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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:
29 June - 28 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to internationally accepted guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Official notice of J MHLW, METI and ME (21 November 2003): YAKUSHOKUHATSU No.1121002, SEIKYOKU No.2, KANPOKIHATSU No. 031121002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diisoamyl phthalate

- Substance type: organic
- Physical state: liquid
- Analytical purity: 100%


- Lot/batch No.: 7920-1-1
- Expiration date of the lot/batch:12 August 2012

- Storage condition of test material: room temperature in the dark

Method

Target gene:
Histidine operon (S. typhimurium)
Tryptophane operon (E. coli)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate (S9 mix)
Species / strain:
E. coli WP2 uvr A pKM 101
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate (S9 mix)
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, 1500, 5000 µg per plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Substance dissolved in DMSO but not in water.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Absence of S9 mix

Migrated to IUCLID6: 2 µg/plate for strains TA100 and TA1535
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Absence of S9 mix

Migrated to IUCLID6: 50 µg/plate for strain TA1537
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Absence of S9 mix

Migrated to IUCLID6: 2 µg/plate for strains TA98
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Absence of S9 mix

Migrated to IUCLID6: 2 µg/plate for strain WP2 uvrA (pKM101)
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene 5 µg/plate for strains TA100 and TA1535 and 10 µg/plate for strain WP2 uvrA (pKM101).
Remarks:
Presence of S9 mix.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Presence of S9 mix

Migrated to IUCLID6: 5 µg/plate for strains TA98 and TA1537
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for test 1; preincubation for test 2

DURATION
- Preincubation period: 0 hours for the first test and 0.5 hours for the second test
- Exposure duration: 72 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system.
Statistics:
No statistical analysis is performed.

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:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
None noted

RANGE-FINDING/SCREENING STUDIES:
None performed

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix. See attachments in background material below for historic control data

RESULTS
First test: No evidence of toxicity was obtained following exposure to diisoamyl phthalate following exposure to diisoamyl phthalate. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to diisoamyl phthalate at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

Second test: No evidence of toxicity was obtained following exposure to diisoamyl phthalate following exposure to diisoamyl phthalate.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to diisoamyl phthalate at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

Any other information on results incl. tables

Results obtained in the absence of metabolic activation: test 1 
Study number: REI0008
Experiment: Test 1
Assay conditions: Plate incorporation assay
Without metabolic activation 
  
Strain Addition Concentration per plate Mean revertants per plate Standard Deviation Fold increase relative to vehicle Individual revertant
            colony counts
  
             
TA98 DMSO 33 4.6 32, 29, 38
Diisoamylphthalate 5 µg 32.3 4.5 1 37, 28, 32
15 µg 30 4.4 0.9 35, 28, 27
50 µg 35.3 3.5 1.1 39, 32, 35
150 µg 35.3 4.5 1.1 40, 31, 35
500 µg 30 2.6 0.9 29, 33, 28
1500 µg 22.3 0.6 0.7 23, 22, 22
5000 µg 28.7 2.1 0.9 28, 31, 27
             
  
             
TA100 DMSO 152.3 16.5 169, 136, 152
Diisoamylphthalate 5 µg 139.7 1.2 0.9 139, 139, 141
15 µg 133.7 5.8 0.9 127, 137, 137
50 µg 128.3 21.2 0.8 143, 138, 104
150 µg 149 6 1 155, 143, 149
500 µg 138 18.2 0.9 128, 127, 159
1500 µg 148.3 7 1 155, 141, 149
5000 µg 135.7 7.8 0.9 138, 127, 142
             
  
             
TA1535 DMSO 26.7 0.6 27, 26, 27
Diisoamylphthalate 5 µg 18.7 5.1 0.7 20, 13, 23
15 µg 20.3 2.9 0.8 17, 22, 22
50 µg 22.7 9 0.9 18, 17, 33
150 µg 20.7 3.2 0.8 22, 17, 23
500 µg 20.7 3.5 0.8 17, 21, 24
1500 µg 25 3.5 0.9 23, 23, 29
5000 µg 18 3.5 0.7 22, 16, 16
             
  
             
TA1537 DMSO 12.3 0.6 12, 13, 12
Diisoamylphthalate 5 µg 14.7 2.3 1.2 12, 16, 16
15 µg 12.3 1.2 1 11, 13, 13
50 µg 11.7 1.2 0.9 11, 13, 11
150 µg 12.3 0.6 1 12, 12, 13
500 µg 11 1 0.9 12, 10, 11
1500 µg 13 3.6 1.1 10, 17, 12
5000 µg 13.3 2.9 1.1 10, 15, 15
             
  
             
WP2 uvrA DMSO 99.7 4.2 101, 95, 103
(pKM101) Diisoamylphthalate 5 µg 100.3 7.6 1 95, 109, 97
15 µg 106.3 28.4 1.1 101, 81, 137
50 µg 106.3 9.5 1.1 106, 116, 97
150 µg 126.3 8.5 1.3 123, 136, 120
500 µg 106 10.1 1.1 104, 97, 117
1500 µg 95.7 18.5 1 117, 86, 84
5000 µg 91.7 3.5 0.9 92, 88, 95
             
