ethyl N-acetyl-3,5-dinitro-L-tyrosinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

L-Tyrosine, N-acetyl-3,5-dinitro-, etyhl ester

Cas Number:

29358-99-4

Molecular formula:

C13H15N3O8

IUPAC Name:

L-Tyrosine, N-acetyl-3,5-dinitro-, etyhl ester

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

The test was performed in two 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.


6.4.13 S9-mix
Phosphate buffer 22.5 mL
0.1M NADP-solution 1.0 mL
1M G6P-solution 0.125 mL
Salt solution 0.5 mL
Rat liver S9 1.0 mL

S9 was obtained by Trinova Biochem GmbH, Gießen.
Batch nos. 4335, 4318
Specification produced from the livers of male Sprague-Dawley rats which were treated with Phenobarbital/5,6-Benzoflavone.

Test concentrations with justification for top dose:

. The following nominal test item concentrations were prepared for experiment 1:
5000, 1500, 500, 150 and 50 µg/plate.
The following nominal test item concentrations were prepared for experiment 2:
5000, 2500, 1250, 625, 313, 156 and 78 µg/plate.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

DMSO and water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and water

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Amino-anthracene, C14H11N, CAS: 613-13-8 4-Nitro-1,2-phenylene diamine, C6H7N3O2, CAS: 99-56-9

Details on test system and experimental conditions:

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 per strain (for details see chapter 6.2.2, page 12).
.
7.2 Conduct of Experiment
7.2.1 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 cultures were checked for growth visually. The incuba-tion 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

ambiguous

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

Test results 3

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

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:

Mean Revertants Experiment 1
Strain TA98 TA100 TA102 TA1535 TA1537
Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Demin.
water Mean 16 21 100 101 233 239 9 9 4 4
sd 1.5 0.6 6.7 3.1 12.9 19.7 0.6 1.0 1.0 1.2
DMSO Mean 17 21 101 102 235 236 11 9 3 4
sd 1.5 0.6 5.1 3.6 6.1 10.6 0.6 0.6 0.6 1.0
0.9% NaCl Mean - - - - 237 - - - - -
sd - - - - 8.3 - - - - -
Positive
Controls* Mean 695 135 500 1197 545 745 189 152 81 59
sd 22.0 14.0 25.0 69.9 34.9 16.2 12.9 14.4 2.6 4.4
f(I) 40.88 6.43 5.00 11.74 2.30 3.16 21.00 16.89 27.00 14.75
5000 µg/plate Mean 15 18 134 128 287 257 8 8 4 4
sd 1.0 1.7 7.0 5.9 16.7 30.0 0.6 0.0 0.0 0.6
f(I) 0.88 0.86 1.33 1.25 1.22 1.09 0.73 0.89 1.33 1.00
1500 µg/plate Mean 14 15 113 117 241 283 11 10 4 4
sd 0.6 0.0 10.8 6.7 18.9 42.8 0.6 1.0 1.5 0.6
f(I) 0.82 0.71 1.12 1.15 1.03 1.20 1.00 1.11 1.33 1.00
500 µg/plate Mean 17 17 108 111 211 164 10 10 4 4
sd 1.5 1.0 9.9 12.7 6.1 41.8 2.0 1.0 1.0 0.6
f(I) 1.00 0.81 1.07 1.09 0.90 0.69 0.91 1.11 1.33 1.00
150 µg/plate Mean 16 18 111 116 221 241 10 11 4 5
sd 0.6 2.5 12.5 10.7 18.0 52.2 0.6 1.5 0.6 1.0
f(I) 0.94 0.86 1.10 1.14 0.94 1.02 0.91 1.22 1.33 1.25
50 µg/plate Mean 17 19 105 105 231 181 10 10 4 6
sd 1.5 2.6 7.0 4.0 28.9 36.3 1.2 0.6 0.6 0.6
f(I) 1.00 0.90 1.04 1.03 0.98 0.77 0.91 1.11 1.33 1.50
sd = standard deviation ±
* Different positive controls were used, see chapter 6.2.4, page 14
s.g.= strong growth, too strong for counting of revertants
n.c. = not calculable
f(I) = increase factor, calculation see chapter 7.4, page 21
- = not tested

