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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-05-11 to 1994-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Limit test:

no

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:

rat

Strain:

other: Bor: WISW (SPFCpb)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Males : 7 weeks Females : 8 weeks
- Weight at study initiation: Males : 167 - 204 g Females : 134 - 163 g
- Fasting period before study: no
- Housing: individually in Macrolon cages, type II, Animal bedding softwood granulation HW 300/500W, supplied by Jelu-Werk, J. Ehrler, D-73494 Rosenberg
- Diet (e.g. ad libitum): ad libitum,ssniff R, special diet for rats, supplied by ssniff Spezialdiaten GmbH, D-59494 Soest
- Water (e.g. ad libitum): ad libitum ,in drinking water quality from the Stadtwerke Halle was provided after filtration by a Pall Sealkleen Filter (Pall Sealkleen Filter with candle, type SLK 7002 ARP, supplied by Pall Filtrationstechnik GmbH, D-63303 Dreieich/Sprendlingen). An automatic watering system with drinking nipples or drinking bottles were used. The water containers providing the watering system were sterilized by autoclave. The drinking nipples were checked for permeability. If necessary, the watering system was sterilized by autoclave or rinsed with hot water (approx. 80°C).
- Acclimation period: Approx. 2 weeks under test conditions before administration of the test substance. Veterinary supervision of the animals before start of the study.

DETAILS OF FOOD AND WATER QUALITY: Contaminant analyses of the diet are performed in appropriate intervals. Certificates of analysis are on file in the testing facility. According to information from the Stadtwerke Halle the water is investigated in appropriate intervals. Certificates of analysis are on file in the testing facility. The known contaminants present in bedding, diet, and water are toxicologically insignificant in the quantities detected for the experiment performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 - 22.5
- Humidity (%): 47 - 80
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Daily preparation of the solutions for administration with different substance concentrations according to the intended doses. The test substance concentrations were prepared with deionized water. Before start of the study stability of the test substance in the solutions for administration at concentrations of approx. 20 mg/mL and 100 mg/mL were examined. According to information from the testing facility the test substance was stable in the administration form at room temperature for 24 hours

VEHICLE
water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the study described herein the stability of the test substance in aqueous solutions was checked. Therefore two solutions with concentrations of approx. 20 mg/mL and 100 mg/mL, respectively, were prepared. The concentration of this solutions was measured after completion of solutions, immediately, and again after 24 hours. In the meantime the solutions were stored at room temperature. The results showed no decrease of concentration during the test period. The HPLC conditions used to analyse the standard- and sample solutions were in accordance with the "Purity" method of DEGUSSA AG. This method was modified in order to accommodate it to this study and to the HPLC-apparatus used. Each standard solutions was injected twice, each sample
solution 3 times.
Column : stainless steel, 125 mm, 4.6 mm ID Spherisorp 5 ODS2, 5µm, Kontron serial number NAK 45338
Column inlet pressure : about 100 bar
Mobile phase : 0.015 mol/L KH2PO4, adjusted to pH 2.6 with phosphoric acid (85%). The aqueous phase is mixed 9:1 with acetonitrile.
Elution : isocratic
Flow : 1.8 mL/min
Temperature : 30°C
Detection wavelengh : 210 nm
Range : 0,5 ABS
Response time : 0.5 sec
Injection volume : 10 µL
Retention time : about 3 minutes (main component)

Duration of treatment / exposure:

4 weeks, half of the high dose group and the control group were observed for a subsequent 6-week recovery without further treatment (recovery period)

Frequency of treatment:

Once daily a.m. (7days per week)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

215 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Male: 10 animals at 0 mg/kg bw/day
Male: 5 animals at 215 mg/kg bw/day
Male: 10 animals at 1000 mg/kg bw/day
Female: 10 animals at 0 mg/kg bw/day
Female: 5 animals at 215 mg/kg bw/day
Female: 10 animals at 1000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a preliminary dose range finder test
- Rationale for selecting satellite groups: as recommended by the guideline
- Post-exposure recovery period in satellite groups: 6 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed daily for the occurrence of toxicity symptoms as well as their severity and duration.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week, starting with pretest period.

FOOD CONSUMPTION AND COMPOUND INTAKE : Once a week, starting with pretest period.

WATER CONSUMPTION AND COMPOUND INTAKE : No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In weeks 1 and 4 a.m. as well as in week 10 from recovery animals. Blood was collected from the retroorbital venous plexus of 1 eye under CO2 anesthesia from all animals.
- Anaesthetic used for blood collection: Yes CO2
- Animals fasted: Not specified
- How many animals: All
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In weeks 1 and 4 a.m. as well as in week 10 from recovery animals. Blood was collected from the retroorbital venous plexus of 1 eye under CO2 anesthesia from all animals.
- Animals fasted: Not specified
- How many animals: All
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected in metabolism cages in week 4 a.m. for 3 hours from all animals. Pretreatment before sampling with tap water (10 mL/kg b.w.).
- Metabolism cages used for collection of urine: No
- Animals fasted: Not specified
- Parameters checked in table [No.1] were examined.

