General Toxicology

Studies in Mice

    Single Dose and Dose Range Finding Study
    Repeated Dose Toxicity Study (up to 52 weeks)

•  Administration Routes: Oral (by Gavage in Drinking Water or in Diet),
   Intravenous (up to 4 weeks), Subcutaneous, Intramuscular (up to 4 weeks),
   Intraperitoneal (at an appropriate dose regimen)
• Toxicokinetic and Recovery Period can be included

Studies in Rats

    Single Dose and Dose Range Finding Study
    Repeated Dose Toxicity Study (up to 52 weeks)

•  Administration Routes: Oral (by Gavage in Drinking Water or in Diet), Dermal,

   Intravenous by Bolus or Continuous Infusion (up to 13 weeks), Subcutaneous

   Intramuscular (up to 4 weeks), Intranasal, Intratracheal, Intraperitoneal

   (at an appropriate dose regimen)

• Toxicokinetic and Recovery Period can be included

Studies in Rabbits

    Single Dose and Dose Range Finding Study
    Repeated Dose Toxicity Study (up to 13 weeks)

• Administration Routes: Oral Gavage, Dermal, Subcutaneous, Intramuscular

  (up to 4 weeks), Intra-articular (at an appropriate dose regimen)

• Toxicokinetic and Recovery Period can be included

Studies in Dogs

    Dose Range Finding Study
    Repeated Dose Toxicity Study (up to 52 weeks)

•  Administration Routes: Oral Gavage, Capsules, Diet, Intravenous by Bolus or
   Continuous Infusion (up to 13 weeks), Subcutaneous, Dermal, Ocular,
   Intramuscular, Intranasal, Intraperitoneal (at an appropriate dose regimen)
• Toxicokinetic and Recovery Period can be included

Studies in Minipigs

    Dose Range Finding Study
    Repeated Dose Toxicity Study (up to 39 weeks)

• Administration Routes: Oral Gavage, Capsules, Intravenous (up to 4 weeks),
  Subcutaneous, Dermal, Ocular, Intramuscular, Intra-articular

  (at an appropriate dose regimen)
• Toxicokinetic and Recovery Period can be included

Studies in Marmosets

    Dose Range Finding Study
    Repeated Dose Toxicity Study (up to 26 weeks)

• Administration Routes: Oral Gavage, Intravenous (up to 4 weeks),
  Subcutaneous, Ocular, Intramuscular
• Toxicokinetic and Recovery Period can be included

 

 Alternative Methods

 

 

 

AREAS OF ACTIVITY

Intelligent Testing Strategies
Compound Screening and Lead Optimisation
Regulatory Testing
Safety/Risk Assessments 

Alternative testing methods have always been pursued in RTC where scientists are trying to Replace and Reduce animal use or Refine traditional animal testing, in the spirit of the 3Rs.RTC answers to the ethical concerns of different regulatory requests from cosmetic to chemical to pharmaceutical, offering a wide variety of in vitro, ex vivo or in vivo tests.

3Rs ethical and scientific principles are completely embraced by European Cosmetic legislation which  aims to ban any animal testing.
With REACH regulation a strong change from a “tick-box approach” to a tiered testing strategy has been introduced also for chemical products with the aim to limit animal use.
The list of test methods, which applicability domain and predictivity are internationally recognized through a formal validation process, is still limited but is continuously growing. RTC is promptly introducing new methods among its services.
For chemical products RTC applies an “intelligent testing strategy” which building blocks are in silico evaluation, read across, grouping, up to a  ‘bottom up’ or ‘top-down’ tiered experimental approach using in vitro screening and “stand-alone” replacement methods for irritation/corrosion or toxicity properties of the products. The purpose is to ensure that use of animals is minimised and in vivo studies are conducted only whether essential.
For pharmaceutical products RTC assists the Sponsor in the evaluation of leading candidates, prioritizing safety tests and identifying those molecules which are least likely to cause attrition (toxicity) during later development phases in order to avoid unnecessary studies and limit costs and in vivo side effects.
The list of RTC services is continuously renewed and, in such an actively evolving field, Sponsors can rely on a sound background knowledge, an excellent experience of regulatory work, continuous up-dating and  flexibility which are key traits of RTC.

