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EC number: 402-920-1 | CAS number: 103300-89-6 N-6-TRIFLUORACETYL-L-LYSYL-L-PROLIN; T.F.A. LYS PRO; TFA-LYS-PRO
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
An Ames test conducted according to OECD TG 471 it is concluded that Epsilon-Trifluoracetyl-L-lysyl-L-proline did not show mutagenic activity in Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 either in the absence or in the presence of the S-9 mix, under the conditions employed in this evaluation.
Link to relevant study records
- 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:
- 1990-06-22 to 1990-07-17
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1983
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His locus
- Species / strain / cell type:
- other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Due to migration, the value was transferred to one of the current document's attachments
- Test concentrations with justification for top dose:
- Concentration range in the preliminary test (with metabolic activation) 0, 5.0, 50.0, 500.0, 5000.0, 50000.0 µg/plate
Concentration range in the preliminary test (without metabolic activation) 0, 5.0, 50.0, 500.0, 5000.0, 50000.0 µg/plate
Concentration range in the main test (with metabolic activation): 0, 620, 1850, 5560, 16670, 50000 µg/plate
Concentration range in the main test (without metabolic activation): 0, 620, 1850, 5560, 16670, 50000 µg/plate - Vehicle / solvent:
- Water
- 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
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
Experiment 1:
TA1535: 3.5E+08 cells/mL
TA1537: 1.5E+08 cells/mL
TA1538: 1.9E+08 cells/mL
TA98: 2.5E+08 cells/mL
TA100: 4.6E+08 cells/mL
Experiment 2:
TA1535: 1.2E+08 cells/mL
TA1537: 4.5E+07 cells/mL
TA1538: 2.5E+08 cells/mL
TA98: 1.8E+08 cells/mL
TA100: 1.8E+08 cells/mL
- Test substance added in agar (plate incorporation)
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 72h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
METHODS FOR MEASUREMENTS OF GENOTOXICIY
A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the vehicle, together with evidence of a dose-response. - Rationale for test conditions:
- As recommended by the guideline
- Evaluation criteria:
- A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the vehicle, together with evidence of a dose-response.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (> 50000 µg/plate)
- 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:
- cytotoxicity
- Remarks:
- (> 50000 µg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (> 50000 µg/plate)
- 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:
- cytotoxicity
- Remarks:
- (> 50000 µg/plate)
- 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:
- cytotoxicity
- Remarks:
- > 50000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES (if applicable):
A preliminary test was performed at the following concentrations:
Concentration range in the preliminary test (with metabolic activation) 0, 5.0, 50.0, 500.0, 5000.0, 50000.0 µg/plate
