4. In Vivo Toxicity Testing

Question 1

What are the different tests for hazard identification?

Answer 1

• Structure-Activity Relationships
• In vitro and short term tests
• Animal bioassays
• Epidemiologic data

Question 2

What part of toxicity testing is animal toxicology part of?

Answer 2

Descriptive toxicology

Question 3

What are the organizations involved in toxicity testing?

Answer 3

• Industry – in house or through contract labs
• Non-government Institutes
• Contract laboratories (CROs)
• Government agencies:
-> Environment Canada
-> Health Canada
-> Environmental Protection Agency (EPA, US)
-> Food & Drug Agency (FDA, US)
-> National Cancer Institute (NCI)
->Occupational Safety and Health Administration (OSHA)
• Academic Institutions

Question 4

What are toxicity tests for? which drugs are tested?

Answer 4

To characterize the toxicity profiles of chemicals (or biologicals).
All drugs developed for human use are tested via:
Safety Pharmacology
Preclinical Toxicology

Question 5

What is the name of the organization that defines the rules companies must follow for toxicity testing for human drugs?

Answer 5

The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH)

Question 6

What is the OECD?

Answer 6

Organization for Economic Co-operation and Development (OECD)
-> Guidelines for the Testing of Chemicals (aside from human drugs)
A collection of the most relevant, internationally agreed, testing methods used by government, industry and independent laboratories to identify and characterize potential hazards of new and existing chemical substances, chemical preparations and chemical mixtures.
Used in regulatory safety testing and subsequent chemical and chemical product notification and chemical registration.
Used to select and rank candidate chemicals during the development of new chemicals and products and in toxicology research.
For anything a company produces in very large quantities and wants to put it in their products.

Question 7

What are the different sections in the OECD guidelines?

Answer 7

Section 1: Physical Chemical Properties
Section 2: Effects on Biotic Systems
Section 3: Environmental Fate and Behaviour
Section 4: Health Effects - about 150 of the most relevant internationally agreed testing methods used by government, industry and independent laboratories to identify and characterise potential hazards of chemicals.
Section 5: Other Test Guidelines

Question 8

What are SIDS? What does the acronym stand for?

Answer 8

SIDS = Screening Information Data Set
->International authorities agree that six basic tests are necessary for a minimum understanding of a chemical’s toxicity, i.e. to screen high production volume chemicals.
If its produced in small quantities it doesn’t have to go through this testing.

We don’t have the SIDS for all of the chemicals out there that are being used or that exist.

Question 9

What are the 6 SIDS tests?

Answer 9

• acute toxicity
• chronic toxicity
• developmental and reproductive toxicity
• mutagenicity (ability to cause DNA damage)
• ecotoxicity (ecosystem)
• environmental fate.

Question 10

What products are required to follow SIDS?

Answer 10

All prescription drugs meet the SIDS requirements, not all products are required to follow SIDS and only 21 of 830 companies (3%) that produce high production volume chemicals have all SIDS tests available for their chemicals.

Question 11

What is the testing scheme for new chemicals?

Answer 11

see L4 slide 13

Question 12

Why is the testing scheme for new chemicals in a specific order?

Answer 12

You do the easier more practical and cheaper tests first (literature review, structure/activity assessment, short-term animal studies - acute short term repeated dose), and you can decide to drop the production of that chemical before having to do the more complicated, time consuming, and expensive tests (reproductive/teratology, chronic toxicity, oncogenicity/carcinogenicity).

Question 13

Why were standardized animal toxicity tests developed and used?

Answer 13

1. chemical exposure can be precisely controlled
2. environmental conditions can be well controlled
3. virtually any type of toxic effect can be evaluated
4. the mechanism by which toxicity occurs can be studied

Requirements today - use humane procedures and the minimum number of animals needed.

Question 14

What variables do we need to know when doing animal toxicity testing?

Answer 14

• Route of exposure
• Age of test animals
• Both sexes
• Dose levels to determine thresholds and dose –response relationships (minimum 3) –> we need to know how much is toxic, not just if something is toxic in large doses.

Question 15

What are the different types of systemic toxicity studies?

