L. For females, the corresponding average intakes were 8 mg/kg bw/d at 100 mg/L, 17 mg/kg bw/d at 200 mg/L and 18 mg/kg bw/d at 300 mg/L. At 100 mg/L, a slight decrease in water and food intake and a slight increase in relative kidney weight was observed. Five dogs died, or were sacrificed because of debilitation, in each of the two higher dosage groups. In the dogs receiving 100 to 300 mg/L in the drinking water, early signs of toxicity included roughening of the coat and, later, retching, and vomiting. Terminal signs of lethargy, weakness, emaciation, and respiratory distress were noted.

 

Repeated dose dermal toxicity

No data are available for the repeated dose toxicity of acrylonitrile by the dermal route; however studies are not required as comprehensive data are available for repeated dose toxicity by the oral and inhalation routes. Testing by the inhalation route is considered to be most relevant (with regard to the likely route of occupational exposure) for a volatile liquid. Based on kinetic considerations, the systemic dermal toxicity of acrylonitrile is not predicted to be fundamentally different to that seen following oral and/or inhalation exposure, therefore specific data for this route are not required. Due to the irritant and sensitising properties of the substance, it is likely that the effects of repeated dose dermal exposure will be dominated by local (site of contact) effects which will severely limit systemic exposure to the substance and consequently limit the relevance of the study. The use of engineering controls and PPE will also minimise dermal exposure to the substance under normal occupational conditions. Testing is therefore not scientifically justified and additionally cannot be supported on grounds of animal welfare.

 

Repeated exposure inhalation toxicity

Nemec et al. (2008) performed a two-generation reproductive toxicity study in Sprague-Dawley rats; the data presented here relate to the repeated dose inhalation toxicity to parental animals. Twenty-five rats/sex/group were exposed to vapour atmospheres of acrylonitrile via whole-body inhalation at concentrations of 0, 5, 15, 45 and 90 ppm, 6 hours daily, on 7 days a week for 10 weeks. Males were exposed for 10 weeks prior to mating and throughout mating until one day prior to termination. Females were exposed for 10 weeks prior to mating and throughout mating, gestation, and lactation until 1 day prior to termination. Exposure of the dams was suspended for 5 days following parturition (lactation days 0 -4) to avoid confounding nesting and nursing behaviour and neonatal survival. Exposure of the dams resumed on Day 5; rats were removed from the litters for 6 hours exposure at about the same time each day. There were no exposure-related mortalities. Bodyweight gain was significantly reduced at 45 and 90 ppm. Food consumption was also reduced at these dose levels, but the difference was only significant at 90 ppm. Clinical signs indicative of the irritant properties of acrylonitrile were observed in rats exposed to 90 ppm throughout the exposure period and within 1 hour of cessation of exposure; the irritant effects of the test material did not generally persist to the following day. Acrylonitrile-related microscopic alterations were limited to morphologically similar nasal lesions in the F0 males and females at 45 ppm, F1 males at 5, 15, and 45 ppm, and the F1 females at 15 and 45 ppm. Four levels of the nasal cavity were examined microscopically for the 5, 15, and 45 ppm groups. Lesions showed a clear exposure-response relationship in incidence and included respiratory/transitional epithelial hyperplasia, sub-acute inflammation, squamous metaplasia, and/or degeneration of the olfactory epithelium. The majority of the lesions were present in the most rostral section (level I) of the nasal tissues examined and are consistent with site-of-contact irritation resulting from exposure to irritant chemicals as reported in the literature by a number of authors. All of the nasal lesions noted in this study are common findings in the nasal epithelium of the rat following sub-chronic to chronic inhalation exposure with an irritating compound and represent the effects of local irritation, rather than a systemic effect. No other treatment-related histopathological findings were noted at any exposure level. Based on the incidence of local irritant effects in the nasal cavity at all exposure levels, a NOAEC cannot be determined for this study. A LOAEC of 5 ppm is determined.