Concentration range in the preliminary test (without metabolic activation) 0, 5.0, 50.0, 500.0, 5000.0, 50000.0 µg/plate
the test substance was not toxic for all strains at all dose levels, thus the highest concentration in the main test was 50000 µg/plate
STUDY RESULTS
- Concurrent vehicle negative and positive control data
Experiment 1:
negative control
-S9: TA1535: 21 ± 2; TA1537: 18 ± 5; TA1538: 27 ± 7; TA98: 48 ± 8; TA100: 78 ± 18
+S9:TA1535: 24 ± 3; TA1537: 23 ± 3; TA1538: 60 ± 10; TA98: 68 ± 3; TA100: 82 ± 3
positive control:
-S9: TA1535: 691 ± 84; TA1537: 1361 ± 310; TA1538: 1367 ± 60; TA98: 857 ± 56; TA100: 839 ± 26
+S9:TA1535: 226 ± 26; TA1537: 216 ± 42; TA1538: 812 ± 17; TA98: 669 ± 47; TA100: 610 ± 46
Experiment 2:
negative control
-S9: TA1535: 66 ± 6; TA1537: 11 ± 4; TA1538: 26 ± 5; TA98: 51 ± 7; TA100: 170 ± 5
+S9:TA1535: 17 ± 1; TA1537: 17 ± 5; TA1538: 47 ± 3; TA98: 72 ± 20; TA100: 149 ± 8
positive control:
-S9: TA1535: 734 ± 113; TA1537: 1820 ± 202; TA1538: 1033 ± 27; TA98: 627 ± 74; TA100: 719 ± 123
+S9:TA1535: 213 ± 25; TA1537: 327 ± 120; TA1538: 643 ± 24; TA98: 420 ± 26; TA100: 544 ± 41
For all test methods and criteria for data analysis and interpretation:
- Statistical analysis; p-value if any : No statistical analysis was reported
Ames test:
- Signs of toxicity yes at > 50000µg/plate
- Individual plate counts :
Experiment 1:
TA1535: 3.5E+08 cells/mL
TA1537: 1.5E+08 cells/mL
TA1538:1.9E+08 cells/mL
TA98: 2.5E+08 cells/mL
TA100: 4.6E+08 cells/mL
Experiment 2:
TA1535: 1.2E+08 cells/mL
TA1537: 4.5E+07 cells/mL
TA1538:2.5E+08 cells/mL
TA98: 1.8E+08 cells/mL
TA100: 1.8E+08 cells/mL
- Mean number of revertant colonies per plate and standard deviation
Experiment 1:
TA1535: 18
TA1537: 15
TA1538:24
TA98: 37
TA100: 89
Experiment 2:
TA1535: 33
TA1537: 18
TA1538:24
TA98: 25
TA100: 182
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data) not reported - Remarks on result:
- other:
- Conclusions:
- From the above findings from a study conducted according to OECD guideline 471 (1983) it is concluded that Epsilon-Trifluoracetyl-L-lysyl-L-prolin did not show mutagenic activity in Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 either in the absence or in the presence of the S-9 mix, under the conditions employed in this evaluation. Thus, the test item does not need to be classified as mutagenic according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).
Reference
Experiments 1: Results
Dose µg/plate |
|
TA1535 |
TA1537 |
TA1538 |
TA98 |
TA100 |
|||||
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
||
0 |
|
22 |
21 |
12 |
21 |
20 |
58 |
42 |
67 |
79 |
80 |
22 |
26 |
19 |
21 |
27 |
52 |
45 |
71 |
95 |
82 |
||
18 |
26 |
22 |
27 |
34 |
71 |
57 |
65 |
60 |
85 |
||
Mean |
21 |
24 |
18 |
23 |
27 |
60 |
48 |
68 |
78 |
82 |
|
SD |
2 |
3 |
5 |
3 |
7 |
10 |
8 |
3 |
18 |
3 |
|
620 |
|
19 |
22 |
17 |
22 |
26 |
62 |
49 |
64 |
67 |
71 |
22 |
26 |
17 |
24 |
22 |
63 |
38 |
73 |
85 |
80 |
||
21 |
29 |
21 |
19 |
32 |
56 |
40 |
67 |
80 |
89 |
||
Mean |
21 |
26 |
18 |
22 |
27 |
60 |
42 |
68 |
77 |
80 |
|
SD |
2 |
4 |
2 |
3 |
5 |
4 |
6 |
5 |
9 |
9 |
|
1850 |
|
22 |
19 |
18 |
25 |
36 |
81 |
38 |
68 |
103 |
103 |
21 |
24 |
19 |
27 |