  
             
TA98 2NF 2 µg 251.7 11.5 7.6 263, 240, 252
TA100 NaN3 2 µg 663 31.1 4.4 656, 636, 697
TA1535 NaN3 2 µg 543 42.8 20.4 573, 494, 562
TA1537 AAC 50 µg 807 36.6 65.4 832, 765, 824
WP2 uvrA (pKM101) NQO 2 µg 1796.3 499 18 1896, 1255, 2238
             
  
Key to Positive Controls        
   
2NF 2-Nitrofluorene
NaN3 Sodium azide
AAC 9-Aminoacridine
NQO 4-Nitroquinoline-1-oxide
Results obtained in the presence of metabolic activation: test 1 
Study number: REI0008
Experiment: Test 1
Assay conditions: Plate incorporation assay
With metabolic activation 
  
Strain Addition Concentration per plate Mean revertants per plate Standard Deviation Fold increase relative to vehicle Individual revertant
            colony counts
  
             
TA98 DMSO 37.3 2.9 34, 39, 39
Diisoamylphthalate 5 µg 36.3 5.1 1 32, 42, 35
15 µg 40.3 4.7 1.1 44, 42, 35
50 µg 45 1 1.2 46, 45, 44
150 µg 38.7 4.7 1 37, 44, 35
500 µg 35.3 4.2 0.9 32, 34, 40
1500 µg 36.7 3.2 1 33, 39, 38
5000 µg 36 5.2 1 42, 33, 33
             
  
             
TA100 DMSO 189.7 9.6 188, 200, 181
Diisoamylphthalate 5 µg 182 20.5 1 183, 202, 161
15 µg 175.7 14 0.9 161, 177, 189
50 µg 146.7 17.5 0.8 142, 132, 166
150 µg 167.3 19.6 0.9 165, 149, 188
500 µg 180 16.7 0.9 198, 177, 165
1500 µg 160.7 14 0.8 175, 147, 160
5000 µg 162 7.2 0.9 170, 156, 160
             
  
             
TA1535 DMSO 28 1 28, 29, 27
Diisoamylphthalate 5 µg 28.3 2.5 1 31, 28, 26
15 µg 26.7 2.5 1 29, 27, 24
50 µg 26.7 5.1 1 31, 28, 21
150 µg 27.7 1.2 1 29, 27, 27
500 µg 22.3 9 0.8 31, 23, 13
1500 µg 24.3 5.5 0.9 18, 28, 27
5000 µg 20 2 0.7 20, 18, 22
             
  
             
TA1537 DMSO 32 3 29, 35, 32
Diisoamylphthalate 5 µg 26.7 1.2 0.8 26, 28, 26
15 µg 26 6.2 0.8 21, 24, 33
50 µg 27.3 1.5 0.9 27, 26, 29
150 µg 27.3 6 0.9 33, 28, 21
500 µg 25.3 4.9 0.8 31, 22, 23
1500 µg 27.3 4.5 0.9 32, 23, 27
5000 µg 25 6.1 0.8 32, 21, 22
             
  
             
WP2 uvrA DMSO 147 2 145, 149, 147
(pKM101) Diisoamylphthalate 5 µg 139 8 0.9 139, 147, 131
15 µg 148.7 14.6 1 155, 159, 132
50 µg 140.7 6.5 1 147, 134, 141
150 µg 150 19.2 1 163, 128, 159
500 µg 109.3 6.5 0.7 109, 103, 116
1500 µg 117.3 7.1 0.8 111, 125, 116
5000 µg 107.3 16.5 0.7 89, 121, 112
             
  
             
TA98 B[a]P 5 µg 199 8 5.3 207, 191, 199
TA100 AAN 5 µg 1664.3 483.9 8.8 2063, 1804, 1126
TA1535 AAN 5 µg 347 21.5 12.4 368, 325, 348
TA1537 B[a]P 5 µg 121.3 19.3 3.8 132, 99, 133
WP2 uvrA (pKM101) AAN 10 µg 480.7 43.1 3.3 434, 519, 489
             
  
Key to Positive Controls        
   
B[a]P Benzo[a]pyrene
AAN 2-Aminoanthracene
Results obtained in the absence of metabolic activation: test 2
Study number: REI0008
Experiment: Test 2
Assay conditions: Pre-incubation assay
Without metabolic activation
Strain Addition Concentration per plate Mean revertants per plate Standard Deviation Fold increase relative to vehicle Individual revertant
            colony counts
             
TA98 DMSO 33.7 1.2 33, 33, 35
Diisoamylphthalate 50 µg 38.7 6.5 1.1 39, 32, 45
150 µg 32.3 4.5 1 32, 37, 28
500 µg 42 3.5 1.2 44, 44, 38
1500 µg 38 3.6 1.1 37, 42, 35
5000 µg 29.3 2.5 0.9 32, 27, 29
             