Answer 15

1. Acute: single dose
2. Subacute: short term repeated dose
3. Subchronic: repeated dose during 10% of their lifespan
4. Chronic: repeated dose during >10% of their lifespan (longest and most expensive - 1-2 years).

Question 16

Specify the testing conditions for SIDS test 1: Acute toxicity in the following categories?

1. Species
2. Age
3. Number of animals
4. Dosage
5. Observation period

Answer 16

1. Species: Rats are preferred for oral and inhalation tests. Rabbits are preferred for dermal tests.
2. Age: young adults
3. Number of animals: 5 of each sex per dose level
4. Dosage: 3 dose levels recommended: exposures are single doses or fractionated doses up to 24hrs for oral and dermal studies and 4hr exposure for inhalation studies.
5. Observation period: 14 days

Question 17

Specify the testing conditions for SIDS test 2: Chronic toxicity in the following categories?

1. Species
2. Age
3. Number of animals
4. Dosage
5. Observation period

Answer 17

1. Species: Two species recommended; rodent and non-rodent (rat and dog)
2. Age: Young adults
3. Number of animals: 20 of each sex for rodents, 4 of each sex for non-rodents per dose level.
4. Dosage: Three dose levels recommended; includes a toxic dose level + NOAEL (Observing the NOAEL would give you a standard in terms of what’s safe). The recommended maximum chronic testing durations for pharmaceuticals are now 6 and 9 months in rodents and non-rodents respectively. (Historically exposures were for 12 months, 24 months for chemicals).
5. Observation period: 12-24 months

Question 18

What is topical toxicity? Where is it done?

Answer 18

Happens in vivo.
Evaluates the effects of chemicals on skin, eyes, and occasionally, oral and vaginal mucous membranes. A compound is placed on the membrane, which is examined for reddening, blistering, or corrosion.
Often done on rabbits
- Eye: administer at day 0 and observe for 21 days
- Skin irritation: administer at day 0 after clipping fur and observe for 14 days

Question 19

What is an assay used for skin sensitization? Explain it.

Answer 19

The murine local lymph node assay (LLNA):
Are you stimulating an immune reaction against the chemical on the rat?
- Put the chemical on the dorsal surface of the ears on days 1, 2, and 3
- Let the rabbit rest on days 4 & 5 for sensitization and lymph node proliferation
- On day 6 inject radioactive 3H-thymidine
- Wait 5 hours so that it is incorporated into the DNA of the dividing cells
- On day 6 dissect the Auricular nodes and prepare a single cell suspension from which you measure radioactive 3H-thymidine incorporation
- This tells you if there is an immune response

Question 20

What are the different tests in the third SIDS requirement for developmental and reproductive toxicity?

Answer 20

1. Single generation developmental/reproductive toxicity studies (including teratology studies)
2. Multigenerational Developmental/Reproductive Toxicity Study
3. Neurobehavioral Effects

Question 21

Explain single generation developmental/reproductive toxicity studies.

Answer 21

Phase 1: General fertility and reproductive performance. Measure of pre-and post-implantation death
- Treat 10 males for 60 days and 20 females for 14 days and then mate them together. Males get treated for longer because spermatogenesis takes a long time. Oocyte formation takes less time in females.
- Looking at fertility in general, if you mate the animals will the females get pregnant? And then see what the progeny look like (do they live? Are they normal?)
Phase 2: Teratology study basic design for mice. 20 inseminated females are treated only during organogenesis period.
- Observe the effects in progeny after birth
Phase 3: Perinatal/postnatal studies
- Treat the mother while she is pregnant (gestation), during organogenesis, and after birth during lactation.
- observe progeny. Sometimes they look normal but behave abnormally

Question 22

Explain Multigenerational Developmental/Reproductive Toxicity Studies.

Answer 22

• F0 (parents) are treated for 60 days and then mated.
• F1 progeny are split into 2 groups:
  F1A. are autopsied at weaning for organ and skeletal defects
  F1B. are weaned off of breastfeeding and are mated with each other to make the next generation F2.
  -This same grouping happens with F2s:
  F2A. autopsied at weaning for organ and skeletal defects
  F2B. mated to make F3s.
• F3A. autopsied at weaning for organ and skeletal defects
• F3B. group is then autopsied for immunohistochemistry to see if the effects are multi-generational.

