Registration Dossier
Registration Dossier
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EC number: 700-008-0 | CAS number: 3753-18-2
Genetic toxicity: in vitro
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:
- The experimental phase of this study was performed between 24 December 2008 and 03 February 2009.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 009
- Report date:
- 2009
Materials and methods
Test guidelineopen allclose all
- 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
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 4,4'-bis(methoxymethyl)-1,1'-biphenyl
- EC Number:
- 700-008-0
- Cas Number:
- 3753-18-2
- Molecular formula:
- C16 H18 O2
- IUPAC Name:
- 4,4'-bis(methoxymethyl)-1,1'-biphenyl
- Reference substance name:
- 4,4-bis(methoxymethyl)-biphenyl
- IUPAC Name:
- 4,4-bis(methoxymethyl)-biphenyl
Constituent 1
Constituent 2
Method
- Target gene:
- Histidine operon for Salmonella
Tryptophan operon for Escherichia
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/betanaphthoflavone
- Test concentrations with justification for top dose:
- Salmonella strains (with and without S9): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
E.coli strain (with and without S9): 50, 150, 500, 1500 and 5000 µg/plate.
Additional dose levels (5 and 15 µg/plate) and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test material. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used:DMSO - dimethul sulphoxide
- Justification for choice of solvent/vehicle: The test material was soluble in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house. The Sponsor indicated that the test material would be insoluble in water, therefore, this solvent was not evaluated.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9 mix
Migrated to IUCLID6: (ENNG)
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA)
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 - 72 hours
- Expression time (cells in growth medium): not appliacble
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 - 72 hours
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: triplicate plating
NUMBER OF CELLS EVALUATED: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency and colony reduction
OTHER EXAMINATIONS: none - Evaluation criteria:
- Acceptance criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal. - Statistics:
- Standard deviation.
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 nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- tested up to the maximum recommended dose level of 5000 µg per plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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 nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- tested up to the maximum recommended dose level of 5000 µg per plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not stated in report
- Effects of osmolality: not stated in report
- Evaporation from medium: not stated in report
- Water solubility: insoluble
- Precipitation: no
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity test:
The test material induced toxicity as weakened bacterial background lawns to TA100 from 1500 µg/plate and was non-toxic to WP2uvrA-. The test material formulation and S9-mix used in this experiment were both shown to be sterile.
COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
ADDITIONAL INFORMATION ON CYTOTOXICITY: - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
The test material caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the revertant colony frequency of all of the Salmonella strains both with and without metabolic activation at and above 1500 µg/plate. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test material varied slightly between strain type, exposures with and without S9-mix and experiment number. The test material caused no visible reduction in the growth of the bacterial background lawns of Escherichia colistrain WP2uvrA-at any dose level. A precipitate (greasy in appearance) was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Table 1 Test Results: Range-Finding Test– Without Metabolic Activation
Test Period |
