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Genetic toxicity in vitro

Description of key information

The genotoxicity of acrylonitrile has been extensively investigated in a large number of standard and non-standard studies in vitro. A number of expert reviews are also available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
No details
Species / strain / cell type:
S. typhimurium, other: TA100, TA 98, TA 1535, TA 1537 and TA 1538
Species / strain / cell type:
E. coli, other: WP2 uvr A and WP2 uvrA/pKM101
Metabolic activation:
with and without
Metabolic activation system:
SD rat (7 week old, male) liver, induced with PCB (KC-500)
Test concentrations with justification for top dose:
-S9 mix / +S9 mix ; 0, 100, 250, 500, 1,000, 2,500, 5,000 μg/plate
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamid(-S9 mix: S. typhimurium TA100, TA98, E.Coli WP2 uvrA and WP2 uvrA/pKM101)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
(-S9 mix : S. typhimurium TA1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
(-S9 mix : S. typhimurium TA1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (+S9 mix: S. typhimurium TA 100, TA 98 and TA 1535 and E. coli WP2 uvr A and WP2 uvrA/pKM101
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
(+S9 mix : S. typhimurium TA 1537, 1538)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation


DURATION
Preincubation : 20 minutes
Temperature : 37 ˚C
Exposure duration: 2 days


NUMBER OF PLATES : Solvent / vehicle controls :3, Positive controls : 2, Test substance : 2

NUMBER OF TEST REPEAT : 2

Evaluation criteria:
A test substance is considered to be mutagenic when a dose-related increase in revertant colony count is observed and the number of revertant colonies per plate with the test substance is more than twice that of the negative control and when a reproducibility of test result is observed. Additionally a test substance for which the results do not meet the above criteria is considered non-mutagenic in this system.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: S. typhimurium TA 100, TA 98, TA 1537, TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not examined
Positive controls validity:
not specified
Species / strain:
E. coli, other: WP2 uvr A and WP2 uvrA/pKM101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not examined
Positive controls validity:
not specified
Remarks on result:
other: strain/cell type: S. typhimurium TA 1535

Results of the bacterial reversion test of acrylonitrile [direct method: -S9]

Test substance dose (μg/plate) Revertant colonies per plate [Mean]
TA100 TA1535 TA98 TA1537 TA1538 WP2 uvrA WP2 uvrA / pKM101
Vehicle control 115, 135, 144

[131]
6, 7, 11

[8]
40, 48, 43

[44]
7, 10, 14

[10]
16, 19, 15

[17]
14, 11, 12

[12]
122, 144, 115

[127]
100 120, 148

[134]
7, 10

[9]
46, 46

[46]
13, 24

[19]
24, 21

[23]
15, 21

[18]
138, 137

[138]
250 120, 121

[121]
13, 11

[12]
27, 41

[34]
13, 7

[10]
17, 24

[21]
17, 18

[17]
104, 125

[115]
500 127, 142

[135]
9, 7

[8]
30, 40

[35]
17, 12

[15]
21, 19

[20]
13, 20

[17]
130, 123

[127]
1,000 146, 140

[143]
7, 10

[9]
39, 38

[39]
20, 21

[21]
33, 23

[28]
18, 12

[15]
117, 145

[131]
2,500 141, 155

[148]
8, 7

[8]
29, 48

[39]
13, 16

[15]
32, 23

[28]
23, 22

[23]
133, 149

[141]
5,000 124, 146

[135]
11, 10

[11]
37, 31

[34]
19, 21

[20]
16, 17

[17]
12, 22

[17]
140, 144

[142]
Positive control substance  AF-2 ENNG AF-2 9AAC 2NF AF-2 AF-2
μg/plate 0.01 5 0.1 80 2 0.01 0.005
colony/plate 565, 532

[549]
565, 743

[654]
479, 366

[423]
742, 607

[675]
281, 219

[250]
91, 104

[98]
938, 1010

[974]

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamid

ENNG : N-ethyl-N-nitro-N-nitrosoguanidine

9AAC : 9-aminoacridine

2NF : 2-nitrofluorene

Table2. Results of the bacterial reversion test of acrylonitrile [direct method: +S9]

Test substance dose (μg/plate) Revertant colonies per plate [Mean] 
TA100 TA1535 TA98 TA1537 TA1538 WP2 uvrA WP2 uvrA / pKM101
Vehicle control 127, 118, 128

[124]
16, 24, 15

[18]
45, 44, 36

[42]
11, 4, 5

[7]
39, 45, 33

[39]
9, 19, 19

[16]
214, 245, 240

[233]
100 117, 112

[115]
13, 23

[18]
55, 38

[47]
7, 6

[7]
36, 38

[37]
10, 15

[18]
220, 236

[228]
250 123, 125

[124]
22, 27

[25]
45, 43

[44]
5, 5

[5]
33, 34

[34]
24, 17

[21]
245, 218

[232]
500 114, 119

[117]
43, 28

[35]
44, 49

[47]
5, 7

[6]
41, 33

[37]
19, 15

[17]
235, 220

[228]
1,000 125, 138

[132]
46, 53

[50]
43, 44

[44]
7, 7

[7]
34, 44

[39]
16, 21

[19]
239, 264

[252]
2,500 121, 150

[136]
89, 62

[76]
26, 51

[39]
3, 6

[5]
35, 33

[34]
25, 24

[25]
279, 256

[268]
5,000 138, 120

[129]
73, 53

[63]
33, 47

[40]
4, 14

[9]
39, 31

[35]
15, 22

[19]
280, 291

[286]
Positive control substance  2AA 2AA 2AA B(a)P B(a)P 2AA 2AA
μg/plate 0.5 2 0.5 5 5 40 1
colony/plate 182, 213

[198]
97, 87

[92]
208, 209

[209]
98, 105

[102]
357, 428

[393]
409, 506

[458]
351, 354

[353]

2AA : 2-aminoanthracene

B(a)P : benzo(a)pyrene

Conclusions:
Acrylonitrile was found to be mutagenic under the conditions of this study [positive: weak positive in TA1535 (+S9)] .
Executive summary:

The mutagenicity of acrylonitrile was examined using preincubation method in S. typhimurium TA 100, TA 98, TA 1535, TA 1537, TA 1538 and E. coli WP2uvr A and WP2 uvrA/pKM101, at concentrations up to 5000 μg/plate with or without a metabolic activation system. In this study, slight positive mutagenic responses were obtained in TA 1535 with metabolic activation. Therefore, acrylonitrile is considered to be mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was performed in for strains of S. typhimurium only and is therefore deficient compared to the current OECD 471 guideline.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1989
Deviations:
yes
Remarks:
Only 4 strains used
Qualifier:
according to
Guideline:
other: NTP protocol
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Reversion to histidine independence
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 97
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rat (5, 10 and 30%) or Syrian hamster liver S9 (5, 10 and 30%)
Test concentrations with justification for top dose:
Five concentrations of between 100 and 10000 µg/plate in the absence of S9 or between 100 and approximately 6666 µg/plate in the presence of S-9 and a negative control (0 µg/plate) were tested. Slight toxicity was observed at the highest concentration tested for most of the strains tested.
Vehicle / solvent:
No vehicle used.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
no vehicle used
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100 & TA1535 without activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
no vehicle used
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA97 without activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
no vehicle used
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
TA98 without activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
no vehicle used
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains with activation
Details on test system and experimental conditions:
The preincubation method was used; acrylonitrile was incubated with the Salmonella strains either in buffer or the S9 metabolic activation mix for 20 minutes at 37oC. Top agar supplemented with L-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. The plates were incubated for 2 days at 37oC. Each trial consisted of triplicate plates of concurrent positive and negative control and acrylonitrile doses.
Evaluation criteria:
Histidine-independent mutant colonies were counted following the 2 day incubation period. A positive response was defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response was defined as an increase in revertants that was not dose-related, not reproducible, or not of sufficient magnitude to suppport a determination of mutagenicty. A negative response was obtained when no increase in revertant colonies was observed following acrylonitrile treatment. There was no minimum percentage or fold increase required for the chemical to be judged positive or weakly positive.
Statistics:
Mean ± standard error from each triplicate are reported
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other:
Remarks:
Either slight toxicity or tested up to at least 5000 µg/plate
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other:
Remarks:
Either slight toxicity or tested up to at least 5000 µg/plate
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight toxicity at highest concentration tested
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight toxicity at highest concentration tested
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No additional information
Remarks on result:
other:
Remarks:
Evidence of mutagenicity in this strain

Acrylonitrile was mutagenic in S. typhimurium strain TA100 in the presence of hamster liver S9, and in strain TA1535 with rat and hamster S9. It was not mutagenic in strain TA97 or TA98 with or without S9.

Results

Strain

Concentration (µg/plate)

Mean number of revertants/plate ± standard error

-S9

Hamster S-9

Rat S-9

5%

10%

10%

30

5%

10%

10%

30%

TA100

0

99 ± 5.6

94 ± 1.5

92 ± 6.7

95 ± 2.1

115 ± 9.3

90 ±10.2

92 ± 6.9

93 ± 4.6

93 ± 3.1

100

96 ± 4.9

89 ± 7.4

106 ± 1.5

98 ± 7.1

126 ± 7.2

97 ± 6.0

88 ± 3.4

100 ± 4.3

100 ± 5.7

333

89 ± 0.9

75 ± 8.7

131 ± 8.1

100 ± 6.3

161 ± 4.2

95 ± 7.1

99 ± 6.6

104 ± 2.6

109 ± 4.4

1000

71 ± 4.7

102 ± 7.8

143 ± 1.8

122 ± 12.5

260 ± 9.1

94 ± 2.4

90 ± 8.4

106 ± 6.9

125 ± 2.0

3333

60 ± 6.9

100 ± 5.5

164 ± 1.2

144 ± 7.3

373 ± 8.1

96 ± 6.0

95 ± 0.9

109 ± 9.2

140 ± 1.5

6666

NT

101 ± 4.3

NT

165 ± 7.6

435 ± 8.2

108 ± 3.8

NT

104 ± 7.0

124 ± 5.1

6667

NT

NT

178 ± 15.9a

NT

NT

NT

71 ± 5.9a

NT

NT

10000

61 ± 4.4a

NT

NT

NT

NT

NT

NT

NT

NT

Positive control

1188 ± 67.7

3138 ± 158.3

917 ± 34.0

1197 ± 90.7

1115 ± 18.3

2871 ± 62.8

1292 ± 62.5

1523 ± 24.1

509 ± 5.6

 

Strain

Concentration (µg/plate)

Mean number of revertants/plate ± standard error

-S9

Hamster S-9

Rat S-9

5%

10%

10%

30

5%

10%

10%

30%

TA1535

0

25 ± 1.0

14 ± 1.2

16 ± 3.5

11 ± 2.3

23 ± 4.2

12 ± 3.4

11 ± 3.1

12 ± 3.5

19 ± 1.2

100

25 ± 1.8

13 ± 1.2

23 ± 3.7

19 ± 2.6

33 ± 2.1

15 ± 0.9

14 ± 1.5

14 ± 3.1

19 ± 1.2

333

23 ± 0.6

18 ± 3.2

33 ± 2.5

30 ± 2.1

67 ± 4.2

16 ± 3.2

21 ± 1.5

23 ± 4.4

32 ± 1.9

1000

18 ± 0.3

24 ± 2.1

79 ± 5.2

53 ± 3.2

161 ± 7.2

25 ± 2.1

27 ± 7.0

23 ± 1.2

51 ± 3.8

3333

10 ± 2.5

36 ± 3.5

94 ± 3.8

95 ± 5.2

364 ± 19.7

27 ± 2.7

24 ± 2.6

47 ± 6.4

98 ± 8.6

6666

NT

43 ± 7.5a

NT

105 ± 3.9

432 ± 13.0

23 ± 1.5

NT

46 ± 3.2a

107 ± 3.5

6667

NT

NT

85 ± 9.2a

NT

NT

NT

21 ± 3.3a

NT

NT

10000

6 ± 0.6a

NT

NT

NT

NT

NT

NT

NT

NT

Positive control

829 ± 35.5

161 ± 5.9

123 ± 11.6

118 ± 9.0

229 ± 14.4

173 ± 10.7

121 ± 2.5

134 ± 2.7

137 ± 7.2

 

Strain

Concentration (µg/plate)

Mean number of revertants/plate ± standard error

-S9

Hamster S-9

Rat S-9

10%

10%

TA97

0

65 ± 3.3

102 ± 1.8

124 ± 8.0

100

82 ± 3.5

101 ± 4.0

120 ± 8.3

333

81 ± 6.5

104 ± 1.7

110 ± 8.4

1000

59 ± 8.5

112 ± 8.9

111 ± 14.6

3333

51 ± 9.0

96 ± 11.3

95 ± 6.0

6667

NT

83 ± 3.5a

NT

10000

25 ± 1.5a

NT

100 ± 10.9a

Positive control

583 ± 103.1

476 ± 36.2

578 ± 15.9

 

Strain

Concentration (µg/plate)

Mean number of revertants/plate ± standard error

-S9

Hamster S-9

Rat S-9

10%

10%

TA98

0

15 ± 0.3

33 ± 2.7

30 ±2.5

100

16 ± 3.5

27 ± 1.2

26 ± 2.7

333

18 ± 0.9

24 ± 1.2

28 ± 3.9

1000

17 ± 3.8

24 ± 1.5

20 ± 4.1

3333

15 ± 0.7

27 ± 7.4

27 ± 5.2

6667

NT

11 ± 2.6a

24 ± 0.6a

10000

6 ± 1.5a

NT

NT

Positive control

1374 ± 46.6

846 ± 19.8

1128 ± 54.0

 

NT: Not tested
a: Slight toxicity observed

Conclusions:
positive with metabolic activation in strains TA100 and TA1535
negative without metabolic activation in strains TA100 and TA1535
negative with and without metabolic activation in strains TA97 and TA98

Acrylonitrile was mutagenic to strains TA100 and TA1535 in the presence of metabolic activation (S9).
Executive summary:

