This presentation deals with the OECD regulatory giudelines foracute,subacte, sub chronic dermal toxicity studies.

1. OECD GUIDELINES FOR TESTING OF
CHEMICALS
ACUTE, SUB ACUTE AND CHRONIC
DERMAL TOXICITY STUDIES
(402, 410, 411)
SUBMITTED BY
GUMMADI HELASRI
(I M.PHARMACY)
Dept. of pharmacology

2. OECD GUIDELINE FORTHETESTING OFCHEMICALS
Acute DermalToxicity: Fixed Dose Procedure (402)
INTRODUCTION
Acute dermal toxicity is the adverse effect caused by a substance
following a single uninterrupted exposure by dermal application over a
short period of time (24 h or less).
• Scientific progress and animal welfare considerations.
• Original acute Dermal Toxicity Guideline TG 402 -1987.
• Harmonised LD50 cut-off values- classification of chemicals.
• A revision of TG 402 was considered timely because
i) testing in one sex (usually females)
ii) in order to estimate confidence intervals (CI)

3. PRINCIPLE OF THE IN VIVO TEST
• Groups of animals, of a single sex, are exposed to the test
chemical in a stepwise procedure using the appropriate
fixed doses as set out in Annex 2.
• The test chemical is applied to the skin in graduated
doses to experimental animals, one dose being used per
group.
• The classification for the test chemical is then defined
based on the outcome observed.

4. DESCRIPTION OF THE METHOD
• Selection of animal species : adult rats(female)
Females should be nulliparous and non-pregnant.
• Weight: (200-300 g)
• Animals with healthy, intact skin are required
• Housing and feeding conditions
• Temperature: 22ºC (±3ºC).
• Relative humidity: 30% 70%
• 12 hours light, 12 hours dark cycle.
• Feeding :conventional laboratory diets &unlimited supply of
drinking water.

5. Preparation of animals
• Acclimatisation: 5days
• On the day before administration of the test chemical, all fur
should be removed from the dorsal/flank area of the test
animals (i.e. at least 10% of the total body surface area) by
closely clipping
Hip bone Scapula
Flank area
(One side)
Dorsal region

6. PROCEDURE
• Administration of doses
• The test chemical should be applied as uniformly as possible
over the exposed area of dorsal/flank skin
• Test chemicals should be held in contact with the skin with a
porous gauze dressing and non irritating tape throughout a
24-hour exposure period.
• Solids- pulverised
• Liquid - generally used undiluted.
• All animals should normally be observed for at least 14 days.

7. Number of animals and dose levels

9. OBSERVATIONS
• At least once during the first 30 minutes, periodically during
the first 24 hours, with special attention given during the
first 2 to 6 hours after the beginning of the exposure period,
and daily thereafter, for a total of 14 days.
• All observations are systematically recorded, with
individual records being maintained for each animal.
• Observations should include changes in
 skin and fur,
 eyes and mucous membranes,
 respiratory, circulatory, ANS & CNS, and
 Somatomotor activity and behaviour pattern.
• Attention should be directed to observations of tremors,
convulsions, salivation, diarrhoea, lethargy, sleep and coma.

10. • Body weight
• Individual weights of animals should be determined before
and after the study.
• Pathology
• All gross pathological changes should be recorded for each
animal.
• Microscopic examination of organs showing evidence of
gross pathology.
DATA AND REPORTING
DATA
• Individual animal data should be provided
• Additionally, all data should be summarised in tabular forms.
TEST REPORT
• The test report must include the following information:
• species/strain used;
• toxic response data by sex and dose;

11. • time of death during the study or whether animals survived to
termination;
• Toxic effects and the time of observation of each abnormal
sign and its subsequent course;
• food and body weight data;
• haematological tests employed and results with relevant
baseline data;
• clinical biochemistry tests employed
• necropsy findings;
• a detailed description of all histopathological findings; and
• statistical treatment of results where appropriate.
• Discussion & interpretation of results
• Conclusion

12. Repeated Dose DermalToxicity: 21/28-day Study
Sub acute dermal toxicity studies (410)
• Adopted: 12 May 1981
• Introductory parameters
• Solid or liquid test substance
• Chemical identification of test substance
• Purity (impurities) of test substance
• Solubility characteristics
• pH (where appropriate)
• Stability, including stability in vehicle when so applied –
Melting point/boiling point.

13. • METHOD
Introduction, Purpose, Scope, Relevance,Application and Limits of Test
• In the assessment and evaluation of the toxic characteristics of a
chemical the determination of subchronic dermal toxicity may
be carried out after initial information on toxicity has been
obtained by acute testing.
• There is sufficient similarity between the considerations
involved in the conduct of a 21day or 28-day repeated dose
dermal study to allow one Guideline to cover both test
durations.

14. • Principle of the test method
• The test substance is applied daily to the skin in graduated
doses to several groups of experimental animals, one dose per
group, for a period of 21/28 days.
• During the period of application the animals are observed daily
to detect signs of toxicity.
• Animals which die during the test are necropsied, and at the
conclusion of the test the surviving animals are sacrificed and
necropsied.

15. DESCRIPTION OF THE TEST PROCEDURE
Experimental animals
• Selection of species adult rat, rabbit or guinea pig may be
• Weights: rats, 200 to 300 g; rabbits, 2.0 to 3.0 kg; guinea pigs,
350 to 450 g.
• Number and sex: At least 10 animals (5 female and 5 male)
with healthy skin should be used at each dose level.
Housing and feeding conditions
• Animals should be caged individually.
• Temperature: 22°C (± 3°) for rodents or 20°C (± 3°) for
rabbits
• Relative humidity: 30-70 per cent
• 12 hours light, 12 hours dark cycle.
• Feeding: conventional laboratory diets & unlimited supply of
drinking water.