From: 20 January 2009 |
To: 23 January 2009 |
||||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||||||||||
Base-pair substitution type |
Frameshift type |
|||||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
||||||||||
- |
0 |
99 96 96 |
(97) 1.7# |
22 25 24 |
(24) 1.5 |
32 31 28 |
(30) 2.1 |
15 20 16 |
(17) 2.6 |
6 5 12 |
(8) 3.8 |
|||
- |
5 |
75 100 91 |
(89) 12.7 |
25 24 20 |
(23) 2.6 |
N/T |
11 16 22 |
(16) 5.5 |
6 5 10 |
(7) 2.6 |
||||
- |
15 |
79 79 108 |
(89) 16.7 |
20 19 20 |
(20) 0.6 |
N/T |
11 21 15 |
(16) 5.0 |
8 15 6 |
(10) 4.7 |
||||
- |
50 |
87 100 104 |
(97) 8.9 |
25 15 22 |
(21) 5.1 |
26 34 27 |
(29) 4.4 |
12 22 15 |
(16) 5.1 |
7 8 8 |
(8) 0.6 |
|||
- |
150 |
105 85 102 |
(97) 10.8 |
22 21 19 |
(21) 1.5 |
22 32 33 |
(29) 6.1 |
11 14 28 |
(18) 9.1 |
16 12 4 |
(11) 6.1 |
|||
- |
500 |
93 85 75 |
(84) 9.0 |
22 19 31 |
(24) 6.2 |
37 37 29 |
(34) 4.6 |
21 22 11 |
(18) 6.1 |
5 8 13 |
(9) 4.0 |
|||
- |
1500 |
48 P 43 P 33 P |
(41) 7.6 |
1 *P 2 *P 3 *P |
(2) 1.0 |
16 P 28 P 20 P |
(21) 6.1 |
5 P 7 P 8 P |
(7) 1.5 |
3 *P 3 *P 3 *P |
(3) 0.0 |
|||
- |
5000 |
1 *P 2 *P 0 *P |
(1) 1.0 |
0 *P 0 *P 0 *P |
(0) 0.0 |
27 P 21 P 14 P |
(21) 6.5 |
3 *P 3 *P 4 *P |
(3) 0.6 |
2 *P 2 *P 1 *P |
(2) 0.6 |
|||
Positive controls
S9-Mix
- |
Name Concentration (μg/plate) No. colonies per plate |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||||
3 |
5 |
2 |
0.2 |
80 |
||||||||||
567 590 588 |
(582) 12.7 |
104 163 168 |
(145) 35.6 |
153 139 170 |
(154) 15.5 |
197 186 168 |
(184) 14.6 |
1178 1375 1157 |
(1237) 120.3 |
|||||
Table 2 Test Results: Range-Finding Test– With Metabolic Activation
Test Period |
From: 20 January 2009 |
To: 23 January 2009 |
||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||||||||
Base-pair substitution type |
Frameshift type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
||||||||
+ |
0 |
106 95 101 |
(101) 5.5# |
14 8 8 |
(10) 3.5 |
36 25 36 |
(32) 6.4 |
26 19 19 |
(21) 4.0 |
8 6 6 |
(7) 1.2 |
|
+ |
5 |
106 94 93 |
(98) 7.2 |
12 12 15 |
(13) 1.7 |
N/T |
29 26 20 |
(25) 4.6 |
9 10 8 |
(9) 1.0 |
||
+ |
15 |
76 99 91 |
(89) 11.7 |
13 13 11 |
(12) 1.2 |
N/T |
22 25 27 |
(25) 2.5 |
8 10 11 |
(10) 1.5 |
||
+ |
50 |
100 93 81 |
(91) 9.6 |
13 10 9 |
(11) 2.1 |
24 33 32 |
(30) 4.9 |
30 27 14 |
(24) 8.5 |
5 5 3 |
(4) 1.2 |
|
+ |
150 |
94 112 75 |
(94) 18.5 |
9 11 12 |
(11) 1.5 |
24 29 35 |
(29) 5.5 |
15 15 20 |
(17) 2.9 |
7 3 7 |
(6) 2.3 |
|
+ |
500 |
105 98 79 |
(94) 13.5 |
14 15 14 |
(14) 0.6 |
31 26 31 |
(29) 2.9 |
20 24 24 |
(23) 2.3 |
6 8 8 |
(7) 1.2 |
|
+ |
1500 |
27 P 30 P 34 P |
(30) 3.5 |
3 P 5 P 2 P |
(3) 1.5 |
27 P 27 P 15 P |
(23) 6.9 |
15 P 6 P 4 P |
(8) 5.9 |
2 P 1 P 1 P |
(1) 0.6 |
|
+ |
5000 |
2 *P 0 *P 1 *P |
(1) 1.0 |
0 P 1 P 0 P |
(0) 0.6 |
29 P 29 P 14 P |
(24) 8.7 |
3 *P 6 *P 3 *P |
(4) 1.7 |
0 *P 0 *P 0 *P |
(0) 0.0 |
|
Positive controls
S9-Mix
+ |
Name Concentration (μg/plate) No. colonies per plate |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 |
2 |
10 |
5 |
2 |
||||||||
3037 3133 3206 |
(3125) 84.8 |
154 221 209 |
(195) 35.7 |
771 775 719 |
(755) 31.2 |
213 194 190 |
(199) 12.3 |
445 555 488 |
(496) 55.4 |
|||
Table 3 Test Results: Main Test– Without Metabolic Activation
Test Period |
From: 31 January 2009 |
To: 03 February 2009 |
|||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
|||||||||||
Base-pair substitution type |
Frameshift type |
||||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
|||||||||
- |
0 |
106 101 119 |
(109) 9.3# |
20 20 25 |
(22) 2.9 |
22 20 31 |
(24) 5.9 |
19 19 22 |
(20) 1.7 |
12 12 14 |
(13) 1.2 |
||
- |
5 |
114 90 100 |
(101) 12.1 |
14 20 21 |
(18) 3.8 |
N/T |
18 25 21 |
(21) 3.5 |
8 8 13 |
(10) 2.9 |
|||
- |
15 |
96 89 102 |
(96) 6.5 |
23 34 21 |
(26) 7.0 |
N/T |
19 18 19 |
(19) 0.6 |
12 10 12 |
(11) 1.2 |
|||
- |
50 |
103 88 100 |
(97) 7.9 |
16 22 19 |
(19) 3.0 |
24 30 18 |
(24) 6.0 |
22 16 18 |
(19) 3.1 |
11 10 13 |
(11) 1.5 |
||
- |
150 |
99 93 93 |
(95) 3.5 |
23 21 23 |
(22) 1.2 |
31 25 21 |
(26) 5.0 |
19 18 23 |
(20) 2.6 |
15 11 9 |
(12) 3.1 |
||
- |
500 |
112 100 115 |
(109) 7.9 |
24 19 19 |
(21) 2.9 |
30 24 25 |
(26) 3.2 |
23 15 19 |
(19) 4.0 |