The mutagenicity of acrylonitrille was determined in four Salmonella typhimurium strains using the preincubation method. The metabolic activation system used was Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver S9. Acrylonitrile (100 to 10000 µg/plate) was mutagenic in S. typhimurium strain TA100 in the presence of hamster liver S9, and in strain TA1535 with rat and hamster S9. It was not mutagenic in strains TA97 or TA98 with or without S9.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
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: Standard of Chromosomal Aberration Test in Cultured Mammalian Cells (18, March 1987) (Ministry of Labor in Japan)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not relevant
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung fibroblasts (CHL cells)
Details on mammalian cell type (if applicable):
The cells used were the 22-45th passage of culture with 25 in modal chromosome number, approximately 15 hours in a cell cycle of the duplication. The cells were defrosted from froozen condition in liquid nitrogen and were used for culture.The cells were cultured in Eagle's MEM with 10 % FCS under 37 ˚C, 5 % CO2 condition.
Metabolic activation:
with and without
Metabolic activation system:
SD Rat (7-week-old, male) liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
-S9 mix :
24h continuous treatment : 0, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035 mg/mL
48h continuous treatment : 0, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035 mg/mL

-S9 mix (short-term treatment) : 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1 mg/mL
+S9 mix (short-term treatment) : 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1 mg/mL
Vehicle / solvent:
Vehicle(s)/solvent(s) used: saline
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
(-S9 mix ; 24 hr treatment, 48 hr treatment)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
(+S9 mix, -S9 mix ; short-term treatment)
Details on test system and experimental conditions:
Exposure duration
continuous exposure: 24 or 48 hr
short-term exposure: 6 hr

NUMBER OF PLATES : 2

NUMBER OF CELLS EVALUATED: 200 cells per dose

DETERMINATION OF CYTOTOXICITY
Method: cell-growth inhibition rate
Evaluation criteria:
For the evaluation of the frequencies of structural aberrations and of polyploidy, the following criteria which are usually used for chromosomal aberration testing with CHL were employed.
Negative(-): less than 5 %
Equivocal(±): 5 % or more, less than 10 %
Positive(+): 10 % or more
Species / strain:
mammalian cell line, other: Chinese hamster lung fibroblasts (CHL cells)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
24h continuous treatment : 0.035 mg/mL, 48h continuous treatment : above 0.030 mg/mL, -S9 mix (short-term treatment) : 0.1 mg/mL, +S9 mix (short-term treatment) : 0.1 mg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Minimum D20 (D20 : Concentration at which will induce chromosome aberrations of 20 %) : 0.020 mg/mL (structural chromosome aberration, 48 hr treatment, without S9 mix)

Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (CHL cells)

Table 1 Chromosomal aberration test on CHL cells treated with acrylonitrile (continuous exposure for 24 hr or 48 hr without S9 mix)

Compound Time of exposure

(hr)
Dose

(mg/mL)
Number of cells analyzed Polyploid cells  Number of cells with structural aberration (%)
 (%) judgement gap ctb cte csb cse oth Total

[-gap]

(%)
Total

[+gap]

(%)
Final judgement
V.C. (saline) 24 10% 200 1.5 - 0.0 0.0 0.5 0.0 0.0 0.0 0.5 0.5 -

* : Positive control (mitomycin C)

ctb: chromatid break, cte:chromatid exchange, csb: chromosome break, cse: chromosome exchange, oth: others

Toxic : mitotic metaphases were not observed.

      

      

Table 2 Chromosomal aberration test on CHL cells treated with acrylonitrile (short-term exposure for 6 hr with or without S9 mix)

Compound S9

mix
Dose

(mg/mL)
Number of cells analyzed Polyploid cells Number of cells with structural aberration (%)
 (%) judgement gap ctb cte csb cse oth Total

[-gap]

(%)
Total

[+gap]

(%)
Final judgement
V.C. (saline) - 10% 200 3.5 - 0.5 0.0 0.5 0.0 0.0 0.0 0.5 1.0 -
Test sub. - 0.01 200 0.0 - 0.5 0.5 0.5 0.0 0.0 0.0 1.0 1.5 -
- 0.02 200 2.0 - 0.0 0.5 0.5 0.0 0.0 0.0 1.0 1.0 -
- 0.04 200 4.5 - 0.0 2.5 2.5 0.0 0.5 0.0 5.0 5.0 ±
- 0.06 200 1.5 - 0.5 6.0 14.0 0.0 0.0 0.0 16.5 16.5 +
- 0.08 200 0.0 - 5.0 17.0 32.0 0.0 0.0 0.0 37.5 38.5 +
- 0.10 Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic
B(a)P* - 0.01 200 3.0 - 0.0 0.5 0.0 0.0 0.0 0.0 0.5 0.5 -
V.C. (saline) + 10% 200 2.5 - 0.0 0.0 0.5 0.0 0.5 0.0 1.0 1.0 -
Test sub. + 0.01 200 1.0 - 0.0 0.0 1.0 0.0 0.0 0.0 1.0 1.0 -
+ 0.02 200 0.5 - 0.0 0.0 3.5 0.0 0.0 0.0 3.5 3.5 -
+ 0.04 200 2.0 - 2.5 10.0 36.5 0.0 0.0 0.0 39.5 41.0 +
+ 0.06 200 0.0 - 4.5 28.0 77.0 0.0 0.5 0.0 82.5 83.0 +
+ 0.08 200 0.0 - 6.5 34.5 81.5 0.0 0.0 0.5 86.0 86.0 +
+ 0.10 Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic
B(a)P* + 0.01 200 4.0 - 1.0 5.0 28.0 0.0 0.0 0.0 30.0 30.5 +

* : Positive control

ctb: chromatid break, cte:chromatid exchange, csb: chromosome break, cse: chromosome exchange, oth: others

Toxic : mitotic metaphases were not observed.

Conclusions:
Interpretation of results: positive

Acrylonitrile was found to be clastogenic under the conditions of this study.

Executive summary:

The clastogenicity of actylonitrile was investigated in CHL cells in vitro. At 48 hours treatment, an increase of the frequency in structural chromosome aberration was observed at above 0.020 mg/mL of the test substance. At a short-term treatment in the absence of a metabolic activation system, an increase of the frequency in structural chromosome aberration was observed at above 0.06 mg/mL of the test substance. In the presence of a metabolic activation system for a short-term treatment, an increase of the frequency in structural chromosome aberration was observed at above 0.04 mg/mL of the test substance. In conclusion, the test substance was judged to be positive for clastogenicity.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
other: NTP protocol
GLP compliance:
not specified
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
Thymidine kinase locus (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cells were maintained as suspension cultures in supplemented Fischer's medium at 37oC. Normal cycling time was approximately 10 hours.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
3.13, 6.25, 12.5, 25, 50 and 100 nL/mL in Experiment 1 and 5, 10, 20, 30, 40 and 50 nL/mL in Experiment 2.
Vehicle / solvent:
Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
250µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
10µg/ml
Details on test system and experimental conditions:
To reduce the number of spontaneously occurring cells resistant to trifluorothymidine (TFT), subcultures were exposed to medium containing thymidine, hypoxanthine, methotrexate and glycine for 1 day, to medium containing thymidine, hypoxanthine and glycine for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added.

Treated cultures contained 6x10e6 cells in 10ml medium. Incubation with acrylonitrile was for 4 hours, at which time the medium plus acrylonitrile was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days. Cell density was monitored so that log phase growth was maintained. After the 2 day period cells were plated in medium and soft agar supplemented with TFT for selection of TFT resistant cells, and cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37oC in 5% CO2 for 10 to 12 days. The test was performed without metabolic activation.

Ethylmethane suflonate was used as a positive control at a concentration of 250µg/ml. Methylmethane sulfonate was also used as a positive control at a concentration of 10µg/ml.

The trial was repeated to ensure reproducibility of results.
Evaluation criteria:
Both trend and peak responses had to be significant (P≤0.05) for the test to be considered positive.
Statistics:
All data were evaluated statistically for trend and peak responses.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduction in relative total growth at highest concentrations tested
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No additional information

Acrylonitrile induced mutations at concentrations of 12.5 nL/mL and higher without activation.

Experiment 1 results: In the absence of S-9

Concentration (nL/mL)

Cloning efficiency (%)

Relative Total Growth (%)

Mutant Count

Mutant fraction

(mutant cells/106clonable cells)

Mean Mutant Fraction

Vehicle control

85

105

58

23

33

69

92

91

44

72

107

55

25

77

96

88

38

3.13

62

69

53

28

31

79

53

78

33

67

53

64

32

6.25

52

68

65

41

37

58

71

61

35

70

63

74

35

12.5

71

47

186

88

71*

86

71

138

53

25

40

10

395

333

212*

72

31

337

156

79

31

346

147

50

46

3

588

428

414*

50

3

500

336

34

3

491

479

100

Lethal

-

-

-

-

Positive control (EMS 250 µg/mL)

54

50

682

421

449*

49

42

686

464

49

50

684

462

* Statistically significant at the 5% level (p≤0.05) when data were compared to the concurrent vehicle control
EMS: Ethylmethane sulfonate

 

 

Experiment 2 results: In the absence of S-9

Concentration (nL/mL)

Cloning efficiency (%)

Relative Total Growth (%)

Mutant Count

Mutant fraction

(mutant cells/106clonable cells)

Mean Mutant Fraction

0

68

83

90

44

34

97

120

83

29

78

97

69

30

5

69

66

45

22

24

45

51

35

26

10

64

69

59

31

23

84

71

52

21

52

61

27

17

20

64

56

95

49

49

55

57

69

42

83

51

142

57

30

78

46

155

67

72*

99

46

226

76

74

41

164

74

40

68

29

226

111

142*

74

24

381

173

50

88

22

407

155

192*

53

7

365

230

Positive control (EMS 250 µg/mL)

59

37

820

465

-

Positive control (MMS 10 µg/mL)

26

6

447

562

-

* Statistically significant at the 5% level (p≤0.05) when data were compared to the concurrent vehicle control
EMS: Ethylmethane sulfonate
MMS: Methylmethane sulfonate

Conclusions:
Interpretation of results: positive without metabolic activation

Acrylonitrile induced mutations at concentrations of 12.5nl/ml and higher without activation.
Executive summary:

The mutagenicity of acrylonitrile was determined in the in vitro mammalian cell gene mutation test, using mouse lymphoma L5178Y cells. Acrylonitrile induced mutations at concentrations of 12.5 nL/mL and higher, without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
other: Expert review / secondary source
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Remarks:
: studies reviewed are largely from the published literature
Type of assay:
bacterial reverse mutation assay
Target gene:
Various: reversion to histidine indepence
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
other: Aspergillus nidulans
Test concentrations with justification for top dose:
Various concentrations were used in the reviewed studies
Vehicle / solvent:
No details
Details on test system and experimental conditions:
Methodological details of individual studies are limited.
Evaluation criteria:
Methodological details of individual studies are limited.
Statistics:
Methodological details of individual studies are limited.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
positive
Species / strain:
E. coli WP2 uvr A
Genotoxicity:
positive
Species / strain:
other: Aspergillus nidulans
Genotoxicity:
positive

The results of the studies in S. typhimurium indicate, overall, that acrylonitrile is a bacterial mutagen. The mutagenicity of acrylonitrile in general appears to be dependent on the presence of exogenous metabolising systems (i.e. rat, mouse or hamster liver S9) and is also more marked in strains sensitive to base-substitution mutagens. The results of studies in E. coli lead to the conclusion that acrylonitrile is a direct acting mutagen in these bacteria.

Conclusions:
Interpretation of results: positive with metabolic activation

Acrylonitrile is weakly mutagenic in reverse mutation assays in Salmonella typhimurium and specific strains of Escherichia coli, the effect generally requiring the presence of metabolic activation, although a number of authors have reported negative results in the Salmonella assay.
Executive summary:

The EU RAR reviews and summarises the extensive dataset on the genetic toxicity of acrylonitrile. Bacterial mutagenicity studies are summarised here.

A large number of bacterial mutagenicity studies have been carried out using a range of strains of Salmonella typhimurium, both with and without metabolic activation.  The results of these assays overall indicate that acrylonitrile is a bacterial mutagen, with mutagenicity generally being dependent on the presence of exogenous metabolising systems (i.e. rat, mouse or hamster liver S9). Responses are also noted to be generally more marked in those strains sensitive to base-substitution mutagens.  Although some of the individual results obtained are considered to be equivocal or negative, interpretation of the datset as a whole indicates that acrylonitrile exhibits mutagenic potential in the Ames test using Salmonella typhimurium in the presence of metabolic activation. While the mutagenic effects of acrylonitrile in E. coli were highly reproducible and statistically significant, the effects seen were weak and reliably demonstrable only over a narrow concentration range when measured using the plate incorporation method. The results of a fluctuation assay confirmed those of the plate incorporation tests, showing mutagenic activity at acrylonitrile concentrations 4-20 fold below levels used in the plate tests in the absence of cytotoxic effects. The differential response of the various tester strains to the mutagenic action suggest that acrylonitrile causes non-excisable mis-repair DNA damage, thought to be associated with the generation of DNA strand breaks.  The results of studies in E. coli lead to the conclusion that acrylonitrile is a direct acting mutagen in these bacteria. The results of studies in Aspergillus nidulans, demonstrated a significant increase in mitotic cross-overs in the plate-incorporation assay at an acrylonitrile concentration of 806 μg/ml, while at 2015 μg/ml a non-statistically significant increase was associated with a marked decrease in survival (10% of control). In the liquid test procedure, acrylonitrile at concentrations of 0.8-4.0 mg/ml induced haploid and diploid non-disjunctional segregants ( up to 10-fold increase on historical control values).

