A detailed knowledge about acute dermal toxicity of synthesized polysaccharide.

1. Acute dermal toxicity of synthesized
polysaccharide according to OECD guidelines
Presented by- Anand Swaroop Gupta
M . Pharm ( PG-1)
Pharmaceutics
Department of Pharmaceutical Technology
Jadavpur University
Kolkata-700032

2. Introduction
 Acute dermal toxicity testing of synthesized polysaccharides can be conducted by using the guidelines set forth by the
Organisation for Economic Co-operation and Development (OECD) Test No. 402.
 Data from an acute dermal toxicity study may serve as a basis for classification and labelling.
 It is an initial step in establishing a dosage regimen in sub-chronic studies and provide information on dermal absorption and
the mode of toxic action of a substance by this route.
 The test involves the application of the substance to the skin of laboratory animals, typically rats or rabbits, under controlled
conditions.
 The animals are closely monitored for signs of skin irritation, such as redness, swelling, and ulceration, as well as signs of
systemic toxicity .
 Based on the recommendations of several expert meetings, international agreement had been reached on harmonised LD50
cut-off values for the classification of chemicals as per Globally Harmonised System (GHS).
 A revision of TG 402 was considered timely because
• testing in one sex (usually females) is generally considered sufficient, and
• in order for a point estimate to be meaningful, there is a need to estimate confidence intervals (CI)
 A biometrical analysis was carried out to assess and compare the performance of multiple test designs for acute dermal toxicity.

3.  Synthesized polysaccharides are those that are chemically produced rather than naturally occurring polysaccharides. They are
used as thickeners, stabilizers, emulsifiers and in pharmaceutical industry as drug delivery system.
 Synthesized polysaccharides have significant potential in skin care including moisturizing, antioxidant, anti-inflammatory,
and maintain the skin's natural hydration levels and prevent damage from environmental stressors, making them an attractive
ingredient for use in a variety of skin care products.
Examples include Carboxy Methyl Cellulose, Hydroxyethyl cellulose, Xanthan gum, Pullulan, Dextran, Alginate,
Glycosaminoglycans( such as Hyaluronic acid), Chitosan.
 While synthesized polysaccharides are generally considered safe and well-tolerated, there is always a possibility that they could
cause dermal toxicity or other adverse effects in certain individuals through
 physical properties of the polysaccharide itself
 high molecular weight or poor solubility
 interaction of the polysaccharide with skin cells and tissues leading to cellular damage
 inflammatory response that exacerbate tissue damage and lead to systemic toxicity.
 The degree of branching, cross-linking, and solubility of the synthesised polysaccharide will all have an impact on its toxicity.

4. Study Design
The original Acute Dermal Toxicity Guideline TG 402 (1) was adopted in 1987. Development of a dermal Fixed Dose Procedure
(FDP) was considered appropriate, This FDP guideline will allow the use of a series of fixed doses for the determination of acute
dermal toxicity in only one sex (usually females).
1) Development of Synthesized Polysaccharides-
•Chemical modification of
polysaccharides by oxidation,
reduction, esterification,
etherification, acylation,
amidation , and grafting.
•Purification can be
done through
filtration,
chromatography,
or precipitation.
Characterization of synthesized
polysaccharide by infrared
spectroscopy, nuclear
magnetic resonance (NMR)
spectroscopy, mass
spectrometry, and X-ray
diffraction.
Selection of
polysaccharide
2) Dose Selection-
• The test substance should be applied uniformly over an area which is approximately 10 % of the total body surface
area.
• Test Substance are administered by dermal route to the group of animals in stepwise procedure using fixed dose of
50,200,1000,2000 mg/kg.
• Dose range should be selected to ensure that a dose-response relationship can be established.

5. 4) Preparation of Animals-
• The animals are acclimatised to the laboratory
conditions for at least five days prior to the start
of the study.
• Approximately 24 hours before the study, fur
should be removed from the dorsal area of the
trunk of the test animals by clipping or shaving.
• Care must be taken to avoid abrading the skin,
which could alter its permeability.
3) Animal Selection-
 Selection of species – Rats, Other species- justification required.
 Animal procurement- Healthy Adult animals should be procured from CPCSEA approved animal Breeding facility.
 Body Weight- Rat (200-300g), Guinea pig(350-450g), Rabbit(1.5-3kg)
 Age- Rats (8 to 12 weeks)
Guinea pig(5 to 6 weeks old)
Rabbits(at least 12 weeks old)
 Sex- Females (nulliparous and non-pregnant)
Males (adequate justification required) but can be used.

6. 5) Approach of the Study-
Sighting Study
Confirmatory Study for appropriate starting dose for main
study((or if a death is seen at the lowest fixed dose).
Main Study
Starting dose should be selected from fixed dose levels of
50,200,1000,2000,mg/kg as dose expected to produce
evident toxicity.
Lowest Fixed Dose (50 mg/kg) administered
Animal dies or exhibit clear signs of toxicity
Study Terminated, assign the substance to GHS Category 1
Second Animal dosed 50mg/kg
Second Animal dies
GHS category 1 will be confirmed
3 Actions Required
Either stop
testing and
assign the
appropriate
hazard
classification
category
Test at higher
fixed dose
Test at lower
fixed dose
Each dose level being studied involve a total of five animals
of one species.
5 Animals = 1 animal from the sighting study dosed at the
chosen dose level + 4 additional animals,
with the exception uncommonly—of cases where a dose level
used on the main research was not included in the sighting
study.