             
TA100 DMSO 160.3 9 166, 165, 150
Diisoamylphthalate 50 µg 147 9.2 0.9 137, 155, 149
150 µg 151 9.2 0.9 141, 159, 153
500 µg 155.3 4.2 1 154, 160, 152
1500 µg 132.7 34.2 0.8 169, 101, 128
5000 µg 133.7 5.5 0.8 134, 128, 139
             
             
TA1535 DMSO 24.7 2.1 23, 27, 24
Diisoamylphthalate 50 µg 17 2.6 0.7 15, 20, 16
150 µg 17.7 0.6 0.7 18, 17, 18
500 µg 19 5.2 0.8 22, 22, 13
1500 µg 20 3 0.8 17, 23, 20
5000 µg 17 1 0.7 17, 18, 16
             
             
TA1537 DMSO 10.3 0.6 11, 10, 10
Diisoamylphthalate 50 µg 14 2.6 1.4 17, 12, 13
150 µg 10.3 2.9 1 7, 12, 12
500 µg 13 0 1.3 13, 13, 13
1500 µg 12.3 4.6 1.2 7, 15, 15
5000 µg 10.3 4.5 1 15, 10, 6
             
             
WP2 uvrA  DMSO 108.7 5.5 103, 109, 114
(pKM101) Diisoamylphthalate 50 µg 113 7.5 1 105, 114, 120
150 µg 103.3 16.5 1 87, 120, 103
500 µg 101.7 11.4 0.9 111, 89, 105
1500 µg 103 6.1 0.9 99, 100, 110
5000 µg 105 6.6 1 111, 98, 106
             
             
TA98 2NF 2 µg 265 12.2 7.9 259, 279, 257
TA100 NaN3 2 µg 800.3 50.5 5 742, 830, 829
TA1535 NaN3 2 µg 667 92.4 27 568, 751, 682
TA1537 AAC 50 µg 1264.3 216.1 122.4 1380, 1398, 1015
WP2 uvrA (pKM101) NQO 2 µg 1836.3 332.4 16.9 1562, 2206, 1741
             
Key to Positive Controls        
   
2NF 2-Nitrofluorene
NaN3 Sodium azide
AAC 9-Aminoacridine
NQO 4-Nitroquinoline-1-oxide
Results obtained in the presence of metabolic activation: test 2
Study number: REI0008
Experiment: Test 2
Assay conditions: Pre-incubation assay
With metabolic activation
Strain Addition Concentration per plate Mean revertants per plate Standard Deviation Fold increase relative to vehicle Individual revertant
            colony counts
             
TA98 DMSO 45 5.6 39, 50, 46
Diisoamylphthalate 50 µg 49.7 5 1.1 49, 45, 55
150 µg 57 2 1.3 55, 59, 57
500 µg 49.7 8.6 1.1 59, 48, 42
1500 µg 55 1 1.2 55, 56, 54
5000 µg 45.3 15.9 1 54, 55, 27
             
             
TA100 DMSO 173.3 10 163, 174, 183
Diisoamylphthalate 50 µg 188.7 12.9 1.1 198, 174, 194
150 µg 210.7 25.4 1.2 233, 183, 216
500 µg 188 43.9 1.1 235, 148, 181
1500 µg 176.3 21 1 175, 156, 198
5000 µg 179.3 24 1 185, 200, 153
             
             
TA1535 DMSO 25 6.2 20, 23, 32
Diisoamylphthalate 50 µg 20.7 0.6 0.8 21, 21, 20
150 µg 21.3 3.1 0.9 22, 24, 18
500 µg 24.7 3.8 1 22, 29, 23
1500 µg 23 2.6 0.9 21, 26, 22
5000 µg 20.3 2.1 0.8 18, 21, 22
             
             
TA1537 DMSO 28.7 2.1 28, 31, 27
Diisoamylphthalate 50 µg 29 2 1 31, 27, 29
150 µg 25.3 3.8 0.9 21, 28, 27
500 µg 25 4.6 0.9 20, 26, 29
1500 µg 26 5 0.9 21, 26, 31
5000 µg 24.7 3.2 0.9 27, 21, 26
             
             
WP2 uvrA DMSO 131.3 5.1 137, 130, 127
 (pKM101) Diisoamylphthalate 50 µg 110.3 11.5 0.8 117, 97, 117
150 µg 110 15.7 0.8 92, 121, 117
500 µg 89 5.6 0.7 84, 95, 88
1500 µg 96 8.2 0.7 103, 87, 98
5000 µg 110 9.6 0.8 117, 99, 114
             
             
TA98 B[a]P 5 µg 285 12.2 6.3 277, 279, 299
TA100 AAN 5 µg 1913.7 129.1 11 1852, 2062, 1827
TA1535 AAN 5 µg 170 5.6 6.8 176, 165, 169
TA1537 B[a]P 5 µg 142.7 14 5 147, 154, 127
WP2 uvrA (pKM101) AAN 10 µg 417 83.4 3.2 458, 321, 472
             
Key to Positive Controls        
   
B[a]P Benzo[a]pyrene
AAN 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that diisoamyl phthalate showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.