Registration Dossier
Registration Dossier
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EC number: 259-226-5 | CAS number: 54553-91-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study without detailed documentation
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Only 4 straines used
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat S9 liver
- Test concentrations with justification for top dose:
- 8; 40; 200; 1000; 5000 Kg/plate
- Vehicle / solvent:
- solvent: dimethyl sulfoxide (CAS No. 67-68-5)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- Details on test system and experimental conditions:
- SYSTEM OF TESTING
- Metabolic activation system:
Aroclor 1254 induced rat S9 liver, obtained from Cytotest Cell Research
GmbH & Co. KG (Batches 280992 and 170593)
ADMINISTRATION:
- Dosing:
main test: 8/40/200/1000/5000 Kg/plate (+/- metabolic activation)
pre-incubation test: 8/40/200/1000/5000 Kg/plate (+/- metabolic activation)
- Number of replicates: 3
- Application: solvent dimethyl sulfoxide (CAS No. 67-68-5)
main test: 50 g/l; pre-incubation test: 100 g/l
- Positive and negative control groups and treatment:
positive, TA 98 and TA 1538: nitrofluorene
positive, TA 100 and TA 1535: sodium azide
positive, TA 1537: aminoacridine
negative: solvent + untreated controls
activity of metabolic system: aminoanthracene / TA 100
- Pre-incubation: 30 minutes at 30 +/- 1 °C
incubation ca. 96 hours at ca. 37 °C - Evaluation criteria:
- mutagenic effects (i.e ratio of revertant rates treated/control >= 2) at <= 5000 Kg/plate with generally positive dose-response relationship in any strain
- 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:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- GENOTOXIC EFFECTS:
- With metabolic activation: None
- Without metabolic activation: None
The positive controls were functional.
PRECIPITATION CONCENTRATION: No precipitation was observed. - Conclusions:
- Based on the available data from this study using four strains of Salmonella typhimurium (TA 1535, TA 1537, TA 1538, TA 98, TA 100) with and without metabolic activation. The Test Item is considered as non-mutagenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 October 2020 - 02 March 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted 30. May 2008
- Deviations:
- yes
- Remarks:
- Study was performed to fill data gaps of in vitro genotoxicity testing performed in 1993. Therefore, only the missing strain TA 102 was tested.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 26. Jun. 2020
- Deviations:
- yes
- Remarks:
- Study was performed to fill data gaps of in vitro genotoxicity testing performed in 1993. Therefore, only the missing strain TA 102 was tested.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9
- Test concentrations with justification for top dose:
- experiment 1: 5000, 1500, 500, 150 and 50 μg/plate.
experiment 2: 5000, 2500, 1250, 625, 313 and 156 μg/plate. - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- other: 2-Amino-anthracene
- Details on test system and experimental conditions:
- 7 PERFORMANCE OF THE STUDY
7.1 Culture of Bacteria
On the day before the start of each experiment, a nutrient broth (Oxoid nutrient broth no. 2) was inoculated with one lyophilizate of Salmonella typhimurium TA102 (for detail see 6.2.2, page 12).
7.1.1 Preparations
Different media and solutions were prepared preliminary (exact production dates are docu-mented in the raw data).
On the day of the test, the bacteria culture was checked for growth visually. The incubation chambers were heated to 37 ± 1 °C. The water bath was turned to 43 ± 1 °C. The table surface was disinfected.
The S9-mix was freshly prepared and stored at 0 °C.
7.1.2 Description of the Method
Per each concentration and control, three plates with (+S9) and three plates without meta-bolic activation (-S9) were used.
The test item solutions were prepared according to chapter 6.1.4, page 11.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ± 1 °C.
Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
100 μL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control). For the positive control MMC 2.5 μL of the stock solu-tion were applied to achieve a final concentration of 0.5 μL/plate.
500 μL S9-mix (see chapter 6.4.13, page 16 for test with metabolic activation) or phos-phate buffer (for test without metabolic activation).
100 μL bacteria suspension (see chapter 6.2.2, page 12, test system, culture of the strains)
2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ± 1 °C.
Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:
100 μL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control).
500 μL S9-mix (see chapter 6.4.13, page 16 for test with metabolic activation) or phos-phate buffer (for test without metabolic activation).
100 μL bacteria suspension (see chapter 6.2.2, page 12, test system, culture of the strains)
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C. - Rationale for test conditions:
- In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized (demin.) water and dimethyl sulfoxide (DMSO). The solid test item was soluble in DMSO, only.
Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations. - Evaluation criteria:
- Five different analysable and non-toxic concentrations were used for the evaluation of the mutagenic potential of the test item.
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, negative control and positive control.
The mean values and standard deviations of each threefold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given.
A result is considered as positive if a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the concentrations occurs in at least one tested strain with or without metabolic activation.
A biologically relevant increase is described as follows:
if in the bacteria strain S. typhimurium TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2.0)
A substance is not mutagenic if it does not meet the criteria above.
If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed. - Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- For Details and Tables please see below or attached Study Report
8.1 Experiment 1
8.1.1 Confirmation of the Criteria and Validity
The strain Salmonella typhimurium TA102 met the criterion of at least 109 bacteria/mL (cor-relating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter 17, page 40 ff.). Both positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
8.1.2 Solubility and Toxicity
In the first experiment, the test item showed no precipitates on the plates in all tested con-centrations.
No signs of toxicity towards the bacteria strain Salmonella typhimurium TA102 could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
8.1.3 Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
8.2 Experiment 2
8.2.1 Confirmation of the Criteria and Validity
Strain Salmonella typhimurium TA102 met the criterion of at least 109 bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter 17, page 40 ff.). Both positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
8.2.2 Solubility and Toxicity
In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strain Salmonella typhimurium TA102 could be observed. The bacterial background lawn was visible and not affected. The number of re-vertant colonies was not reduced.
8.2.3 Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
9 VALIDITY
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
The positive controls showed f(I) values > 2 (strain specific threshold, see chapter 7.3., page 20) which demonstrated the mutagenic potential of the diagnostic mutagens (historical data of the laboratory see chapter 17, page 40).
The confirmation tests of the genotype performed by Trinova BioChem GmbH did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL. In the sterility control no growth of bacteria could be detected.
Since all criteria for acceptability have been met, the study is considered valid. - Conclusions:
- The Test Items was tested for genotoxicity in an OECD 471 and GLP compliant study with the Salmonella typhimurium test strain TA102 with and without metabolic activation. Based on this study, the Test Item is non-mutagenic under the conditions of the test.
In both experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 μg/plate.
The test item showed no signs of toxicity towards the bacteria in both the presence and the absence of metabolic activation in both experiments.
The results of both experiments showed that the test item caused no increase in the number of revertants compared to the solvent control, in both the presence and absence of metabolic activation in the tested strain Salmonella typhimurium TA102. The test item did not induce a dose-related increase in the number of revertant colonies, neither in the presence nor in the absence of metabolic activation.
Based on the results of this study it is concluded that the Test item is not mutagenic in the Salmonella typhimurium strain TA102 in the presence and absence of metabolic activation under the experimental conditions in this study. - Executive summary:
Findings and Results:
The test item was tested in the Bacterial reverse mutation assay and on demand of the sponsor only with the strain Salmonella typhimurium TA102.
The study procedures described in this report were based on the most recent Guideline OECD 471 (2020) and EU Method B.13/14 (2008).
The test was performed in two valid experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation.
Experiment 1:
In the first experiment, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9 mix in the strain Sal-monella typhimurium TA102 using the plate incorporation method.
The test item showed no precipitates on the plates at any of the concentrations.
The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed. Thus, the test item showed no signs of toxicity towards the bacteria in both the presence and the absence of metabolic activation.
The results of this experiment showed that the tested concentrations showed neither a significant nor a dose-related increase in the number of revertants in the tested strain, in the presence and the absence of metabolic activation.
Experiment 2:
Based on the results of the first experiment, the test item was tested up to concentrations of 5000 μg/plate in the presence and absence of S9 mix in the strain Salmonella typhimurium TA102 using the pre-incubation method.
