biosafty project

1. BIO-SAFETY
STUDIES
IONPs Project
Biosafety study Proposal ID 43766 1

2. ACUTE DERMAL
IRRITATION/CORROSION
[OECD 404]
Biosafety study Proposal ID 43766 2

3. Acute Dermal Irritation/Corrosion [OECD
404]
■ PRINCIPLE OF THE TEST
The test chemical to be tested is applied in a
single dose to the skin of an experimental
animal; untreated skin areas of the test animal
serve as the control.
Biosafety study Proposal ID 43766 3

4. Acute Dermal Irritation/Corrosion [OECD 404]
PREPARATIONS FOR THE TEST
Selection of
animal
species
• The albino rabbit is the preferable laboratory
animal, and healthy young adult rabbits are used
Preparation
of the
animals
• Approximately 24 hours before the test, fur should be removed
by closely clipping the dorsal area of the trunk of the animals.
Care should be taken to avoid abrading the skin, and only
animals with healthy, intact skin should be used. Some strains of
rabbit have dense patches of hair that are more prominent at
certain times of the year. Such areas of dense hair growth
should not be used as test sites.
TEST
PROCEDURE
• The test chemical should be applied to a small area
(approximately 6 cm2) of skin and covered with a gauze patch,
which is held in place with non-irritating tape. The patch should
be loosely held in contact with the skin by means of a suitable
semi-occlusive dressing for the duration of the exposure period.
If the test chemical is applied to the patch, it should be
attached to the skin in such a manner that there is good contact
and uniform distribution of the test chemical on the skin. Access
by the animal to the patch and ingestion or inhalation of the
test chemical should be prevented.
Biosafety study Proposal ID 43766 4

5. Acute Dermal Irritation/Corrosion [OECD
404]
■ Dose level
A dose of 0.5 mL of
liquid or 0.5 g of solid
or paste is applied to
the test site
■ Observation period
The duration of the observation
period should be sufficient to
evaluate fully the reversibility of
the effects observed. However,
the experiment should be
terminated at any time that the
animal shows continuing signs of
severe pain or distress. To
determine the reversibility of
effects, the animals should be
observed up to 14 days after
removal of the patches. If
reversibility is seen before 14
days, the experiment should be
terminated at that time.
 Clinical observations and
grading of skin reactions
All animals should be
examined for signs of
erythema and edema, and
the responses scored at 60
minutes, and then at 24, 48
and 72 hours after patch
removal. Dermal reactions
are graded and recorded
according to the grades in
the Table below.
Histopathological
examination should be
considered to clarify
equivocal responses.
Biosafety study Proposal ID 43766 5

6. Acute Dermal Irritation/Corrosion [OECD 404]
■ Table 1: Erythema and Eschar Formation
Observation Score
No erythema 0
Very slight erythema (barely
perceptible)
1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to
eschar formation preventing grading of
erythema
4
Biosafety study Proposal ID 43766 6

7. Acute Dermal Irritation/Corrosion [OECD 404]
■ Table 2: Edema Formation
Observation Score
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well
defined by definite raising)
2
Moderate oedema (raised
approximately 1 mm)
3
Severe oedema (raised more than 1 mm
and extending beyond area of
exposure)
4
Biosafety study Proposal ID 43766 7

8. ■ 1. Dermal irritation is the production of reversible damage of the skin
following the application of a test chemical for up to 4 hours.
■ 2. Dermal corrosion is the production of irreversible damage of the skin;
namely, visible necrosis through the epidermis and into the dermis, following
the application of a test chemical for up to four hours. Corrosive reactions are
typified by ulcers, bleeding, bloody scabs, and, by the end of observation at
14 days, by discoloration due to blanching of the skin, complete areas of
alopecia, and scars. Histopathology should be considered to evaluate
questionable lesions
Biosafety study Proposal ID 43766 8

9. BACTERIAL REVERSE
MUTATION TEST
[OECD 471]
Biosafety study Proposal ID 43766 9

10. Bacterial Reverse Mutation Test [OECD
471]
■ PRINCIPLE OF THE TEST
■ Suspensions of bacterial cells are exposed to
the test substance in the presence and in the
absence of an exogenous metabolic activation
system. In the plate incorporation method,
these suspensions are mixed with an overlay
agar and plated immediately onto minimal
medium
Biosafety study Proposal ID 43766 10