Question 23

Explain neurobehavioral testing,

Answer 23

Monitor the effects of a chemical on cognitive function during development and in the adult.
- The offspring might look normal but there can be neural behaviour effects.
- Not everything is visible in terms of external malformations.
Ex: exposure to alcohol in utero can affect education and learning.

Question 24

What were the 3 groups that the brominated flame retardants (BFRs) were tested on in animal studies?

Answer 24

1) BFR Exposure of Adult Males♂
2) BFR Exposure of Females prior to/during pregnancy♀
3) Effects of in utero and postnatal BFR exposures on F1 progeny

Question 25

In the BFR animal study, what were the assumptions to determine the conversion of house dust in a human to a rat? What was the conclusion?

Answer 25

Assumptions:
1. Daily dust ingestion: 100mg (Health Canada)
2. 18 month-old average bodyweight: 11 kgs (EPA)
3. Surface area ratio: Human to Rat is 1:6.2 (FDA)
Conclusion: In rats – a dose of 0.02 - 0.06 mg/kg/day is similar to exposure from house dust for an 18 month old

Question 26

Explain the testing procedure of the BFR exposed adult male rats.

Answer 26

• 15 adult rats (300-350g) per diet group (5)
  Diet of BFR for 70 days (Enough time to go through a cycle of spermatogenesis):
  Group 1: 0 mg/kg=control
  Group 2: 0.02 mg/kg,
  Group 3: 0.2 mg/kg,
  Group 4: 2 mg/kg,
  Group 5: 20 mg/kg
• The histology (micro-anatomy of cells, tissues, and organ microscope observation) and reproductive system were observed for 5 rats per diet group (total of 25 males)
• Blood and tissue collection and analysis and reproductive system thyroid function were observed for 10 males per diet group (total of 50 males).

Question 27

Explain the testing procedure of the BFR exposed adult female rats.

Answer 27

• Females (200-250g) fed a control diet for one week
• For 2-3 weeks, compare a control group vs the BFR-supplemented groups.
  Groups: 0, 0.06, 20, and 60 mg/kg/day where there is 15 females per group.
  During this time, the females are monitored to ensure they are eating property (measure body weight) and that they keep their menstrual cycle (estrus cycle)
• Mate the females with proven breeders because we want to know if they can get pregnant
• Continue the BFR diet
• After gestation, assess pregnant females and assess progeny. You can also continue to assess during lactation

Question 28

What are the conclusions on the BFR animal study on the adult male rats?

Answer 28

No effects on male reproduction in adult rats
Altered Thyroid hormone physiology
Induced liver drug metabolizing enzymes

Question 29

What are the conclusions on the BFR animal study on the female rats prior to/during pregnancy?

Answer 29

• Induced dam (pregnant female) and fetal liver enzymes
• No effects on estrus cyclicity/fertility/most measures of pregnancy outcome
• Increased fetal digit and skeletal anomalies
• Dam ovary -altered folliculogenesis (rate of development of oocyte in ovary is abnormal)

Question 30

What are the conclusions on the BFR animal study on the in utero and postnatal exposures on F1 progeny?

Answer 30

Increased liver weights
Altered serum biochemistry
Effects on skeletal ossification on PND (post natal day) 4
Altered timing of puberty – delay in ♂, accelerated in ♀
Effects on F1 ovaries on PND 46 (after puberty)

Question 31

What are the different mutagenicity tests (SIDS test 4)?

Answer 31

1. Micronucleus test for DNA damage
2. Ames test for bacterial mutagenicity
3. Carcinogencity

Question 32

Explain the micronucleus test for DNA damage.

Answer 32

Want to know if chemical is a mutagen? You look at DNA damage.

We’re looking at individual cells that are exposed (ex: radiation or a chemical). If the chemical is mutagenic it will cause chromosome fragmentation and/or cause chromosomes to lag in cell division. Therefore you are looking for micronucei (extra-nuclear bodies that contain damaged chromosome fragments and/or whole chromosomes that were not incorporated into the nucleus after cell division) which is a sign of DNA damage.