The test item showed no precipitates on the plates at any of the test item concentrations.
The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed. The test item showed no signs of tox-icity towards the bacteria in both the presence and the absence of metabolic activation.
The results of this experiments showed that the test item caused no increase in the number of revertants compared to the solvent control, in both the presence and absence of metabolic activation. The test item did not induce a dose-related increase in the number of revertant colonies in the tested strain, in the presence and absence of metabolic activation.
Conclusion:
Based on the results of this study it is concluded that the test item
is not mutagenic in the Salmonella typhimurium strain TA102 in the presence and absence of metabolic activation under the experimental conditions in this study.
Referenceopen allclose all
The detailed data of the two experiments are listed in the annex (Exp. 1 see chapter14, page28ff., Exp. 2 see chapter15, page33ff.).
First Experiment
The strain Salmonella typhimuriumTA102 met the criterion of at least 109bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter17, page40ff.). Both positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strain Salmonella typhimurium TA102 could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment.
To verify this result, a further experiment (exp. 2) was performed.
The mean revertant values of experiment 1 are shown in Table 8.1‑a.
The mean revertant values of the three replicates are presented in the following table.
Concentrations of the test item are stated as nominal concentrations, for real concentrations see chapter18, page 41).
Table 8.1‑a Mean Revertants Experiment 1
Strain |
TA102 |
|||
Induction |
-S9 |
+S9 |
||
DMSO |
Mean |
247 |
243 |
|
sd |
15.1 |
22.0 |
||
NaCl |
Mean |
245 |
-- |
|
sd |
8.3 |
-- |
||
Positive |
Mean |
581 |
737 |
|
sd |
24.4 |
31.1 |
||
f(I) |
2.37 |
3.03 |
||
5000 µg/plate |
Mean |
228 |
235 |
|
sd |
12.0 |
28.4 |
||
f(I) |
0.92 |
0.97 |
||
1500 µg/plate |
Mean |
241 |
232 |
|
sd |
8.3 |
10.6 |
||
f(I) |
0.98 |
0.95 |
||
500 µg/plate |
Mean |
248 |
251 |
|
sd |
8.0 |
18.9 |
||
f(I) |
1.00 |
1.03 |
||
150 µg/plate |
Mean |
240 |
235 |
|
sd |
13.09 |
18.5 |
||
f(I) |
0.97 |
0.97 |
||
50 µg/plate |
Mean |
228 |
233 |
|
sd |
6.9 |
9.2 |
||
f(I) |
0.92 |
0.96 |
||
sd = standard deviation±
* Different positive controls were used, see chapter6.2.4, page13
f(I) = increase factor, calculation see chapter7.3, page20
-- = not tested
Second Experiment
StrainSalmonella typhimuriumTA102 met the criterion of at least 109bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter 17, page 40ff.). Both positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strain Salmonella typhimurium TA102 could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment. The mean revertant values of experiment 2 are shown in Table 8.2‑a. The mean revertant values of the three replicates are presented in the following table. Concentrations of the test item are stated as nominal concentrations, for real concentrations see chapter 18, page 41).
