Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP condition

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Colorless or pale purple crystal (At the time of the receipt: White powder)
- purity: 99.0%
- Impurities (identity and concentrations): Water(0.16%), 9 ppm Diaminodiphenyl ether
- Lot/batch No.: 89-021 (Mitsui Toatsu Chemicals LTD.)

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
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; 0, 62.5, 125, 250, 500, 1000 and 2000 μg/plate (TA100, TA1535 and TA1537)
0, 156, 313, 625, 1250, 2500 and 5000 μg/plate (TA98)
0, 313, 625, 1250, 2500 and 5000 μg/plate (WP2 uvrA)
+S9 mix; 0, 156, 313, 625, 1250, 2500 and 5000 μg/plate (TA100, TA1535 and TA1537)
+S9 mix: 0, 313, 625, 1250, 2500 and 5000 μg/plate (TA98 and WP2 uvrA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No data
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix; AF-2 (TA100, TA98 and WP2 uvrA), Sodium azide (TA1535) and 9-Aminoacridine (TA1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: +S9 mix; 2-Aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation
DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF PLATES: 3
NUMBER OF REPLOCATES: 2

DETERMINATION OF CYTOTOXICITY
- Method: Growth inhibition
Evaluation criteria:
The chemicals were considered to be mutagenic when a dose-related increase in revertant colony count was observed and the number of revertant colonies per plate with the test substance was more than twice that of the negative control and when a reproducibility of test result was observed. A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test.
Statistics:
Statistical analysis was not conducted.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Concentration range: 50 - 5000 μg/plate (common ratio: 3)
Cycotoxic conc.:
-S9 mix: >=1500 μg/plate (TA100, TA1535, TA1537); 5000 μg/plate(TA98)
+S9 mix: 5000 μg/plate(TA100, TA1535, TA1537)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

[1st trial] (See Table1)

-S9 mix: Negative (all tester strains)

+S9 mix: Negative (all tester strains)

 

[2nd trial] (See Table2)

-S9 mix:

Negative: TA100, TA1535, TA1537, and TA98

Positive: WP2 uvrA (=1250 μg/plate)

+S9 mix: Negative (all tester strains)

 

[Confirmation test] (See Table3)

Test strain: E. coli WP2 uvr A

Concentration range: 850, 1250, 1650, 2050, 2450 μg/plate (400 μg/plate)

Result: Negative

Table1-1. Mutagenicity of 4-aminophenol in reverse mutation test (1st trial) on bacteria.

With(+) or Without(-)
S9 mix
Test substance dose (μg/plate) Revertant colonies per plate [Mean ± S.D.] 
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
S9 mix (-) 0     36, 20, 25
[27 ± 8.2]
22, 15, 17
[18 ± 3.6]
 
156     - 22, 16, 14
[17 ± 4.2]
 
313     16, 23, 13
[17 ± 5.1]
9, 20, 21
[17 ± 6.7]
 
625     24, 34, 20
[26 ± 7.2]
28, 21, 28
[26 ± 4.0]
 
1250     52, 43, 42
[46 ± 5.5]
14, 17, 21
[17 ± 3.5]
 
2500     35, 37, 50
[41 ± 8.1]
0*, 0*, 0*
[0 ± 0.0]
 
5000     21, 37, 35
[31 ± 8.7]
0*, 0*, 0*
[0 ± 0.0]
 
S9 mix (+) 0 124, 128, 127
[126 ± 2.1]
12, 6, 16
[11 ± 5.0]
33, 29, 27
[30 ± 3.1]
25, 28, 22
[25 ± 3.0]
8, 9, 8
[8 ± 0.6]
156 145, 145, 160
[150 ± 8.7]
12, 10, 12
[11 ± 1.2]
- - 11, 9, 7
[9 ± 2.0]
313 145, 141, 143
[143 ± 2.0]
13, 12, 18
[14 ± 3.2]
32, 28, 27
[29 ± 2.6]
27, 19, 30
[25 ± 5.7]
12, 4, 13
[10 ± 4.9]
625 110, 122, 119
[117 ± 6.2]
7, 12, 11
[10 ± 2.6]
24, 34, 25
[28 ± 5.5]
27, 15, 39
[27 ± 12.0]
11, 12, 8
[10 ± 2.1]
1250 84, 78, 72
[78 ± 6.0]
3, 4, 4
[4 ± 0.6]
36, 34, 48
[39 ± 7.6]
19, 27, 23
[23 ± 4.0]
13, 9, 16
[13 ± 3.5]
2500 27, 12, 29
[23 ± 9.3]
2, 2, 2
[2 ± 0.0]
46, 48, 53
[49 ± 3.6]
23, 11, 24
[19 ± 7.2]
7, 9, 10
[9 ± 1.5]
5000 22*, 60*, 36*
[39 ± 19.2]
0*, 0*, 0*
[0 ± 0.0]
10, 20, 12
[14 ± 5.3]
8, 7, 6
[7 ± 1.0]
0*, 1*, 3*
[1 ± 1.5]
Positive control
S9 mix (-)
Chemical name     AF-2 AF-2  
Dose (μg/plate)     0.01 0.1  
Number of colonies/plate     280, 281, 302
[288 ± 12.4]
640, 613, 635
[629 ± 14.4]
 
