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Description of key information

Oral repeated dose administration did not lead to adverse effects in OECD TG 422 study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study according to GLP and OECD guideline
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: US EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
equivalent or similar to guideline
Guideline:
other: US EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents
GLP compliance:
yes (incl. QA statement)
Remarks:
testing lab.
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males approx. 11 weeks, females approx. 12 weeks
- Housing: groups of 5 animals of the same sex (pre-mating), females were caged with males on a one-to-one basis (mating), males were housed in home cage at a maximum of 5/cage, females were individually housed (post-mating)
- Diet: ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%):40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
400, specific gravity 1.125
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe
- Amount of vehicle (if gavage): 5ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (18 February 2015). The concentrations analyzed in the formulations were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of the high and low dose groups were assessed for homogeneity and were found to be homogenous. Formulations were stable at room temperature for at least 6 hours.
Duration of treatment / exposure:
males: 31 days (2 weeks prior to mating, during mating, and up to the day before necropsy)
females: 40-53 days (2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation); exception: 3 females in group 4 were not dosed on day 21 or 22 post coitum since they were littering at the time of dosing.
Frequency of treatment:
daily, approximately at the same time each day
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:

No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a 14-day dose range finding study, up to 1000 mg/kg bw/d were tested without dose limiting effects
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION :
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight)
- How many animals: 5 animals/sex/group
- Parameters examined: white blood cells, differential leukocyte count, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, MCV, MCH, MCHC, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters examined: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, phosphate, bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males during week 4 of treatment, females from lactation day 4 onward (before blood sampling)
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: sensory activity / grip strength / motor activity

OTHER:
- pup mortality was determined on day 1 of lactation and daily thereafter.
- cliinical signs for pups were recorded at least once daily
- body weight of live pups was determined on Days 1 and 4 of lactation
- sex was determined for all pups on days 1 and 4 of lactation
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- males at completion of mating period (at least 28 days of dose administration), females which deliver: lactation days 5-7, females which fail to deliver: post-coitum days 25-27
- organ weights: 5 animals/sex/group, adrenal glands, brain, epididymides (all males), heart, kidneys, liver, ovaries, spleen, testes (all males), thymus, uterus (including cervix), prostate (weighed when fixed for at least 24 hours), seminal vesicles including coagulation glands (weighed when fixed for at least 24 hours), thyroid including parathyroid

HISTOPATHOLOGY: Yes
- The following organs were preserved for all animals, and organs and tissues of the selected 5 animals/sex of Groups 1 and 4 were analyzed except for those organs listed in parentheses: adrenal glands, (aorta), brain, caecum,cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes, mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes, ovaries, (pancreas), peyer's patches, (esophagus), pituitary gland, preputial gland, prostate gland, rectum, (salivary glands), sciatic nerve, seminal vesicles, sceletal muscle, (skin), spinal cord, spleen, sternum, stomach, testes, thymus, thyroid, (tongue), trachea, urinary bladder, uterus, vagina, all gross lesions.
- The additional slides of the testes of all males of Groups 1 and 4 and all males that failed to sire to examine staging of spermatogenesis and histopathology of interstitial cell structure.
- The liver of the selected males of Groups 2 and 3 based on suspected treatment-related microscopic findings in this organ.
- All gross lesions of all animals (all dose groups).
- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and viagina) of all animals of Groups 1 and 4 and all males that failed to sire and all females that failed to deliver healthy pups.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 4) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weight was increased in males in dose-dependent manner from 100 mg/kg bw/d.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal hepatocellular hypertrophy was detected in 2/5 males at 300 mg/kg bw/d and 4/5 males in 1000 mg/kg bw/d
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
study according to guideline and GLP, Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A combined repeated dose/reproductive toxicity testing was conducted according to OECD testing guideline 422 (BASF 2015). Male and female rats received daily doses of 0, 100, 300, or 1000 mg/kg bw/d Ligand TFME in polyethylene glycol 400 via gavage (10 animals per sex per dose). Two weeks after start of treatment, males and females of the same dose level were mated on a one-to-one basis for a maximum of two weeks, pairs were separated after verification of mating. Males were analyzed after 31 days continuous treatment, while females were allowed to litter and were sacrificed on postnatal day 5-7 after 40-53 days of continuous treatment. The animals were subjected to gross pathology and organs were fixed and embedded for histopathology. Treatment did not result in mortality or signs of systemic toxicity, and no effects on body weight gain, food consumption, hematology or clinical chemistry were observed. Neurological testing did not reveal any differences compared to the control group. The liver weights in males were increased starting at 100 mg/kg bw/d and minimal hepatocellular hypertrophy was detected in 2/5 males at 300 mg/kg bw/d and 4/5 males in 1000 mg/kg bw/d. In the absence of any other signs of toxicity, increased liver weights in males only, together with minimal hepatocellular hypertrophy are considered an adaptive change and are not regarded as adverse effect. Therefore, the NOAEL for subacute repeated dose toxicity, rat, was determined to be ≥1000 mg/kg bw/d.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
study according to guideline and GLP

Justification for classification or non-classification

The current data do not fulfill the criteria for classification according to Regulation (EC) 1272/2008 or Regulation 67/548/EEC, thus a non-classification for Ligand TFME-DPP is warranted.