11. Bacterial Reverse Mutation Test [OECD
471]
DESCRIPTION OF THE METHOD
Medium
■ Agar (e.g.
containing Vogel-
Bonner minimal
medium E and
glucose) and an
overlay agar
containing
histidine and biotin
or tryptophan, to
allow for a few
cell divisions, is
used.
Bacteria
■ 1Fresh cultures of bacteria should be grown up to the late exponential or
early stationary phase of growth (approximately 10 cells per ml). The
recommended culture temperature is 37°C.
■ At least five strains of bacteria should be used. These should include four
strains of S. typhimurium (TA1535; TA1537 or TA97a or TA97; TA98; and
TA100) that have been shown to be reliable and reproducibly responsive
between laboratories. These four S. typhimurium strains have GC base
pairs at the primary reversion site and it is known that they may not
detect certain oxidising mutagens, cross-linking agents and hydrazines.
Such substances may be detected by E.coli WP2 strains or S. typhimurium
TA102 (19) which have an AT base pair at the primary reversion site.
Therefore the recommended combination of strains is:
1. S. typhimurium TA1535, and
2. S. typhimurium TA1537 or TA97 or TA97a, and
3. S. typhimurium T A98, and
4. S. typhimurium T A100, and
5. E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102.
Biosafety study Proposal ID 43766 11

12. Bacterial Reverse Mutation Test [OECD
471]
DESCRIPTION OF THE METHOD
Test substance/Preparation
■ IONPs should be dissolved or suspended in appropriate solvents (DMSO) or vehicles
and diluted if appropriate prior to treatment of the bacteria. Liquid test
substances may be added directly to the test systems and/or diluted prior to
treatment.
■ At least five different analysable concentrations of IONPs should be used with
approximately half log (i.e. √10) intervals between test points for an initial
experiment.
■ Concurrent strain-specific positive and negative (solvent or vehicle) controls, both
with and without metabolic activation, should be included in each assay.
■ The suitable positive controls for assays with metabolic activation:
Cyclophosphamide (monohydrate) [CAS no. 50-18-0 (CAS no. 6055-19-2)].
■ The suitable positive controls for assays without metabolic activation: Mitomycin
Biosafety study Proposal ID 43766 12

13. Bacterial Reverse Mutation Test [OECD
471]
DESCRIPTION OF THE METHOD
Test substance/Preparation
■ Negative controls, consisting of solvent alone: DMSO
■ IONPs solution is preincubated with the test strain (containing approximately
108 viable cells) and sterile buffer or the metabolic activation system (0.5 ml)
for 20 min. or more at 30°-37°C prior to mixing with the overlay agar and
pouring onto the surface of a minimal agar plate. Usually, 0.05 or 0.1 ml of
test substance/test solution, 0.1 ml of bacteria, and 0.5 ml of S9-mix or
sterile buffer, are mixed with 2.0 ml of overlay agar. Tubes should be aerated
during pre-incubation by using a shaker.
Biosafety study Proposal ID 43766 13

14. Mammalian Erythrocyte Micronucleus
Test (OECD 474)
■ Principle of the test
■ Animals are exposed to the test chemical by an appropriate route. If bone marrow is used, the
animals are humanely euthanized at an appropriate time(s) after treatment, the bone marrow is
extracted, and preparations are made and stained
■ Number and sex of animals
■ In general, the micronucleus response is similar between male and female animals and, therefore,
most studies could be performed in either sex. Group sizes at study initiation should be
established with the aim of providing a minimum of 5 analysable animals of one sex.
■ Controls
■ Negative control group animals should be included at every sampling time and otherwise handled
in the same way as the treatment groups, except for not receiving treatment with the test
chemical. If a solvent/vehicle is used in administering the test chemical, the control group should
receive this solvent/vehicle.
■ Positive control substances should reliably produce a detectable increase in micronucleus
frequency over the spontaneous level. [Cyclophosphamide (monohydrate) [CASRN 50-18-0
(CASRN 6055-19-2)]
Biosafety study Proposal ID 43766 14

15. Mammalian Erythrocyte Micronucleus
Test (OECD 474)
■ Dose levels
■ If a preliminary range-finding study is performed because there are no suitable
data already available to aid in dose selection
■ Treatment schedule
■ Preferably, 2 or more treatments are performed, administered at 24-hour
intervals, especially when integrating this test into other toxicity studies.
■ Bone marrow
■ Bone marrow cells are usually obtained from the femurs or tibias of the animals
immediately following humane euthanasia. Commonly, cells are removed,
prepared using fetal bovine serum. bone marrow cells should be immediately
stained supravitall, smear preparations are made and then stained for microscopy,
or fixed and stained appropriately. he uses of a DNA specific stain (Giemsa for
microscopic analysis)
Biosafety study Proposal ID 43766 15

