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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
1985, Ames test, TA98, TA100, TA1535, TA1537 and TA1538, not mutagenic
Link to relevant study records
Reference
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:
March 05, 1985 - May 06, 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
performed with the Salmonella typhimurium strains TA98, TA 100, TA 1535, TA 1537 and TA 1538
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
Investigation of the potential gene mutagenic activity of Pyridin-2-ethanol according to the plate incorporation test of Ames et al.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his-, obtained from B.N. Ames, University of California, Berkeley CA, 94720, USA, May 1981.

Characterization of strains:
The strains used are mutants derived from Salmonella typhimurium LT2 and have the following genotypes:
S. typhimurium TA 1535 his G46 rfa- uvrB-
S. typhimurium TA 1537 his C3076 rfa- uvrB-
S. typhimuriumTA 1538 his D3052 rfa- uvrB-
S. typhimurium TA 98 his D3052 rfa- uvrB- R +
S. typhimurium TA 100 his G46 rfa- uvrB- R +
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial fraction prepared from the livers of rats treated with the enzyme-inducing agent Aroclor 1254.
Test concentrations with justification for top dose:
1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 micrograms per plate (mcg/pl.). Each concentration, including the controls, was tested in triplicate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (bidistilled water)
- Justification for choice of solvent/vehicle: On the day of the experiment, the test article was dissolved in bidistilled water at the highest investigated dose. The other doses were dilutions from this stock solution with the solvent in half-log intervals. All control data were established on the same day as the experiment.
Untreated negative controls:
yes
Remarks:
Bidistilled water, solvent of the test article
Negative solvent / vehicle controls:
yes
Remarks:
Bidistilled water
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
methylmethanesulfonate
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: the plates were incubated at 37 degrees centigrade in the dark for 3 days.

OTHER: To avoid any light effects on the test article, all experiments were performed under yellow light.

Storage:
The strain cultures are stored in sterile 0.5 ml ampoules (0.45 mL bacterial culture and 0.05 mL dimethylsuIfoxide) at -70°C.
Pre-culture of strains:
The bacteria were grown in a shaking water bath for 16 hours overnight at 37 °C in 2 .5% Nutrient Broth No. 2. After centrifugation, the bacteria were resuspended to a concentration of approximately 1 x 10 exp. 8 to 2 x 10 exp.9 cells per milliliter in 0 .16 % Nutrient Broth and 0.5% sodium chloride. The concentration of germs was controlled photometrically and determined in an experimental test with histidine-rich potassium chloride solution on selective agar plates.
Evaluation criteria:
A material is identified as a mutagen in this test system if there was a reproducible demonstration of a dose effect relation with a 2-fold increase in the number of revertants over the controls in a minimum of one strain. With the strain TA 100, a 1.5-fold increase was the criterion for a positive result. These criteria are generally in accordance with the international used standards for the evaluation of Ames Test results.
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:
In the toxicity experiment with Pyridin-2-ethanol, neither quantitative nor qualitative evidence of a cytotoxic effect was observed
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
Control plates with the solvent (negative control) showed numbers of spontaneous revertant colonies per plate which were within the normal range of those cited in the literature. In the second experiment, the rate of spontaneous revertants in the strain TA 100 were at the lower limit of the normal range cited in the literature and compared to historical controls, gathered in the testing laboratory.

Control plates with reference mutagens (positive controls) showed a distinct increase of the revertant colonies with the tester strains. This confirms the reversion properties of each strain. The positive results of the mutagens 2-Amino-anthracene and Benzo(a)pyrene indicate that the metabolizing system was functioning.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxic effect of the test article was observed.
Remarks on result:
other: strain/cell type: all strains/cell types tested (in addition S. typhimurium TA1538)
Remarks:
Migrated from field 'Test system'.

Individual results:

Table 1: Salmonella test without S-9 mix (first experiment)
Number of revertant colonies per plate with group means
Dose/plate TA 98 TA 100 TA 1535 TA 1537 TA 1538
0 mcg control 25 30 122 108 42 26 7 10 14 14
29 111 17 8 12
35 91 20 15 15
1,58 mcg 25 27 135 114 29 27 4 8 18 17
29 95 28 7 19
28 111 25 14 14
5 mcg 31 27 99 108 22 24 12 10 14 12
23 118 26 11 12
26 108 25 8 17
15.8 mcg 27 30 114 112 21 24 9 8 10 12
37 116 35 10 12
26 107 16 6 13
50 mcg 31 25 119 103 26 22 6 7 12 13
23 97 24 4 14
21 92 15 10 13
158 mcg 19 23 120 114 16 18 9 9 12 13
27 109 22 5 14
24 112 15 13 12
500 mcg 25 27 122 119 16 20 5 9 15 13
31 118 26 6 12
26 116 18 16 11
1580 mcg 20 23 119 114 14 17 13 11 14 13
30 95 13 7 12
18 129 23 14 14
5000 mcg 19 22 124 109 15 16 8 6 15 13
24 95 18 8 13
23 108 14 3 12
MMS 500 mcg --- --- 324 421 --- --- --- --- --- ---
--- 450 --- --- ---
--- 489 --- --- ---
9-AMA 40 mcg --- --- --- --- --- --- 53 59 --- ---
--- --- --- 56 ---
--- --- --- 69 ---
2-NF 5 mcg 718 698 397 372 ___ --- 38 29 1054 1086
766 347 --- 24 1115
610 371 --- 24 1088
ENNG 10 mcg 124 121 1027 1057 623 742 --- --- --- ---
119 1102 661 --- ---
121 1042 943 --- ---
 --- Not determined
Table 2: Salmonella test with S-9 mix (first experiment)
Number of revertant colonies per plate with group means
Dose/plate TA 98 TA 100 TA 1535 TA 1537 TA 1533
0 mcg control 21 27 114 131 12 14 30 26 27 28
38 144 15 30 30
23 135 14 19 28
1,58 mcg 19 25 103 102 14 18 30 27 28 23
26 95 20 28 22
31 108 21 24 18
5 mcg 20 24 101 111 16 14 36 31 19 25
25 122 14 30 27
28 110 11 28 28
15.8 mcg 24 24 138 138 21 21 27 33 25 27
22 127 16 39 29
27 148 26 33 26
50 mcg 30 29 107 119 16 15 27 28 27 26
30 113 12 29 21
28 138 16 28 30
158 mcg 23 29 128 120 26 21 33 31 31 32
32 106 20 30 36
32 127 16 31 28
500 mcg 34 29 115 112 14 15 26 29 19 22
29 98 17 30 24
24 123 15 30 22
1580 mcg 31 28 115 119 17 15 29 30 25 26
27 106 13 29 25
25 137 16 32 28
5000 mcg 29 27 126 116 11 14 33 29 19 24
24 95 19 28 28
27 128 12 26 25
2-AA 0.5 mcg 91 86 185 217 --- --- --- --- 115 107
91 233 --- --- 97
77 232 --- --- 109
2-AA 1 mcg --- --- --- --- 150 133 --- --- --- ---
--- --- 126 --- ---
--- --- 122 --- ---
2-AA 10 mcg --- --- --- --- --- --- 168 165 --- ---
--- --- --- 182 ---
--- --- --- 145 ---
BaP 5 mcg 295 288 480 462 --- --- 142 143 129 144
285 460 --- 162 143
284 446 --- 124 160
 --- Not determined
---
TABLE 3  : Toxicity test without S-9 mix (first experiment)
DOSE MEAN NUMBERS OF REVERTANT COLONIES RELATIVE SURVIVAL RATE   
TA 1537 TA 1537 + RTA   
0 mcg control 10 369 1,00   
1.58 mcg 8 353 0,96   
5 mcg 10 355 0,96   
15.8 mcg 8 376 1,03   
50 mcg 7 388 1,06   
158 mcg 9 375 1,02   
500 mcg 9 349 0,95   
1580 mcg 11 353 0,95   
5000 mcg 6 393 1,08   
---
TABLE 4 : Toxicity test with S-9 mix (first experiment)
DOSE MEAN NUMBERS OF REVERTANT COLONIES RELATIVE SURVIVAL RATE   
TA 1537 TA 1537 + RTA   
0 mcg control 26 339 1,00   
1.58 mcg 27 315 0,92   
5 mcg 31 326 0,94  
15.8 mcg 33 336 0,97  
50 mcg 28 370 1,09  
158 mcg 31 334 0,97   
500 mcg 29 321 0,93   
1580 mcg 30 315 0,91  
5000 mcg 29 335 0,98   