Positive control
S9 mix (+)
Chemical name 2AA 2AA 2AA 2AA 2AA
Dose (μg/plate) 1 2 10 0.5 2
Number of colonies/plate 528, 616, 700
[615 ± 86.0]
324, 360, 340
[341 ± 18.0]
597, 594, 622
[604 ± 15.4]
331, 311, 295
[312 ± 18.0]
337, 317, 325
[326 ± 10.1]

*: Inhibition was observed against growth of the bacteria.

-: Not tested

AF-2; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

SA; Sodium azide

9AA; 9-Aminoacridine

Table1-2. Mutagenicity of 4-aminophenol in reverse mutation test (1st trial) on bacteria.

With(+) or Without(-)
S9 mix
Test substance dose (μg/plate) Revertant colonies per plate [Mean ± S.D.] 
Base-pair substitution type Frameshift type
TA100 TA1535     TA1537
S9 mix (-) 0 102, 139, 118
[120 ± 18.6]
15, 11, 6
[11 ± 4.5]
    4, 5, 8
[6 ± 2.1]
62.5 122, 133, 150
[135 ± 14.1]
10, 8, 8
[9 ± 1.2]
    9, 9, 8
[9 ± 0.6]
125 133, 126, 124
[128 ± 4.7]
11, 6, 6
[8 ± 2.9]
    7, 7, 6
[7 ± 0.6]
250 147, 119, 145
[137 ± 15.6]
5, 12, 7
[8 ± 3.6]
    8, 11, 4
[8 ± 3.5]
500 116, 103, 110
[110 ± 6.5]
9, 8, 6
[8 ± 1.5]
    6, 3, 4
[4 ± 1.5]
1000 97*, 86*, 81*
[88 ± 8.2]
4*, 2*, 8*
[5 ± 3.1]
    5, 6, 8
[6 ± 1.5]
2000 60*, 51*, 70*
[60 ± 9.5]
0*, 0*, 0*
[0 ± 0.0]
    0*, 0*, 0*
[0 ± 0.0]
Positive control
S9 mix (-)
Chemical name AF-2 SA     9AA
Dose (μg/plate) 0.01 0.5     80
Number of colonies/plate 682, 778, 785
[748 ± 57.6]
296, 336, 339
[324 ± 24.0]
    1040, 539, 811
[797 ± 250.8]

*: Inhibition was observed against growth of the bacteria.

-: Not tested

AF-2; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

SA; Sodium azide

9AA; 9-Aminoacridine

Table2-1. Mutagenicity of 4-aminophenol in reverse mutation test (2nd trial) on bacteria.

With(+) or Without(-)
S9 mix
Test substance dose (μg/plate) Revertant colonies per plate [Mean ± S.D.] 
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
S9 mix (-) 0     24, 26, 20
[23 ± 3.1]
21, 12, 24
[19 ± 6.2]
 
156     - 22, 17, 20
[20 ± 2.5]
 
313     27, 23, 21
[24 ± 3.1]
20, 17, 22
[20 ± 2.5]
 
625     40, 31, 34
[35 ± 4.6]
14, 19, 26
[20 ± 6.0]
 
1250     48, 41, 51
[47 ± 5.1]
9, 23, 22
[18 ± 7.8]
 
2500     51, 34, 45
[43 ± 8.6]
0*, 0*, 0*
[0 ± 0.0]
 
5000     36*, 29*, 32*
[32 ± 3.5]
0*, 0*, 0*
[0 ± 0.0]
 
