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Body Weight in Toxicology Studies

Dr R B Cope BVSc BSc(Hon 1) PhD cGLPCP DABT ERT

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Learning Objectives

• To understand what body weight measurements are used in toxicology studies

• Understand the factors affecting body weight and growth• Understand the potential artifacts affecting body weight and

growth measurements• Understand the critical design factors pertaining to the design

of diets for toxicology studies• The importance of control data and the types of control data

available• Understand the factors involved in determining treatment-

related and adverse effects• Understand the factors involved in determining abnormal

thresholds for body weight and growth in toxicology studies

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General Concepts of Interpretation

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Common Parameters Measured

• Growth (body weight change over time)• Absolute body weight• Food consumption relative to body weight• Efficiency of food utilization (body weight gain per 100 g of

food consumed)• Cumulative food consumption (total food consumed since the

start of the study divided by the days on study)• Cumulative food consumption relative to body weight

(cumulative daily food consumption divided by average body weight)

Body weight profile of a female mouse in the control group from a 21-month carcinogenicity study and the interval body weights calculated by averaging body weights in the analysis

intervals.

Hoffman W P et al. Toxicol. Sci. 2002;66:313-319

© 2002 Society of Toxicology

Upper panel, interval body weight (least squares mean ± SEM) versus week for females from a mouse carcinogenicity study.

Hoffman W P et al. Toxicol. Sci. 2002;66:313-319© 2002 Society of Toxicology

Other Indices:Upper panel, interval daily food consumption relative to body weight (least squares mean ±

SEM) versus week for females from a mouse carcinogenicity study.

Hoffman W P et al. Toxicol. Sci. 2002;66:313-319© 2002 Society of Toxicology

Upper panel, interval body weight (least squares mean ± SEM) versus week for females from a mouse carcinogenicity study.

Hoffman W P et al. Toxicol. Sci. 2002;66:313-319

© 2002 Society of Toxicology

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Interpretation of Body Weight and Body Weight Gain in Repeat Dose Studies

• Body weight, and in particular growth (body weight gain) is often a particularly sensitive general endpoint in toxicology

• In theory, long term changes in body weight are dependent upon:– Genetic potential for growth

• Hopefully equivalent across study groups because of inbred/line-bred strains and random allocation to groups

• Critical that the same strain should be used in all experimental groups

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Interpretation of Body Weight and Body Weight Gain in Repeat Dose Studies

• In theory, long term changes in body weight are dependent upon:– Stage of development span over which the measurements

are taken (e.g. juvenile to adult, mature adult only etc..)

• Critical that the same batch of animals (same generation, same age range, same rearing etc.) be used across all experimental groups

• Critical that there is genuinely random allocation to treatment group

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Interpretation of Body Weight and Body Weight Gain in Repeat Dose Studies

• In theory, long term changes in body weight are dependent upon:

– Balance between caloric intake and caloric expenditure over the long term

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Interpretation of Body Weight and Body Weight Gain in Repeat Dose Studies

• Some factors that need to be considered are:– Energy demand

• Room temperature• Individually or group housed (heat loss)• Presence of a hair coat and type of hair coat

– Food palatability– Dietary caloric density– Food availability

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Interpretation of Body Weight and Body Weight Gain in Repeat Dose Studies

• Some factors that need to be considered are:– Injuries that may inhibit eating/drinking– Nutrient deficiencies induced by the test article or due to

dilution of essential nutrients by high concentrations of the test article

– Capacity and opportunity to exercise– Neurological/neurobehavioral effects of the test article– General ill thrift

• Rodents that are a little sick tend not to eat become mildly hypothermic quickly (high metabolic rate per unit volume) become less likely to eat become more hypothermic etc.