43 |
70 |
33 |
73 |
106 |
97 |
||
24 |
18 |
22 |
21 |
40 |
61 |
50 |
64 |
107 |
120 |
||
Mean |
22 |
20 |
20 |
24 |
40 |
71 |
40 |
68 |
105 |
107 |
|
SD |
2 |
3 |
2 |
3 |
4 |
10 |
9 |
5 |
2 |
12 |
|
5560 |
|
24 |
22 |
25 |
27 |
28 |
44 |
35 |
82 |
105 |
95 |
17 |
22 |
19 |
30 |
31 |
69 |
50 |
76 |
74 |
85 |
||
21 |
29 |
19 |
27 |
30 |
65 |
51 |
72 |
80 |
83 |
||
Mean |
21 |
24 |
21 |
28 |
30 |
59 |
45 |
77 |
86 |
88 |
|
SD |
4 |
4 |
3 |
2 |
2 |
13 |
9 |
5 |
16 |
6 |
|
16670 |
|
24 |
15 |
9 |
19 |
16 |
55 |
34 |
96 |
72 |
89 |
17 |
17 |
13 |
29 |
22 |
57 |
44 |
111 |
77 |
105 |
||
29 |
19 |
14 |
30 |
27 |
64 |
36 |
82 |
86 |
93 |
||
Mean |
23 |
17 |
12 |
26 |
22 |
59 |
38 |
96 |
78 |
96 |
|
SD |
6 |
2 |
3 |
6 |
6 |
5 |
5 |
15 |
7 |
8 |
|
50000 |
|
18 |
14 |
9 |
19 |
27 |
42 |
14 |
52 |
127 |
128 |
11 |
18 |
10 |
13 |
24 |
39 |
20 |
63 |
123 |
115 |
||
19 |
13 |
17 |
17 |
19 |
36 |
22 |
39 |
100 |
116 |
||
Mean |
16 |
15 |
16 |
16 |
23 |
39 |
19 |
51 |
117 |
120 |
|
SD |
4 |
3 |
3 |
3 |
4 |
3 |
4 |
12 |
15 |
7 |
|
Pos. C. |
|
613 |
223 |
025 |
245 |
1436 |
826 |
908 |
624 |
840 |
567 |
780 |
201 |
635 |
235 |
1338 |
793 |
797 |
664 |
813 |
659 |
||
679 |
253 |
422 |
168 |
1326 |
816 |
865 |
718 |
865 |
605 |
||
Mean |
691 |
226 |
361 |
216 |
1367 |
812 |
857 |
669 |
839 |
610 |
|
SD |
84 |
26 |
310 |
42 |
60 |
17 |
56 |
47 |
26 |
46 |
Experiment 2: Results:
Dose µg/plate |
|
TA1535 |
TA1537 |
TA1538 |
TA98 |
TA100 |
|||||
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
||
0 |
|
28 |
17 |
12 |
17 |
22 |
43 |
43 |
94 |
174 |
157 |
17 |
17 |
7 |
12 |
24 |
48 |
57 |
64 |
165 |
148 |
||
20 |
18 |
14 |
22 |
22 |
49 |
54 |
57 |
172 |
141 |
||
Mean |
22 |
17 |
11 |
17 |
26 |
47 |
51 |
72 |
170 |
149 |
|
SD |
6 |
1 |
4 |
5 |
5 |
3 |
7 |
20 |
5 |
8 |
|
620 |
|
25 |
19 |
13 |
21 |
20 |
43 |
39 |
62 |
174 |
184 |
37 |
14 |
16 |
8 |
22 |
53 |
48 |
61 |
159 |
165 |
||
36 |
16 |
23 |
22 |
32 |
54 |
48 |
78 |
168 |
150 |
||
Mean |
33 |
16 |
17 |
17 |
25 |
50 |
45 |
67 |
167 |
166 |
|
SD |
7 |
3 |
5 |
8 |
6 |
6 |
5 |
10 |
8 |
17 |
|
1850 |
|
33 |
33 |
12 |
15 |
25 |
57 |
53 |
76 |
177 |
160 |
29 |
28 |
12 |
21 |
36 |
57 |
39 |
89 |
212 |
177 |
||
30 |
25 |
12 |
20 |
30 |
61 |
40 |
73 |
182 |
191 |
||
Mean |
31 |
29 |
12 |
19 |
30 |
58 |
44 |
79 |
190 |
176 |
|
SD |
2 |
4 |
0 |
3 |
6 |
2 |
8 |
9 |
19 |
16 |
|
5560 |
|
17 |
23 |
18 |
22 |
27 |
54 |
34 |
74 |
182 |
191 |
34 |
25 |
13 |
20 |
27 |
49 |
39 |
65 |
190 |
209 |
||
25 |
16 |
24 |
23 |
34 |
47 |
33 |
73 |
172 |
194 |
||
Mean |
25 |
21 |
18 |
22 |
29 |
50 |
35 |
71 |
181 |
198 |
|
SD |
9 |
5 |
6 |
2 |
4 |
4 |
3 |
5 |
9 |
10 |
|
16670 |
|
11 |
21 |
16 |
16 |
15 |
54 |
56 |
73 |
168 |
158 |
30 |
22 |
12 |
16 |
21 |
53 |
48 |
57 |
162 |
169 |
||
26 |
21 |
18 |
18 |
21 |
44 |
52 |
80 |
180 |
156 |
||
Mean |
22 |
21 |
15 |
17 |
19 |
50 |
52 |
70 |
170 |
161 |
|
SD |
10 |
1 |
3 |
1 |
3 |
6 |
4 |
12 |
9 |
7 |
|
50000 |
|
21 |
20 |
12 |
26 |
15 |
41 |
25 |
65 |
146 |
172 |
11 |
16 |
2 |
18 |
37 |
35 |
26 |
43 |
156 |
170 |
||
19 |
12 |
15 |
15 |
36 |
43 |
24 |
49 |
154 |
163 |
||
Mean |
17 |
16 |
10 |
20 |
29 |
40 |
25 |
52 |
152 |
168 |
|
SD |