It is concluded that acrylonitrile is weakly mutagenic in assays performed in S. typhimurium, with effects more marked in strains sensitive to base-pair substitution. Positive responses are generally also dependent on the presence of metabolic activation. Data suggest that acrylonitrile is a direct-acting mutagen in E. coli, however effects were weak.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
other: Expert review / secondary source
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The EU RAR summarises and critically reviews the available data on the clastogenicity in vitro of acrylonitrile.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The EU RAR summarises and reviews the results of a number of studies of clastogenicity in vitro.
GLP compliance:
no
Remarks:
: reviews of largely published literature studies
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not relevant to this assay type
Species / strain / cell type:
other: various cell types
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Various
Vehicle / solvent:
Not repored
Genotoxicity:
positive

The potential of acrylonitrile to induce sister chromatid exchange (SCE) and the induction of DNA single breaks was investigated in adult human bronchial epithelial cells obtained from autopsy specimens passage.  Cytotoxicity (measured by colony forming efficiency) was marked at the highest concentration of 600 μg/mL; lower concentrations were not associated with cytotoxicity. SCEs were significantly increased at concentrations of 150 and 300 μg/mL. A 2-fold increase in the SCE frequency over solvent control at the highest exposure concentration of 30 μg/ml; frequencies were also slightly increased at lower exposure levels. Effects were only seen in cultures harvested after a delay of 12 hours in addition to the standard harvest time of approximately 28 hours, consistent with the observation that acrylonitrile caused a significant cell cycle delay. Chromosomal aberrations were also increased. In a similar study, CHO cells were exposed for 1 hour to 1, 2 or 4 mM acrylonitrile (53, 106 or 212 μg/mL) and fixed between 25 and 36 hours after treatment for evaluation of SCE.  Exposure to 2 mM (+S9) produced an increase in SCE to 19.3 per cell from the control value of 11.9, but no effect was seen either at the lower concentration or in the absence of S9. Cytotoxicity at 4 mM resulted in very limited cell survival. An increase in chromosomal aberrations is also reported in cells exposed to acrylonitrile at 4 mM for 1 hour and fixed at three different times 13 to 19 hours after treatment. The significance of this finding is considered to be questionable given the cytotoxicity seen at this exposure level; there was no reliable evidence of aberrations at the lower exposure levels. In a further study, 14.5% of Chinese hamster liver fibroblast cell line CH1-L cells exposed to the highest investigated concentration of 25 μg/mL acrylonitrile (for 2 hours, -S9) showed chromosomal aberrations, compared to 1% of controls. Smaller (but statistically significant) increases were also noted at concentrations of 2.5 and 12.5 μg/mL but not at the other investigated concentration of 6.25 μg/mL. No effect on chromosome number was observed, indicating that acrylonitrile does not induce aneuploidy. Findings were confirmed in a further study in which CH1-L cells were exposed to 2.5-25 μg/mL acrylonitrile. In contrast an additional study reports that acrylonitrile at concentrations of 5 and 50 mM inhibited microtubule assembly in microtubule preparations from Drosophila melanogaster and mouse brain, indicating a potential aneuploidy effect. A study using Chinese hamster lung fibroblast (CHL) cells also demonstrated that acrylonitrile could induce chromosomal aberrations in the absence of S9. Cells were exposed for 24 or 48 hours to 3.13, 6.25 or 12.5 μg/mL with higher levels being cytotoxic. At the highest concentration, the number of cells showing aberrations, excluding gaps, was 19% at 24 hours and 30.5% at 48 hours, compared with 0.3-0.5% in saline controls. The number of polyploid cells was also increased. Chromosome aberrations, SCE and polyploidy were also investigated in the metabolically competent RL4 rat liver cell line. Cells were exposed to acrylonitrile at concentrations of 1.25, 2.5, 5 or 10 μg/mL; cytotoxicity (50% reduction in plating efficiency) was seen at an exposure level of 5-10 μg/mL. The authors found no evidence of SCE induction or chromosomal aberration. Cell cycle delay was noted at 10 μg/mL, with an associated reduction in the number of 2nd division metaphases. However this was not compensated for and thus potential SCEs or chromosome aberrations induced by acrylonitrile may have been missed in this study. Exposure of human lymphocytes to 0.5 μM acrylonitrile (26.5 μg/mL) resulted in a significant increase in SCE, however another study using concentrations of 1 and 10 μg/ml (24 hour exposure –S9; 1 hour +S9) reports negative results. A study of micronuclei induction in CHO cells reports positive result being obtained both in the presence and absence of S9.

Conclusions:
Interpretation of results: positive

Acrylonitrile induces sister chromatid exchanges, micronuclei and chromosomal aberrations in in vitro studies.
Executive summary:

The EU RAR reviews and summarises the numerous studies of chromosomal aberration, SCE and micronuclei induction performed in vitro in mammalian cells. Acrylonitrile induces sister chromatid exchanges, micronuclei and chromosomal aberrations in in vitro studies. The number of positive responses noted, leads to the conclusion that acrylonitrile is clastogenic in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
other: Expert review / secondary source
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This independently peer-reviewed review of the toxicology of acrylonitrile includes an overview of the available in vitro clastogenicity studies for acrylonitrile.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The authors provide an overview of the available in vitro data on clastogenicity.
GLP compliance:
no
Remarks:
: review of literature studies
Type of assay:
other: review of various clastogenicity studies in vitro
Target gene:
Not relevant.
Test concentrations with justification for top dose:
Various
Species / strain:
other: rat liver and CHO cell lines
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
SCE
Species / strain:
other: Chinese hamster lung, liver, and ovary cells
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks on result:
other: all strains/cell types tested

In vitro test systems using mammalian cell lines exposed to acrylonitrile gave negative results for SCE without metabolic activation and positive results with activation.

Conclusions:
Interpretation of results: positive with metabolic activation

Positive results are reported in various in vitro clastogenicity assays with acrylonitrile.
Executive summary:

The Sapphire Group reviewed the available data on the clastogenicity in mammalian cells in vitro. In vitro test systems using mammalian cell lines (rat liver and CHO cells) exposed to acrylonitrile gave negative results for SCE without metabolic activation and positive results with activation.  Chromosomal aberrations were induced in vitro in Chinese hamster lung, liver, and ovary cells in the absence of metabolic activation.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
other: Expert review / secondary source
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
Summaries and critical reviews of the available DNA damage/repair studies evaluated in the EU RAR are included. The studies are largely non-standard.
GLP compliance:
no
Remarks:
: review of largely non-standard studies
Type of assay:
other: various studies of DNA damage and repair in vitro
Target gene:
Not relevant: studies investigated DNA repair.
Species / strain / cell type:
other: various
Metabolic activation:
not specified
Test concentrations with justification for top dose:
The reviewed studies used various test concentrations.
Vehicle / solvent:
No data
Species / strain:
other: mammalian cells
Metabolic activation:
not specified
Genotoxicity:
other: generally negative results
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: other: HeLA, rat hepatocytes and human mammary epithelial cells

Negative responses have generally been obtained in DNA repair assays using rat hepatocytes and human mammary epithelial cells in vitro, however both negative and positve responses are reported for HeLa cells.

Conclusions:
Athough the results of DNA repair assays performed in cultured cells are generally negative, a small number of positive responses are also reported. Findings indicate that the positive responses noted in some studies may be attributable to the metabolite CEO.
Executive summary:

aThe EU RAR has summarised and reviewed the extensive genotoxicity dataset for acrylonitrile; studies of DNA damage and repair in vitro are reported here. Studies included investigations of unscheduled DNA synthesis using various techniques in HeLA cells, primary cultures of rat hepatocytes and human mammary epithelial cells. While the majority of studies report negative results, positive or equivocal responses are reported in a number of studies. It is notable that one study reports a negative response for acrylonitrile (even at concentrations causing cytotoxicity) but a positive response for the metabolite CEO. Findings therefore indicate that positive reponses seen in some assays may be attributable to CEO rather than acrylonitrile itself.

Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: other: all types
Type of information:
other: Expert review / secondary source
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published review of genotoxicity data
Qualifier:
no guideline available
Principles of method if other than guideline:
The authors review the genotoxicity of acrylonitrile and consider the role of genotoxic or other mechanisms in the carcinogenicity of acrylonitrile
GLP compliance:
no
Remarks:
: published review
Type of assay:
other: literature review
Target gene:
Various
Species / strain / cell type:
other: various
Test concentrations with justification for top dose:
Various
Vehicle / solvent:
Not specified
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Species / strain:
other: various
Genotoxicity:
ambiguous

In vitro genotoxicity test assays of ACN have yielded mixed results, without any consistent effect of metabolic activation apparent. Some positive genotoxicity data for ACN appear to result from artifacts or from non-DNA-reactive mechanisms. In vivo micronucleus, chromosome aberration, and autoradiographic unscheduled DNA synthesis assays were negative for ACN.

Conclusions:
Interpretation of results: positive in vitro
Executive summary:

The author critically reviews the extensive dataset available for the genetic toxicity of acrylonitrile, and additionally considers the data in relation to a mode of action for the carcinogenicity of acrylonitrile noted in chronic rodent toxicity studies. In rats, acrylonitrile exposure is associated with tumours in the brain, Zymbal's gland, and mammary gland. Adducts affecting base pairing are reported to have been formed in isolated DNA exposed in vitro to the acrylonitrile metabolite cyanoethylene oxide (CEO), while DNA from liver (not shown to be a target organ for acrylonitrile carcinogenicity in rats) contained low levels of 7-(2-oxoethyl)guanine, an adduct believed not to interfere with base pairing.  Studies have not shown adducts in brain DNA from acrylonitrile-exposed rats, suggesting that the brain tumours in these animals may have arisen by mechanisms other than direct DNA reactivity. The results of numerous genotoxicity assays performed with acrylonitrile do not indicate a particular mechanism. Positive reverse mutagenesis in Salmonella typhimurium HisG46 base substitution tester strains by acrylonitrile is attributable to the metabolite CEO. Other in vitro assays with acrylonitrile have yielded mixed results, without any consistent effect of metabolic activation apparent. Some positive studies appear to be attributable to artefacts or to non-DNA-reactive mechanisms. In vivo micronucleus, chromosome aberration, and autoradiographic unscheduled DNA synthesis assays with acrylonitrile are negative. The author notes that the comparative genotoxicity of vinyl chloride and acrylonitrile indicates that (despite other similarities) they cause rodent tumours by different mechanisms.  Importantly, the absence of DNA adduct formation in the brains of rats exposed to acrylonitrile suggests the operation of epigenetic mechanisms in the carcinogenicity of this substance.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
other: Expert review / secondary source
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The EU RAR summarises and critically reviews the results of a number of mammallian cell mutation assays performed with acrylonitrile.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The EU RAR summarises the results of a number of studies of various experimental designs.
GLP compliance:
no
Remarks:
: largely published studies
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (tk), hprt, ouabain resistance (oua)
Species / strain / cell type:
mammalian cell line, other: various
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Various test concentrations were used
Vehicle / solvent:
No details
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with
Genotoxicity:
positive

Mouse lymphoma L5178Y cells exposed for 2 hours to concentrations of 12.5-200 μg/ml acrylonitrile in the presence or absence of S9 (from Aroclor 1254-induced rat liver) showed a significant increase in the number of mutations at the 6-thioguanine locus. Positive results are also reported in mouse lymphoma L5178Y cells at the thymidine kinase (TK) locus following a treatment time of 2 hours and expression time of 4 days at acrylonitrile concentrations of 80-225 μg/ml in the presence and absence of exogenous metabolic activation (S9 from Aroclor 1254-induced rat liver). Similar results were achieved in another study using the L5178Y(TK) assay with treatment for 3 hours and expression time of 48 hours at concentrations of 5-69 μg/ml (with S9 fraction from non-induced rat liver) and concentrations of 22-43 μg/ml (without activation). This result was confirmed in a further L5178Y/TK assay, using concentrations of 10-24 μg/ml, an exposure period of 4 hours and an expression time of 48 hours: acrylonitrile induced a positive dose-related mutagenic response in the absence of S9.

In contrast, other authors observed only a weakly positive response in the L5178Y/TK assay, with or without S9 from Aroclor-induced male F344 rats with an exposure period of 4 hours, expression time of 48 hours and acrylonitrile concentrations of 1-60 μg/ml. A negative result is also reported for acrylonitrile in the L5178Y/TK assay using concentrations of 12.5-100 μg/ml, an exposure period of 2 hours, and an expression period of 48 hours, with or without S9 from Aroclor-induced Sprague Dawley rats. Negative results with acrylonitrile were also seen at the oua locus at concentrations up to 100 μg/ml and under similar experimental conditions.

A marked positive response is reported in mouse lymphoma P388F/TK cell line, with a 20-fold increase in the mutation frequency seen at the highest concentration of 161 μg/ml in the presence of metabolic activation but no effects in the absence of metabolic activation even at concentrations (up to 80 μg/ml) resulting in 50% cytotoxicity.

 

A positive response both in the presence and absence of metabolic activation (S9 fraction from Aroclor-induced rat liver was seen in TK6 human lymphoblasts (TK locus), at concentrations of 5-50 μg/ml using an exposure period of 3 hours (+S9) or 20 hours (-S9) and with an expression period of 72 hours.  The same authors also report a positive response in the metabolically competent AHH-1 cell line (HPRT locus) at concentrations of 5-25 μg/ml, an exposure period of 28 hours and an expression period of 6 days.

 

A study in the TK human lymphoblast cell line reports a negative response for acrylonitrile in the absence of S9, with less than a 2-fold increase in mutation frequency seen over a concentration range of 0.4-1.5 mM (21-80 μg/ml) but a significant positive response (4-fold increase) seen at the highest exposure concentration of 1.4 mM (74 μg/ml). In contrast, a marked positive response (17-fold increase in mutation frequency) was seen following exposure to 100 μM CEO without metabolic activation. 

Conclusions:
Interpretation of results: positive with metabolic activation

Positive results are reported in studies in mammalian cell lines, generally in the presence of metabolic activation and frequently only at cytotoxic concentrations.
Executive summary:

The EU RAR reviews and summarises the extensive database available for the mammalian cell mutation of acrylonitrile; studies invetigating effects at the Tk, oua and hprt loci are available. The results of these studies are mixed, however a number of studies report positive responses. The positive results reported in mammalian cell lines are generally seen in the presence of metabolic activation and are frequently only reported at cytotoxic concentrations. The results from these experiments confirm that acrylonitrile is weakly mutagenic in mammalian cells, while the mutagenicity exhibited by CEO suggests that this may be the ultimate mutagenic metabolite of acrylonitrile.

Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: gene mutation
Type of information:
other: Expert review / secondary source
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
This review summarises the available data on the in vitro mutagenicity of acrylonitrile in bacterial and mammalian cell systems
GLP compliance:
no
Remarks:
: literature review
Type of assay:
other: review of in vitro mutagenicity studies performed in bacteria and mammalian cell systems
Target gene:
Various
Species / strain / cell type:
other: various systems in vitro
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Various

Consitently positive responses are reported in S. typhimurium strains TA1530 and TA1535 in the presence of metabolic activation; results in other strains are less consistent, however the general requirement for metabolic activation to yield a positive response indicates a role for oxidative metabolites in the mutagenic responses observed. Positive responses are reported in E. coli; mixed responses are reported for S. cerevisae. Positive responses are noted in mammalian cell systems and a largely dependent on metabolic activation.