LIST OF SERVICES

In silico Investigation

    • OECD Toolbox
    • DEREK (partnered)

In vitro Models

    • Metabolism (interspecies comparative assays)

    • Protein binding

 

In vitro cellular models

    • Balb/3T3 Toxicity assay including assessment of starting dose for acute oral toxicity

    • Balb/3T3 Phototoxicity assay

    • Barrier permeability (e.g. Caco-2 cells - partnered)

    • Organ toxicity screening:     

- Liver toxicity (e.g. Primary rat hepatocytes or HepG2 cells)       

- Kidney toxicity (e.g. LLC-RK1 cells)      

- Lung epithelial toxicity (e.g. A549 cells)      

- Haematotoxicity

         (e.g. CFU murine Granulocyte/Macrophage Assay or in vitro

         Blood Cell Haemolysis test)

    • Genotoxicity screening assays (e.g. mini-Ames test, micronucleus test)

 

3D and ex vivo models 

    • Skin corrosion/Irritation

- Corrositex (OECD 435)
- Episkin (OECD 439)

    • Ocular irritation and corrosion

       - Bovine Corneal Opacity and Permeability Assay  (BCOP - OECD 437)
       - Human Corneal epithelium (hCE )

 

In vivo refinement

    • LLNA methods

      - Non-radioactive (BrdU: ELISA)       - Reduced LLNA approach (BrdU: ELISA)

 

  Genetic Toxicology

 

Areas of Activity

Project Design
Regulatory Testing
Mechanistic Studies
Compound Screening
Safety/Risk Assessments

Mutagenicity data are of fundamental importance during early toxicological evaluation being usefull indicators  of potential chronic effects of test compounds. Since the mid 1970s  the RTC genetic toxicology group has been performing all relevant in vitro and in vivo assays. Studies are designed according to international guidelines, including OECD, EC, OPPTS, ICH or Japanese guidelines.

The introduction of in vivo comet assay and micronucleus assay combined into one protocol has completed our regulatory testing package.

RTC is in close  contact with the scientific community and collaborates with academia and industries on new development projects. Our senior genetic toxicologists have received advanced training by the most recognised international experts and can look back to decades of hands-on experience. They play an important role in international scientific associations and are contributing to the harmonisation of regulatory testing procedures.

The expertise of our genetic toxicology group and the quality of our  studies  are a guarantee of a  scientifically sound, rapid and well communicated service to our clients.

List of Services

Bacterial Mutation Assay
•    Standard Method
•    Prival Modification Method
•    Modified Method for Evaluation of Peptides and Amino acids containing material

Mammalian Cell Gene Mutation  (TK and HPRT mutations)  

Chromosomal Damage in vitro
(Chinese Hamster Cells, Human Lymphocytes)
•    Micronucleus Test
•    Chromosome Aberration Test
•    Sister Chromatid Exchange Test

Chromosomal Damage in vivo
•  Micronucleus Test
•  Metaphase Analysis

In vivo and in vitro Comet Assay
In vivo and in vitro UDS
Photomutagenicity
Mutagenicity Screening

  Development and Reproductive Toxicology

 

Areas of Activity

Project Design
Compound Screening
Regulatory Testing
Safety/Risk Assessments

RTC has more than 30 years of experience in conducting reproduction toxicology studies, offering data interpretation and advising on the
significance of the results obtained.
RTC facility is well equipped for all specimen processing and uses dedicated software for data capture and handling.
The regulatory testing package offered to pharmaceutical clients includes all ICH compliant studies: embryo-foetal, fertility and
early embryonic development, pre- and post-natal development and toxicokinetic studies in pregnant animals. For various sectors
of the chemical industry, fertility screening tests, teratology and multigeneration studies (sometimes combined with toxicological
studies) are included in RTC’s programme and are performed in accordance with international requirements.
RTC’s services also cover reproduction/development toxicity screening, combined with repeated dose toxicity as required.
RTC also performs single and repeated dose toxicity and pharmacokinetic studies in neonatal and juvenile rats and minipigs.
RTC has been involved in the OECD interlaboratory assessment to evaluate the uterotrophic assay which is of interest to chemical
industry clients.