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Interpretation of Body Weight and Body Weight Gain in Repeat Dose Studies

• Some factors that need to be considered are:– Test article has the capacity to alter metabolic rate

• Most notably, to disturb the hypothalamic-pituitary-thyroid axis

– Test article has the capacity to increase or decrease heat loss (e.g. peripheral vasoconstrictors/vasodilators)

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Guidance from the FDA Redbook:• When the test substance has no caloric value and constitutes a substantial

amount of the diet (e.g., more than 5%), both caloric and nutrient densities of the high dose diet would be diluted in comparison to the diets of the other groups. As a consequence, some high dose animals may receive higher test substance doses than expected because animals fed such diluted diets ad libitum may eat more than animals in other dosed groups to compensate for the differences in energy and nutrient content of the high dose diets. Such circumstances make it especially important that feed consumption of these animals be as accurately and closely monitored as possible in order to determine whether changes observed could be due to overt toxicity of the test substance or to a dietary imbalance. To further aid in this assessment, two control groups can be used; one group would be fed the undiluted control diet and a second group would be fed the control diet supplemented with an inert filler (e.g., methylcellulose) at a percentage equal to the highest percentage of the test substance in the diet.

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Guidance from the FDA Redbook:• When the vehicle for the test substance is expected to have caloric and/or nutritional values,

which are greater than that of the control ration, an adjustment in the caloric and/or nutritional components may be necessary.

• When administration of the test substance is expected to have an effect on feed intake because of its unpleasant taste or texture, other feeding regimens or experimental designs may be necessary.

• When the test substance interferes with the absorption of nutrients, leading to nutritional deficiencies or changes in nutrient ratios, this can confound assessment of the toxicological endpoints under consideration. For example, fat soluble vitamins may preferentially partition with a mineral oil or fat substitute which is largely unabsorbed, such that a potential deficiency in these vitamins may result. This potential may be eliminated by additional nutrient fortification of the feed for those groups receiving the test substance. Appropriate levels of nutrient fortification should be determined experimentally.

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Guidance from the FDA Redbook:

• If the above guidance has not been adhered to, it makes it extremely difficult to interpret the findings of a study and to determine what effects are adverse, what effects are test article-related and what effects are artifact

• If the study does not adhere to these guidelines it should be rejected and a better study performed

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Common Artifacts Affecting Growth and Body Weight in Toxicology Studies

• High levels of the test article are used in the experimental diets which distorts their nutritional characteristics and caloric density– Control diet and test diets are not isocaloric and

isonutritive– Common things that I personally seen which are not

directly related to the pathophysiology of the test article:• Iron deficiency due to dilution of the diet formulation

by high levels of the test article• B-group vitamin deficiencies due to dilution of the diet

formulation by high levels of the test article

Remember: toxicology studies often use young (6 week old) rapidly growing animals tat have a high nutritional demand. Under these circumstances, the effects of dietary deficiencies are often more pronounced than in adult animals

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Common Artifacts Affecting Growth and Body Weight in Toxicology Studies

• High levels of the test article are used in the experimental diets which distorts their nutritional characteristics and caloric density– Control diet and test diets are not isocaloric and

isonutritive– Common things that I personally seen which are not

directly related to the pathophysiology of the test article:• Iron deficiency due to dilution of the diet formulation

by high levels of the test article• B-group vitamin deficiencies due to dilution of the diet

formulation by high levels of the test article

Remember: toxicology studies often use young (6 week old) rapidly growing animals tat have a high nutritional demand. Under these circumstances, the effects of dietary deficiencies are often more pronounced than in adult animals

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Common Artifacts Affecting Growth and Body Weight in Toxicology Studies

• Not changing the cages or water bottles of all the experimental groups on the same day in relation to the measurement of body weight

• Leaving the water bottles out of the cages before performing body weight measurements

• Using different scales with different calibrations to measure different experimental groups

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Common Artifacts Affecting Growth and Body Weight in Toxicology Studies

• Different experimental groups housed in different rooms or in different parts of the same room – different environmental conditions (notably temperature, ventilation, noise levels etc.)

• Different experimental groups subjected to different lighting patterns

• Contaminants in the dietary formulations (notably aflatoxins and other mycotoxins in corn-based AIN diets)

• Unexpected contaminants in the test article

• Changing the test article batch half-way through the study

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Common Artifacts Affecting Growth and Body Weight in Toxicology Studies

• Control and experimental diets stored under different conditions

• Mixing and batch errors with diets (should always have analytical data on the concentration of the test article in the final formulated diets and stability data regarding the test article once it is incorporated in the diet)

• Lack of retention samples – makes it really hard to figure out what when wrong if you can’t go back and analyze the test article or the test diets