Conclusions:
Interpretation of results: positive with metabolic activation

The authors of the review note positive responses in various systems. The dependency on metabolic activation for a positive response and the greater activity of the metabolite CEO leads to the conclusion that the metabolite may be responsible for the observed mutagenic activity of acrylonitrile.
Executive summary:

In an independently peer-reviewed paper, The Sapphire Group present a review and critical assessment of the extensive dataset on the bacterial and mammalian cell mutagenicity of acrylonitrile. In bacterial test systems, acrylonitrile is consistently reported to be mutagenic to Salmonella typhimurium strains TA1530 and TA1535 as long as a metabolic activation system (S9) was used. Mixed results are noted in strain TA100 with S9; negative results in TA98, TA102, TA1537, and TA1538, and generally negative results are seen in all strains in the absence of metabolic activation. The general requirement for metabolic activation to yield a positive response indicates a role for oxidative metabolites in the mutagenic responses observed. Unlike S. typhimurium, acrylonitrile tested positive in three out of four strains of Escherichia coli. Variable results are reported for the mutagenic effects of acrylonitrile on fungi (Saccharomyces cerevisiae). Both positive and negative results have been obtained for gene conversion, forward and reverse mutation, and aneuploidy with and without metabolic activation using this test system. In mammalian test systems, positive results for acrylonitrile-induced gene mutation in mouse L5178Y lymphoma cells (with and without activation) have been reported by a number of workers. In contrast, other studies report negative results when testing mouse lymphoma (oubain resistant) cells for gene mutation. Similarly, negative results are also reported for acrylonitrile in the mouse L5178Y lymphoma cells and in acrylonitrile in mouse P388F lymphoma cells without activation; and positive results with activation. In human lymphoblastoid cells, acrylonitrile produced a positive mutagenic response in the presence (3.5-fold increase) and absence (2-fold increase) of metabolic activation. In the absence of S9, the increased mutational frequency was associated with marked cytotoxicity at this exposure level (18% survival).  Other reports state that acrylonitrile produces a positive mutagenic response but required metabolic activation. CEO, on the other hand, did not require activation to increase mutation frequency. The authors concluded that CEO was 15-fold more potent that acrylonitrile in producing mutations, and that the amount of CEO produced by S9 could account for the positive response for acrylonitrile.

Endpoint:
genetic toxicity in vitro, other
Remarks:
Type of genotoxicity: other: the results of various genetic effects are investigated in studies in yeast cells
Type of information:
other: Expert review / secondary source
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
The results of various genotoxic studies in yeast are reviewed in the RAR
GLP compliance:
no
Remarks:
: review of literature studies
Type of assay:
other: various studies are reported
Target gene:
Various
Species / strain / cell type:
Saccharomyces cerevisiae
Test concentrations with justification for top dose:
Various
Genotoxicity:
positive

Positive responses were seen for the induction of mitotic aneuploidy, point mutation and mitotic recombination inSaccharomyces cerevisiae(strains D61-M, D6 and D7) following exposure to aqueous acrylonitrile at concentrations up to 5000 μg/ml without metabolic activation.

 

Acrylonitrile at concentrations of 0.27-0.99 μl/ml (without exogenous metabolic activation) induced a dose-dependent increase in total cycloheximide-resistant colonies (approximately 10-fold at the highest concentration of 0.99 μ/ml) and was considered to be genetically active in Saccharomyces cerevisiae strain D61-M.

 

Acrylonitrile was also demonstrated to have mutagenic activity in three strains of Saccharomyces cerevisiae exposed to concentrations of 0.8, 8, 80 or 800 μg/ml in the presence or absence of Aroclor 1254-induced rat liver S9 fraction. Mutation frequency at the highest concentrations ranged from 10- to 20-fold of controls; cytotoxicity was very marked at 800 μg/ml. Two further studies confirmed the potential of acrylonitrile to induce mutations in Saccharomyces both with or without metabolic activation. Following treatment with 6.25-50 μg/ml acrylonitrile, a significant increase in the incidence of gene conversions (tryptophan-prototrophic colonies) was seen in Saccharomyces cerevisiae D7, in the absence of metabolic activation at concentrations of 25 and 50 μg/ml, while a level of 100 μg/ml had an inhibitory effect on growth. Acrylonitrile concentrations of 30 or 60 μg/ml also produced a large increase in respiratory-deficient (petite) mutations in this yeast strain under conditions optimising endogenous metabolic activation.

 

Acrylonitrile at a concentration of 800 μg/ml in the presence of Aroclor 1254-induced female Wistar rat liver S9 caused an significant increase in illegitimate mating in Saccharomyces cerevisiae; no effects were noted at lower concentrations (0.8, 8, 80 μg/ml).  Mitotic recombination was also increased. This genetic activity was shown to require metabolic activation.

 

Another study also demonstrated that acrylonitrile produced significant increases in the frequency of mitotic gene conversion (up to 10-fold increase in prototrophy at a concentration of 500 μg/ml) in the stationary- and log-phases of yeast culture (Saccharomyces cerevisiae JD1) in the presence of Aroclor 1254-induced rat liver S9 and in the optimised yeast P-450 assay. In contrast no dose-related increase in frequency of mutation was seen in the forward-mutation system in Schizosaccharomyces pombe P1 at concentrations of 16-250 μg/ml in the absence and presence of phenobarbital/beta-naphthoflavone-induced rat liver S9. Other authors used a forward-mutation assay in Schizosaccharomyces pombe in the growth phase, involving incorporation of acrylonitrile at dose levels of 0.2 to 250 μg/plate in the presence and absence of S9 fraction from the livers of phenobarbital or Aroclor 1254-induced rats. Mutation frequency was 3 times greater than that of the controls at concentrations of 0.2, 0.5 and 1.0 μg/plate (-S9), 3 times greater at doses of 0.2, 0.5, 1.0 and 10.0 μg/plate in the presence of S9 from phenobarbital-induced rats and 5 times greater at the same concentrations in the presence of S9 from Aroclor-induced rats.

Conclusions:
Interpretation of results: positive

A number of studies indicate positive results for acrylonitrile in yeast assays.
Executive summary:

A number of investigators have used various strains of Saccharomyces cerevisiae with the aim of establishing whether acrylonitrile has the potential to induce such effects as forward and reverse mutation, mitotic chromosome loss, mitotic recombination and other genetic effects. These studies, although not a standard requirement, are summarised and reviewed in the EU RAR.A number of studies indicate positive results in yeast assays.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The genotoxicity of acrylonitrile has been extensively investigated in a large number of standard and non-standard studies in vivo. A number of expert reviews are also available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: High quality, published NTP study
Qualifier:
according to
Guideline:
other: NTP protocol
Principles of method if other than guideline:
NTP protocol: investigation of micronuclei formation in mouse peripheral blood NCEs following sub-chronic treatment. Study performed as an adjunct to a 14-week range-finding study. MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.D. (1990). The in vivo erythrocyte micronucleus test: Measurement at steady state increases assay efficiency and permits integration with toxicity studies. Fundam. Appl. Toxicol. 14, 513-522.
GLP compliance:
yes
Remarks:
FDA GLP
Type of assay:
micronucleus assay
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 72±3oF, 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 exposure:
Acrylonitrile was administered in deionised water by gavage. The dosing volume was 10ml/kg bw.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 days per week
Post exposure period:
No post exposure period
Dose / conc.:
0 mg/kg bw/day
Remarks:
Gavage; 14-week study
Dose / conc.:
5 mg/kg bw/day
Remarks:
Gavage; 14-week study
Dose / conc.:
10 mg/kg bw/day
Remarks:
Gavage; 14-week study
Dose / conc.:
20 mg/kg bw/day
Remarks:
Gavage; 14-week study
Dose / conc.:
40 mg/kg bw/day
Remarks:
Gavage; 14-week study
Dose / conc.:
60 mg/kg bw/day
Remarks:
Gavage; 14-week study
No. of animals per sex per dose:
10 animals per dose group per sex (where there were 10 animals surviving).
Control animals:
yes, concurrent vehicle
Positive control(s):
No positive control
Tissues and cell types examined:
Polychromatic erythrocytes in peripheral blood samples taken at the end of the 14 week toxicity study.
Details of tissue and slide preparation:
Smears from peripheral blood were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.
Evaluation criteria:
Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in up to 10 animals per dose group. An individual trial was considered positive in the trend test P value was less than or eual to 0.025 of if the P value for any single dosed group was less than or equal to 0.025 divided by the number of dosed groups.
Statistics:
The mean of the pooled results from all animls within a treatment group ± standard error of the mean. The frequency of micronucleated cells among NCEs was analysed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the control group (ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
No increase in the frequency of micronucleated NCEs was observed in peripheral blood samples from male or female mice administered
acrylonitrile by gavage for 14 weeks.

No increase in the frequency of micronucleated NCEs was observed in peripheral blood samples from male or female mice administered acrylonitrile by gavage for 14 weeks.

Conclusions:
Interpretation of results: negative
No increase in the frequency of micronucleated NCEs was observed in peripheral blood samples from male or female mice administered acrylonitrile by gavage for 14 weeks.
Executive summary:

Groups of male and female mice (10/sex) were gavaged with acrylonitrile (in deionised water) at dose levels of 0, 5, 10, 20, 40 or 60 mg/kg bw/d acrylonitrile on 5 days/week for 14 weeks. Terminal peripheral blood samples were collected and analysed for the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs). No increase in the frequency of micronucleated NCEs was observed in peripheral blood samples following 14 weeks oral acrylonitrile exposure.

Endpoint:
in vivo insect germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
other: expert review / secondary source
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
The EU RAR summarises the findings of a number of Drosophila genotoxicity studies of various designs.
GLP compliance:
no
Remarks:
: largely published studies
Type of assay:
other: various studies in Drosophila
Species:
Drosophila melanogaster
Route of administration:
other: injection, feeding, inhalation
Duration of treatment / exposure:
Various
Frequency of treatment:
Various
Post exposure period:
Various
No. of animals per sex per dose:
Various
Tissues and cell types examined:
Various
Genotoxicity:
positive

In a study on eukaryotic gene mutation in vivo, in which the occurrence of sex-linked recessive lethal mutations in Drosophila melanogaster was examined following administration of 0.1% acrylonitrile by intra-abdominal injection, the results proved negative.

 

Other workers tested acrylonitrile for activity in a Drosophila mitotic recombination and somatic mutation (SRM) assay using white and white-coral as genetic markers. Female Drosophila melanogaster were given food containing 5-20 mM acrylonitrile during an egg-laying period of 4 days and developing offspring were cultured for 10-11 days. Hatching females were scored for the chosen genetic markers. Acrylonitrile at a concentration of 5 mM in the food produced a 3.5-fold increase in eye mutations, the incidence of single spot mosaics rising to 1.24% compared with 0.33% in controls. Twin mosaic spots were also increased. Higher concentrations of 10 and 20 mM in food resulted in lethality and sterility.

 

An additonal study also used a Drosophila mitotic recombination and somatic mutation (SRM) assay to examine the mutagenic potential of acrylonitrile, the genetic markers chosen being wing cell spots. The Drosophila used in the study were of two types, DNA repair-proficient and excision-repair-deficient. The various treatments in this study included exposure of 48- or 72-hour-old Drosophila melanogaster larvae to gaseous acrylonitrile at a level of 1 μl in a 1,150 ml chamber (0.8 ppm) for 0.5 or 1 hours, acute feeding of 48 or 72-hour larvae with 15 or 80 mM acrylonitrile in food for 2 hours, and chronic feeding with 1.5 mM over a 96-hour period. The authors concluded that acrylonitrile was a weak mutagen (marginally positive) in the Drosophila wing spot test, on the basis of a positive response in both the DNA repair-proficient (51.9% wings with spots, compared with 28.7% in controls) and excision-repair-deficient larvae proficient (90% wings with spots, compared with 74% in controls) following chronic feeding with 1.5 mM over a 96-hour period. Results in the other exposure regimes were not consistent, some positive and some negative results being obtained. Fujikawa et al (1985, IPCS collaborative study) examined the potential of acrylonitrile to induce sex chromosome aneuploidy in the Drosophila melanogaster ZESTE system. Developing Drosophila larvae were exposed to acrylonitrile for 4 days. Exposure was accomplished by addition of 1 ml of 1, 2, 4 or 8 mM acrylonitrile onto the surface of the culture vessel. The larvae were then allowed to develop and the eyes of adult males examined for colour mutations. These authors observed a significant increase in red spot eyes indicative of a somatic mutation, the frequency being 0.39% in the cultures exposed to 8 mM acrylonitrile, compared with 0.1% in control. No increase was seen at lower dose levels.

 

A final study also used the Drosophila melanogaster ZESTE system. Groups of 20 adult female Drosophila aged 2-3 days were exposed to 2.7 ppm acrylonitrile vapour for 0, 10, 30, 50 or 70 minutes and were reunited with groups of 10 males and permitted to deposit eggs for 2 days. Acrylonitrile was found to be relatively non-toxic at the concentration tested, with 13% females being killed at this level after 70 minutes exposure. In this study, there was some evidence of chromosome loss, as evidenced by the appearance of small numbers (3-4) of white-eyed males, following 50 or 70 minutes exposure to acrylonitrile, and a single observation of chromosome gain (zeste-eyed female), following 30 or 70 minutes exposure. The mutations occurred predominantly in Brood A, obtained from the first sampling period.

Conclusions:
Interpretation of results: positive
The overall conclusion to be drawn from these Drosophila studies is that acrylonitrile is capable of producing mutations in vivo in this organism.
Executive summary:

The EU RAR summarises and critically reviews a number of studies of the genetic toxicity of acrylonitrile in Drosophila. The studies assessed various mutational endpoints, with mostly positive results. The overall conclusion to be drawn from these Drosophila studies is that acrylonitrile is capable of producing mutations in vivo in this organism.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
Assessment of genetic toxicity in the mouse micronucleus assay and DNA strand break assay
GLP compliance:
no
Remarks:
: published studies
Type of assay:
micronucleus assay
Species:
mouse
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
No details
Route of administration:
intraperitoneal
Vehicle:
No information available
Details on exposure:
Acrylonitrile was administered i.p. at a dose of 20 mg/kg bw
Duration of treatment / exposure:
Single i.p. injection
Frequency of treatment:
Single i.p. injection
Post exposure period:
Not applicable
Dose / conc.:
20 mg/kg bw (total dose)
Remarks:
Single intraperitoneal injection
No. of animals per sex per dose:
No information available
Control animals:
not specified
Positive control(s):
ENU, ethylene dibromide
Tissues and cell types examined:
Bone marrow PCEs (micronuclei) and liver (alkali-labile sites)
Evaluation criteria:
The number of DNA strand breaks and the number of micronucleated erythrocytes
Statistics:
No information available
Key result
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Acrylonitrile was negative in the bone marrow test. No single-strand breaks were detected in rat liver, but alkali-labile sites were produced at one order of magnitude lower at least than with ethylene dibromide or ENU.