9 11 9 |
(10) 1.2 |
||
- |
1500 |
69 *P 69 *P 66 *P |
(68) 1.7 |
5 *P 5 *P 4 *P |
(5) 0.6 |
26 P 25 P 29 P |
(27) 2.1 |
13 P 13 P 9 P |
(12) 2.3 |
5 *P 2 *P 2 *P |
(3) 1.7 |
||
- |
5000 |
9 *P 5 *P 4 *P |
(6) 2.6 |
0 *P 0 *P 0 *P |
(0) 0.0 |
26 P 25 P 30 P |
(27) 2.6 |
4 *P 3 *P 3 *P |
(3) 0.6 |
1 *P 0 *P 0 *P |
(0) 0.6 |
||
Positive controls
S9-Mix
- |
Name Concentration (μg/plate) No. colonies per plate |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||||
3 |
5 |
2 |
0.2 |
80 |
|||||||||
1183 1514 1387 |
(1361) 167.0 |
1680 1967 1918 |
(1855) 153.5 |
1864 1830 1710 |
(1801) 80.9 |
196 206 228 |
(210) 16.4 |
1070 1184 1389 |
(1214) 161.6 |
||||
Table 4 Test Results: Main Test– With Metabolic Activation
Test Period |
From: 31 January 2009 |
To: 03 February 2009 |
||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||||||||
Base-pair substitution type |
Frameshift type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
||||||||
+ |
0 |
82 95 95 |
(91) 7.5# |
14 15 11 |
(13) 2.1 |
26 29 36 |
(30) 5.1 |
31 26 25 |
(27) 3.2 |
13 13 9 |
(12) 2.3 |
|
+ |
5 |
97 95 89 |
(94) 4.2 |
15 11 15 |
(14) 2.3 |
N/T |
27 26 29 |
(27) 1.5 |
10 11 9 |
(10) 1.0 |
||
+ |
15 |
88 85 95 |
(89) 5.1 |
10 16 18 |
(15) 4.2 |
N/T |
22 23 19 |
(21) 2.1 |
11 10 11 |
(11) 0.6 |
||
+ |
50 |
97 92 110 |
(100) 9.3 |
15 13 11 |
(13) 2.0 |
26 30 31 |
(29) 2.6 |
23 24 23 |
(23) 0.6 |
15 10 10 |
(12) 2.9 |
|
+ |
150 |
84 91 91 |
(89) 4.0 |
16 11 7 |
(11) 4.5 |
29 26 26 |
(27) 1.7 |
24 24 20 |
(23) 2.3 |
12 13 9 |
(11) 2.1 |
|
+ |
500 |
93 84 93 |
(90) 5.2 |
10 11 11 |
(11) 0.6 |
29 31 31 |
(30) 1.2 |
21 23 20 |
(21) 1.5 |
8 10 14 |
(11) 3.1 |
|
+ |
1500 |
48 *P 47 *P 59 *P |
(51) 6.7 |
4 *P 3 *P 3 *P |
(3) 0.6 |
30 P 27 P 32 P |
(30) 2.5 |
14 *P 12 *P 15 *P |
(14) 1.5 |
4 P 5 P 4 P |
(4) 0.6 |
|
+ |
5000 |
7 *P 3 *P 4 *P |
(5) 2.1 |
0 *P 0 *P 0 *P |
(0) 0.0 |
27 P 32 P 22 P |
(27) 5.0 |
5 *P 3 *P 3 *P |
(4) 1.2 |
0 *P 1 *P 0 *P |
(0) 0.6 |
|
Positive controls
S9-Mix
+ |
Name Concentration (μg/plate) No. colonies per plate |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 |
2 |
10 |
5 |
2 |
||||||||
3606 3248 3213 |
(3356) 217.5 |
125 140 155 |
(140) 15.0 |
617 530 493 |
(547) 63.7 |
379 224 245 |
(283) 84.1 |
474 361 420 |
(418) 56.5 |
|||
BP2AAN/TP*#
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
N/T Not tested at this dose level
P Precipitate
* Partial absence of bacterial background lawn
# Standard deviation
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
N/T Not tested at this dose level
P Precipitate
* Partial absence of bacterial background lawn
# Standard deviation
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
N/T Not tested at this dose level
P Precipitate
* Partial absence of bacterial background lawn
# Standard deviation
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
N/T Not tested at this dose level
P Precipitate
* Partial absence of bacterial background lawn
# Standard deviation
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
The test material was considered to be non-mutagenic under the conditions of this test. - Executive summary:
The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co‑factors). The dose range was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type. The experiment was repeated on a separate day using the same dose ranges as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.
Additional dose levels (5 and 15 µg/plate) and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test material.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the revertant colony frequency of all of the Salmonella strains both with and without metabolic activation at and above 1500 µg/plate. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test material varied slightly between strain type, exposures with and without S9-mix and experiment number. The test material caused no visible reduction in the growth of the bacterial background lawns of Escherichia coli strain WP2uvrA-at any dose level. A precipitate (greasy in appearance) was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.
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