Question 33

Explain the Ames test for for bacterial mutagenicity.

Answer 33

• Inject a rat with a chemical to induce activating enzymes in the supernatant fraction of the liver (S9) and extract them
  3 different test tubes:
• S9 + bacteria A + chemical to be tested
• S9 + bacteria B + chemical to be tested
• S9 + bacteria A or B + control
  Observe the growth of bacteria on a petri dish
• The bacterial strains used in the test are engineered so that they cant grow without folic acid. We don’t put folic acid on the plate so they won’t be able to grow unless the chemical mutates them. Therefore, a chemical that causes mutations will cause more colonies to grow.
• There is a spontaneous rate of mutation so the control can have a small amount of growth.

Question 34

Describe the carcinogenicity test.

Answer 34

• Look at tissues long term to see if you’re getting any tumours. You might not have to do this long term carcinogenicity assay if you already see there is DNA damage in the other test…you can eliminate the chemical without going through all the steps.
• Carcinogenesis/mutagenesis studies measure the potential for a compound to produce tumours. In theory, animals are exposed to the compound throughout their lifetimes and resulting tumours are evaluated. In fact, animal studies are rare because of the expense.

Question 35

What is ecotoxicity? What organisms can it be tested on?

Answer 35

Looking at the toxicity effects on the whole ecosystem, the biological variety is huge.
Tests can be done on Drosophila, round worms, zebra fish, etc.
Ex: All of these organisms go through many of equivalent stages so we can look for chemicals that cause physical abnormalities in zebra fish and get a very good idea of how it can affect these organisms. Using roundworms for looking at effects on somatogenesis or oocyte development so you can see the effects on germ cells this way. Can use these to see what’s going on in other vertebrates and humans.

Question 36

What is the canadian council for animal care?

Answer 36

If an organisation is under the CCAC, then they need to run their research methods by this council in order for it to be approved ethical to proceed.

Question 37

What is the point of departure? (POD)

Answer 37

It is the starting point from which your reference dose is determined. The POD can be the LOAEL, the NOAEL, or the BMD10. Depending on the data you have, you need to choose which one to use. If you have the choice, the best option for determining the reference dose is the BMD10.

Question 38

Why is it important to have a high dose range when doing toxicity tests?

Answer 38

Because you need to have enough dose points to see where the toxic effect is observed.

Question 39

What happens between the NOAEL and the LOAEL?

Answer 39

Between NOAEL and LOAEL, we do not have data points, only curve approximation —> model data. Use data to find the BMD10.

Question 40

What is the BMD10?

Answer 40

It is the bench mark dose. The dose that has an adverse effect in 10% of the population. It is NOT a dose group, rather, it is calculated.

Question 41

What is the process of deriving toxicity values?

Answer 41

1. Test on humans, animals, or in vitro
2. Integrate data and evaluate studies to decide if they are valid, evaluate end points, or evaluate data-sets for dose-response assessment
3. Derive the appropriate POD
   - If the data is adequate for modeling: use the BMD
   - If there is inadequate data: use the NOAEL or the LOAEL as the POD
4. Apply uncertainty factors
5. Derive toxicity values: determine the reference dose from the above information

Question 42

What is the reference dose? (RfDs)

Answer 42

The safe dose. An estimate of the maximum acceptable oral dose of a toxic substance.

Question 43

How is the reference dose calculated?

Answer 43

RfD (mg/kg/day) = Any of the PODs (BMD>NOAEL>LOAEL) (mg/kg/day) / (UF-inter)(UF-intra)(UF-other)
UFinter = interspecies uncertainty factor
UFintra = intraspecies
UFother = uncertainty in NOAEL, other data insufficiencies

Question 44

Each country has a different method of determining which chemicals need to be tested on. What is Canada’s approach?

Answer 44

There are way too many chemicals that need to be tested on still, it is impossible to test all of them. Therefore, Canada has a domestic substances list in order to categorize and prioritize the chemicals.
There are 500 prioritized chemicals for safety testing, some are identified as data rich and some as data poor, meaning we need to do more research on them.