Acrylonitrile was negative in the bone marrow test. No single-strand breaks were detected in rat liver, but alkali-labile sites were produced at one order of magnitude lower at least than with ethylene dibromide or ENU.

Conclusions:
Interpretation of results: negative
Acrylonitrile was negative in the bone marrow test. No single-strand breaks were detected in rat liver, but alkali-labile sites were produced at one order of magnitude lower at least than with ethylene dibromide or ENU.
Executive summary:

Acrylonitrile was administered i.p. to mice at 20 mg/kg. Acrylonitrile was negative in the bone marrow micronucleus test. No single-strand breaks were detected in rat liver, but alkali-labile sites were produced at one order of magnitude lower at least than with ethylene dibromide or ENU.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
Investigation of clastogenicity in mouse bone marrow in vivo
GLP compliance:
no
Remarks:
: older, published study
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
Male NMRI mice
Route of administration:
intraperitoneal
Vehicle:
No information available
Details on exposure:
Acrylonitrile was administered to mice in a single dose of 20 or 30 mg/kg i.p.
Duration of treatment / exposure:
Single i.p. injection; induction of chromosome aberrations was evaluated at 6, 18, 24, 48 and 72 hours after injection, and polychromatic erythrocytes were examined 24, 30 and 48 hours after injection.
Frequency of treatment:
Single i.p. injection
Post exposure period:
Not applicable
Dose / conc.:
20 mg/kg bw (total dose)
Remarks:
Single intraperitoneal injection
Dose / conc.:
30 mg/kg bw (total dose)
Remarks:
Single intraperitoneal injection
No. of animals per sex per dose:
No information available
Control animals:
yes
Positive control(s):
Not examined
Tissues and cell types examined:
Somatic and germ cells.
Details of tissue and slide preparation:
Bone marrow cells and polychromatic erythrocytes were examined. Chromosome aberrations were detected in meiotic and postmeiotic germ cells.
Evaluation criteria:
The percentage of chromosomal aberrations in bone marrow cells, and the percentage of micronuclei in polychromatic erythrocytes was compared between treated mice and controls.
Statistics:
No information available.
Sex:
male
Genotoxicity:
negative
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not examined
Additional information on results:
The percentage of aberrations and micronuclei did not differ between treated and control mice.

The percentage of aberrations and micronuclei did not differ between treated and control mice.

Conclusions:
Interpretation of results: negative
No evidence of genetic toxicity was seen under the conditions of this study.
Executive summary:

The clastogenic potential of acrylonitrile was investigated in somatic and germ cells of male NMRI mice. Induction of chromosome aberrations was followed in bone marrow cells 6, 18, 24, 48 and 72 hours after a single intraperitoneal injection of 20 or 30 mg/kg bw acrylonitrile, and polychromatic erythrocytes were examined for the presence of micronuclei 24, 30 and 48 hours after injection. The percentage of aberrations and micronuclei did not differ between treated and control mice. It was therefore concluded by the authors that acrylonitrile has no clastogenic effects on male mouse cells in vivo.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
Dominant lethal assay in rats
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
The animals were Fischer 344 male rats
Route of administration:
oral: gavage
Vehicle:
Normal saline
Details on exposure:
Acrylonitrile was administered daily for 5 days by gavage at a dose of 60 mg/kg in normall saline, acrylamide (acting as a positive control) was administered at 30 mg/kg in normal saline and a negative control group were given vehicle only. (50 rats), receiving the vehicle only, was tested in parallel. An additional group of 20 males received a single i.p. injection of 0.2 mg/kg bw triethylenemelamine (TEM) (positive control) on the afternoon of day 5.
Duration of treatment / exposure:
5 days
Frequency of treatment:
Daily for 5 days
Post exposure period:
10 weeks (for mating)
Dose / conc.:
60 mg/kg bw/day
Remarks:
Gavage dosing: 5 days
No. of animals per sex per dose:
50 males per group
Control animals:
yes, concurrent vehicle
Positive control(s):
Acrylamide 30 mg/kg in saline
Tissues and cell types examined:
Germ cells.
Details of tissue and slide preparation:
Beginning on day one after dosing, each male was caged with one untreated female weekly for 10 weeks. Females were removed after six days and replaced by a new female one day later. Females were sacrificed 13 days after the end of each respective mating week and examined for numbers of viable foetuses, early foetal deaths (resorptions), late foetal deaths, and corpora lutea.
Evaluation criteria:
Pre-implantation losses were calculated from the number of corpora lutea minus the total number of implants, while post-implantation losses were considered to be the sum of both early and late foetal deaths.
Statistics:
No information available
Sex:
male
Genotoxicity:
negative
Remarks:
acrylonitrile
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Acrylonitrile did not cause increases in either index during the 8 weeks study period. Neither compound reduced the mating rate. Acrylonitrile demonstrated no fertility effects and does not appear to be a dominant lethal mutagen in male rat germ cells in vivo. Decreases in male body weight were present for three to four weeks after dosing.

Acrylonitrile did not cause increases in post-implantation loss during the 8 weeks study period and did not reduce the mating rate. Acrylonitrile demonstrated no fertility effects and does not appear to be a dominant lethal mutagen in male rat germ cells in vivo.

Conclusions:
Interpretation of results: negative
Acrylonitrile did not demonstrate any effects on fertility, and did not appear to be a dominant lethal mutagen in male rat germ cells in vivo.
Executive summary:

The study was performed to investigate the ability of acrylonitrile to induce dominant lethal mutations in the germ cells of male F344 rats.  Groups of 50 male rats were gavaged daily for 5 days with 60 mg/kg bw acrylonitrile (in saline) or vehicle only. A positive control group of 20 males received a single intraperitoneal injection of 0.2 mg/kg triethylenemelamine (TEM) on the afternoon of Day 5.  From one day after exposure, males were mated (1:1) with untreated female per week for 4 weeks (positive controls) or 10 weeks (other groups). Females were necropsied 13 days after the end of the appropriate mating week and the amount of pre- and post-implantation loss calculated. Mating rates were reduced only during Week 1 in the positive control group. Acrylonitrile treatment of male rats induced no increases in either pre- or post-implantation loss in females in any of the 10 weeks post-exposure examined. The positive control caused both indices to increase during all 4 weeks examined. The authors concluded that acrylonitrile has neither fertility nor dominant lethal effects in the male Fischer 344 rat after oral administration. The authors further concluded that acrylonitrile may not pose a significant mutagenic risk to germ cells.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
other: expert review / secondary source
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
Review and summary of published chromosomal aberration studies.
GLP compliance:
no
Remarks:
: review of published studies
Type of assay:
chromosome aberration assay
Species:
mouse
Strain:
other: NMRI, C75B1/6
Route of administration:
intraperitoneal
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
16-24 hours
Remarks:
Doses / Concentrations:
20, 30, 60-100 mg/kg bw
Basis:
other: actual dose
No. of animals per sex per dose:
Not reported
Tissues and cell types examined:
Bone marrow
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

The results of the study of Leonard et al (1981) indicate that neither the percentage of chromosomal aberrations nor micronuclei differed from controls. Sharief et al (1986) noted a slight increase in SCEs at a dose level of 30 mg/kg bw, however overall the result is not considered to be indicative of induction of SCE. The same study did not detect an increase in chromosome aberrations in the bone marrow cells of mice dosed with 60-100 mg/kg bw acrylonitrile, a positive response was however obtained with the positive control cyclophosphamide.

Conclusions:
Interpretation of results: negative
No evidence of clastogenicity was reported under the conditions of these studies.
Executive summary:

The available data on the clastogenicity of acrylonitrile in vivo are summarised in the EU RAR. Studies investigated clastogenicity in the bone marrow of mice administered acrylonitrile by intraperitoneal injection at dose levels of 20, 30, 60 or 100 mg/kg bw. The results of the studies do not provide any evidence of clastogenicity in vivo.

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: other: Chromosomal and gene mutation
Type of information:
other: expert review / secondary source
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline available
Principles of method if other than guideline:
The paper reviews all the transgenic mutagenicity studies conducted up to July 2004, with recommendations for development of an OECD test guideline on transgenic rodent mutation assays.
GLP compliance:
not specified
Type of assay:
transgenic rodent mutagenicity assay
Species:
mouse
Strain:
other: CD2F1 (BALB/C_DBA2)
Sex:
male
Details on test animals and environmental conditions:
The test animals were lacZ transgenic mice of the strain CD2F1 (BALB/C_DBA2), obtained from Covance Research products (Denver, USA). The mice are commercially available as Muta™ mouse. Male animals were definately used as collected tissues included seminiferous tubules, it is not clear if female mice were also included in the experiment.
Route of administration:
oral: drinking water
Vehicle:
No information available.
Details on exposure:
Mice were exposed to acrylonitrile in drinking water for 28 days. Total doses were 505, 1620 and 2350 mg/kg (equivalent to approximate daily doses of 18.04, 57.86 and 83.93 mg/kg, respectively). The mice then remained untreated for a further 49 days to allow the tissues to reach the full mutant frequency.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily - in drinking water
Post exposure period:
49 days
Remarks:
Doses / Concentrations:
Total dose: 505, 1620 and 2350 mg/kg (equivalent to approximate daily doses of 18.04, 57.86 and 83.93 mg/kg, respectively)
Basis:
nominal in water
No. of animals per sex per dose:
7-8 control mice per experiment, 6-10 treated mice per experiment (see Table 1).
Control animals:
yes
Positive control(s):
The review compares the results between 163 chemicals, including substances that are employed in guideline tests as positive controls e.g. cyclophosphamide.
Tissues and cell types examined:
Any tissue can be collected from the mice that allows extraction of high molecular weight genomic DNA. The tissues used in the studies with acrylonitrile reported in the review are: splenic lymhocytes, lung, seminiferous tubules, brain and bone marrow.
Details of tissue and slide preparation:
49 days after completion of the 28 day exposure period, DNA was isolated from tissues obtained from the exposed transgenic mice, packaged into phage heads and absorbed onto E. coli.
Evaluation criteria:
Mutant frequency.
Statistics:
No information available.
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not applicable
Additional information on results:
Acrylonitrile was negative in 15 Muta™ mouse assays (Table 1).

Table 1. Results and treatment conditions in the Muta™ mouse assays with acrylonitrile

Tissue

Result

MF (control)

MF (treated)

Fold increase

IMF

Exposure period

Total consumed dose

Sampling time

Splenic lymphocytes

Negative

6.5 (8/4.5)

6.4 (10/3.8)

0.98

-0.1

28 days

1620 mg/kg

49 days

Lung

Negative

7.4 (7/3.3)

7.6 (9/5.1)

1.03

0.2

1620 mg/kg

Seminiferous tubules

Negative

3.5 (8/4.1)

2.5 (9/4.7)

0.71

-1

2350 mg/kg

Seminiferous tubules

Negative

3.5 (8/4.1)

3.2 (10/4.5)

0.91

-0.3

1620 mg/kg

Splenic lymphocytes

Negative

6.5 (8/4.5)

6.8 (9/4.3)

1.05

0.3

2350 mg/kg

Splenic lymphocytes

Negative

6.5 (8/4.5)

6 (6/3.6)

0.92

-0.5

505 mg/kg

Lung

Negative

7.4 (7/3.3)

6.8 (9/4)

0.92

-0.6

2350 mg/kg

Brain

Negative

4 (8/3.3)

4.7 (9/2.1)

1.18

0.7

2350 mg/kg

Brain

Negative

4 (8/3.3)

4.5 (9/4.9)

1.13

0.5

1620 mg/kg

Brain

Negative

4 (8/3.3)

4.8 (6/2.8)

1.2

0.8

505 mg/kg

Bone marrow

Negative

5.4 (8/4)

4.9 (9/4.1)

0.91

-0.5

2350 mg/kg

Bone marrow

Negative

5.4 (8/4)

4.5 (9/4.4)

0.83

-0.9

1620 mg/kg

Bone marrow

Negative

5.4 (8/4)

4.7 (6/2.8)

0.87

-0.7

505 mg/kg

Lung

Negative

7.4 (7/3.3)

10 (6/3.1)

1.35

2.6

505 mg/kg

Seminiferous tubules

Negative

3.5 (8/4.1)

3.3 (6/2.4)

0.94

-0.2

505 mg/kg

MF = mutation frequency x 10-5

IMF = induced mutation x 10-5

Numbers in parentheses indicate number of animals per group / number plaques per group x106

Conclusions:
Interpretation of results: negative splenic lymphocytes, lung, seminiferous tubules, brain and bone marrow
Acrylonitrile was negative in the transgenic rodent mutation model, tested up to a total dose of 2350 mg/kg (approx. 83.93 mg/kg/day for 28 days). Tissues examined were splenic lymphocytes, lung, seminiferous tubules, brain and bone marrow.
Executive summary:

The paper reviewed all the transgenic rodent mutation studies conducted up until July 2004. Results were published in the review for 163 chemicals, including acrylonitrile. Acrylonitrile was evaluated in the Muta™ mouse assay. Mice were exposed to acrylonitrile in the drinking water for 28 days, giving total consumed doses of 505, 1620 and 2350 mg/kg (controls received water alone). Following completion of the exposure period, mice went untreated for a further 49 day to allow the tissues tor each the maximum mutant frequency. Mice were then sacrificed for collection of tissues (splenic lymphocytes, lung, seminiferous tubules, brain and bone marrow). DNA was extracted from these tissues, packaged into phage heads and absorbed into E. coli. The bacteria were then plated and the number of mutant colonies determined. Acrylonitrile did not cause an increase in mutant frequeny compared to controls, in any of the tissues examined or doses levels tested. It can be concluded that acrylonitrile is negative in this transgenic rodent mutation model.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
other: expert review / secondary source
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
The EU RAR reviews the findings of a number of studies of UDS in vivo.
GLP compliance:
no
Remarks:
: largely published studies
Type of assay:
unscheduled DNA synthesis
Species:
other: rat and mouse
Strain:
other: various
Sex:
male/female
Route of administration:
other: various
Vehicle:
Various protocols are used in the reviewed studies.
Details on exposure:
Various protocols are used in the reviewed studies.
Duration of treatment / exposure:
Various protocols are used in the reviewed studies.
Frequency of treatment:
Various protocols are used in the reviewed studies.
Post exposure period:
Various protocols are used in the reviewed studies.
No. of animals per sex per dose:
Various protocols are used in the reviewed studies.
Tissues and cell types examined:
Liver, spermatocytes, lung tissue.
Sex:
male/female
Genotoxicity:
ambiguous

Conflicting results have been obtained in studies of unscheduled DNA synthesis.