Table 8.2‑a Mean Revertants Experiment 2
Strain |
TA102 |
||||||
Induction |
-S9 |
+S9 |
|||||
Demin. water |
Mean |
267 |
271 |
||||
sd |
6.1 |
14 |
|||||
DMSO |
Mean |
267 |
276 |
||||
sd |
10.1 |
6.9 |
|||||
NaCl |
Mean |
263 |
-- |
||||
sd |
12.2 |
-- |
|||||
Positive |
Mean |
659 |
624 |
||||
sd |
58.6 |
42.3 |
|||||
f(I) |
2.51 |
2.26 |
|||||
5000 µg/plate |
Mean |
279 |
284 |
||||
sd |
10.1 |
10.6 |
|||||
f(I) |
1.04 |
1.03 |
|||||
2500 µg/plate |
Mean |
280 |
276 |
||||
sd |
12.0 |
16.0 |
|||||
f(I) |
1.05 |
1.00 |
|||||
1250 µg/plate |
Mean |
280 |
264 |
||||
sd |
12.0 |
24.3 |
|||||
f(I) |
1.05 |
0.96 |
|||||
625 µg/plate |
Mean |
275 |
285 |
||||
sd |
32.6 |
20.5 |
|||||
f(I) |
1.03 |
1.03 |
|||||
313 µg/plate |
Mean |
259 |
272 |
||||
sd |
22.7 |
21.2 |
|||||
f(I) |
0.97 |
0.99 |
|||||
156 µg/plate |
Mean |
272 |
260 |
||||
sd |
18.3 |
22.3 |
|||||
f(I) |
1.02 |
0.94 |
|||||
sd = standard deviation±
* Different positive controls were used, see chapter 6.2.4, page 13
f(I) = increase factor, calculation see chapter 7.3, page 20
-- = not tested
Mutagenicity of Test item
The study was performed with the plate incorporation (experiment 1) and pre-incubation method (experiment 2) in the absence and presence of a metabolic activation system (S9). Under these conditions the influence of the test item on the bacterial test strainSalmonella typhimuriumTA102was evaluated in two experiments (exp. 1 and exp. 2).The test item Chemark 1355 showed no relevant increase in the number of revertants in the Salmonella typhimurium test strain TA102 in both evaluated experiments.
Based on the results of this study it is concluded thatChemark 1355is not mutagenic in theSalmonella typhimuriumtest strain TA102 in the absence and presence of metabolic activation under the experimental conditions of the present study.
ANNEX 2: DATA OF EXPERIMENT 1 (For Details see attached Study Report)
Determination of Titre
Criterion: The determination of titre should give a number of at least 109cells/mL, correlating to 100 colonies/plate after dilution.
Exact colony counts could not be determined due to the strong growth of the bacteria, but by visual inspection it was obvious that the values exceed 100 colonies/ plate
Table14.1‑a Titre Values (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
s.g. |
s.g. |
Repl. 2 |
s.g. |
s.g. |
Assessment |
ok |
ok |
s.g.= strong growth, too strong for counting of colonies
ok= the criterion was fulfilled
Sterility Control
Criterion: No colony per plate may grow.
Table14.2‑a Sterility (colonies per plate)
|
DMSO |
NaCl |
Repl. 1 |
0 |
0 |
Repl. 2 |
0 |
0 |
Repl. 3 |
0 |
0 |
Repl. 4 |
0 |
0 |
Assessment |
ok |
ok |
ok= the criterion was fulfilled
Spontaneous Revertants
The determined values were within the normal range of the laboratory.
DMSO
Table14.3‑a Spontaneous Revertants DMSO (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
236 |
228 |
Repl. 2 |
240 |
232 |
Repl. 3 |
264 |
268 |
Mean |
247 |
243 |
sd |
15.1 |
22.0 |
sd = standard deviation±
NaCl
Table14.3‑b Spontaneous Revertants NaCl (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
236 |
-- |
Repl. 2 |
248 |
-- |
Repl. 3 |
252 |
-- |
Mean |
245 |
-- |
sd |
8.3 |
-- |
sd = standard deviation±
-- = not tested
Positive Controls
¨ Without
metabolic activation:
Mitomycin C (MMC) in NaCl
¨ With
metabolic activation:
2-Amino anthracene (2-AA) in DMSO
For used concentrations see chapter6.2.4, page13.(attached full study Report)
Table14.4‑a Diagnostic Mutagens (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Substance |
MMC |
2-AA |
Repl. 1 |
560 |
712 |
Repl. 2 |
576 |
728 |
Repl. 3 |
608 |
772 |
Mean |
581 |
737 |
sd |
24.4 |
31.1 |
f(I) |
2.37 |
3.03 |
Rev. abs. |
336 |
494 |
sd = standard deviation±
