This presentation explains OECD Test Guidelines 204, which describes the dermal toxicity test used to assess the effects of chemical on laboratory animals. The ppt covers the objective of the test, principle, procedure, observation and interpretation of results. OECD TG 204 is widely used in environmental risk, assessment, toxicology studies and regulatory safety evaluation of chemicals, pesticides and pharmaceuticals. This ppt is useful for pharmacy, life science, toxicology and environmental science students as well as for exam preparation and academic reference.

1. OECD GUIDELINES
402 : ACUTE DERMAL TOXICITY (FIXED DOSE
PROCEDURE)
SUBMITTED BY – MANJARI SRIVASTAVA SUBMITTED TO: MS.NEELAM PAINULY
COURSE – M.Pharm (Pharmacology) DESIGNATED: ASSOCIATE PROFESSOR
ERP ID: 25MPHARCO0009

2. INTRODUCTION
• OECD Guidelines for the Testing of Chemicals are regularly updated to reflect scientific progress and
improve animal welfare.
• Acute dermal toxicity (TG 402) was first adopted in 1987 to assess harmful effects of chemicals after
single skin exposure.
• Recent studies on many chemicals showed that oral toxicity data are equal to or more severe than
dermal toxicity data in over 98% of cases.
• This raised doubts about the routine need for acute dermal toxicity testing, as it often adds little
regulatory value.
• OECD Guidance Document 237 provides criteria for waiving dermal toxicity tests, helping to reduce
unnecessary animal use.
• Dermal toxicity testing is now recommended only in exceptional cases with strong scientific
justification.
• International experts agreed on harmonized LD cut-off values under the Globally Harmonized
₅₀
System (GHS).

3. • The revised TG 402 supports testing in one sex (usually females) and improved study designs to reduce
animal numbers.
• Biometrical evaluation of alternative test methods helped select more reliable and humane testing
approaches.
• The updated TG 402 balances human health protection, regulatory needs, and ethical animal testing
practices.
 Alternative approaches such as Fixed Dose Procedure, Acute Toxic Class, and Up-and-Down methods
were evaluated.
 The revised guideline improves regulatory decision-making while maintaining scientific accuracy.
 Results of dermal toxicity studies may still be important for environmental and ecological risk
assessments.

4. GENERAL CONSIDERATION
 In vivo acute dermal toxicity testing should be done only after reviewing all existing information.
 A weight-of-the-evidence approach is used to avoid unnecessary animal testing.
 Information on the chemical identity, structure, and physico-chemical properties must be evaluated
first.
 Data from other in vitro and in vivo toxicity studies should be considered before dermal testing.
 Available QSAR data and data from structurally similar chemicals should be reviewed.
 The intended use of the chemical and the probability of human skin exposure must be assessed.
 The regulatory purpose of generating dermal toxicity data should be clearly justified.
 This information helps decide whether the test is needed and to select an appropriate starting dose.
 Only doses expected to cause moderate toxicity should be used; lethal doses should be avoided.
 Opportunities to waive the dermal study should be checked using OECD Guidance Document 237.
 Acute dermal toxicity data are mainly used for hazard classification and labelling.
 For mixtures and formulated products, dermal toxicity is usually not greater than oral toxicity, so
testing can often be waived.

5. PRINCIPLES OF TEST
•Groups of animals of one sex only (usually females) are used for the test.
•Animals are exposed to the test chemical using a stepwise fixed-dose procedure.
•Fixed dose levels are selected as given in Annex 2 of the guideline.
•A range-finding study may be done first if there is little or no existing information.
•The starting dose is chosen to produce clear signs of toxicity but not severe effects or death.
•Additional groups may receive higher or lower doses, depending on observed toxicity or deaths.
•Testing continues until a dose causing toxicity or no more than one death is identified.
•The study may also stop if no effects are seen at the highest dose or deaths occur at the lowest dose.
•The test chemical is applied to the skin of animals, with one dose per group.
•Animals are carefully observed for toxic effects and mortality during the study.
•Animals that die during the test are examined (necropsy).
•At the end of the study, surviving animals are humanely sacrificed and necropsied, and the chemical is
classified based on the results.