 

A 2-year inhalation study (Quast et al., 1980) demonstrated treatment-related non-neoplastic changes in Sprague- Dawley (Spartan) rats exposed to 20 or 80 ppm acrylonitrile for six hours/day, five days/week for 104 weeks. Findings consisted of effects on bodyweight and early mortality in both sexes at 80 ppm and in females at 20 ppm. As a result of irritation, inflammatory and degenerative changes (hyperplasia and metaplasia of the respiratory epithelium) were present in the nasal turbinates of both exposure groups. A significantly increased number of rats exposed to 80 ppm also showed focal gliosis and perivascular cuffing in the brain. The key toxicological findings in this study were considered to be local irritant effects in the nasal epithelium comprising suppurative rhinitis, hyperplasia, focal erosions, and squamous metaplasia of the respiratory epithelium, with hyperplasia of the mucus-secreting cells. Effects were seen at the lowest exposure level of 20 ppm. The EU RAR suggests that, as effects were due to local irritancy and the other systemic, non-neoplastic findings in treated rats were secondary to carcinogenicity rather than direct systemic toxicity, the application of an uncertainty factor of five to the LOAEC of 20 ppm could be used to derive a suggested NOAEC of 4 ppm (9 mg/m3).

 

Maltoni et al. (1977) exposed male and female rats to acrylonitrile vapour at concentrations of 0, 5, 10, 20 or 40 ppm for 4 hours per day, 5 days per week for 12 months. Rats were not sacrificed and instead kept under observation without acrylonitrile exposure, following the 12 month period until spontaneous death. Slight increases in tumour incidences were observed, but none were statistically significant. A significant increase in malignant and total number of tumours was seen in the 5 ppm females. There was no increase in mortality in the treated rats or bodyweight effects and borderline effects on carcinogenicity were seen in females only at 5 ppm. The study focused on neoplastic changes and provided little useful information for the assessment of chronic toxicity of acrylonitrile. A NOAEC of between 5 -10 ppm can be estimated for this study.

 

Dudley et al. (1942) investigated the effects of repeated inhalation exposure to acrylonitrile were in a series of studies in various species (rats, rabbits, guinea pigs, dogs, cats and rhesus monkeys). The experiment was split into three studies: Study 1: 4 weeks exposure (4 hours/day, 5 days/week) to 56 ppm acrylonitrile in air, dogs and monkeys; Study 2: 8 weeks exposure (4 hours/day 5 days/week) to 100 ppm acrylonitrile in air, rats, guinea pigs, rabbits and cats; Study 3: 8 weeks exposure (4 hours/day 5 days/week) to 153 ppm acrylonitrile in air, rats, guinea pigs, rabbits, cats and monkeys. One dog (of 2) died following the fourth exposure to 56 ppm. One cat (of 4) died following the eleventh exposure to 100 ppm. Four adult rats (of 8) died during the third and fourth week of exposure to 153 ppm, and five young rats (of 8) died during the third and fifth exposure weeks. Three guinea pigs (of 16) died during the fifth week of exposure to 153 ppm. One rabbit (of 4) died during the first week of exposure to 153 ppm. One cat (of 4) died after the second exposure, and one monkey (of 2) died during week 6 of exposure. The experiments demonstrated that dogs are more susceptible to acrylonitrile than monkeys. Cats were shown to be more sensitive to acrylonitrile than rodents and rabbits. Repeated exposures to 153 ppm were toxic to guinea pigs, rats, rabbits, and were less toxic to monkeys and cats. Exposure to 153 ppm resulted in irritation of the eyes and nose, loss of appetite, gastro-intestinal disturbances, and an incapacitating weakness of the hind legs from which the animals recovered relatively rapidly. Target organs identified were the nervous system (transitory limb weakness and/or paralysis in dogs and cats), the kidney (histopathological changes in rats and rabbits), the upper respiratory tract (nasal irritation in all species studied) and the lung (bronchopneumonia in all species except cats).

 

