N-(N6-trifluoroacetyl-L-lysyl)-L-proline

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

N-(N6-trifluoroacetyl-L-lysyl)-L-proline

EC Number:

402-920-1

EC Name:

N-(N6-trifluoroacetyl-L-lysyl)-L-proline

Cas Number:

103300-89-6

Molecular formula:

C13H20F3N3O4

IUPAC Name:

(2S)-1-[(2R)-2-amino-6-(2,2,2-trifluoroacetamido)hexanoyl]pyrrolidine-2-carboxylic acid

Test material form:

solid: particulate/powder

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).