---
Table 5: Salmonella test without S-9 mix (second experiment)
Number of revertant colonies per plate with group means
Dose/plate TA 98 TA 100 TA 1535 TA 1537 TA 1533
0 mcg control 32 30 93 81 38 31 9 10 24 19
30 63 29 8 18
29 86 26 12 15
1,58 mcg 29 31 116 101 36 31 11 11 16 15
30 89 30 10 13
34 99 26 12 15
5 mcg 24 25 76 87 38 32 13 9 21 19
30 98 25 9 14
22 87 32 6 22
15.8 mcg 27 22 91 105 29 27 14 14 18 18
21 109 24 10 16
17 115 28 18 19
50 mcg 27 26 115 120 28 34 6 7 20 21
26 134 29 7 21
25 112 46 8 22
158 mcg 18 25 121 116 30 30 12 10 19 21
31 117 25 10 18
25 110 34 8 25
500 mcg 26 25 87 96 24 30 9 11 16 19
23 108 38 8 20
26 94 27 16 22
1580 mcg 36 25 84 81 26 27 7 9 15 19
18 74 32 9 23
20 86 22 11 19
5000 mcg 33 27 130 107 32 37 13 9 17 16
21 99 42 8 11
26 93 36 5 19
MMS 500 mcg --- --- 482 515 --- --- --- --- --- ---
--- 494 --- --- ---
--- 569 --- --- ---
9-AMA 40 mcg --- --- --- --- --- --- 98 99 --- ---
--- --- --- 73 ---
--- --- --- 126 ---
2-NF 5 mcg 891 899 613 612 ___ --- 45 39 1144 1178
870 550 --- 35 1142
935 674 --- 36 1247
ENNG 10 mcg 214 231 1737 1699 1576 794 --- --- --- ---
241 1845 1327 --- ---
238 1516 1889 --- ---
 --- Not determined
---
Table 6: Salmonella test with S-9 mix (second experiment)
Number of revertant colonies per plate with group means
Dose/plate TA 98 TA 100 TA 1535 TA 1537 TA 1533
0 mcg control 48 49 97 68 19 21 23 34 30 31
48 59 21 36 31
50 48 23 44 33
1,58 mcg 62 55 69 73 16 17 33 27 33 33
49 73 17 27 32
54 77 17 20 33
5 mcg 46 56 94 90 7 10 34 35 25 27
66 83 13 41 27
57 92 11 31 30
15.8 mcg 57 53 67 78 14 10 29 27 34 32
57 88 7 26 31
46 80 10 26 30
50 mcg 57 51 95 109 16 14 19 22 38 27
52 110 21 32 27
43 121 6 15 16
158 mcg 54 52 108 104 16 15 37 31 33 29
55 115 14 32 34
48 88 15 25 20
500 mcg 48 53 97 106 6 12 31 31 34 29
58 112 14 26 30
53 110 15 37 23
1580 mcg 49 46 99 115 19 14 16 21 29 30
45 133 13 28 29
44 113 11 19 33
5000 mcg 38 46 91 114 22 18 31 31 29 30
50 120 16 28 34
51 132 16 33 26
2-AA 0.5 mcg 90 104 147 152 --- --- --- --- 69 72
110 147 --- --- 68
113 161 --- --- 78
2-AA 1 mcg --- --- --- --- 70 70 --- --- --- ---
--- --- 67 --- ---
--- --- 73 --- ---
2-AA 10 mcg --- --- --- --- --- --- 188 207 --- ---
--- --- --- 215 ---
--- --- --- 217 ---
BaP 5 mcg 270 251 298 325 --- --- 115 112 99 102
266 341 --- 113 98
217 335 --- 109 109
 --- Not determined
---
TABLE 7  : Toxicity test without S-9 mix (second experiment)
DOSE MEAN NUMBERS OF REVERTANT COLONIES RELATIVE SURVIVAL RATE   
TA 1537 TA 1537 + RTA   
0 mcg control 10 346 1,00   
1.58 mcg 11 333 0,96   
5 mcg 9 346 1,00   
15.8 mcg 14 339 0,97  
50 mcg 7 339 0,99  
158 mcg 10 329 0,95  
500 mcg 11 350 1,01   
1580 mcg 9 349 1,01   
5000 mcg 9 362 1,05  
---
TABLE 8  : Toxicity test with S-9 mix (second experiment)
DOSE MEAN NUMBERS OF REVERTANT COLONIES RELATIVE SURVIVAL RATE   
TA 1537 TA 1537 + RTA   
0 mcg control 34 349 1,00   
1.58 mcg 27 333 0,97   
5 mcg 35 335 0,95   
15.8 mcg 27 332 0,97   
50 mcg 22 336 1,00   
158 mcg 31 311 0,89   
500 mcg 31 326 0,94   
1580 mcg 21 319 0,95   
5000 mcg 31 336 0,97  