6. DESCRIPTION OF THE METHOD
Selection of the animal species
 Adult rats are the preferred species for acute dermal toxicity studies.
 Females are normally recommended, as sex differences are minimal and females may be slightly
more sensitive.
 Males may be used only with scientific justification if related compounds show higher male
sensitivity.
 Animals should be healthy young adults (8–10 weeks old) from commonly used laboratory strains.
 Females must be nulliparous and non-pregnant, with body weight 200–300 g (±20% of mean).
 Animals must have healthy, intact skin suitable for dermal exposure.

7. Housing and Feeding Conditions
 The temperature in experimental animal room should be 22֯C (±3֯C).
 Lighting should be artificial (12 hr light & 12 hr dark ).
 For feeding, convectional laboratory diet should be given with unlimited supply of drinking water.
Preparation of animals
 Animals should be acclimatised to laboratory conditions for at least 5 days before the study begins.
 Animals are randomly selected and individually marked for identification.
 One day before dosing, fur is removed from the dorsal/flank area, covering at least 10% of total body
surface area.
 Fur removal is done by close clipping; anaesthetics may be used to reduce stress during handling.

8. PROCEDURE
Administration of doses
 The test chemical is applied uniformly over the dorsal/flank skin, covering at least 10% of total
body surface area; a smaller area may be used for highly toxic substances.
 The application site is covered with a porous gauze dressing and non-irritating tape and kept in
contact with the skin for a 24-hour exposure period.
 The test site is further secured to prevent ingestion of the test chemical; restraint may be used if
necessary, but without immobilising the animal.
 During the exposure period, animals may be housed individually to prevent oral ingestion by cage
mates.
 Solid test chemicals are moistened (preferably with water or a suitable vehicle) to ensure skin
contact; liquids are generally applied undiluted, and vehicle volume (usually 0.5–1 mL) is recorded.
 After 24 hours, residual test chemical is removed using water or an appropriate solvent, and animals
are returned to group housing as soon as practicable to minimize stress.

9. Number of animals and doses level
 The test chemical is administered sequentially to single animals, with two animals per selected
dose level in the main study.
 Acute dermal toxicity is often unknown or high when a study is required (e.g. LD < 200 mg/kg
₅₀
body weight).
 If toxicity information is absent or insufficient, a dose-range finding study using one animal at
200 mg/kg body weight is recommended.
 If prior information exists but waiver is not applicable, alternative starting doses (e.g. 50, 1000,
or 2000 mg/kg body weight) may be selected.
 A minimum 48-hour interval is allowed between dosing of animals; all animals are observed for
at least 14 days, with progression only after survival is confirmed.

10. OBSERVATIONS
• Animals are observed immediately after dosing, during the first 30 minutes, periodically in the first
24 hours (especially 2–6 hours), and daily for at least 14 days or longer if required.
• The onset, duration, and disappearance of clinical signs are recorded individually, and moribund or
severely distressed animals are humanely euthanised with the time of death documented.
• Observations include changes in skin, fur, eyes, mucous membranes, and effects on the respiratory,
circulatory, nervous systems, behaviour, and signs such as tremors, convulsions, salivation, diarrhoea,
lethargy, or coma.
•Body weights are recorded on or before dosing, weekly thereafter, and at study termination before
humane euthanasia of survivors.
• All animals (including those that die or are euthanised) undergo gross necropsy, with gross lesions
recorded and microscopic examination of affected organs or treatment sites considered.

11. DATAAND REPORTING
•Individual animal data must be recorded and reported for all test animals.
•All results should be summarized in tabular form for each test group.
•Tables should include: number of animals used, animals showing toxicity, animals dead or
euthanised, time of death, nature and time course of toxic effects, reversibility, and necropsy
findings.
•Based on the range-finding study, the main study follows one of three actions: repeat the same dose,
test a higher dose, or test a lower dose.
•Doses causing death in range-finding studies are not repeated, and results from the main study (2
animals per dose) confirm the final classification with no further testing required.

12. REFERENCE
 OECD (2017), Test No. 402: Acute Dermal Toxicity, OECD Guidelines for the Testing
of Chemicals, Section 4, OECD Publishing, Paris,
https://doi.org/10.1787/9789264070585-en.

13. THANK YOU