7. Sighting Study Main Study
• Same procedure will be followed for 200,1000 and 2000
mg/kg dose based on GHS category for acute dermal
toxicity.
• For reasons of animal welfare concern, testing of animals in
GHS Category 5 ranges (2000-5000 mg/kg) is discouraged.
As shown in Figure , the primary study is terminated when one of the
following outcomes is observed:
• Some fixed dose causes obvious toxicity with no more than one
death,
• No death or obvious toxicity is observed at some fixed dose
• Lower than that causing mortality of a single sighting study animal
or two or more animals at a main study dose,
• death occurs at the lowest fixed dose, or no effects are observed at
the highest dose.

8. CATEGORY LD50 mg/kg INDICATION ACTION ON SKIN
I LD50 ≤ 50 mg/kg -
II 50 mg/kg ≤ LD50≤ 200 mg/kg DANGER Fatal in contact with Skin
III 200 mg/kg ≤ LD50 ≤ 1000 mg/kg DANGER Toxic in contact with Skin
IV 1000 mg/kg ≤ LD50 ≤ 2000 mg/kg WARNING Harmful in contact with Skin
V 2000 mg/kg < LD50 < 5000 mg/kg WARNING Harmful in contact with the
Skin
6) Evaluation Parameters-
Observations should include:-
• changes in skin and fur,
• eyes and mucous membranes,
• respiratory, circulatory, autonomic and central nervous systems,
• somatomotor activity and behaviour pattern.
• Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
In addition, the treatment site may be observed at 24, 48 and 72 hours after removal of test chemical using the Draize criteria, as these data may be
useful for waiving the need for a separate in vivo skin irritation study.
Body weight - Individual weights of animals should be determined on the day of, or immediately prior to, the administration of the test chemical
and at least weekly thereafter
GHS CRITERIA FOR ACUTE DERMAL TOXICITY-

9. 7) Histopathology-
• All test animals (including those that die during the test or are removed from the study for animal welfare reasons) should be
subjected to gross necropsy.
• The liver, kidney, brain, and intestine were removed, and their weight were recorded, and brain/body weight ratios, liver/body
weight ratios, and kidney/body weight ratios of each animals were calculated.
Methods
I. Selection of species – Rats, Other species- justification required.
II. All the approval will be taken with Institutional Animal Ethics Committee( IAEC).
III. 20 rats selected for sighting study at each dose.
IV. A group of Rats (usually females) are acclamized minimum for 5 days and randomized manually.
V. Synthesized polysaccharides are prepared ( Solid or liquid)
VI. Approximately 24 hours before the study, fur should be removed by shaving the dorsal area of the trunk.
VII.Individual weights of animals should be determined .
VIII.Synthesized polysaccharide should be applied uniformly over an area which is approximately 10 % (about 40 𝑐𝑚2)of the total
body surface area.

10. IX. Animals are observed
• Immediately after dosing at least once during the first 30 minutes,
• Periodically during the first 24 hours, Special attention in first 2 to 6 hours after exposure period
• Daily for total 14 days
X. Outcomes
• A-Death
• B-Evident Toxicity
• C-No Toxicity
XI. Administered Synthesized polysaccharides toxicity classified under Global Harmonised System in different
Categories(1,2,3,4&5)
XII. Surviving animals are weighed and then humanely killed for histopathological analysis.

11. Sample Size
Sighting Study-
No. of Animals Dose (mg/kg) body weight
1 50mg/kg
1 200mg/kg
1 1000 mg/kg
1 2000mg/kg
Main Study-
No. of Animals Dose (mg/kg) body weight
5 50mg/kg
5 200mg/kg
5 1000 mg/kg
5 2000mg/kg

12. Implementation Strategy
General steps that could be taken:
 Identifying the specific polysaccharide being tested, the route of exposure and intended use of the polysaccharide.
 Only rat studies were selected- To reduce uncertainty associated with comparing results across species.
 Focus on studies from last decade- When data on multiple formulation types were available for particular active ingredient.
 Toxicity study should include at least three dose levels, including high, mid and low dose.
 Dose levels should be selected based on physicochemical properties of polysaccharide.
 Observations for any sign of toxicity such as changes in behavior, clinical signs and mortality.
 Determination of Median Lethal Dose (LD50), if LD50 is higher than exposure limit, the polysaccharide is considered safe.
 Interpret the result.
 Report and communicate the result.
Overall , the implementation strategy for accessing the acute dermal toxicity of synthesized polysaccharide should follow best
practices for animal testing and comply with applicable regulatory requirements.

13. All the values were expressed as mean + Standard Deviation. Comparisons were made between control and treatment groups using
SPSS 16 using one-way ANOVA. A value of P < 0.05 was considered as statistically significant.
Statistical Analysis
Ethical Issues
Since no human is used IAEC approval should be required for preclinical studies.
And all the experiment protocol will be carried out as the regulation of CPCSEA (Committee for the Purpose of
Control and Supervision of Experiments on Animals) guideline.
To obtain the ethical approval the researcher must provide-
• Detailed description of study protocol
• Including rationale for the testing
• Methods of substance administration
• Endpoints to be measured
• Statistical methods to be used for data analysis.