In the toxicity experiment with PYRIDIN-2-ETHANOL, neither quantitative nor qualitative evidence of a cytotoxic effect was observed.

In the described bacterial mutagenicity tests, no relevant increase of the revertant colony numbers was obtained in the Salmonella typhimurium strains used at all dose levels tested when compared with the corresponding controls. The presence of liver microsomal activation did not influence these findings.

These results were confirmed in a second, independent experiment for the strains TA 98, TA 1535, TA 1537 and TA 1538. The increased rate of revertants in the strain TA 100 in the second experiment compared with the corresponding controls are considered to be not relevant.This must be concluded because the control rates are at the lower limit of control rates cited in the literature and of historical controls gathered in the testing laboratory. There is also no clear-cut dose dependency, and the revertant rates after application of the test article are in the medium range compared to revertant rates of historical controls.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced no point mutations by base-pair changes or frameshifts in the genome of the strains used.

Therefore, PYRIDIN-2 -ETHANOL is not considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The study was performed according to the OECD Guideline 471 and EU Method B13/14 with deviations (5 Salmonella strains tested) and still considered to be of high quality quality (reliability Klimisch 2). The vehicle and the positive control substances fulfilled validity criteria of the test system. The test was performed with and without liver microsomal activation. The test article was tested at the following concentrations: 1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 micrograms per plate. Each concentration, including the controls, was tested in triplicate.
No toxic effect of the test article was observed.
Executive summary:

Pyridin-2-ethanol was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per plate on five Salmonella typhimurium LT2 mutants (TA98, TA 100, TA 1535, TA 1537 and TA 1538). The study was performed according to the OECD Guideline 471 and EU Method B13/14 with deviations (five Salmonella strains tested) and considered to be of the high quality (reliability Klimisch 2). The strains have the following genotypes : S. typhimurium TA 1535 his G46 rfa- uvrB-, S. typhimurium TA 1537 his C3076 rfa- uvrB-, S. typhimurium TA 1538 his D3052 rfa- uvrB-, S. typhimurium TA 98 his D3052 rfa- uvrB- R + and S. typhimurium TA 100 his G46 rfa- uvrB- R +. The bacteria were treated with the test material using the Ames plate incorporation method at up to eight dose levels (1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg per plate), in triplicate, both with and without the addition of a rat liver homogenate metabolising system (cofactor-supplemented post-mitochondrial fraction prepared from the livers of rats treated with the enzyme-inducing agent Aroclor 1254.). Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. The vehicle (water) control plates gave counts of revertant colonies within the normal range. The positive controls  methyl methanesulfonate (MMS), 9 -Aminoacridine (9 -AMA), 2 -nitrofluorene  (2 -NF), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), 2-Aminoanthracene (2-AA) and benzo(a)pyrene (BaP) had a marked mutagenic effect, as was seen by a biologically relevant increase of induced revertant colonies compared to the corresponding negative controls. So all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated. No cytotoxic effect of the test article was observed. In the described bacterial mutagenicity tests, no relevant increase of the revertant colony numbers was obtained in the Salmonella typhimurium strains used at all dose levels tested when compared with the corresponding controls. The presence of liver microsomal activation did not influence these findings.

These results were confirmed in a second, independent experiment for the strains TA 98, TA 1535, TA 1537 and TA 1538. The increased rate of revertants in the strain TA 100 in the second experiment compared with the corresponding controls are considered to be not relevant.This must be concluded because the control rates are at the lower limit of control rates cited in the literature and of historical controls gathered in the testing laboratory. There is also no clear-cut dose dependency, and the revertant rates after applicationof the test article are in the medium range compared to revertant rates of historical controls.In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced no point mutations by base-pair changes or frameshifts in the genome of the strains used.

Therefore, PYRIDIN-2 -ETHANOL is not considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

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

Additional information

Additional information from genetic toxicity in vitro:

The test substance Pyridin-2-ethanol was investigated for its potential to cause gene mutation in Salmonella typhimurium strains (TA98, TA100, TA1537, TA1535 and TA1538) (Banduhn,1985).The study was performed according to the OECD Guideline 471 with deviations (Salmonella strain TA1538 instead of TA 102) according to the principles of the good laboratory practice and therefore considered to be of high quality (reliability Klimisch 2). The vehicle and the positive control substances fulfilled validity criteria of the test system. Under the conditions of this experiment Pyridin-2-ethanol was found to cause no toxic effects. Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain in comparison with the corresponding controls. The presence of microsomal activation did not influence these findings. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material caused neither base-pair substitutions, nor frameshift mutations. Therefore the test results with Pyridin-2-ethanol revealed no indication of gene mutagenic activity.


Justification for selection of genetic toxicity endpoint
well documented GLP-Guideline study according to OECD 471

Justification for classification or non-classification

According to the European regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification and will not require labelling as a mutagen.