The document summarizes several OECD guidelines for dermal toxicity testing: - Guideline 402 describes methods for acute dermal toxicity testing using rats. Animals are exposed to test chemicals at fixed doses and observed for 14 days. - Guidelines 410, 411, and 434 describe methods for repeated dose dermal toxicity testing over 21/28 days, 90 days, and other durations to evaluate sub-chronic effects. Rats, rabbits or guinea pigs are exposed daily and observed for signs of toxicity. - Guideline 435 describes an invitro membrane barrier method for evaluating skin corrosion potential without using live animals.

1. OECD Guideline For Dermal Toxicity Testing
Presenting
M201013 Priya Jadhav
Subject Incharge
Dr. Anagha Damre Mam

2. OECD Guideline For Dermal Toxicity
Testing

3. OECD Guidelines For Dermal Toxicity
Guideline Number Name of Guideline Year adopted
402 Acute Dermal Toxicity 24 Feb 1987
410 Repeated Dose Dermal Toxicity 12 May 1981
411 Sub chronic Dermal Toxicity 12 May 1981
434 Acute Dermal Toxicity- fixed
dose procedure
14 May 2004(first version)
435 Invitro membrane barrier
method for skin corrosion
28 July 2015

4. OECD 402 24 Feb 1987
Acute Dermal Toxicity: Fixed Dose Procedure
 The original acute Dermal Toxicity Guideline TG 402 was adopted in 1987.
 Developed by The Office of Prevention, Pesticides and Toxic substances US-
EPA. Acute Dermal Toxicity- Adverse effects occurring within a short time of
dermal application of single dose or multiple doses of a substance within 24
hours period.
 A revision of TG 402 was considered timely because
 i) testing in one sex (usually females) is generally considered sufficient
 ii) in order for a point estimate to be meaningful, there is a need to estimate
confidence intervals.
 Adequately separated doses enable a test chemical to be ranked for hazard
classification purposes according to the Globally Harmonized System (GHS) for
the classification of chemicals without a point estimate of toxicity (10).

5.  OBJECTIVES: To provide the information about health hazardous on short term
dermal exposure
 PRINCIPLE: Groups of animal of a single sex are exposed to the test chemical
in a stepwise procedure using the appropriate fix doses are set out in Annex 2
the taste chemical is applied to the skin is a graduated doses to the
experimental animal one dose being used per group the classification for the
test chemical is then defined based on the outcome observed

7. DESCRIPTION OF THE METHOD
 Selection of animal species: - The adult rat is the preferred species to be
used. Females should be nulliparous and non-pregnant.
 Each animal, at the commencement of its dosing, should be a young adult (at
least 8-10 weeks old) with a size which facilitates the conduct of the test
(200-300 g) and its weight should fall within an interval of ±20 % of the mean
weight of any previously dosed animals.
 Housing and feeding conditions: - The temperature in the experimental
animal room should be 22ºC (±3ºC). Although the relative humidity should be
at least 30% and preferably not exceed 70% other than during room cleaning
the aim should be 50-60%. Lighting should be artificial, the sequence being 12
hours light, 12 hours dark. For feeding, conventional laboratory diets may be
used with an unlimited supply of drinking water.

8. Preparation of animals
 Acclimatization: 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

9. ROCEDURE
 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- pulverized Liquid - generally used undiluted.
 All animals should normally be observed for at least 14 days.

10. 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 Somatoform
activity and behavior pattern.
 Attention should be directed to observations of tremors, convulsions,
salivation, diarrhea, lethargy, sleep and coma.

11. 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
 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;

12. 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
 statistical treatment of results where appropriate.
 Discussion & interpretation of results.
 Conclusion

13. OECD 41012 May 1981
Repeated Dose Dermal Toxicity: - 21/28-day Study
 Prerequisites
 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

14. 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 sub chronic dermal toxicity may be carried out after
initial information on toxicity has been obtained by acute testing. It provides
information on possible health hazards likely to arise from repeated exposures
by the dermal route over a limited period of time.
 There is sufficient similarity between the considerations involved in the
conduct of a 21-day or 28-day repeated dose dermal study to allow one
Guideline to cover both test durations. The main difference lies in the time
over which dosing takes place

15. PRINCIPLE OF 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.

16. 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 cent12 hours light, 12 hours dark cycle.
 Feeding: conventional laboratory diets & unlimited supply of drinking water.