S9 mix (+) 0 157, 14, 161
[153 ± 10.6]
11, 12, 17
[13 ± 3.2]
24, 27, 20
[24 ± 3.5]
25, 29, 22
[25 ± 3.5]
13, 14, 12
[13 ± 1.0]
156 146, 157, 151
[151 ± 5.5]
9, 11, 13
[11 ± 2.0]
- - 8, 11, 13
[11 ± 2.5]
313 182, 178, 184
[181 ± 3.1]
7, 10, 5
[7 ± 2.5]
23, 33, 28
[28 ± 5.0]
27, 35, 26
[29 ± 4.9]
8, 11, 7
[9 ± 2.1]
625 155, 154, 161
[157 ± 3.8]
8, 7, 8
[8 ± 0.6]
18, 24, 28
[23 ± 5.0]
20, 30, 25
[25 ± 5.0]
11, 15, 4
[10 ± 5.6]
1250 101, 97, 91
[96 ± 5.0]
7, 6, 8
[7 ± 1.0]
43, 49, 40
[44 ± 4.6]
28, 18, 25
[24 ± 5.1]
5, 5, 6
[5 ± 0.6]
2500 22, 19, 23
[21 ± 2.1]
1, 0, 1
[1 ± 0.6]
41, 38, 42
[40 ± 2.1]
22, 15, 20
[19 ± 3.6]
3, 7, 4
[5 ± 2.1]
5000 42*, 57*, 40*
[46 ± 9.3]
0*, 0*, 0*
[0 ± 0.0]
6, 11, 14
[10 ± 4.0]
5, 9, 11
[8 ± 3.1]
0*, 0*, 1*
[0 ± 0.6]
Positive control
S9 mix (-)
Chemical name     AF-2 AF-2  
Dose (μg/plate)     0.01 0.1  
Number of colonies/plate     290, 311, 324
[308 ± 17.2]
629, 623, 632
[628 ± 4.6]
 
Positive control
S9 mix (+)
Chemical name 2AA 2AA 2AA 2AA 2AA
Dose (μg/plate) 1 2 10 0.5 2
Number of colonies/plate 760, 868, 809
[812 ± 54.1]
270, 281, 304
[285 ± 17.3]
643, 534, 563
[580 ± 56.5]
297, 294, 301
[297 ± 3.5]
262, 275, 290
[276 ± 14.0]

*: Inhibition was observed against growth of the bacteria.

-: Not tested

AF-2; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

SA; Sodium azide

9AA; 9-Aminoacridine

Table2-2. Mutagenicity of 4-aminophenol in reverse mutation test (2nd trial) on bacteria.

With(+) or Without(-)
S9 mix
Test substance dose (μg/plate) Revertant colonies per plate [Mean ± S.D.] 
Base-pair substitution type Frameshift type
TA100 TA1535     TA1537
S9 mix (-) 0 115, 148, 138
[134 ± 16.9]
11, 10, 10
[10 ± 0.6]
    12, 12, 11
[12 ± 0.6]
62.5 119, 125, 99
[114 ± 13.6]
8, 5, 14
[9 ± 4.6]
    12, 9, 8
[10 ± 2.1]
125 137, 132, 135
[135 ± 2.5]
8, 5, 10
[8 ± 2.5]
    10, 10, 7
[9 ± 1.7]
250 127, 142, 128
[132 ± 8.4]
11, 9, 5
[8 ± 3.1]
    8, 13, 8
[10 ± 2.9]
500 118, 129, 135
[127 ± 8.6]
8, 10, 8
[9 ± 1.2]
    10, 4, 8
[7 ± 3.1]
1000 106*, 101*, 100*
[102 ± 3.2]
6*, 3*, 3*
[4 ± 1.7]
    16, 4*, 6*
[9 ± 6.4]
2000 0*, 0*, 0*
[0 ± 0.0]
0*, 0*, 0*
[0 ± 0.0]
    0*, 0*, 0*
[0 ± 0.0]
Positive control
S9 mix (-)
Chemical name AF-2 SA     9AA
Dose (μg/plate) 0.01 0.5     80
Number of colonies/plate 788, 741, 788
[772 ± 27.1]
315, 319, 336
[323 ± 11.2]
    932, 845, 841
[873 ± 51.4]

*: Inhibition was observed against growth of the bacteria.

-: Not tested

AF-2; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

SA; Sodium azide

9AA; 9-Aminoacridine

Table3. Mutagenicity of 4-aminophenol in confirmation test of reverse mutation test on bacteria.

With(+) or Without(-)
S9 mix
Test substance dose (μg/plate) Revertant colonies per plate [Mean ± S.D.] 
Base-pair substitution type  
    WP2uvrA    
S9 mix (-) 0     19, 24, 31
[25 ± 6.0]
   
850     25, 16, 31
[24 ± 7.5]
   
1250     31, 46, 43
[40 ± 7.9]
   
1650     41, 42, 38
[40 ± 2.1]
   
2050     41, 36, 30
[36 ± 5.5]
   
2450     32, 41, 35
[36 ± 4.6]
   
Positive control
S9 mix (-)
Chemical name     AF-2    
Dose (μg/plate)     0.01    
Number of colonies/plate     280, 235, 254
[256 ± 22.6]
   

AF-2; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Applicant's summary and conclusion