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

Statistics:

Mean values of all parameters and -where indicated - standard deviations were calculated separately for each group and sex.
For statistical evaluation of food consumption, body weights, and organ weights the DUNNETT-Test (4) was used. For values of hematological and clinical chemistry examinations the DUNNETT-Test was used in case of normal distribution, otherwise the STEEL-Test was employed. Significant differences between mean values of control group 1 and dose groups 2 and 3 were marked with * or ** (significance level of p < 0.05 or p < 0.01 according to DUNNETT) or + (significance level p < 0.05 according to STEEL), respectively. Results of urinalysis are only given as individual animal data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The behaviour and general condition of the rats remained unaffected. As the only symptoms during the study individual animals (3 males, 1 female) of the treatment groups exhibited a red incrustration at the neck probably due to scratches and one female rat of the control group showed moderate focal alopezia. These findings are considered as incidental.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights exhibited no differences between treated and control rats.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of treated animals was not affected. In males food consumption was minimally (less than 1 g/animal/day) lower than control values. In females food consumption was insignificantly higher than the control value. One animal of the low dose group (No. 39) spoiled a lot of food and therefore had to be excluded from evaluation.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No substance related effects were noticed.

Haematological findings:

no effects observed

Description (incidence and severity):

No substance related changes were detected.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of clinical chemistry parameters in week 1 revealed alterations in triglycerides, cholesterol, and albumin. Cholesterol concentrations were reduced while albumin was increased in male and female animals. Triglycerides were also reduced but the change was more pronounced in the low dose group and only males were affected. The changes in the relative concentrations of a1- and β-globulins probably were an artefact due to the increase of albumin. All values are only marginally out of the normal range and therefore regarded as a sign of adaption to the altered nutrition. The statistically significant differences in iron (high dose, females) and creatinine (high dose, females) concentrations are considered as toxicologically irrelevant, as the values are well within the normal range. The second determination during week 4 of the treatment period indicated almost the same alterations in cholesterol, triglycerides, and albumin in males. In females albumin was increased also, but cholesterol and triglyceride concentrations were not altered significantly. Additionally males of the high dose group showed minimal increases in creatinine, alanine aminotransferase (increased also in low dose males), and alkaline phosphatase. Calcium concentrations were marginally reduced. In high dose females statistically significant increases were detected in alkaline phosphatase, aspartate aminotransferase, and urea (low dose also). All of these changes were of only border line extent and are therefore considered as incidental findings of no toxicological relevance. The third examination at the end of the recovery period (week 10) did not reveal any changes except for an incidentally low value of alkaline phosphatase activity in males.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The investigation during week 4 of the treatment period did not reveal any significant differences between treated and control rats.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The measurement of the absolute and relative organ weights during necropsy indicated a minimal increase in liver weights of male and female rats of the high dose group. At the end of the recovery period the differences between treated and control animals had disappeared. The finding is considered as an adaptive reaction to the high doses of peptide. This judgement also is supported by the absence of any morphological change in the livers during microscopical examination.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related alterations were detected during necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment related histologic changes were observed in the stomach.
Stomach
The glandular mucosa of animals treated with the test substance showed elongated gastric pits which were covered by hyperplastic surface epithelial cells. The finding was observed only in the region of the fundic mucosa and showed a diffuse distribution pattern.
The cytoplasm of the hyperplastic cells had an increased basophilia. As demonstrated by the PAS stain it contained only minimal amounts of PAS positive mucus substances. The other cell types of the gastric mucosa remained unchanged. Diffuse hyperplasia of surface epithelial cells was observed in all group 2 and 3 male rats and also in 1/5 control animals. In females 3/5 group 2 and 5/5 group 3 rats were affected. The finding was graded as minimal to moderate and showed a dose related increase in grade. The mean severity was 1.2 and 2.2 in male rats of groups 2 and 3, respectively (1 = minimal; 2 = slight; 3 = moderate). In the 1/5 control rats which had diffuse hyperplasia of the surface epithelial cells it was graded as minimal. In group 2 female animals the mean severity was 1.0 and in group 3 females 2.2. All other histopathological findings, e. g. focal endocardiosis of the heart or lymphoid hyperplasia of the spleen are regarded as incidental and not related to the treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the dose levels tested

Remarks on result:

other:

Remarks:

no adverse effects observed at the dose levels tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The results of the present 4-week oral toxicity study conducted according to OECD guideline 407 (1981) on ε-Trifluoroacetyl-L-lysyl-L-proline in Wistar rats indicate, that the test substance produces only mild, adaptive effects even at very high doses and after prolonged exposure. Although there are minor effects even at the low dose, these effects are not interpreted as toxicity, but as adaption to the unusually high peptide supply or the slight local irritant effects. The liver and the stomach displayed the highest sensitivity to the presence of the test substance.
The NOAEL is estimated as above 1000 mg/kg body weight.