17.  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

18. 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.

19. Observations
 A careful clinical examination should be made at least once each day.
Additional observations should be made daily with appropriate actions taken
to minimize 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
 Hematological parameters
 Clinical biochemistry determination
 Gross necropsy Histopathology

20. DATA AND REPORTING DATA-
 Individual animal data should be provided
 Additionally, all data should be summarized 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;
 hematological tests employed and results with relevant baseline data; clinical
biochemistry tests; necropsy findings.
 Discussion & interpretation of results.
 Conclusion

21. Sub chronic Dermal Toxicity: 90-day
Study (411)
• Sub chronic 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.

22. 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.

23. 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; guineapigs, 350 to 450 g.
 Number and sex: At least 20 animals (10 female and 10male) 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 cent12 hours light, 12 hours dark cycle.
 Feeding: conventional laboratory diets & unlimited supply of drinking water.

24.  Test conditions Dose levels
 At least three dose levels, with a control and, whereas 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

25. 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.

26. Observations
 A careful clinical examination should be made at least once each day.
Additional observations should be made daily with appropriate actions taken
to minimize 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 Hematological
parameters Clinical biochemistry determination
 Gross necropsy Histopathology

27. DATA AND REPORTING
 Data
 Individual animal data should be provided. Additionally, all data should be
summarized 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 animal survived to termination ; Toxic effects and the time
of observation of each abnormal sign and its subsequent course & food and body
weight data;
 hematological tests
 Discussion & interpretation of results and conclusion.

28. Acute Dermal Toxicity – Fixed Dose Procedure 434
 This guideline was suggested by the British Toxicological Society in 1984.In
this guideline death of animal as an endpoint concept is removed &evident
toxicity is considered as an end point. Initial Considerations
 All available information on the test substance should be considered prior to
conducting the study.
 Principle: Only moderatory toxic doses are used & the dose levels that are
expected to be lethal or causing marked pain & distress should be avoided.
Moribund animals are humanely killed & are considered in the results.
 Groups of animals of single sex are given fixed doses.
 Test method :Selection of animal species
 Housing & feeding condition Preparation of animals

29.  Administration of doses :Substance applied as thin, uniform layer, Porous
gauze dressing & non-irritant tape till 24 hour exposure period. Moistening of
test substance if in Solid form, with water or suitable vehicle. Liquids used
undiluted. Removal of test substance at the end of exposure period
 Limit Test : Only used when the experimenter has the information that the
test material is non-toxic. Information can be gained from the test data of
similar compounds. If information is absent then directly main test is
performed. For the limit test the sighting study begins at 2000 mg/Kg
followed by subsequent dosing of 4 animals.

31. Observation
 For signs of toxicity animals to be observed. Immediately after dosing during
first 30 minutes. Periodically during first 24 hours. Daily thereafter for a total
of 14 days.
 Other observations - changes in fur, skin colour, eyes n mucus membranes,
respiratory system, ANS(salivation, diarrhoea etc.),CNS(tremors, convulsions
etc.)Body Weight & Pathology.

32. Data and Reporting :
Individual data for each animal in tabular form showing:
 No. used animals.
 Animals showing toxicity.
 Animals found dead during test or killed.
 Time of death of individual animals.
 Time course of Toxic effects. Necropsy findings.

33.  Test report –
 Test substances physical nature, purity, and, where relevant, physio-chemical
properties
 Vehicle (including isomerisation);− identification data, including CAS number (if
available).
 Test Animal species/strain used;
− microbiological status of the animals, when known;
− number, age and sex of animals (including, where appropriate, a rationale for
use of males instead of females)
− source, housing conditions, diet, historical data etc.
− date and time of death if prior to scheduled sacrifice.

34. Test conditions:
 details of test substance formulation, including details of the physical form
of the material administered;
 details of the administration of the test substance including dosing volumes
and time of dosing;
 details of food and water quality (including diet type/source, water source);
 the rationale for the selection of the starting dose;
 method of randomization in animal selection.