5 |
4 |
7 |
6 |
12 |
4 |
1 |
11 |
5 |
5 |
|
Pos. C. |
|
604 |
190 |
1590 |
440 |
1002 |
627 |
542 |
450 |
818 |
586 |
801 |
240 |
1905 |
201 |
1051 |
631 |
674 |
404 |
581 |
543 |
||
798 |
210 |
1966 |
341 |
1045 |
670 |
665 |
406 |
758 |
504 |
||
Mean |
734 |
213 |
1820 |
327 |
1033 |
643 |
627 |
420 |
719 |
544 |
|
SD |
113 |
25 |
202 |
120 |
27 |
24 |
74 |
26 |
123 |
41 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In an in vivo mouse micronucleus study conducted according to EU Method B.12 (1992) , ε-Trifluoroacetyl-L-Iysyl-L-proline did not induce any chromosome mutations in mice by damage to the chromosomes or the mitotic apparatus at 24- or 48-hour intervals alter the animals had received a single intraperitoneal dose of 2150 mg/kg bw. Therefore, ε-TrifluoroacetyI-L-lysyl-L-proline is considered non-mutagenic in the reported in the vivo mouse micronucleus test.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1994-06-14 to 1995-05-18
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 1992
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: males : 27 - 33 g females: 24 - 28 g
- Assigned to test groups randomly: yes, under following basis:using computer generated random numbers.
- Fasting period before study: 16h before start of the experiments
- Housing: individually in Macrolon cages, type II with Animal bedding softwood granulation HW 300/500W supplied by Jelu-Werk, J. Ehrler, D-73494 Rosenberg
- Diet (e.g. ad libitum): Standard diet ad libitum, ssniff® M, "Special diet for Mice", supplied by SSNIFF Spezialdiaten GmbH, D-59494 Soest
- Water (e.g. ad libitum):Water ad Iibitum in drinking-water quality from the Stadtwerke Halle/Westfalen was provided after filtration by a Pall Seal- kleen Filter (Pall Sealkleen Filter with candle, type SLK 7002 ARP, supplied by Pall Filtrationstechnik GmbH, D-63303 Dreieich/Sprendlingen). Water was provided in drinking-bottles.
- Acclimation period: 6 days under test conditions with veterinary care.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 22.0
- Humidity (%): 58 - 63
- Photoperiod (hrs dark / hrs light): 12/12
- Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: water
- Concentration of test material in vehicle: 100 mg/mL
- Amount of vehicle (if gavage or dermal): 21.5 mL/kg b.w. i.p. - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
An orientating study of the acute toxicity of epsilon-Trifluoroacetyl-L-Iysyl-L-proline in mice after single intraperitoneal administration with the test substance batch used in the present experiment was performed. In this study, solely the maximum dose to administer intraperitoneally according to the guideline (2150 mg/kg body weight) caused first signs of toxicity. Therefore, this dose was used in the micronucleus test. Administration Volume: 21.5 mL/kg b.w. - Duration of treatment / exposure:
- At 24- and 48-hour intervals after treatment, 6 mice each per sex of the negative control group and 7 mice each per sex of the test material group were sacrificed to prepare bone marrow smears. All animals of the positive control group were sacrificed 24 hours after treatment.