• Test article tested is not the same as the test article being registered

Note: it is very common with new chemicals that the initial tox testing is performed on relatively impure substances and the later tox testing is performed on progressively more pure preparations of the substance. This can make it difficult at times to work out what specifically is causing the effects and how these relate to the same substance produced by different suppliers or with different industrial grades of the same substance. Petroleum solvents are notorious for this problem

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The Importance of Control Data in Assessing Body Weight and Growth

• The control data that should (ideally) be available:

– Historical growth rate curve (with SD/95% CI) for the particular species and strain from the animal supplier

– Historical growth rate curve (with SD/95% CI) for the particular species and strain from the experimental facility using the same control diet formulation

– Data from the negative control group in the study

– Feed consumption data from the control and experimental groups

– Analytical data on diet/water impurities (part of GLP)

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Interpreting Body Weight and Growth Data• Is the effect related to treatment (i.e. cause and effect)?

A difference is less likely to be treatment-related if:– There is no obvious dose response (provided that there is appropriate

dose spacing; body weight and growth effects are almost always statistically continuous data)

– The effect is due to one or more animals that could be considered outliers

– Measurement is inherently imprecise (i.e. not repeatable)– Any effect is within normal biological variation (i.e. within ± 3 SD of the

mean assuming normal distribution) based on relevant historical control values

– There is a lack of biological plausibility – i.e. is there a likely mode of action for the effects on weight and growth? Are the effects similar to other substances in the same chemical class?

– There are artifacts in the experimental design or inadequate controls (control artifacts)

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Interpreting Body Weight and Growth Data• Is the observe effect adverse?

A difference is less likely to be adverse if:– There is no alteration in general function of the animal or any relevant

organs/tissues– There is evidence of transiency or adaptation e.g. short period of

mildly decreased growth/weight loss while adapting onto the diet followed by a recovery (quite common)

– The severity is less than the threshold of concern (see below)– The effects are isolated or independent from other adverse effects e.g.

body weight/growth change does not appear to affect survival or functioning in the long-term

– The effect is peculiar to the particular species or strain– The effect is not a precursor to other adverse effects– The effect on body weight/growth is secondary to another effect e.g.

reduced palatability, test article induces pain when eaten etc..

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Interpreting Body Weight and Growth Data• So what is the threshold of concern for body weight and

growth?– In reproductive/developmental studies any statistically

significant reduction in body weight and growth that is maintained over the course of development is generally adverse, even if not associated with specific adverse changes to other parameters

– Reduced body weight or growth in the pre-weaning phase of development is almost always adverse and of concern

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Interpreting Body Weight and Growth Data• So what is the threshold of concern for body weight and

growth?– Small changes (statistically significant) which remain within

the normal range of relevant historical control data are likely not adverse

– Changes outside of the normal range that are consistent over time (i.e. repeatable over time) are likely adverse

– Changes that are associated with abnormal feed consumption are potentially adverse

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Maximum Tolerated Dose• Definition:

– highest dose of a xenobiotic that will produce the desired effect without unacceptable toxicity (excessive deaths)

– Typically in toxicology studies, the threshold for the MTD is the dose is expected to produce ≤ 10% decrease growth compared with control animals in a chronic near life-time study based on data derived from a sub-chronic (90 day) study

• There is evidence to suggest that a > than 10% decrease in growth will result in distortion of the results of chronic studies (notably carcinogenesis) as well as producing adverse effects are associated with the effects of decreased growth/weight loss rather than the test article per se.

• Suggests that a 10% decrease in growth or body weight in a 90 day study is more than likely to be adverse (controversial)

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Feed/Water Consumption• Requirements

– If the test article is administered in the food, food consumption should be measured at the same interval as body weights

– If the test article is administered in the water, water consumption should be measured at the same interval as body weights

– It is impossible to accurately estimate the dose unless you have the above data

• There is generic data on feed consumption for experimental species that can be used as a last resort, but often this is HIGHLY inaccurate and can lead to serious under or over estimation of the doses

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Feed Consumption• Under most circumstances, feed consumption parallels

growth and growth rate.

• Under most circumstances, water consumption parallels growth, growth rate and feed consumption– Renal disease – reduced or increased depending on the type and stage

of the disease– Diabetes mellitus– Level of salt in the diet

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Questions?