16. Test conditions
• Dose levels
• At least three dose levels, with a control and, where
appropriate, a vehicle control, should be used.
• Except for treatment with the test substances, animals in the
control group should be handled in an identical manner to
the test group subjects.
• The highest dose level should result in toxic effects but not
produce an incidence of fatalities which would prevent a
meaningful evaluation.
• The lowest dose level should not produce any evidence of
toxicity.
• If application of the test substance produces severe skin
irritation, the concentration may be reduced

17. • Procedure
• The animals are treated with the test substance, ideally for at
least 6 hours per day on a 7-day per week basis, for a period
of 21/28 days.
• application on a 5-day per week basis is considered to be
acceptable.
• Animals in a satellite group scheduled for follow-up
observations should be kept for a further 14 days without
treatment to detect recovery from, or persistence of, toxic
effects.

18. Observations
• A careful clinical examination should be made at least once
each day. Additional observations should be made daily with
appropriate actions taken to minimise loss of animals to the
study, e.g. necropsy or refrigeration of those animals found
dead and isolation or sacrifice of weak or moribund animals
Clinical observations
• Haematological parameters,
• Clinical biochemistry determination
• Gross necropsy
• Histopathology

19. DATA AND REPORTING
DATA
• Individual animal data should be provided
• Additionally, all data should be summarised in tabular forms.
TEST REPORT
• The test report must include the following information:
• species/strain used & toxic response data by sex and dose;
• time of death during the study or whether animals survived to
termination;
• Toxic effects and the time of observation of each abnormal
sign and its subsequent course & food and body weight data;
• haematological tests employed and results with relevant
baseline data; clinical biochemistry tests ;necropsy findings.
• Discussion & interpretation of results
• Conclusion

20. Subchronic DermalToxicity: 90-day Study (411)
• Subchronic dermal toxicity is the adverse effects occurring as
a result of the repeated daily dermal application of a chemical
to experimental animals for part (not exceeding 10 per cent) of
a life span.
• Adopted: 12 May 1981
• Introductory parameters
• Solid or liquid test substance
• Chemical identification of test substance
• Purity (impurities) of test substance
• Solubility characteristics
• pH (where appropriate)
• Stability, including stability in vehicle when so applied –
Melting point/boiling point.

21. PRINCIPLE OF THE TEST
• The test substance is applied daily to the skin in graduated
doses to several groups of experimental animals, one dose per
group, for a period of 90 days.
• During the period of application the animals are observed
daily to detect signs of toxicity.
• Animals which die during the test are necropsied, and at the
conclusion of the test the surviving animals are sacrificed and
necropsied.

22. DESCRIPTION OF THE TEST PROCEDURE
Experimental animals
• Selection of species adult rat, rabbit or guinea pig may be
• Weights: rats, 200 to 300 g; rabbits, 2.0 to 3.0 kg; guinea
pigs, 350 to 450 g.
• Number and sex: At least 20 animals (10 female and 10
male) with healthy skin should be used at each dose level.
Housing and feeding conditions
• Animals should be caged individually.
• Temperature: 22°C (± 3°) for rodents or 20°C (± 3°) for
rabbits
• Relative humidity: 30-70 per cent
• 12 hours light, 12 hours dark cycle.
• Feeding: conventional laboratory diets & unlimited
supply of drinking water.

23. Test conditions
• Dose levels
• At least three dose levels, with a control and, where
appropriate, a vehicle control, should be used.
• Except for treatment with the test substances, animals in the
control group should be handled in an identical manner to
the test group subjects.
• The highest dose level should result in toxic effects but not
produce an incidence of fatalities which would prevent a
meaningful evaluation.
• The lowest dose level should not produce any evidence of
toxicity.
• If application of the test substance produces severe skin
irritation, the concentration may be reduced

24. PROCEDURE
• The animals are treated with the test substance, ideally for at
least 6 hours per day on a 7-day per week basis, for a period
of 90 days.
• However, based primarily on practical considerations,
application on a 5-day per week basis is considered to be
acceptable.
• Animals in a satellite group scheduled for follow-up
observations should be kept for at least a further 28 days
without treatment to detect recovery from, or persistence of,
toxic effects.

25. • Observations
• A careful clinical examination should be made at least
once each day. Additional observations should be made
daily with appropriate actions taken to minimise loss of
animals to the study, e.g. necropsy or refrigeration of
those animals found dead and isolation or sacrifice of
weak or moribund animals
Clinical observations
• Ophthalmological examination
• Haematological parameters
• Clinical biochemistry determination
• Gross necropsy
• Histopathology

26. DATA AND REPORTING
Data
• Individual animal data should be provided
• Additionally, all data should be summarised in tabular
forms.
Test report
• The test report must include the following information:
• species/strain used & toxic response data by sex and
dose;
• time of death during the study or whether animals
survived to termination;
• Toxic effects and the time of observation of each
abnormal sign and its subsequent course & food and body
weight data;
• haematological tests
• Discussion & interpretation of results and conclusion.

27. STUDY DAYS (to be
conduct)
ANIMALS
REQUIRED
ACUTE TOXICITY 14 1-2
SUB ACUTE TOXICITY 28 10
CHRONIC TOXICITY 90 20

28. REFERENCES
• OECD Testing of Chemicals Actue , Sub Acute, Sub Chronic
Dermal Toxicity Studies TG -402, 410, 411.
• Gobal Harmonised System(GHS) Guidelines.
• Evaluation of Acute And Sub-acute Dermal Toxicity Studies
of Ethanolic Leaf Extract of Lawsonia Inermis In Rats , Ali
Khairullah Zahi etal.,volume6 ,number1, journalofadvances
inbiotechnology,2017.
• www.google.com for images.