f(I) = increase factor, calculation see chapter7.3, page20
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
Test Item Chemark 1355
Concentrations of the test item are stated as nominal concentrations, for real concentrations see chapter 18, page 14
Mutagenicity Test
Table14.5‑a Concentration 5000 µg/plate (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
216 |
204 |
Repl. 2 |
228 |
240 |
Repl. 3 |
240 |
260 |
Mean |
228 |
235 |
sd |
12.0 |
28.4 |
f(I) |
0.92 |
0.97 |
Rev. abs. |
-19 |
-8 |
sd = standard deviation±
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
f(I) = increase factor, calculation see chapter7.3, page20
Table14.5‑b Concentration 1500 µg/plate (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
232 |
220 |
Repl. 2 |
244 |
236 |
Repl. 3 |
248 |
240 |
Mean |
241 |
232 |
sd |
8.3 |
10.6 |
f(I) |
0.98 |
0.95 |
Rev. abs. |
-6 |
-11 |
sd = standard deviation±
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
f(I) = increase factor, calculation see chapter7.3, page20
Table14.5‑c Concentration 500µg/plate (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
240 |
236 |
Repl. 2 |
248 |
244 |
Repl. 3 |
256 |
272 |
Mean |
248 |
251 |
sd |
8.0 |
18.9 |
f(I) |
1.00 |
1.03 |
Rev. abs. |
1 |
8 |
sd = standard deviation±
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
f(I) = increase factor, calculation see chapter7.3, page20
Table14.5‑d Concentration 150 µg/plate (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
232 |
224 |
Repl. 2 |
232 |
224 |
Repl. 3 |
256 |
256 |
Mean |
240 |
235 |
sd |
13.9 |
18.5 |
f(I) |
0.97 |
0.97 |
Rev. abs. |
-7 |
-8 |
sd = standard deviation±
f(I) = increase factor, calculation see chapter7.3, page20
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
Table14.5‑e Concentration 50µg/plate (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
220 |
228 |
Repl. 2 |
232 |
228 |
Repl. 3 |
232 |
244 |
Mean |
228 |
233 |
sd |
6.9 |
9.2 |
f(I) |
0.92 |
0.96 |
Rev. abs. |
-19 |
-10 |
sd = standard deviation±
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
f(I) = increase factor, calculation see chapter7.3, page20
Annex 3: Data of Experiment 2 (for Detail please see attached full Study Report)
Determination of Titre
Criterion: The determination of titre should give a number of at least 109cells/mL, correlating to 100 colonies/plate after dilution.
Exact colony counts could not be determined due to the strong growth of the bacteria, but by visual inspection it was obvious that the values exceed 100 colonies/ plate.
Table15.1‑a Titre Values (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
s.g. |
s.g. |
Repl. 2 |
s.g. |
s.g. |
Assessment |
ok |
ok |
s.g. = strong growth, too strong for counting of colonies
ok= the criterion was fulfilled
Sterility Control
Criterion: No colony per plate may grow.
Table15.2‑a Sterility (colonies per plate)
|
DMSO |
NaCl |
Repl. 1 |
0 |
0 |
Repl. 2 |
0 |
0 |
Repl. 3 |
0 |
0 |
Repl. 4 |
0 |
0 |
Assessment |
ok |
ok |
ok= thecriterion was fulfilled
Spontaneous Revertants
The determined values were within the normal range of the laboratory.
DMSO
Table15.3‑a Spontaneous Revertants DMSO (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
256 |
272 |
Repl. 2 |
268 |
272 |
Repl. 3 |
276 |
284 |
Mean |
267 |
276 |
sd |
10.1 |
6.9 |
sd = standard deviation±
NaCl
Table15.3‑b Spontaneous Revertants NaCl (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
252 |
-- |
Repl. 2 |
260 |
-- |
Repl. 3 |
276 |
-- |
Mean |
263 |
-- |
sd |
12.2 |
-- |
sd = standard deviation±
-- = not tested
Positive Controls
¨ Without
metabolic activation:
Mitomycin C (MMC) in NaCl
¨ With
metabolic activation:
2-Amino anthracene (2-AA) in DMSO
¨ For used concentrations see chapter6.2.4, page13.