- Frequency of treatment:
- once
- Dose / conc.:
- 2 150 mg/kg bw/day (nominal)
- Remarks:
- as recommended by the guideline
- No. of animals per sex per dose:
- Control group: 12 males and 12 females
Positive Control group: 6 males and 6 females
Test group 14 males and 14 females - Control animals:
- yes
- Positive control(s):
- cyclophosphamide
- Justification for choice of positive control(s): according to guideline
- Route of administration: i.p.
- Doses / concentrations:31.6 mg/kg bw - Tissues and cell types examined:
- bone marrow
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: according to guideline
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Three groups of mice, the negative control group containing 12 males and 12 females, the positive control group containing 6 males and 6 females, and the test material group with 14 males and 14 females each received a single intraperitoneal administration (all groups: diet withdrawal: 16 h before treatment).
Group 1, the negative control, received physiological saline solution (0.9%).
Group 2, the test material group, received 2150 mg/kg body weight (males and females) of the test material. The test material was given as a freshly prepared solution in aqua ad iniectabilia.
Group 3, the positive control, received Cyclophosphamide(31.6 mg/kg b.w.) dissolved in physiological saline solution (0.9%).
The animals were observed for up to 7.5 hours after administration for the occurrence of toxicity symptoms; on days 2 and 3 only once daily in the morning. At 24- and 48-hour intervals after treatment, 6 mice each per sex of the negative control group and 7 mice each per sex of the test material group were sacrificed to prepare bone marrow smears. All animals of the positive control group were sacrificed 24 hours after treatment. After sacrifice , in group 2 animals the abdominal cavity was opened for optical control of the resorption of the test material solution.
DETAILS OF SLIDE PREPARATION: All mice were sacrificed by CO2 overdose. Both femurs were removed from each mouse and the bone marrow cells flushed into a labelled centrifuge tube with approximately 1.5 mL of fetal calf serum (Art. No. 210463, Boehringer Mannheim GmbH, D-68305 Mannheim). The tubes were centrifuged at approx. 180 x g for 5 minutes (Eppendorf Zentrifuge 5415, Eppendorf- Netheler-Hinz GmbH, D-22331 Hamburg), after which the supernatant serum was discarded and the bone marrow cells suspended upon a thin layer of serum. A small drop of the marrow serum suspension was smeared on a slide, which was identified by study number, animal number, species, sex, and date of preparation, and allowed to dry overnight. At least two slides per animal were prepared. The following day, the smears were stained using the panoptic stain method developed by PAPPENHEIM. Before evaluation, the slides were coded separately per sex. So they could not be assigned to animals of a specific group or sampling time during the microscopical examination.
METHOD OF ANALYSIS: For each sampling time the bone marrow smears from the first 5 animals per sex and group were used for evaluation. One slide per animal was examined. The remaining smears of each sex and group per interval were evaluated if macroscopical examination of the first smears revealed technical imperfections which precluded accurate microscopical analysis. From each animal one thousand polychromatic erythrocytes (PCE) were scored under the microscope (magnification 650- 1000 x, C. ZEISS, D-73447 Oberkochen) for the incidence of polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was calculated, based on 1000 erythrocytes (PCE + NCE) scored per slide, as a measure of the toxic efficacy of the test material. - Evaluation criteria:
- If a test material produced neither a statistically significant and reproducible positive response nor a dose related statistically significant response at any one of the test points compared to the negative control group, it is considered non-mutagenic in this system (significance level: 5%; p < 0.05).
- Statistics:
- The frequencies of polychromatic erythrocytes with micronuclei of the test material group and of the positive control group were compared with those of the negative control group at each sampling time (positive control only at 24 hours). A POISSON test (6) was applied. The data from each treatment group for each sex as well as for both sexes combined were compared with the respective negative control group data by means of a software developed by the Biometrical Department (FB) of ASTA Medica AG using a VAX 4000-400 computer (Digital Equipment Corp., D-81927 München). The values of the first 5 animals of each sex and sampling time were used for statistical evaluation.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): negative
- Ratio of PCE/NCE (for Micronucleus assay): please refer to any other information on results incl. tables.