The EU RAR summarises a number of additional studies investigating the repeated exposure inhalation toxicity of acrylonitrile. The studies are not of standard design or are considered to be of questionable quality, and therefore are not considered to be of critical relevance to the risk assessment. The studies are included for completeness. Gut et al. (1985) studied the effect on intermediary metabolism in rats following repeated exposure to acrylonitrile via inhalation. Male Wistar rats were exposed to acrylonitrile at a concentration of 130 ppm (280 mg/m3) for 8 hours/day for 5 days. Bhooma et al. (1992) studied the effect of acrylonitrile on the procoagulant activity (PCA) of rat lung. In this study six rats were exposed to acrylonitrile levels of 100 ppm (225 mg/m3) for 5 hours/day for 5 days. The lungs (together with other organs) were removed. The lungs were lavaged 6 times with 5 ml cold, sterile, isotonic saline, with collection of the lavage effluent. Cells were counted in a haemocytometer chamber and viability was determined. Levels of PCA in macrophages and bronchoalveolar lavage (BAL) fluid were measured on day 1, 3, 5, 7, 14 and 28 days after acrylonitrile exposure. 90-day subacute inhalation studies (Brewer, 1976) were carried out in groups of 6 beagle dogs (3 male and 3 female) exposed to mean atmospheric concentrations of 0, 24 (54 mg/m3) and 54 ppm (121.5 mg/m3) acrylonitrile, and in 40 albino rats and 30 albino CD-1 mice exposed to 0, 24 (54 mg/m3), 54 ppm (121.5 mg/m3 and 108 (243 mg/m3). The exposure regime was 6 hours per day, 5 days per week for a total of 57 exposures. Microscopic examinations of brain (cerebrum, cerebellum and pons), bronchi, small intestine, gonads, gall bladder (dogs only), heart, kidney, liver, lungs, lymph nodes, spleen, trachea and thyroid were carried out in untreated controls (dog) and dogs at 24 and 54 ppm (54 mg/m3 and 121.5 mg/m3), untreated controls (rats and mice) and rats and mice at 108 ppm (243 mg/m3). The study of Gut et al (1985) in rats indicated that blood glucose could be a good marker as an indicator of exposure to acrylonitrile, but whether this indicator is sufficiently sensitive in humans needs to be researched further.

 

The study of Bhooma et al. (1992) demonstrates findings on pro-coagulant activity consistent with the irritant effects of acrylonitrile on the lung. The EU RAR notes that, since acrylonitrile is a known irritant it could be anticipated that a degree of lung irritation would occur at levels associated with an elevation of the macrophage PCA level. The EU RAR concludes that the quality of the Brewer studies is questionable due to the presence of chronic respiratory disease in the rodents and the high mortality in all exposure groups. The value of this study for risk assessment purposes was therefore concluded to be limited. The results suggest that the main effect of acrylonitrile inhalation in dogs was irritation of the lungs, to which the females seemed to be more sensitive than males. As with acute exposure, dogs were more sensitive to acrylonitrile than rats or mice, while the two rodent species seemed to be equally sensitive. The results indicate that the subchronic no observed adverse effect level (N(A)OEL) is less than 24 ppm (54 mg/m3) in the most sensitive species, the dog, as lung irritation was still seen at this low-dose level. For the purposes of risk assessment (while recognising the limitations of the studies) this exposure level may be considered to be an LO(A)EL.

 

Summary

Repeated exposure to acrylonitrile results in damage to the kidney, gastrointestinal tract, central nervous system and adrenal gland. The respiratory tract is also affected following repeated exposure by inhalation. Dogs appear to be the most sensitive species to exposure to acrylonitrile by inhalation, with mortalities being seen at exposure levels causing no deaths in other species, however no reliable long-term oral study has been carried out in the dog. In relation to target organ toxicity, the central nervous system appears to be a primary target organ, with neurofunctional changes being observed, although the evidence for frank neurotoxicity is limited. Nephrotoxicity is observed at high dose levels. Gastrointestinal lesions seen following oral dosing may in part be due to a local irritant effect. The neurotoxicity of acrylonitrile can partly be explained by cyanide released during metabolism. Other effects may occur through the alkylation of molecules in the central nervous system by the reactive epoxide metabolite CEO. Additionally, acrylonitrile itself is capable of non-enzymatically cyanoethylating essential functional groups in the body. All of these factors may contribute to the overall toxicity of acrylonitrile.

 

For repeated dose toxicity by the oral route, the key study is the F344 rat drinking water study of Johannsen & Levinskas (1980), from which a NOAEL of 3 ppm (equivalent to average daily dose levels of 0.25 mg/kg bw/d in males and 0.36 mg/kg bw/d in females) was derived. For repeated dose inhalation toxicity, the key study is the 2 -generation rat study of Nemec et al. (2008), from which a LOAEC of 5 ppm was determined based on irritant effects on the nasal mucosa.

 

With regard to the effects of repeated exposure to acrylonitrile in humans, the EU RAR concludes that the data are difficult to assess in relation to establishment of a dose-response relationship, however many of the findings seen in animal studies (notably neurological and irritant effects) reflect the reported findings in workers. It concludes that the respiratory tract appears to be a key target organ following the inhalation of acrylonitrile, both in humans and in experimental animals.

Justification for classification or non-classification

No classification is required, based on the results of repeated dose oral and inhalation toxicity studies. Acrylonitrile is classified as acutely toxic by all routes of exposure; effects seen at high levels of exposure in repeated dose studies are considered to be manifestations of the same toxicity, therefore additional classification (STOT RE) is not proposed.