Conclusions:
Interpretation of results: ambiguous : negative results in studies using autoradiography
The RAR concludes that conflicting results have been obtained in UDS studies. Negative results have been obtained in studies using rat liver hepatocytes ex vivo and in rat spermatocytes using preferred autoradiographical techniques, while UDS has been reported in rat lung and in the gastrointestinal tract in vivo, in studies uisng the less reliable method of scintillation counting.
Executive summary:

The results of the various in vivo UDS studies are critically reviewed and summarised in this document.

The study of Hogy & Guengerich (1986) measured in vivo DNA repair by incorporation of 3H-thymidine during unscheduled DNA synthesis (UDS) occurring in the presence of hydroxyurea suppression of replicative DNA synthesis. Two hours after an oral dose of 50 mg/kg to male F344 rats, acrylonitrile produced a 3-fold increase in incorporation of 3H-thymidine in liver, indicative of an effect on DNA repair, but there was no concomitant increase in the brain. Butterworth et al (1992) used autoradiography to determine UDS in spermatocytes of rats exposed to acrylonitrile at a single oral dose of 75 mg/kg, or during 5 days to an oral dose of 60 mg/kg.  Incorporation of thymidine incorporation into nuclear DNA was not significantly different between treated and control animals. The authors concluded that UDS was not induced by acrylonitrile.

 

A number of other authors (Tardif et al, 1987; Ahmed et al, 1992) have examined unscheduled DNA synthesis or repair in lung tissue. These workers dosed young male Sprague-Dawley rats orally with a single dose of 46.5 mg/kg of unlabeled acrylonitrile and then measured the replicative DNA synthesis and UDS in lung DNA by the ratio of tritiated thymidine incorporated following hydroxyurea suppression. They observed a 1.5-fold increase of thymidine incorporation into lung DNA 30 minutes after dosing, rising to a 3.3-fold increase after 24 hours. At all time points, a significant decrease in lung DNA synthesis was observed in acrylonitrile-treated animals compared to controls. The DNA replicative index was significantly lower than 1.0 at all time points, thus implying a significant decrease in lung DNA replication resulting from a single oral dose of acrylonitrile. As replicative DNA synthesis is blocked by hydroxyurea, the authors attributed the thymidine incorporation to DNA repair. However the EU RAR cautions that the lack of a validated UDS assay in the lung means that the results of these studies must however be interpreted with caution. The methodology employed (determination of radioactivity by liquid scintillation counting) is also not regarded as the most reliable means of establishing evidence of UDS, preference being given to autoradiographical techniques. Ahmed et al (1994) also studied acrylonitrile-induced gastric DNA damage and UDS in adult male Sprague-Dawley rats in the presence and absence of the P450 inhibitor SKF 525-A, which slows acrylonitrile oxidation to CEO. UDS in animals exposed orally to 23 or 46 mg/kg was increased in a dose and time dependent manner, up to a maximum of 6-fold at 2 hours post exposure, with a slow exponential decrease to baseline at 24 hours. SKF treatment prior to acrylonitrile exposure decreased the UDS observed to 35% of that observed in SKF untreated animals, consistent with an important role for CEO in the induction of UDS in acrylonitrile-exposed animals.

The RAR concludes that conflicting results have been obtained in UDS studies. Negative results have been obtained in studies using rat liver hepatocytes ex vivo and in rat spermatocytes using preferred autoradiographical techniques, while UDS has been reported in rat lung and in the gastrointestinal tract in vivo, in studies uisng the less reliable method of scintillation counting.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study examined sister chromatid exchange and chromosomal aberrations in bone marrow cells from male mice
GLP compliance:
no
Type of assay:
sister chromatid exchange assay
Species:
mouse
Strain:
C57BL
Sex:
male
Details on test animals and environmental conditions:
Male C57Bl/6 mice
Route of administration:
intraperitoneal
Vehicle:
No vehicle is reported, but controls were given saline only injections
Details on exposure:
Mice were administered a single i.p. injection of 30, 45 or 60 mg/kg acrylonitrile. Bromodeoxyuridine (BrdUrd) was administered as a subcutaneous implant 30 minutes prior to acrylonitrile dosing.
Duration of treatment / exposure:
Single i.p. injection; mice were killed 24 hours after dosing for SCE analysis, and 16 hours after dosing for analysis of chromosome aberrations.
Frequency of treatment:
Single injection
Post exposure period:
Not applicable
Dose / conc.:
30 mg/kg bw (total dose)
Remarks:
Single intraperitoneal dose
Dose / conc.:
45 mg/kg bw (total dose)
Remarks:
Single intraperitoneal dose
Dose / conc.:
60 mg/kg bw (total dose)
Remarks:
Single intraperitoneal dose
No. of animals per sex per dose:
4 males per group
Control animals:
yes
Positive control(s):
Cyclophosphamide - chromosome aberrations
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
Bone marrow cells were evaluated for sister chromatid exchange and chromosome aberration.
Evaluation criteria:
30 metaphases were scored per animal.
Statistics:
No information available
Key result
Sex:
male
Genotoxicity:
ambiguous
Remarks:
SCEs at 30 mg/kg
Toxicity:
yes
Remarks:
at 45 and 60 mg/kg
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Weakly positive results were achieved for inducttion of SCE at the 30 mg/kg dose level; 4.7 ± 0.62 SCEs per cell were seen in treated mice compared to 4.0 ± 0.79 in untreated mice and 3.5 ± 0.34 in saline controls. Only 1 animal per group survived at the two higher dose levels; the mouse that survived at 45 mg/kg displayed 7.3 ± 4.00 SCEs per cell, whilst the mouse that survived at 60 mg/kg had a comparable incidence of SCEs to controls. Some decrease in mitotic index was apparent at the higher dose levels, but no effect was apparent and 30 mg/kg. There were no increases in the number of chromosome aberrations, however a positive result was achieved with the positive control cyclophosphamide.

Weakly positive results were achieved for the induction of SCE at the 30 mg/kg dose level; 4.7 ± 0.62 SCEs per cell were seen in treated mice compared to 4.0 ± 0.79 in untreated mice and 3.5 ± 0.34 in saline controls. Only 1 animal per group survived at the two higher dose levels; the mouse that survived at 45 mg/kg displayed 7.3 ± 4.00 SCEs per cell, whilst the mouse that survived at 60 mg/kg had a comparable incidence of SCEs to controls. Some decrease in mitotic index was apparent at the higher dose levels, but no effect was apparent and 30 mg/kg. There were no increases in the number of chromosome aberrations, however a positive result was achieved with the positive control cyclophosphamide.

Conclusions:
Interpretation of results: ambiguous - weakly positive at an intermediate dose level
A slight increase in SCEs were noted in male mice at the lower dose level of 30 mg/kg bw.
Executive summary:

The authors examined the induction of sister chromatid exchange (SCE) and chromosomal aberrations in bone marrow cells from male C57BL/6 mice (4 per group) administered a single intraperitoneal injection at dose levels of 60-100 mg/kg bw acrylonitrile in physiological saline. Bromodeoxyuridine (BrdU) was administered to the animals as a subcutaneous implant 30 minutes before dosing with acrylonitrile. Animals were killed 24 hours after dosing for the analysis of SCE and at 16 hours after dosing for the analysis of chromosome aberrations. A total of 30 metaphases were examined for each animal.  A slight increase in the incidence of SCE was detected at a dose level of 30 mg/kg bw (4.7 SCEs per cell compared with 4.0 in untreated animals and 3.5 in saline controls. Statistical significance (p <0.05) is cited for this result, but the justification for this claim is not clear, and overall the result is not considered to be indicative of induction of SCE. One animal per group survived at each of the higher dose levels of 45 and 60 mg/kg bw, with an increase in SCE (7.3) being seen in the animal receiving 45 mg/kg while the animal receiving 60 mg/kg had a comparable incidence of SCE to control. Some toxicity (decreased mitotic index) was apparent at these higher dose levels, but no effect was apparent at dose levels of 30 mg/kg bw and below. No increase in chromosome aberrations in the bone marrow cells of mice dosed with 60-100 mg/kg bw acrylonitrile was seen; a positive response was however obtained with the positive control cyclophosphamide. Overall, given the limitations of this study, the result is considered to be of limited value.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The authors studied gastric DNA damage and unscheduled DNA synthesis (UDS) in rats using liquid scintillation counting (LSC)
Qualifier:
no guideline followed
Principles of method if other than guideline:
The authors studied gastric DNA damage and unscheduled DNA synthesis (UDS) in rats
GLP compliance:
no
Remarks:
: published study
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Adult male Sprague-Dawley rats
Route of administration:
oral: unspecified
Vehicle:
No information available
Details on exposure:
Rats were exposed orally to 23 or 46 mg/kg acrylonitrile, in the presence or absence of the P450 inhibitor SKF 525-A
Duration of treatment / exposure:
A single oral dose was adminstered
Frequency of treatment:
A single oral dose was adminstered
Post exposure period:
UDS was evaluated between 2 and 24 hours after dosing
Dose / conc.:
23 mg/kg bw (total dose)
Remarks:
Single gavage dose
Dose / conc.:
46 mg/kg bw (total dose)
Remarks:
Single gavage dose
No. of animals per sex per dose:
No information available
Control animals:
not specified
Positive control(s):
No information available
Tissues and cell types examined:
Gastric mucosa
Sex:
male
Genotoxicity:
positive
Additional information on results:
UDS was increased in a dose and time dependent manner, up to a maximum of 6-fold at 2 hours post exposure, with a slow exponential decrease to baseline at 24 hours. SKF 525-A treatment prior to acrylonitrile exposire decreased the UDS observed to 35% of that observed in SKF 525-A untreated animals.

UDS was increased in a dose and time dependent manner, up to a maximum of 6-fold at 2 hours post exposure, with a slow exponential decrease to baseline at 24 hours. SKF 525-A treatment prior to acrylonitrile exposire decreased the UDS observed to 35% of that observed in SKF 525-A untreated animals.

Conclusions:
Interpretation of results: positive : gastric mucosal cells
Gavage administration of acrylonitrile to rats caused UDS in gastric mucosal cells; results indicate that effects may be mediated by the metabolite, CEO.
Executive summary:

Male Sprague-Dawley rats were exposed orally to 23 or 46 mg/kg bw acrylonitrile, in the presence or absence of the P450 inhibitor SKF 525-A (which slows acrylonitrile oxidation to CEO). UDS was evaluated between 2 and 24 hours after dosing. UDS was increased in a dose and time dependent manner, up to a maximum of 6-fold at 2 hours post exposure, with a slow exponential decrease to baseline at 24 hours. SKF 525-A treatment prior to acrylonitrile exposire decreased the UDS observed to 35% of that observed in SKF 525-A untreated animals. The authors concluded that CEO plays an important role in the induction of UDS in acrylonitrile-exposed animals.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
UDS was measured using liquid scintillation counting (LSC)
Qualifier:
no guideline followed
Principles of method if other than guideline:
The authors measured in vivo DNA repair in the liver and brain by incorporation of 3H-thymidine during unscheduled DNA synthesis (UDS)
GLP compliance:
no
Remarks:
: published study
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
The animals were male Fischer 344 rats, obtained from Harlan Industries IN., and weighed approximately 300 g.
Route of administration:
oral: gavage
Vehicle:
Saline
Details on exposure:
Rats recived an oral dose by gavage of 50 mg/kg bw acrylonitrile alone, acrylonitrile (50 mg/kg bw) followed immediately by hydroxyurea (500 mg/kgbw i.p. at 2 hour intervals), hydroxyurea alone, or remained untreated (controls). Other animals were administered CEO (6 mg/kg bw). Two hours after, treatment [methyl-³H]thymidine (71 Ci/mmol) was administered at a dose of 1.2 mCi/kg bw s.c. This dose was repeated after 2 hours, and half the dose was given again after 2 more hours for a total of 3.0 mCi/kg. The rats were killed 2 hours after the last ³H-thymidine dose.
Duration of treatment / exposure:
2 hours
Frequency of treatment:
Single oral dose
Post exposure period:
Not examined
Dose / conc.:
50 mg/kg bw (total dose)
Remarks:
Single oral dose
No. of animals per sex per dose:
12 male rats per group
Control animals:
yes, concurrent no treatment
Positive control(s):
Not examined.
Tissues and cell types examined:
Brain and liver
Details of tissue and slide preparation:
The rats were killed 2 hours after the last ³H-thymidine dose and brains and livers were frozen. Organs from randomly selected groups of 3 rats were pooled to provide a total of 4 samples per treatment. DNA was isolated, dissolved in 1.5 mM sodium citrate buffer (pH 7.0) containing 15 mM NaCl, assayed and counted as a gel in Beckman MP scintillation fluid.
Evaluation criteria:
The amount of radioactivity in the samples was compared between groups.
Statistics:
t-test
Sex:
male
Genotoxicity:
positive
Remarks:
liver
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not examined
Sex:
male
Genotoxicity:
negative
Remarks:
brain
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not examined
Additional information on results:
Acrylonitrile produced a 3-fold increase in incorporation of ³H-thymidine in liver, but there was no concomitant increase in the brain.