Table15.4‑a Diagnostic Mutagens (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Substance |
MMC |
2-AA |
Repl. 1 |
596 |
592 |
Repl. 2 |
668 |
608 |
Repl. 3 |
712 |
672 |
Mean |
659 |
624 |
sd |
58.6 |
42.3 |
f(I) |
2.51 |
2.26 |
Rev. abs. |
396 |
348 |
sd = standard deviation±
f(I) = increase factor, calculation see chapter7.3, page20
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
Test Item Chemark 1355 Exp 2
Concentrations of the test item are stated as nominal concentrations, for real concentrations see chapter18, page 41
Mutagenicity Test
Table15.5‑a Concentration 5000µg/plate (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
268 |
276 |
Repl. 2 |
280 |
280 |
Repl. 3 |
288 |
296 |
Mean |
279 |
284 |
sd |
10.1 |
10.6 |
f(I) |
1.04 |
1.03 |
Rev. abs. |
12 |
8 |
sd = standard deviation±
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
f(I) = increase factor, calculation see chapter7.3, page20
Table15.5‑b Concentration 2500µg/plate (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
268 |
260 |
Repl. 2 |
280 |
276 |
Repl. 3 |
292 |
292 |
Mean |
280 |
276 |
sd |
12.0 |
16.0 |
f(I) |
1.05 |
1.00 |
Rev. abs. |
13 |
0 |
sd = standard deviation±
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
f(I) = increase factor, calculation see chapter7.3, page20
Table15.5‑c Concentration 1250µg/plate (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
268 |
236 |
Repl. 2 |
280 |
276 |
Repl. 3 |
292 |
280 |
Mean |
280 |
264 |
sd |
12.0 |
24.3 |
f(I) |
1.05 |
0.96 |
Rev. abs. |
13 |
-12 |
sd = standard deviation±
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
f(I) = increase factor, calculation see chapter7.3, page20
Table15.5‑d Concentration 625µg/plate (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
252 |
268 |
Repl. 2 |
260 |
280 |
Repl. 3 |
312 |
308 |
Mean |
275 |
285 |
sd |
32.6 |
20.5 |
f(I) |
1.03 |
1.03 |
Rev. abs. |
8 |
9 |
sd = standard deviation±
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
f(I) = increase factor, calculation see chapter7.3, page20
Table15.5‑e Concentration 313µg/plate (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
240 |
248 |
Repl. 2 |
252 |
280 |
Repl. 3 |
284 |
288 |
Mean |
259 |
272 |
sd |
22.7 |
21.2 |
f(I) |
0.97 |
0.99 |
Rev. abs. |
-8 |
-4 |
Rev.abs. = absolute revertants, calculation see chapter7.3, page20
f(I) = increase factor, calculation see chapter7.3, page20
sd = standard deviation±
Annex 4: Data of the Cytotoxicity Test
The toxicity of the following concentration was tested: 5000 µg/plate.
Two plates with and without metabolic activation were incubated with the corresponding dose of the test item on maximal soft agar.
Experimental Parameters
Date of treatment:
06. Jan. 2021
Tested strain:
TA102
Test item concentrations:
5000 µg/plate
Incubation time: 48 h
Incubation temperature:
37±1 °C
Method: Plate
incorporation method
Determination of Titre
Criterion: The determination of titre should give a number of at least 109cells/mL, correlating to at least 100 colonies/plate after dilution.
Exact colony counts could not be determined due to the strong growth of the bacteria, but by visual inspection it was obvious that the values exceed 100 colonies/ plate
Table16.2‑a Titre Values (colonies per plate)
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
s.g. |
s.g. |
Repl. 2 |
s.g. |
s.g. |
Mean |
s.g. |
s.g. |
sd |
n.c. |
n.c. |
Assessment |
ok |
ok |
s.g. = strong growth, too strong for counting of colonies
sd = standard deviation ±
n.c. = not calculable
ok= the criterion was fulfille
Toxicity Control
The test item is considered non-toxic, if the quotient titre/toxicity is below 2.