35. Results
 Tabulation of response data and dose level of each animal.
 Weight of animals.
 Time course of onset of toxicity symptoms (Reversible or not).
 Necropsy and histopathological findings
 Discussion & Interpretation of results
 Conclusions

36. Invitro membrane barrier method for skin
corrosion 435
 Skin corrosion :It refers to the production of irreversible damage to the skin
as visible necrosis through the epidermis & into the dermis, following the
application of a test material.
 Advantages of this method.In vitro test that utilizes an artificial membrane,
so no living animal is required & this reducing & refining the use of animal
testing.It is an alternative for the standard in vivo rabbit skin procedure. and
also avoiding the pain, distress that might occurs if animals are used.
 Disadvantages of this method Validation studies are carried out to check
reliability of test results. Many non corrosive chemicals & their mixtures &
some corrosive chemicals & their mixtures may not qualify. Aqueous
substances with a pH range of 4.5 to 8.5 often do not qualify.

37. Initial considerations :- Identification and GHS classification of corrosive
agents
Corrosive Category
(category 1)
(applies to authorities
not using subcategories)
Potential Corrosive
Subcategories
(only applies to some
authorities)
Corrosive in >1 of 3 animals
Exposure Observation
Corrosive Corrosive Subcategory 1A
Corrosive Subcategory 1B
Corrosive Subcategory 1C
Less than 3 min NMT 1 hr
3 min to 1 hr NMT 14 days
1 to 4 hrs NMT 14 days

38. Principle:-
The entire test system is composed of two main components:
a) Macromolecular bio-barrier.
b) Chemical detection system (CDS)
Membrane barrier damage after the application of corrosive substances
detected.
Penetration of membrane barrier is measured by a no. of procedures, including a
change in the colour of a pH indicator dye.
The membrane barrier should be determined valid

39. Procedure:
 The following is a generic description of the components and procedures of an
artificial membrane barrier test method for corrosivity assessment. The
membrane barrier and the compatibility/indicator and categorization
solutions can be constructed, prepared or obtained commercially, e.g.,
Corrositex®.A sample test method protocol for the validated reference test
method can be obtained at. Testing should be performed at ambient
temperature (17-25°C) and the components should comply with the following.
1) Test substance compatibility test.
Whether the compound is detectable by CDS or not. The CDS & the exposure
conditions used in this test should reflect the exposure in MBT.
2) Test substance categorization test
It is a type of screening test to distinguish weak and strong acids/bases. Two
different time scales used for GHS classification

40.  MB test method components: MB consists of 2 components:
A)Proteinaceous macromolecular aqueous (PMA)gel. Composed of protein e.g.
keratin, collagen or protein mixtures. Acts as target for the test substance. It
should be of equal thickness & without air bubbles as it could affect its
functional integrity.
B) Permeable supporting membrane (SM).It provides mechanical support.
 The CDS
It is an indicator solution used to detect the passage of test substance through
the barrier. Various pH indicator dyes & their combinations e.g. cresol red,
methyl orange are used. The measurement system can be Electronic or visual.

41. Test performance
 Assembly of the test method components:
 MB positioned in vial or tube containing indicator solution.
 Application of Test Chemical:
1) 500 μL of liquid or 500 mg solids (finely powdered).
2) Applied to upper surface of MB.
3) Replicates for each TS & its corresponding controls.
4) Time of application is noted.

42. Measurement of barrier penetration:
 Monitoring each vial.
 Time of first change in indicator solution "Barrier Penetration "is noted.
 Time elapsed b/w Application and Penetration is determined.
Study acceptability criteria :
 Time elapsed is used to predict corrosivity of the test substance.
 For study acceptability
a)+ve control = expected penetration response time.
b)-ve control = neither be corrosive & not should alter the corrosive potential of
test substance.
c)12 proficiency chemicals = demonstrates the technical proficiency/skill.
Interpretation of results & corrosivity classification of TS.
Time in minutes is used.

44. Data And Reporting
 All data should be reported in a tabular form Means & standard deviations for
each trial is also recorded.
 Test report: Test & control substances. Test condition.
 Results
1) Tabulation of data from individual test samples.
2) Description of:
a) Effects observed.
b) Evaluation and classification criteria.
 Discussion of results
 Conclusions

45. THANK YOU….