Radioactivity detected in samples of liver and brain

Treatment

dpm/µg of DNA

Liver

Brain

Control

164 ± 41a

13.1 ± 1.6

Control + hydroxyurea

5.61 ± 0.54

1.26 ± 0.55

Acrylonitrile

168 ± 53

10.4 ± 2.1

Acrylonitrile + hydroxyurea

17.7 ± 5.8

0.80 ± 0.26

 

Correction factor – acrylonitrile treated/control

1.03 ± 0.52

0.79 ± 0.26

Repair ration – acrylonitrile + hydroxyurea/(control + hydroxyurea x correction factor)

3.07 ± 1.61b

0.80 ± 0.41c

aMean ± SD

bsignificantly greater than unity (p < 0.005, t-test one-tailed)

cNot significantly different from unity (p < 0.10, t-test, two-tailed)

Conclusions:
Interpretation of results: positive
Two hours after an oral dose of 50 mg/kg bw acrylonitrile, there was a significant increase in incorporation of ³H-thymidine in liver
Executive summary:

The authors used several approaches to investigate the mechanism of acrylonitrile carcinogenicity in rats. Groups of 12 male Fischer 344 rats received an oral dose by gavage of 50 mg/kg bw acrylonitrile alone, acrylonitrile (50 mg/kg bw) followed immediately by hydroxyurea (500 mg/kg i.p. at 2 hour intervals), hydroxyurea alone, or remained untreated (controls). Addtional groups were administered CEO (6 mg/kg bw by ip injection). Two hours after acrylonitrile treatment [methyl-³H]thymidine was administered at a dose of 1.2 mCi/kg bw s.c. This dose was repeated after 2 hours, and half the dose was given again after 2 more hours for a total of 3.0 mCi/kg. The rats were killed 2 hours after the last ³H-thymidine dose and the brains and livers frozen. Organs from randomly selected groups of 3 rats were pooled to provide a total of 4 samples per treatment. DNA was isolated and assayed, and radioactivity was estimated by scintillation counting. Two hours after an oral dose of 50 mg/kg bw acrylonitrile, there was a significant increase in incorporation of ³H-thymidine in liver, indicative of an effect on DNA repair, but there was no concomitant increase in the brain. Acrylonitrile did not appear to increase the rate of DNA synthesis resulting from tissue injury and regeneration either in the liver (a non-target organ) or the brain (a target organ) of treated rats. Acrylonitrile did, however, cause a slight increase in unscheduled DNA synthesis in rat liver but not brain. The epoxide metabolite of acrylonitrile (CEO) was formed in perfused rat liver; this metabolite accumulated in the perfusate as long as acrylonitrile was available to the organ. When radiolabelled CEO was administered to rats by intraperitoneal injection, covalent binding to both liver and brain protein was found, but no covalent binding to nucleic acids could be detected (at the level of 0.3 alkylations/10e6 bases. No 1,N6-ethenoadenosine or 1,N6-ethenodeoxyadenosine was found in liver nucleic acids after administration of either acrylonitrile or CEO, with the limits of detection being 0.3 alkylations/10e6 RNA bases and 1 alkylation/10e6 DNA bases. However, low levels of N7-(2-oxoethyl)guanine were detected in the livers of these rats (0.014-0.032 alkylations/10e6 DNA bases. In the brains of the treated rats the levels of N7-(2-oxoethyl)guanine were not above the limit of detection.

 

The authors conclude that the results of this study indicate that acrylonitrile has some limited potential for genotoxicity in vivo and that the metabolite CEO, with its ability to leave the liver and possibly to enter the brain, can interact with nucleic acids to a limited degree.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
UDS was measured using liquid scintillation counting (LSC)
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vivo UDS in the lung
GLP compliance:
no
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Young male Sprague-Dawley rats
Route of administration:
oral: gavage
Vehicle:
No vehicle reported
Details on exposure:
A single oral dose of 46.5 mg/kg was administered
Duration of treatment / exposure:
A single oral dose of 46.5 mg/kg was administered; the lungs were examined 0.5, 3, 6, 12, or 72 hours after administration
Frequency of treatment:
Single dose
Post exposure period:
Lungs were examined 0.5, 3, 6, 12, or 72 hours after administration
Dose / conc.:
46.5 mg/kg bw (total dose)
Remarks:
Single oral dose
No. of animals per sex per dose:
No information available
Control animals:
yes
Positive control(s):
Not examined
Tissues and cell types examined:
Lung tissue
Details of tissue and slide preparation:
No information available
Evaluation criteria:
Replicative DNA synthesis and UDS in lung DNA was measured by the ratio of ³H–thymidine incorporated following hydroxyurea suppression.
Statistics:
No information available
Key result
Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Remarks:
Hyperplasia of Clara cells and occasional focal perivascular edema were seen 24 hours after acrylonitrile administration.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
There was a 1.5-fold increase in thymidine incorporation into lung DNA 30 minutes after dosing, rising to a 3.3-fold increase after 24 hours. At all time points, a significant decrease in lung DNA synthesis was observed in acrylonitrile treated animals compared to controls. Covalent binding of acrylonitrile was detected in lung DNA.
Hyperplasia of Clara cells and occasional focal perivascular oedema were seen 24 hours after acrylonitrile administration.

The DNA replicative index, calculated as the amount of ³H-thymidine incorporated in DNA of acrylonitrile-treated animal/control, was significantly lower than 1.0 at all time points, thus implying a significant decrease in lung DNA replication resulting from a single oral dose of acrylonitrile. As replicative DNA synthesis is blocked by hydroxyurea, the authors attributed the thymidine incorporation to DNA repair.

Conclusions:
Interpretation of results: positive
The authors conclude that acrylonitrile demonstrates genotoxic effects in the lung following a single oral dose in rats
Executive summary:

Acrylonitrile was administered to Sprague-Dawley rats in a single oral dose of 46.5 mg/kg bw. Replicative DNA synthesis and UDS in lung DNA was measured by the ratio of tritiated thymidine incorporated following hydroxyurea suppression at 0.5, 3, 6, 12, or 72 hours after administration. The authors reported decreases in replicative DNA synthesis of more than 50% at 0.5, 6 and 24 hours after acrylonitrile administration, and an increased rate of DNA repair at 0.5 and 6 hours; they conclude that acrylonitrile demonstrates genotoxic effects following a single oral dose. The EU RAR (2004) advises that results of studies employing these methods must be interpreted with caution, as there is no validated UDS assay using lung as a target tissue.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
The authors evaluated UDS in rat spermatocytes and hepatocytes in vivo by the (preferred) autoradiographic method
GLP compliance:
no
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
not specified
Sex:
male
Details on test animals and environmental conditions:
Male rats
Route of administration:
oral: gavage
Vehicle:
No information available
Duration of treatment / exposure:
Acrylonitrile was administered orally by gavage at a single dose of 75 mg/kg bw, or 60 mg/kg bw/day for 5 days.
Frequency of treatment:
Acrylonitrile was administered orally by gavage at a single dose of 75 mg/kg bw, or 60 mg/kg bw/day for 5 days.
Post exposure period:
Not examined
Dose / conc.:
60 mg/kg bw/day
Remarks:
Gavage treatment for 5 days
Dose / conc.:
75 mg/kg bw (total dose)
Remarks:
Single gavage dose
No. of animals per sex per dose:
No information available
Control animals:
yes
Positive control(s):
No information available
Tissues and cell types examined:
Rat spermatocytes and hepatocytes were examined at 2, 4 or 12 hours following the last dose.
Details of tissue and slide preparation:
No information available
Evaluation criteria:
Incorporation of ³H-thymidine into spermatocyte DNA measured by the autoradiographic method
Statistics:
No information available
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
No UDS activity was seen in the DNA repair assay: there was no significant difference in thymidine incorporation into spermatocyte or hepatocyte DNA in treated and control animals at 2, 4 or 12 hours following the last dose.

No UDS activity was seen in the DNA repair assay with acrylonitrile: there was no significant difference in thymidine incorporation into spermatocyte or hepatocyte DNA in treated and control animals at 2, 4 or 12 hours following the last dose.

Conclusions:
Interpretation of results: negative no evidence of UDS for acrylonitrile; positive for CEO
No UDS activity was seen in the DNA repair assay: there was no significant difference in thymidine incorporation into spermatocyte or hepatocyte DNA in treated and control animals at 2, 4 or 12 hours following the last dose.
Executive summary:

The study used the preferred (autoradiography) method for the assessment of UDS in the spermatocytes and liver of rats exposed to a single oral dose of 75 mg/kg bw or to five repeated daily doses of 60 mg/kg bw/d. Acrylonitrile did not induce DNA repair in hepatocyte DNA repair assays, either in vitro or in vivo. Cyanoethylene oxide (CEO), a metabolite of acrylonitrile, did not yield a DNA repair response in this assay but was highly toxic and could not be tested at equivalent levels.  The authors concluded that these results show a highly tissue-specific pattern of genotoxic activity for acrylonitrile that correlates with the tissue-specific pattern of carcinogenicity, and that the induction of DNA repair by CEO confirms the genotoxic potential of this metabolite.

Mode of Action Analysis / Human Relevance Framework

In the most recent expert review of the genotoxicity of acrylonitrile (Albertini et al., 2016), the authors consider the evidence for direct and indirect (as a consequence of oxidative stress) damage in the context of the clearly demonstrated carcinogenicity of acrylonitrile in rodent studies; specifically the rat brain. The authors also take into account toxicokinetic, mechanistic and epidemiological data. They conclude that the available data clearly demonstrate that acrylonitrile can induce both mutations and oxidative stress.  The data indicate that the mutagenic potential of acrylonitrile is weak; most in vitro studies reporting positive results having used comparatively high concentrations and sensitive test systems. The lack of induction of sex-linked recessive lethal mutations in Drosophila is considered to be comparable with findings for other weak mutagens where mutagenic effects are inhibited by effective repair. Notably, there is also a remarkable disparity between the potential of acrylonitrile to induce chromosome level mutations in vitro and in vivo, with no evidence being demonstrated in studies in vivo, even in studies using high dose levels and non-physiological routes of administration. Acrylonitrile does, however, induce gene mutations in vivo in rodents. Furthermore, some molecular epidemiological studies in humans report gene and chromosome level mutations in exposed worker populations; however it is important to note that it impossible to exclude confounder effects in some studies and the use of inappropriate methods in others.  Despite the clear findings of carcinogenicity in rodent species, there is no convincing evidence of human carcinogenicity from epidemiological studies. There is clear evidence from studies performed with radiolabelled acrylonitrile that both acrylonitrile and the active metabolite CNEO are widely distributed in vivo and reach cancer targets. The radioactivity is, however, primarily bound to proteins. Specific DNA adducts (N7OEG) have been demonstrated only in a single study and in a non-target tissue (the liver), indicating that adducts are not efficiently produced and/or are rapidly repaired. The lack of evidence for specific DNA adducts in the target tissue (brain) suggests that direct mutagenicity is not the mode of action. In contrast, there is ample evidence for the induction of 8oxoG adducts in brain tissue at tumorigenic dose levels, implying that if mutation is a critical early event in the production of brain tumours, this is most likely secondary to indirect mutagenicity. Furthermore, several target tissues for acrylonitrile carcinogenicity in the mouse have been evaluated for somatic mutations in mice, and none have been found, arguing against direct mutagenicity. It is concluded that the mode of action for rodent carcinogenicity is probably complex and is, in part, mediated through mutagenicity. Mutagenicity, may, however, have two underlying mechanisms - direct and indirect - with current evidence indicating that indirect mutagenicity may be more prominent. Furthermore, both direct and indirect mutagenicity may be influenced by associated tissue effects such as increased cell proliferation and glutathione depletion. Non-genotoxic effects may also modulate the rodent carcinogenicity of acrylonitrile in a tissue-specific manner.  Despite the clear findings of carcinogenicity in rodent species, there is no convincing evidence of human carcinogenicity from epidemiological studies, further suggesting differences in species sensitivity.

Additional information

The genetic toxicity of acrylonitrile has been extensively investigated over many years in both standard and non-standard investigative studies in vitro and in vivo; in isolated DNA, cell cultures, experimental animal and in worker exposure studies. The majority of the studies are non-standard and, individually, many may be considered limited in terms of design. As a consequence of the extensive and historical dataset, it is inevitable that there is some inconsistency between the findings of individual studies. Interpretation of the dataset therefore requires consideration of the weight of evidence from the available studies. The acrylonitrile genotoxicity dataset has previously been reviewed by (among others), Whysner (1998), in the EU RAR (2004) and by The Sapphire Group (2004), subsequently in an update by Strother (2010) and most recently by Albertini (2016). An overview of the dataset is given below.  The significance of the findings of the various studies with regard to the mode of action of carcinogenicity of acrylonitrile seen in chronic rodent bioassays is specifically discussed. For completeness, findings of human studies are also reported.

 

Studies in vitro

Non-mutation studies 

The results of studies in vitro with acrylonitrile demonstrate binding to proteins and isolated DNA. DNA binding by acrylonitrile is relatively slow and was enhanced by liver microsomes but not by brain microsomes. In contrast, the reactive metabolite 2-cyanoethylene oxide (CEO) was shown to bind rapidly to DNA without the need for metabolic activation. Subsequent investigations have indicated that the apparent low level of binding of acrylonitrile to DNA may be attributable to protein contamination or binding to DNA-associated proteins. Acrylonitrile is capable of producing adducts in isolated DNA, albeit only following very long exposures at biologically irrelevant temperatures, to massive concentrations not representative of those achievable in vivo. In contrast, CEO has been shown to produce DNA adducts following much shorter exposure periods and at lower concentrations. The induction of 8oxoG adducts by acrylonitrile exposure has been demonstrated in rat astrocyte DNA but not in rat hepatocyte DNA, and has also been demonstrated in human astrocyte DNA, but only following exposure to much higher concentrations. Many investigators have demonstrated that acrylonitrile and CEO exposure can result in DNA strand breaks, however the methods used in many of these studies had the potential of converting alkali labile sites to DNA stand breaks. Contrasting results in standard and modified (Fapy-G) Comet assays indicate a lack of direct DNA damage by acrylonitrile. A single rec assay in B. subtilis shows a positive response for acrylonitrile in the presence of metabolic activation, indicating the induction of DNA strand breaks. The ability of acrylonitrile to induce UDS in vitro has been investigated in a number of assays using both autoradiography and liquid scintillation techniques, the former being the preferred technique as it enables scheduled (replicative) DNA synthesis to be excluded more reliably. Using liquid scintillation counting, positive responses are reported in cultured human peripheral blood lymphocytes and primary rat hepatocytes; a negative response has been reported in HeLa cells. In contrast, studies using autoradiography report negative responses in rat hepatocytes and human mammary epithelial cells, even at cytotoxic concentrations. A positive response is noted for acrylonitrile and CEO in a study using human mammary epithelial cells and autoradiography; the positive response with acrylonitrile was seen only at very high concentrations. The potential for acrylonitrile to induce sister chromatid exchange (SCE) has been investigated in a number of cell types, although it should be noted that this type of change is without genetic consequence. Positive responses have been reported in CHO cells (with and without metabolic activation) and in cultured human bronchial cells; negative responses are reported in metabolically competent rat liver cells and in human peripheral blood lymphocytes (both with and without metabolic activation). 