List of Services

For Pharmaceuticals (ICH Guidelines)
• Fertility and Early Embryonic Development to Implantation Study in Rat/Mouse
• Pre- and Post-natal Development Study in Rat/Mouse
• Embryo-Foetal Development Study in Rat/Mouse/Rabbit
• Fertility and Embryo-Foetal Development Study in Rat (Combined)/Mouse

For Chemicals (OECD, EU, OPPTS, METI)

For Food Additives (Red Book)
• Reproduction/Development Toxicity Screening Study in Rat
• One Generation Reproduction Toxicity Study in Rat
• Two Generation Reproduction Toxicity Study in Rat
• Prenatal Development Toxicity Study in Rat/Rabbit
• Combined Repeated Dose Toxicity Study with the Reproduction/Development Toxicity

  Screening Test

• Extended One Generation Study Reproductive Toxicity Study in Rats
• Neurotoxicity Study in Pregnant Rat
• Uterotrophic Assay in Immature Female Rat

Technical details
Routes of administration

Neonatal and Juvenile studies
• Pharmacokinetic and Toxicity Studies in Rat and Minipig by oral,
  subcutaneous and intravenous routes.

   Biologicals

 

 

 

Areas of Activity

Safety evaluation

•    Recombinant proteins and peptides
•    Monoclonal antibodies
•    Biosimilars
•    Vaccines
•    Gene therapy
•    Somatic cell therapy

Toxicology for Biologicals and Advanced Therapies

In the last decade RTC has acquired a considerable expertise in the development of biologicals and products for advanced therapies working on simple and conjugated peptides, recombinant proteins, monoclonal antibodies, biosimilars and products for gene therapy and somatic cell therapy.

The scientists at RTC assist the client in tailoring the most suitable preclinical development according to product specifications and different therapeutic indications. Furthermore, RTC can support the Sponsor during presentation of the project to regulatory authorities. The experimental design is prepared in compliance with the updated versions of the most relevant international guidelines and in accordance with the most recent scientific publications.

In order to select the relevant species RTC performs Tissue Cross Reactivity studies on human and animal tissues.

RTC offers the possibility to perform toxicology testing on a wide range of animal models, including the small non-human primate, Callithrix jaccus (commonly known as marmoset) which allows to proceed to clinical phase I with a limited amount of product.

RTC can provide  assistance for any bioanalytical activities, which play a key role in the development of a biological product. RTC has a solid experience in customised methods which require specific reagents and challenging set-up and validation. We are in close contact with the scientific community and  collaborate with scientists from academia and industries  for the development and transfer of immunological methods.

Central laboratory activity
•    Set-up, validation of PK methods and sample analysis
•    Set-up, validation of immunogenicity methods and sample analysis
•    Management and logistics of central lab

Activities with external partners  
Selection of the relevant species  
•    Characterisation of mAb affinity and dissociation rate by BIACORE
•    Production of specific reagents for analytical purposes
•    Biodistribution analysis by qPCR

  Minipig Toxicology

 

Regulatory Agencies require pharmaceutical products to be tested both in rodent and non-rodent species, in order to assure their safe use in humans. The non-rodent species most frequently used is the dog, but the selection must be based on the prediction potential of the experimental model and not only on a traditional approach. In comparison with humans, dogs have some unique physiologic attributes that can affect the disposition of drugs; besides, ethical reasons may limit their use. Different reasons, from anatomical similarities to humans to  functional similarities and availability of disease models, support the selection of minipig as a suitable alternative non-rodent species for example:

• hepatic P450 cytochromes are comparable between pigs and humans supporting

  the usefulness of minipigs as experimental animals to predict biotransformation

  pathways in man;

• the gastrointestinal system of the minipig offers some anatomical advantages

  (in terms of similarity to humans) and functional advantages (in terms of absorption,

  metabolism and reduced tendency to vomit) when compared to dogs;

• based on the similarities of the minipig and human skin, the minipig is the species

  of choice in dermal studies to evaluate both local tolerance and possible systemic

  toxicity after dermal application.

Minipigs are also a valuable model for safety testing of medical device.  

Furthermore, political and societal support to limit the use of dogs and primates as non-rodent species encourages the selection of this animal model which is widely accepted by international regulatory authorities and scientifically justified for many toxicity tests.

Minipig as an alternative approach in Toxicity Testing

RTC introduced the pig as an animal model in 1993 to perform pre-clinical studies in this species.  
The first experience was gained within the field of veterinary pharmaceutical compounds. As a natural consequence of the husbandry, handling and procedural experience acquired with conventional swine, in 1996 RTC introduced the Goettingen minipig as an alternative non-rodent species.