- PCEs with MN:
Negative control:
24h application: Males: mean: 1.8 (0.84) n=5, females: mean: 1.2 (0.45) n=5
48h application:Males: mean: 2.2 (0.84) n=5, females: mean: 1.0 (1.00) n=5
Test item:
24h application: Males: mean: 1.6 (1.14) n=5, females: mean: 2.2 (1.64) n=5
48h application:Males: mean: 2.6 (1.67) n=5, females: mean: 2.4 (1.67) n=5
Positive control:
24h application: Males: mean: 29.0 (11.68) n=5, females: mean: 11.4 (5.18) n=5
- Statistical evaluation: please refer to any other information on results incl. tables. - Conclusions:
- In a study conducted according to EU Method B.12 (1992) , ε-Trifluoroacetyl-L-Iysyl-L-proline induced no chromosome mutations in mice by damage to the chromosomes or the mitotic apparatus at 24- or 48-hour intervals alter the animals had received a single intraperitoneal dose of 2150 mg/kg body weight. Therefore, ε-TrifluoroacetyI-L-lysyl-L-proline is considered non-mutagenic in the reported in vivo mouse micronucleus test and does not need to be classified according to Regualtion (EC)No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).
Reference
Statistical Evaluation of the Data (POISSON Test)
|
24h |
48h |
||
No. Of PCEs with MN |
m |
f |
m |
f |
verum |
8 |
11 |
13 |
12 |
negative control |
9 |
6 |
11 |
5 |
test statistic F |
0.800 |
1.5714 |
1.0833 |
2.000 |
degrees of freedom |
20,16 |
14,22 |
24,26 |
12,24 |
p-value |
0.685 |
0.166 |
0.419 |
0.072 |
No. of PCEs with MN |
|
|
|
|
positiv control |
145 |
57 |
|
|
negativ control |
9 |
6 |
|
|
test statistic F |
14.5000 |
8.1429 |
|
|
degrees of freedom |
20,290 |
14,114 |
|
|
p-value |
0.000 |
0.000 |
|
|
Results (Negative control)
24 h application |
48 h application |
||||||
Animal No. |
PCEs with MN |
|
Ration PCE/NCE |
Animal No. |
PCEs with MN |
|
Ration PCE/NCE |
|
N |
% |
|
|
N |
% |
|
Males |
|
|
|
|
|
|
|
1 |
3 |
0.3 |
1.95 |
7 |
3 |
0.3 |
2.55 |
2 |
2 |
0.2 |
2.38 |
8 |
2 |
0.2 |
2.36 |
3 |
1 |
0.1 |
2.60 |
9 |
3 |
0.3 |
1.98 |
4 |
1 |
0.1 |
2.29 |
10 |
2 |
0.2 |
1.74 |
5 |
2 |
0.2 |
2.60 |
11 |
2 |
0.2 |
1.81 |
Mean |
1.8 |
|
|
|
2.2 |
|
|
SD |
0.84 |
|
|
|
0.84 |
|
|
N |
5 |
|
|
|
5 |
|
|
Females |
|
|
|
|
|
|
|
13 |
1 |
0.1 |
2.56 |
19 |
2 |
0.2 |
1.95 |
14 |
1 |
0.1 |
2.77 |
20 |
1 |
0.1 |
1.91 |
15 |
2 |
0.2 |
1.84 |
21 |
0 |
0 |
2.08 |
16 |
1 |
0.1 |
2.45 |
22 |
0 |
0 |
2.62 |
17 |
1 |
0.1 |
2.34 |
23 |
2 |
0.2 |
3.18 |
Mean |
1.2 |
|
|
|
1.0 |
|
|
SD |
0.45 |
|
|
|
1.00 |
|
|
N |
5 |
|
|
|
5 |
|
|
Mean |
|
|
|
|
|
|
|
M+F |
1.5 |
|
|
|
1.6 |
|
|
SD |
0.45 |
|
|
|
1.07 |
|
|
N |
10 |
|
|
|
10 |
|
|
Results Test item