 

In summary, studies of non-mutational endpoints in vitro have demonstrated that acrylonitrile is capable of binding to DNA and proteins. Protein binding is more extensive and DNA binding is seen only following lengthy exposure to very high concentrations and findings may be attributable to protein contamination. In contrast, the metabolite CEO is shown to bind much more rapidly and avidly to DNA. Acrylonitrile has, however, been shown to induce the formation of oxidative damage specific DNA adducts, with mutagenic potential. A number of studies have noted the ability of acrylonitrile or CEO to cause DNA strand breakage, however findings may be attributable to the presence of alkali-labile sites. UDS is noted in a number of studies using liquid scintillation counting but generally not in studies using the preferred autoradiography method. The results of SCE studies with acrylonitrile are variable and only positive at unrealistically high concentrations.

 

Mutation studies

The mutagenicity of acrylonitrile has been investigated in a large number of bacterial mutation assays. The results of studies in Salmonella strains sensitive to frameshift mutation (TA97, TA98, TA1537, TA1538) are almost entirely negative, whereas mostly positive results are reported in Salmonella strains (TA100, TA1530, TA1535, TA1950) carrying the hisG46 allele and sensitive to GC to AT base pair substitution. It is notable that studies in TA102, which is considered to be sensitive to oxidative damage, have proved to be largely negative. Studies of bacterial mutation in E. coli strains have given mixed results, although more recent studies in strains WP2, WP2uvrA, and WP2(PKM101) have more consistently reported positive results in the presence of metabolic activation. WP2 tester strains include an AT base pair as the critical site. Fungal studies in S. cerevisiae and Schizosaccharomyces pombe have given mixed results for gene mutation endpoints but more consistently positive results for chromosomal level mutation, both with and without metabolic activation. A positive result has also been reported for aneuploidy/non-disjunction in Aspergillus nidulans.

 

In mammalian cell studies, a number of positive results are reported for acrylonitrile in L5178Y mouse lymphoma cells (Tk locus) both with and without metabolic activation; negative results are reported for this cell line at the oua locus. L5178Y cells are particularly sensitive to mutations, in part because they have a mutation in the P53 tumour suppressor gene, but also because they may be especially sensitive to oxidative damage. The results of studies in other cell lines are variable, with both negative and positive results reported. There is no consistent association with metabolic activation; some studies report positive results with activation only, others both with and without activation. Molecular analyses indicate that point mutations (for CEO involving AT and GC pairs) may predominate over deletion mutations. In mammalian cells, the potential of acrylonitrile to induce clastogenicity has been investigated in human peripheral blood lymphocytes, CHO, CHL and metabolically competent rat liver RL4 cell lines. Many studies have reported positive results for the induction of structural aberrations, with most requiring metabolic activation. There is no evidence for the induction of numerical aberrations.

 

Studies in vivo

Studies in Drosophila

Two kinds of mutation have been investigated in Drosophila studies with acrylonitrile; studies of sex-chromosome aneuploidy and gave a positive result, however further studies with this system show that nitriles can induce aneuploidy due to effects on spindle formation. Two studies of heritable SLRL gene mutation with acrylonitrile have been negative. It is notable that concentrations of acrylonitrile used in the Drosophila studies are high compared to those used in mammalian studies.

 

Non-mutation studies 

A number of studies in vivo have demonstrated that acrylonitrile and CEO are capable of binding to cellular macromolecules. Early studies may not have sufficiently differentiated DNA binding from binding to protein contaminants. Other studies have shown the induction of specific guanine adducts in liver DNA but not in brain DNA; protein binding in these organs was found to be comparable. DNA adducts specific for oxidative damage (8oxoG) have been identified in the rat, most markedly (out of the organs investigated) in the brain.  Other studies show that the induction of adducts is associated with lipid peroxidation, ROS generation and reduced levels of glutathione in the brain but not in the liver. DNA fragmentation has been reported in the brains of rats administered acrylonitrile, with this effect diminished by antioxidant (taurine) co-treatment. Increased levels of lipid peroxidation products have also been reported in the brain and plasma of treated animals. Several studies have reported the induction of UDS responses in rats administered acrylonitrile either by intraperitoneal injection or oral gavage. It is notable that all of the positive studies used liquid scintillation counting to measure UDS. One study showed that the induction of UDS response was associated with glutathione depletion, was increased by depleting glutathione prior to administration and was inhibited by pre-treatment with sulphydryl compounds. A single study using autoradiography (the preferred method) to assess UDS showed a negative response in testis and liver DNA following gavage with acrylonitrile. A single study reports an essentially negative (weak positive) SCE response in mice following the intraperitoneal injection of acrylonitrile.

 

Mutation studies

 Investigation of mutagenicity and clastogenicity in appropriate animal models is of most relevance in terms of carcinogenic potential; the models used generally incorporate relevant toxicokinetic, toxicodynamic and metabolic factors all of which could potentially influence the genetic toxicity potential of the test substance.

 

Exposure of rats by inhalation to acrylonitrile at concentrations of up to 500 ppm for 90 days did not result in observable effects on cells of the bone marrow (Johnson et al., 1978). No effects were observed in the bone marrow cells of mice administered acrylonitrile by gavage at dose levels of up to 21 mg/kg bw/d for up to 30 days, following intraperitoneal injection with dose levels of up to 20 mg/kg bw/d for up to 30 days; similarly no effects were seen in the bone marrow of rats administered acrylonitrile by gavage at a dose level of 40 mg/kg bw/d for 16 days (Rabello-Gay & Ahmed, 1980). Leonard et al., (1981)showed no induction of bone marrow micronuclei or chromosomal aberrations following the intraperitoneal injection of a single dose of acrylonitrile at a dose level of 20 or 30 mg/kg bw. No increase in the proportion of bone marrow cells was demonstrated in mice following inhalation exposure to dose levels of up to 140 mg/kg bw/d equivalent (Zhurkov et al., 1983) or following a single intraperitoneal injection of up to 60 mg/kg bw (Sharief et al., 1986). Similar negative effects were seen in mice administered acrylonitrile by single or repeated intraperitoneal injection (10 mg/kg bw) or by single (5, 10 mg/kg bw) or repeated (20 mg/kg bw) gavage dosing (Nesterova et al., 1999). The high quality NTP study (NTP, 2001) also showed no evidence of increased micronuclei formation in the peripheral blood NCEs of mice in a 14-week gavage study at dose levels of up to 60 mg/kg bw/d.

 

A small number of dominant lethal studies performed with acrylonitrile have reported negative results following administration by intraperitoneal injection in mice (Leonard et al., 1981), inhalation exposure of mice (Zurkov et al., 1983) and in rats following gavage administration (Working et al., 1987).

 

An unpublished abstract of a study of the induction of Hprt mutations in the splenic lymphocytes of mice administered acrylonitrile by gavage for 6 weeks (Walker & Ghanayem, 2003) reports positive results in normal mice at the highest dose level tested of 20 mg/kg bw/d and in CYPE2E1 knock-out mice at the highest dose level tested of 60 mg/kg bw/d (which was lethal to normal mice). Results indicate the requirement for metabolic (or enhancement by) oxidative metabolic activation of mutagenicity and also the involvement of mechanisms other than direct DNA-reactive mutagenicity. An study of Lac Z mutagenicity in the Mutamouse model using administration of acrylonitrile in the drinking water at dose levels of up to 750 ppm for 4 weeks and with a 7-week expression period reports negative findings in all tissues investigated (bone marrow, lung, splenic lymphocytes, male germ cells and brain). This assay detects point mutations, therefore indicating that the positive response in the previous study is attributable to large scale changes.

 

Cell transformation assays

Several cell transformation assays performed in various cell types with acrylonitrile report positive findings. Findings in SHE cells demonstrate a requirement for the oxidative metabolism of acrylonitrile to CEO for a positive response and an association with ROS generation, reductions in catalase and SOD activity, glutathione depletion and the formation of 8oxoG DNA adducts. Inhibition of P450 activity with ABT prevented cell transformation. Treatment with antioxidants inhibited both the markers of oxidative stress and also cell transformation, strongly suggesting that the cell transformation properties of acrylonitrile are secondary to the induction of oxidative stress. The oxidative metabolism of acrylonitrile generates cyanide, and it is notable that similar findings (oxidative stress and cell transformation) have been reported in CHO cells treated with cyanide. Acrylonitrile has also been shown to inhibit gap junctions in murine lung fibroblasts.

 

Human studies

A small number of molecular epidemiological studies of acrylonitrile-exposed populations have been performed. The ability of acrylonitrile to bind to specific nucleophilic cites on haemoglobin is utilised in these studies as a biomarker of exposure and a metric of internal dose; CEVal haemoglobin adducts have been used by a number of studies as a sensitive biomarker of exposure. Studies in acrylonitrile-exposed human populations have investigated the induction of chromosome aberration, micronuclei induction, DNA strand breaks, sex chromosome aneuploidy and investigation of sperm parameters as markers of effect. The results of studies investigating effects at the chromosomal level are mixed; one study reports a positive finding but with a response pattern which does not indicate exposure to acrylonitrile as the causative factor.  Another positive study reporting chromosomal aberrations is confounded by high levels of illness attributable to other chemical exposures. Other studies report a change in the pattern of chromosomal aberration with no overall increase, or do not provide sufficient detail to facilitate interpretation. A study of Hprt mutations in acrylonitrile-exposed workers used a method known to produce artefacts and is therefore not considered to be reliable.

 

EU RAR conclusion (EU RAR, 2004)

 

The EU RAR concludes that acrylonitrile has been shown to be weakly mutagenic in in vitro systems, indicative of a genotoxic potential.  However it was not considered that these findings were reliably reflected in the in vivo situation, suggesting that acrylonitrile or its active metabolites do not reach target tissuesin vivo, possibly due to the detoxification of the epoxide metabolite CEO via a glutathione conjugation pathway which may not exist inin vitrotest systems. Nevertheless it was concluded that, the body of evidence on the in vitro mutagenicity of acrylonitrile, together with the positive results in Drosophila, leads to the conclusion that for the purposes of risk assessment, acrylonitrile could be regarded as ‘genotoxic or at least mutagenic’.

 

Mechanistic considerations

The extensive dataset clearly indicates that acrylonitrile is genotoxic in vitro. Acrylonitrile is, however, a weak mutagenic agent. Most positive findings are reported in vitro and involve high exposure levels and test organisms selected for their susceptibility to mutagenesis. In vivo, however, there is a dramatic paucity of positive studies. There is only one report of weak gene mutation induction in mice and scattered positive results in humans, many of which can be questioned. The lack of induction of sex-linked recessive lethal mutations in Drosophila is comparable with other weak mutagens whose mutagenic effects are inhibited by effective repair systems. Acrylonitrile has the capacity to induce oxidative stress and oxidative DNA damage. This effect (and consequent indirect genotoxicity) may account for the genotoxicity profile of acrylonitrile, indicating a threshold response. The threshold is due to the fact that mutagenicity will only be apparent following exposure to sufficient acrylonitrile to generate oxidative stress and ROS and overwhelm natural cellular defences. Acrylonitrile can also produce other (non-genotoxic) cellular effects associated with carcinogenesis, including cell transformation and the inhibition of gap junctions. Some of the carcinogenicity of acrylonitrile in rodent studies in vivo is likely to depend on these non-genotoxic effects, which are also likely to be tissue-dependent. A high incidence of spontaneous tumours in a tissue or organ may reflect a high number of spontaneously initiated cells which require needing only promotion to produce tumours. The genotoxicity profile of acrylonitrile, considered in toto, is most compatible with an indirect genotoxic MoA for cancer induction in rodents, and not a direct, DNA-reactive MoA. There are exposure levels below which no oxidative stress, and no oxidative DNA damage, has be detected. These factors together with the non-linear tumor dose response observed in rodents and the absence of a causal link between acrylonitrile exposure and cancer in extensive occupational epidemiology investigations support a view that acrylonitrile is a threshold rodent carcinogen.

Justification for classification or non-classification

Based on the large body of available information, acrylonitrile is not classified with regard to germ cell mutagenicity according to the CLP Regulation.

 

The extensive dataset clearly indicates that acrylonitrile is weakly genotoxic in vitro. Studies of genotoxic effects in exposed workers have reported mostly negative results; studies that have produced indications of effects have limitations which argue against their use for classification decisions.  Adequate in vivo experimental data exist to support a decision on the classification of acrylonitrile.  All published peer reviewed studies of mutational effects in mammalian species have reported negative results, including studies of germ cell mutagenicity. These include investigations of both chromosome aberrations and micronucleus inductions in rat and mouse somatic cells and studies of heritable chromosome level changes, such as dominant lethal effects, in both species, and chromosome level changes in murine spermatocytes.  These studies were conducted independently by several laboratories, spanned a period of over 20 years, and involved exposure by a variety of routes including inhalation, oral and intraperitoneal injection. Acrylonitrile exposure concentrations (although not durations) were at levels comparable to or greater than those that have produced cancer in the various bioassays available. There are unpublished reports of slight increases in hprt mutant frequencies in lymphocytes of rats and mice treated with acrylonitrile. These results warrant verification given that negative results for LacZ mutations were published from the same mice.  Somatic cell mutations and male germ cell aneuploidy have been reported in Drosophila at very high exposure levels, while a third mutational endpoint evaluated in Drosophila, i.e. that reflecting sex-linked recessive lethal heritable mutations, has been negative as were tests of induction of reciprocal translocations.  Nitriles and a wide range of other chemicals have been shown to induce aneuploidy in Drosophila sperm. This finding does not appear to correlate with other genotoxicity findings or to be relevant to carcinogenicity.