16. Mammalian Erythrocyte Micronucleus
Test (OECD 474)
■ Analysis (manual)
■ All slides or samples for analysis, including those of positive and negative
controls, should be independently coded before any type of analysis and
should be randomized so the manual scorer is unaware of the treatment
condition; such coding is not necessary when using automated scoring systems
which do not rely on visual inspection and cannot be affected by operator
bias. The proportion of immature among total (immature + mature)
erythrocytes is determined for each animal by counting a total of at least 500
erythrocytes for bone marrow and 2000 erythrocytes for peripheral blood. At
least 4000 immature erythrocytes per animal should be scored for the
incidence of micronucleated immature erythrocyte.
Biosafety study Proposal ID 43766 16

17. Bacterial Reverse Mutation Test [OECD
471]
MATERIALS
Item Quantity Vendor Contact details
Animals
(Rabbits)
12 rabbits VACSERA
Disposables Panorama
chemicals
01151233001
Ames test kit 1 kit EBPI AWitham@biotox
icity.com
Tissue
processing
chemicals
(Alcohol,
Xylene, paraffin)
2 bottles each Panorama
chemicals
01151233001

18. Bacterial Reverse Mutation Test [OECD
471]
MATERIALS
CPK 1 kit Endomedix 01007642228
LDH 1 kit Endomedix 01007642228
AST 1 kit Endomedix 01007642228
ALT 1 kit Endomedix 01007642228
Creatinine 1 kit Endomedix 01007642228
MDA 1 kit Endomedix 01007642228
Urea 1 kit Endomedix 01007642228
GxP 1 kit Endomedix 01007642228

19. BUDGET
ACUTE
DERMAL
IRRITATION
STUDY
Items
Qty per test
substance
Total
Qty
Total
Price
Vendor Comment
Animals (Rabbits) 6 36 3600 VACSERA
Self
controlled
Shaving Machine - 1 500
Alcohol absolute - 3L 270
Panorama
Chemicals
Xylene - 1L 200
Parafin - 20K 200
Formalin - 1L 200
Microtom - 72
Slide
1080 Essam
15LE per
slide
H&E stain -
Histopatholist -
72
Slide
2160 Dr. Adel Baker
30 LE per
Slide
Total 8210
Biosafety study Proposal ID 43766 19

20. BUDGET
HISTOPATHOLOGICAL
ASSESSMENT
Items
Qty per test
substance
Total
Qty
Total
Price
Vendor Comment
Animals (Rabbits) 6 42 4200 VACSERA Controlled study
Alcohol absolute - 15L 810
Panorama
Chemicals
6 organs X 42
animals
Xylene - 5L 1000
Parafin - 20K 200
Formalin - 5L 1000
Microtom - 252
Slide
18144 Essam 15LE per slide
H&E stain -
Histopatholist -
252
Slide
7560 Dr. Adel Baker 30 LE per Slide
cpk kit 100
Delta Medical Lab
LDH kit 100
AST kit 100
ALT kit 100
Creatinine kit 100
MDA kit 100
Urea kit 100
Gxp kit 100
Disposables 1000
Panorama
Chemicals
Total 34714
Biosafety study Proposal ID 43766 20

21. BUDGET
MTT&
AMES
TEST
Items
Qty per test
substance
Total
Qty
Total
Price
Vendor Comment
Primary
Lymphocytes cell
line
- - - VACSERA
MTT test kit - - 600
Germany-
ebpi
Ames test kit 74575.2
Panorama
Cyclophosphamide 100
Mitomycin C 250
Total 75525.2
Biosafety study Proposal ID 43766 21

22. BUDGET
MICRONUCLEUS
TEST
Items
Qty per test
substance
Total
Qty
Total
Price Vendor Comment
Animals (Rabbits) 6 48 4800 VACSERA 2 Controls
KCL - 50
Panorama
Glacial acetic acid 100
Cyclophosphamide 100
PBS 250
Gemsa Stain 1000
Feotal Bovine
serum
1500
Total 4800
Biosafety study Proposal ID 43766 22

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24. Biosafety study Proposal ID 43766 24

25. Biosafety study Proposal ID 43766 25

26. Biosafety study Proposal ID 43766 26