24 h application |
48 h application |
||||||
Animal No. |
PCEs with MN |
|
Ration PCE/NCE |
Animal No. |
PCEs with MN |
|
Ration PCE/NCE |
|
N |
% |
|
|
N |
% |
|
Males |
|
|
|
|
|
|
|
101 |
2 |
0.2 |
2.17 |
108 |
4 |
0.4 |
2.14 |
102 |
2 |
0.2 |
1.44 |
109 |
0 |
0 |
1.70 |
103 |
1 |
0.1 |
2.26 |
110 |
2 |
0.2 |
2.64 |
104 |
0 |
0 |
2.16 |
111 |
4 |
0.4 |
2.57 |
105 |
3 |
0.3 |
2.25 |
112 |
3 |
0.3 |
2.95 |
Mean |
1.6 |
|
|
|
2.6 |
|
|
SD |
1.14 |
|
|
|
1.67 |
|
|
N |
5 |
|
|
|
5 |
|
|
Females |
|
|
|
|
|
|
|
115 |
1 |
0.1 |
1.24 |
122 |
1 |
0.1 |
2.50 |
116 |
2 |
0.2 |
1.60 |
123 |
3 |
0.3 |
1.97 |
117 |
2 |
0.2 |
3.46 |
124 |
5 |
0.5 |
2.27 |
118 |
5 |
0.5 |
1.82 |
125 |
2 |
0.2 |
1.46 |
119 |
1 |
0.1 |
1.78 |
126 |
1 |
0.1 |
2.42 |
Mean |
2.2 |
|
|
|
2.4 |
|
|
SD |
1.64 |
|
|
|
1.67 |
|
|
N |
5 |
|
|
|
5 |
|
|
Mean |
|
|
|
|
|
|
|
M+F |
1.9 |
|
|
|
2.5 |
|
|
SD |
13.37 |
|
|
|
1.58 |
|
|
N |
10 |
|
|
|
10 |
|
|
Results Positive control
24 h application |
|||
Animal No. |
PCEs with MN |
|
Ration PCE/NCE |
|
N |
% |
|
Males |
|
|
|
401 |
49 |
4.9 |
3.65 |
402 |
29 |
2.9 |
4.99 |
403 |
20 |
2.0 |
3.52 |
404 |
25 |
2.5 |
2.41 |
405 |
22 |
2.2 |
3.57 |
Mean |
29.0 |
|
|
SD |
11.68 |
|
|
N |
5 |
|
|
Females |
|
|
|
407 |
9 |
0.9 |
1.98 |
408 |
5 |
0.5 |
1.95 |
409 |
11 |
1.1 |
1.65 |
410 |
13 |
1.3 |
2.03 |
411 |
19 |
1.9 |
3.20 |
Mean |
11.4 |
|
|
SD |
5.18 |
|
|
N |
5 |
|
|
Mean |
|
|
|
M+F |
20.2 |
|
|
SD |
12.59 |
|
|
N |
10 |
|
|
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
In an in vivo mouse micronucleus study conducted according to EU Method B.12 (1992) , ε-Trifluoroacetyl-L-Iysyl-L-proline did not induce any chromosome mutations in mice by damage to the chromosomes or the mitotic apparatus at 24- or 48-hour intervals alter the animals had received a single intraperitoneal dose of 2150 mg/kg bw. Therefore, ε-TrifluoroacetyI-L-lysyl-L-proline is considered non-mutagenic in the reported in the vivo mouse micronucleus test. Thus, it does not need to be classified according to Regualtion (EC)No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).
An Ames test conducted according to OECD TG 471 it is concluded that Epsilon-Trifluoracetyl-L-lysyl-L-proline did not show mutagenic activity in Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 either in the absence or in the presence of the S-9 mix, under the conditions employed in this evaluation. Thus, the test item does not need to be classified as mutagenic according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).
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