Table16.3‑a 5000µg/plate on maximal-soft-agar with culture diluted by 106
Strain |
TA102 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
s.g. |
s.g. |
Repl. 2 |
s.g. |
s.g. |
Mean |
s.g. |
s.g. |
sd |
n.c. |
n.c. |
Titre/Tox |
< 2 |
< 2 |
Assessment |
non- toxic |
non- toxic |
s.g. = strong growth, too strong for counting of colonies
sd = standard deviation±
n.c. = not calculable
The quotient titre/toxicity was not calculable, but it was obvious, that the test item did not reveal any cytotoxic properties towards the bacterial test strainSalmonella typhimuriumTA102.
Assessment
The test item did not show cytotoxicity towards the strainSalmonella typhimuriumTA102.
On the base of these results, both experiments were performed with 5000 µg/plate as maximum dose.
COMPARISON WITH HISTORICAL DATA
In the following table, the history of the spontaneous revertants and positive controls of the performed experiments with the strainSalmonella typhimuriumTA102 up to 05. Jan. 2021 is stated in comparison with the experiments performed within this study. Only experiments which were performed before the performance of the study were considered.
For the historical data, the plate incorporation method and the pre- incubation method were used.
Table 17‑a Historical Data of Spontaneous Revertants
Strain |
|
TA102 |
|
Induction |
|
- S9 |
+ S9 |
DMSO |
334 |
341 |
|
Min |
215 |
201 |
|
Max |
593 |
573 |
|
sd |
71 |
77 |
|
Exp 1 |
247 |
243 |
|
Exp 2 |
267 |
276 |
|
NaCl |
Mean |
433 |
-- |
Min |
188 |
-- |
|
Max |
544 |
-- |
|
sd |
143 |
-- |
|
Exp 1 |
245 |
-- |
|
Exp 2 |
263 |
-- |
|
Positive Controls* |
Mean |
971 |
n.c. |
Min |
555 |
451 |
|
Max |
1287 |
s.g. |
|
sd |
235 |
n.c. |
|
Exp 1 |
581 |
737 |
|
Exp 2 |
659 |
624 |
|
sd = standard deviation±
s.g.= strong growth, too strong for counting of colonies
n.c. = not calculable
* Different positive controls were used, see chapter6.2.4, page13
-- = not tested
All values lie inside the range of the historical data.
Annex 6: Real Concentrations in the Study
In the following table, the nominal concentrations and the real concentrations (based on weights used) in the experiments are compared.
Table 18-a Experiment 1
Experiment1 |
|
Nominal concentrations |
Real concentrations |
5000 µg/plate |
5014 µg/plate |
1500 µg/plate |
1504 µg/plate |
500 µg/plate |
501 µg/plate |
150 µg/plate |
150 µg/plate |
50 µg/plate |
50 µg/plate |
Table 18-b Experiment 2
Experiment 2 |
|
Nominal concentrations |
Real concentrations |
5000 µg/plate |
5016 µg/plate |
2500 µg/plate |
2508 µg/plate |
1250 µg/plate |
1254 µg/plate |
625 µg/plate |
627 µg/plate |
313 µg/plate |
314 µg/plate |
156 µg/plate |
157 µg/plate |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
The substance did not show genotoxic effects in an Ames tests with Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and 1538 (50 - 5000 µg/plate), all with and without metabolic activation. In addition, a second Ames test with with Salmonella typhimurium TA 102 was performed. The test substance was determined to be non genooxic under the condistions of the test (with and without metabolic activation, 50 - 5000 µg/plate).
In the attached OECD Toolbox study (read across) for effects in V79 Chinese hamster lung cells, with and without metabolic activation, the results were:
In the first test set with no info on metabolic activation PDPI scored -0.6, ie negative for genotoxicity and cytotoxicity
In the second test set with metabolic activation PDPI scored +0.2, ie ambiguous
In the third test set without metabolic activation PDPI scored -1, ie negative for genotoxicity and cytotoxicity
In addition, the parent substances of this salt (pyromellitic acid and 2 -phenyl-2 -imidazoline) are both negative.
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
No effects observed. Therefore, the Test Substance must not be classified.
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