Experimental Designs

 Routes of administration

•    Oral (by gavage or capsules)
•    Intravenous (bolus or slow infusion)
•    Subcutaneous
•    Intramuscular
•    Dermal
•    Ocular
•    Intra-articular

 Local tolerance studies  

•    Dermal and subcutaneous administration
•    Single and repeated dose

 Wound healing studies

•    Full-thickness wounds with removal of dermis and epidermis

 Pharmacokinetic studies

•    All available routes of administration
•    Analysis of different biological matrices (blood, urine, tissues)

 Immunological methods applied

•    Set-up and validation of PK/TK methods for large molecules (ELISA, RIA)
•    Set-up and validation of immunogenicity methods for Anti-Drug Antibodies
•    Study sample analysis
•    Immunophenotyping

 Systemic toxicity

•    Repeated dose up to 39 weeks
•    Toxicokinetics
•    All in vivo and post-mortem examinations performed in-house

 Neonatal and juvenile toxicity and kinetics

•    Single and repeated dose
•    Neonatal minipigs from 7 days of age up to weaning (28 days)
•    Juvenile minipigs starting from post-weaning age

  Services for Reach

 

The Scientific Staff at RTC includes specialists in different regulatory disciplines. The following consultancy services are offered for the chemical industry:

• Evaluation of regulatory requirements, existing studies and literature for any

  product type

• Revision/evaluation of available toxicological data, SAR/QSAR evaluation, waiving

  strategies and testing proposal

• Design of complete testing programmes, including cost and time evaluation

• Monitoring of outsourced studies

  (Physico-chemical properties and ecotoxicology  studies)

• Preparation of scientific and regulatory dossier documents (IUCLID 5 files, Robust

  summaries, CSA/CSR, Technical dossier; US EPA/Japan METI dossier preparation)

• Risk assessment with evaluation of exposure scenarios and evaluation of safety

  procedures

• Direct contact and liaison with regulatory authorities

• Hazard classification and labelling proposal under  GLP ; preparation of PPORD

  and MSDS

Experimental Services for Reach

RTC’s services cover all  studies required for assessment of effects on human health described in Annexes VII to X of REACH Legislation.

RTC can manage entire projects, by allocating and monitoring all the studies for the evaluation of physico-chemical and ecotoxicological properties to selected partner laboratories.
Examples of relevant studies performed  at RTC are:

In Vitro Studies and Genetic Toxicology

•    Bacterial gene mutation
•    Mammalian cell gene mutation (HPRT, TK)
•    In vitro and in vivo micronucleus test and metaphase analysis
•    In vitro and in vivo UDS
•    In vitro and in vivo comet assay
•    Stand alone methods alternative to animal testing

     (in vitro skin and eye irritation/corrosion)
•    Toxicity in Balb 3T3 cell line
•    Mechanistic studies for endocrine disruptors
•    Photosafety studies

Toxicology Studies

•    Acute, subacute, subchronic/chronic, oncogenicity
•    In vivo irritation and sensitisation studies
•    Standard study designs according to OECD, OPPTS
•    Specific studies: neurotoxicity studies in rats
•    Species: rodents and non-rodents

Reproduction Toxicology Studies

•    Full range of required protocols
•    Standard study design according to OECD, OPPTS, METI including combined

     toxicity – reproduction/developmental toxicity tests
•    Specific studies: uterotrophic assay in immature female rats
•    Species: rats and rabbits

Drug Exposure Evaluation (non radioactive)

•    Toxicokinetics

With REACH Legislation, new testing programmes and regulatory procedures have been  set up.  Strong attention was paid to intelligent testing strategies such as preference for in vitro tests, optimised in vivo tests, use of in silico methods (SARs, QSARs and biokinetic models), use of Thresholds of Toxicological Concern (TTC), grouping substances through read-across and chemical categories, exposure assessment or exposure-based waiving.

 Bioanalytical Services

Production, Purification and Characterization of Product-Specific Antibodies
Set-up of Bioanalytical Methods via Ligand Binding Assays:

• ELISA
• RIA
• Dot-Blot
• Western-Blot

Quantitative Validation and Analysis of PK/TK Samples by ELISA or RIA

Semi-Quantitative Qualification and Analysis of Immunogenicity

Samples by ELISA, RIA, FACS, Dot-Blot or Western-Blot
Immunogenicity Assessment in Animal and Human Samples by Validated Methods
Cross-Reactivity Studies in FDA Panel of Tissues

(Rat, Mouse, Rabbit, Minipig, Dog tissues are collected in RTC Facilities; Non-Human Primate and HumanTissues are provided by Qualified Partners)

• Immunohistochemistry (IHC) on Frozen and Formalin-Fixed Paraffin

  Embedded Sections
• Western-Blot