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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471: negative

Read Across:

OECD 473: negative

OECD 476: negative

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
Study period:
Aug 04, 1999 - Feb 16, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
none
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)TRY operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
his G 428, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
E. coli WP2
Details on mammalian cell type (if applicable):
trp C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
Test concentrations with justification for top dose:
The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan: 5-5000 µg/plate have been used in the 1st and 2nd series respectively.
Vehicle / solvent:
DMSO
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Positive controls:
yes
Positive control substance:
9-aminoacridine
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9
Positive controls:
yes
Positive control substance:
other: daunomycine
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:
-----------------------------------------------------------------------------------------Mean Number of Colonies Maximal Mean Number of Colonies over the Actual(Solvent Control) Solvent Control (Test Material)-----------------------------------------------------------------------------------------<=10 <=9 >=30<=30 <=19 >=40<=80 <=29 >=80<=200 <=49 >=120<=500 <=79 >=200Assessment No increase Clear increase-----------------------------------------------------------------------------------------All further results, ranging between "no" and "clear", are assessed as "weak in-creases".Interpretations:A test material is defined as non-mutagenic in this assay if "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)A test material is defined as mutagenic in this assay if: - a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Precipitation occured at concentrations or equal 500 µg/plate. Toxicity to bacteria was not observed.

First Series


Test Material
 Concentration / [µg/plate]
 +/-
S9
mix
 Mean Revertants per plate
TA98
 TA100
 TA102
 TA1535
 TA1537
 WP2
Solvent control
-
 16
 78
 205
 8
 6
 65









Test material
 5
 -
 13
 82
 186
 10
 4
 75
15.8
 -
 13
 69
 177
 9
 3
 74
50
 -
 18
 68
 187
 12
 5
 69
150
 -
 8
 76
 184
 11
 5
 61
500
 -
 9
 73
 222
 10
 6
 69
1580
 -
 9
 70
 205
 12
 4
 65
5000
 -
 12
 68
 202
 9
 6
 58









Solvent control
+
 18
 80
 252
 11
 5
 70









Test material 5
 +
 15
 82
 249
 10
 5
 75
15.8
 +
 17
 77
 246
 12
 8
 66
50
 +
 15
 87
 248
 8
 6
 73
158
 +
 19
 81
 242
 8
 5
 69
500
 +
 19
 80
 257
 9
 5
 74
1580
 +
 20
 77
 264
 10
 6
 73
5000
 +
 19
 82
 296
 12
 7
 72









Postive Controls
 Name
 -
 DAUN
 ENNG
 CUM
 ENNG
 9-AA
 ENNG
Concentration / [µg/plate]
 2
 5
 200
 10
 50
 5
Revertants per plate
 317
 510
 992
 274
 541
 1521
Name
 +
 2-AA
 2-AA
 B(a)P
 2-AA
 2-AA
 2-AA
Concentration / [µg/plate] 1
 1
 10
 1
 2
 10
Revertants per plate 115
 283
 804
 54
 55
 915


Second Series


Test Material
 Concentration / [µg/plate]
 +/-
S9
mix
 Mean Revertants per plate
TA98
 TA100
 TA102
 TA1535
 TA1537
 WP2
Solvent control
-
 18
 91
 205
 8
 4
 79









Test material
 50
 -
 11
 81
 233
 6
 5
 79
158
 -
 13
 78
 230
 9
 3
 85
500
 -
 14
 80
 195
 7
 3
 71
1580
 -
 11
 85
 181
 6
 2
 80
5000
 -
 11
 74
 224
 5
 4
 73









Solvent control
+
 23
 112
 211
 12
 6
 89









Test material 50
 +
 19
 100
 217
 9
 7
 92
158
 +
 22
 112
 214
 9
 6
 87
500
 +
 20
 119
 245
 10
 6
 96
1580
 +
 23
 113
 220
 9
 5
 105
5000
 +
 22
 108
 210
 8
 6
 90









Postive Controls
 Name
 -
 DAUN
 ENNG
 CUM
 ENNG
 9-AA
 ENNG
Concentration / [µg/plate]
 2
 5
 200
 10
 50
 5
Revertants per plate
 197
 631
 523
 231
 202
 1160
Name
 +
 2-AA
 2-AA
 B(a)P
 2-AA
 2-AA
 2-AA
Concentration / [µg/plate] 1
 1
 10
 1
 2
 10
Revertants per plate 101
 338
 720
 93
 80
 1005



Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.
Executive summary:

Purpose

The purpose of this in vitro reverse gene mutation test is to identify agents that cause mutations in bacteria cells thus providing information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA pkM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed for all strains. In the series with S9 mix, 10 % or 30 % S9 in the S9 mix were used inn the 1st and 2nd series, respectively.

Results

The test material was dissolved in DMSO and tested at concentrations ranging from 5.00 to 5000 pg/plate. Precipitation of the test material on the agar plates and toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitroso-guanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

No increase in revertant colonies was obtained after treatment with the test material up to the highest concentrations investigated, thus showing the absence of mutagenic potential in vitro with and without external metabolizing system.

Conclusions

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD TG 476. A detailed read across justification is provided in chapter 13 of this dossier.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 88.9 µg/L (without S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Executive summary:

For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD TG 476. A detailed read across justification is provided in chapter 13 of this dossier.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD TG 473. A detailed read across justification is provided in chapter 13 of this dossier.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Executive summary:

For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD TG 473. A detailed read across justification is provided in chapter 13 of this dossier.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the results provided, the test item is not classified for mutagenicity according to Regulation (EC) No 1272/2008.