Subacute toxicity study These slides will be helpful for M.Pharm 2nd semester Pharmacology students to prepare for the subject " Toxicological Screening Methods" as per PCI new syllabus regulations

1. Repeated Dose Toxicity studies
(Subacute/Sub-chronic toxicity study)
Oral, Dermal and Inhalation routes
Dr. K. Nandakumar
Professor, Department of Pharmacology
MCOPS, MAHE, Manipal

2. Objectives of this Presentation
• The presentation will help the students of Master of Pharmacy (Pharmacology
and related disciplines) to comprehend the topic– ”Sub-acute toxicity studies” ,
in their curriculum. [PCI syllabus: Toxicological Screening Methods (MPL202T)].
• To describe how subacute toxicity studies are performed in industries, to meet
regulatory requirements.
• Describe OECD 407, 410 and 412 guidelines pertaining to repeated dose toxicities
of test chemicals administered via oral, dermal and inhalation routes.

3. Generally we adopt OECD guidelines
(1) The initial guidelines were published in 1981
(2) Revised in 1995 to include neuro and immunotoxicities.
(3) Ammended in 1998, to obtain information related to endocrine
disruptors
Oral route – Guideline no. 407 [28 d], 408 [90 d] (Rodents), 409 (Non-rodents)
Dermal route – Guidelines no. 410 [28 d], 411 [90 d],
Inhalation route – Guidelines no. 412 [28 d], 413 [90 d].

4. Principle of the Test
OECD 407 OECD 410 OECD 412
Test substance administered once
daily, by oral route, for 28 d.
Animals observed closely, each
day for signs of toxicity
Test substance administered dermally
for 28 days. Observed closely, each
day for signs of toxicity
Test substance - inhalation administered
for a defined period. Observed closely,
each day for signs of toxicity
Establish dose dependent relation-ship for the toxic effect
Allows to determination of the No-Observed Adverse Effect Level (NOAEL) and provide information on selection
of doses for long term studies

5. Selection and Preparation of Animals
OECD 407 OECD 410 ECD 412
Rats are the preferred species.
Lower species - increase variability: dissecting smaller organs.
Other species are accepted under suitable justification.
Young healthy adult animals of commonly used laboratory strains should be employed.
Females should be nulliparous and non pregnant.
The strain which has be adopted shall be in accordance to the chronic study.
Dosing should be initiated as early after the weaning period. At any case dosing should not be
delayed more than 9 weeks old.
Body weight variation should not exceed greater than 20%

6. Housing and Selection of doses
Husbandry conditions similar to Acute toxicity studies:
Temperature – 19 to 25o C, RH – 55%- 65% ; Light : dark- 12:12, Free
access to standard pellet and potable drinking water
Selection of doses: (Minimum 3 doses)
First High dose is decided
-
- This dose should induce
toxic effect but not severe
toxicity or death
- Toxicokinetic data, SAR,
Previous experience,
Efficacy study, LD10 may
help in predicting this
High DoseLow Dose Medium Dose
- Usually 2 to 4 times
reduced as compared to
the highest dose levels.
- Preferentially falls on the
human exposure dose.
- Should not produce any
observed toxic effect
- May be 10 times lower
than the medium dose
levels or higher dose
levels

7. Grouping and number of animals
25 males + 25 Females
Control
(5 M+ 5F)
Low dose
(5 M+5F)
Medium dose
(5M+5F)
High dose
(5M + 5F)
Satellite receives
high dose
(5M+5F)
5 groups
Additional animals can be kept in each group if interim sacrifices are planned. Exposure to various routes
is as per acute toxicity exposure.
Satellite groups helps to identify delayed type of toxicity and withdrawal effects. Limit dose studies can be
done at 1000 mg/kg

8. Mode of exposure of Test substance
OECD 407
Oral route
OECD 410
Dermal route
OECD 412
Inhalation route
Test substance - orally
administered for a defined period.
Observed closely, each day for
signs of toxicity
Test substance - skin administered
for a defined period. Observed
closely, each day for signs of toxicity
Test substance - inhalation administered
for a defined period. Observed closely,
each day for signs of toxicity

9. Observations
Daily observations:
Clinical observations, General health record of animals,
Mortality and morbidity
– Preferably at the same time on each day and at anticipated peak
period
Weekly observations:
- Body weight changes, Food and water intake
- Behavioral, neurological and autonomical profiles
End observations:
- Functional observations like grip strength, auditory, visual stimuli
etc.
- Vaginal smears, Blood collection, Gross necropsy and organ
collection
- Additional Blood sampling can be done to establish Toxicokinetics
and pharmacokinetics as per ICH guidelines

10. Parameters assessed
- Hematological parameters
- Clinical biochemistry
- Targeting to assess liver, kidney and heart functions
- Metabolic profiles and electrolyte balance
- Hormones and Endocrine related endpoints
- Wet weight of organs
- All the major weighable organs shall be weighed
- Expressed in terms of organ indices
- Gross necropsy and Histology of all the Organs.
- Satellite group will undergo the same procedure after 14 days of post
treatment. [28 +14 d]

11. THANK YOU