4. In Vivo Toxicity Testing Flashcards

Question

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 1

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

Answer 2

- 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).

Answer 3

- 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

Answer 4

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

Answer 5

- 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)

Answer 6

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)

Answer 7

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

Answer 8

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.

Answer 9

- 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.

Answer 10

- 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.

Answer 11

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.

Answer 12

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.

Answer 13

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.

Answer 14

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

Answer 15

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

Answer 16

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.

Answer 17

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

Answer 18

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

Answer 19

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

Answer 20

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.