Mean Revertants Experiment 2
Strain TA98 TA100 TA102 TA1535 TA1537
Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Demin.
water Mean 17 18 85 85 223 224 10 10 4 5
sd 1.5 1.0 5.3 4.7 4.6 12.0 1.5 1.0 1.2 1.5
DMSO Mean 17 18 75 86 217 231 10 9 6 6
sd 0.6 2.0 3.5 1.5 9.2 2.3 1.5 1.2 1.2 0.6
0.9% NaCl Mean - - - - 229 - - - - -
sd - - - - 6.1 - - - - -
Positive
Controls* Mean 564 169 436 1504 656 716 239 161 92 42
sd 30.2 10.1 30.3 48.0 36.7 20.0 11.5 6.1 2.6 4.0
f(I) 33.18 9.39 5.13 17.49 2.86 3.10 23.90 17.89 15.33 7.00
5000 µg/plate Mean 14 17 105 105 229 231 6 8 3 5
sd 2.1 1.0 4.2 3.1 12.9 2.3 1.5 1.5 1.2 2.0
f(I) 0.82 0.94 1.40 1.22 1.06 1.00 0.60 0.89 0.50 0.83
2500 µg/plate Mean 17 18 93 97 231 233 7 9 4 4
sd 1.5 2.6 5.0 6.4 8.3 8.3 0.6 1.2 2.5 3.2
f(I) 1.00 1.00 1.24 1.13 1.06 1.01 0.70 1.00 0.67 0.67
1250 µg/plate Mean 13 17 81 89 215 220 8 8 4 5
sd 1.5 3.1 5.5 8.3 4.6 6.9 1.0 1.0 0.6 0.6
f(I) 0.76 0.94 1.08 1.03 0.99 0.95 0.80 0.89 0.67 0.83
625 µg/plate Mean 13 19 87 89 224 225 7 8 3 5
sd 1.2 1.5 4.2 2.3 10.6 14.0 1.5 1.5 1.0 2.3
f(I) 0.76 1.06 1.16 1.03 1.03 0.97 0.70 0.89 0.50 0.83
313 µg/plate Mean 17 19 75 79 228 236 10 11 3 5
sd 3.5 1.5 5.0 2.1 4.0 6.9 0.0 0.6 0.6 1.0
f(I) 1.00 1.06 1.00 0.92 1.05 1.02 1.00 1.22 0.50 0.83
156 µg/plate Mean 14 15 74 76 229 224 12 11 4 6
sd 1.5 2.6 4.6 4.0 10.1 16.0 1.2 1.0 1.0 1.7
f(I) 0.82 0.83 0.99 0.88 1.06 0.97 1.20 1.22 0.67 1.00
78 µg/plate Mean 14 16 76 72 232 237 13 10 4 5
sd 2.6 4.0 4.0 7.5 6.9 12.2 1.7 1.0 0.6 0.6
f(I) 0.82 0.89 1.01 0.84 1.07 1.03 1.30 1.11 0.67 0.83
sd = standard deviation ±
* Different positive controls were used, see chapter 6.2.4, page 14
s.g.= strong growth, too strong for counting of revertants
n.c. = not calculable
f(I) = increase factor, calculation see chapter 7.4, page 21
- = not tested

Applicant's summary and conclusion

Conclusions:

The test item L-Tyrosine, N-acetyl-3,5-dinitro-, ethyl ester, dissolved in DMSO was tested in the Bacterial Reverse Mutation test by performing two individual experiments (plate in-corporation and pre-incubation method). Both experiments were performed in the absence and presence of a metabolic activation system (S9).
The test item showed no precipitation and no toxicity at any of the tested concentrations up to 5000 µg/plate in experiment 1 and 2 (see chapter 8.1.2 and 8.2.2).
In experiment 1 (plate incorporation method), none of the tested concentrations showed a relevant increase (increase factor f(I) ≥ 2 or 3) in the number of revertants in all tested strain.
However, for TA100 (+/-S9) and TA102 (-S9) a dose-related increase over the tested con-centrations could be observed, and therefore, the test item is stated as likely mutagenic under the conditions of this experiment.
To verify this result, a further experiment (exp. 2) was performed with the pre-incubation method and adapted test item concentrations.
In experiment 2, the test item again caused no relevant increase (increase factor f(I) ≥ 2 or 3) in the number of revertants in all bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation and for TA100 (+/-S9) a dose-related increase over the tested concentrations could be observed.
In the lower concentrations (Exp. 1: 150 and 50 µg/plate, Exp.2: 313, 156, 78 µg/plate), the number of revertants is not decreasing to lower numbers, because these values are near the number of the spontaneous revertants in DMSO.
According to Dan D.Levy et al. (see Literature in chapter 5) the historical control data of the solvent controls are a further support to evaluate the result together with clear increase and dose-dependency.
In experiment 1 the number of spontaneous revertants of TA100 of the solvent DMSO were higher than the mean of the historical control data (mean -S9: 66, mean +S9: 69), but with-in the control data range (mean ± 3 SD -/+S9: 36-102). The test item concentrations reached no increase with f(I) ≥ 2, but at all concentrations the mean number of revertants was beyond the historical control range (upper limit mean ± 3 SD: 102) with and without metabolic activation. And also, above the 95.5% (mean ± 2 SD) control limits (-S9: 42-90; +S9: 47-91). In combination with the high solvent control values of DMSO, the increase of the test item values is not strong and only slightly higher than an f(I) value of 1.0.

In experiment 2 the number of spontaneous revertants of TA100 of the solvent DMSO were slightly higher than the mean of the historical control data (mean -S9: 66, mean +S9: 69). The test item concentrations reached no increase with f(I) ≥ 2, but at the highest concentra-tion (5000 µg/plate) the number of revertants was beyond the historical control range (up-per limit: 102) with and without metabolic activation. The revertant colony counts at 2500 g/plate were also near the upper limit of the historical control data range and above the 95.5% control limits.
The dose dependency could be verified, but the clear increase over the historical data of the solvent control was not reproducible, resulting in no clearly positive result under the conditions of the study and therefore it is assessed as equivocal, but with a tendency for the test item to show weak mutagenic effects.

Based on the results of this study it is concluded that L-Tyrosine, N-acetyl-3,5-dinitro-, ethyl ester tested in the Salmonella typhimurium strains, in the presence and absence of metabolic activation under the experimental conditions in this study led to an inconclusive outcome.

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 bacterial test strains was evaluated in both experiments. The test item L-Tyrosine, N-acetyl-3,5-dinitro-, ethyl ester showed no relevant increase in the number of revertants (f(I) ≥ 2 or 3) in the Salmonella typhimurium test strains TA98, TA100, TA102, TA1535 and TA1537 in both evaluated experiments, but for the strain TA100 a dose-related increase was found with and without metabolic activa-tion in experiment 1 and 2.
Based on the results of this study it is concluded that L-Tyrosine, N-acetyl-3,5-dinitro-, ethyl ester tested in the Salmonella typhimurium strains, in the presence and absence of metabolic activation under the experimental conditions in this study led to an inconclusive outcome (see discussion in chapter 10)

Executive summary:

Findings and Results:

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 item L-Tyrosine, N-acetyl-3,5-dinitro-, ethyl ester was tested in the Bacterial reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537).

The test was performed in two 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 strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.

The test item showed no precipitates and no signs of toxicity on the plates at any of the concentrations in both the presence and absence of metabolic activation (for details see chapter 8.1.2, page 22).

The results of this experiment showed that none of the tested concentrations showed a relevant increase (increase factor f(I) ≥ 2 or 3) in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

For TA100 (+/-S9) and TA102 (-S9) a tendency of a dose-related increase over the tested concentrations could be observed.

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 all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the test item concentrations.

For TA1537 (-S9) a slight decrease in the number of revertants was observed in all tested concentrations, but no further signs of toxicity could be observed (for detail see chapter 8.2.2, page 24).

The results of this experiment showed that the test item caused no relevant increase (increase factor f(I) ≥ 2 or 3) in the number of revertants in all bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation. For TA100 (+/-S9) a dose-related increase over the tested concentrations could be observed.

 

 

Conclusion:

Based on the results of this study it is concluded that L-Tyrosine, N-acetyl-3,5-dinitro-, ethyl ester tested in the Salmonella typhimurium strains, in the presence and absence of metabolic activation under the experimental conditions in this study led to an inconclusive outcome (see discussion in chapter 10 for further details)

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 bacterial test strains was evaluated in both experiments. The test item L-Tyrosine, N-acetyl-3,5-dinitro-, ethyl ester showed no relevant increase in the number of revertants (f(I) ≥ 2 or 3) in the Salmonella typhimurium test strains TA98, TA100, TA102, TA1535 and TA1537 in both evaluated experiments, but for the strain TA100 a dose-related increase was found with and without metabolic activation in experiment 1 and 2.

Based on the results of this study it is concluded that L-Tyrosine, N-acetyl-3,5-dinitro-, ethyl ester tested in the Salmonella typhimurium strains, in the presence and absence of metabolic activation under the experimental conditions in this study led to an inconclusive outcome (see discussion in chapter 10)