Document JJKDqMqOpvR3OVzJK4zdmwEGr
AR226-3139
CONFIDENTIAL
SPONSOR
ElfAtochemS.A. Cours Michelet La Defense 10
92091 Paris-la-Defense CEDEX
France
IFM recherche
SNC W LWrAt DC S iSO 000 (
TEST SUBSTANCE 6
STUDY TITLE ACUTE ORAL TOXICITY
IN RATS
STUDY DIRECTOR Xavier Manciaux
STUDY COMPLETION DATE
13 December 1999
PERFORMING LABORATORY
err
Centre International de Toxicologie BP 563 - 27005 Evreux - France
LABORATORY STUDY NUMBER 18745 TAR
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CENTRE INTERNATIONAL DE TOXICOLOGIE
B. P. SS3 2700S Evreux Cedex France
V^J. A-f ULUUT I. ^V> A U f "T/1
CONTENTS
STATEMENT OF THE STUDY DIRECTOR
4
OTHER SCIENTISTS INVOLVED IN THIS STUDY
4
STATEMENT OF QUALITY ASSURANCE UNIT
5
SUMMARY
6
RESUME
7
1. INTRODUCTION
8
2. MATERIALS AND METHODS
2.1 TEST SUBSTANCE
2.1.1 Identification 2.1.2 Formulation procedure
2.2 TEST SYSTEM
9
2.2.1 Animals
9
2.2.2 Environmental conditions
9
2.2.3 Food and water
9
2.3 TREATMENT
10
2.3.1 Fasting of the animals
10
2.3.2 Administration of the test substance
10
2.3.3 Chronology of the study
10
2.4 CLINICAL EXAMINATIONS
10
2.4.1 Clinical signs and mortality
10
2.4.2 Body weight
10
2.5 NECROPSY
11
2.6 DATA EVALUATION
II
2.7 PROTOCOL ADHERENCE
11
2.8 ARCHIVING
3.RESULTS
12
3.1 CLINICAL EXAMINATIONS
12
3.1.1 Clinical signs and mortality (table 1)
12
3.1.2 Body weight (figures 1 and 2, tables 2 and 3)
12
3.2 PATHOLOGY (table 4)
12
4. CONCLUSION
12
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Figure 1: Body weight of treated rats
13
Figure 2: Body weight ofCIT historical control rats
14
Table 1: Individual clinical signs and mortality
15
Table 2: Individual and mean body weight and weekly body weight change (g)
16
Table 3: Mean body weight and weekly body weight change ofCIT historical control rats
17
Table 4: Individual macroscopic examinations at necropsy
18
APPENDICES 1. Test article description and analytical certificate 2. Diet formula
19
20 23 and 24
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STATEMENT OF THE STUDY DIRECTOR
The study was performed in compliance with the principles of Good Laboratory Practice as
described in:
. OECD Principles on Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98) 17.
. Decret N 90-206 du 7 mars 1990 concemant les Bonnes Pratiques de Laboratoire (Journal
Officiel du 9 mars 1990), Ministere de 1'Industrie et de 1'Amenagement du Territoire. . Council Directive 87/18/EEC of 18 December 1986 on the harmonization of laws,
regulations or administrative provisions relating to the application of the Principles of Good Laboratory Practice and the verification of their applications for tests on chemical substances (OJ No. L 15 of 17.1.87).
I declare that this report constitutes a true and faithful record of the procedures undertaken and the results obtained during the performance of the study.
This study was performed at CIT, Centre International de Toxicologie, BP 563, 27005 Evreux,
France.
. Toxicology
/M^
X. Manciaux
Study Director
Doctor of Pharmacy
Date: 13 December 1999
OTHER SCIENTISTS INVOLVED IN THIS STUDY
For Pharmacy:
P.O. Guillaumat Doctor of Pharmacy
For Toxicology: C. Pelcot Study Supervisor
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STATEMENT OF QUALITY ASSURANCE UNIT
Type of inspections
Protocol Report
Inspections
4 June 1999 10 November 1999
Dates
Reported to Study
Director (*)
4 June 1999 9 December 1999
Reported to
Management (*)
4 June 1999 10 December 1999
m addition to the above-mentioned inspections, at about the same time as the study described in the present report, "process-based" and routine facility inspections of critical procedures relevant to this study type were also made by the Quality Assurance Unit. The findings of these inspections were reported to the Study Director and to CTT Management.
The inspections were performed in compliance with CIT Quality Assurance Unit procedures and the Good Laboratory Practice.
The reported methods and procedures were found to describe those used and the results to constitute an accurate and complete reflection of the study raw data.
C>^c^^^_.
L. Valette-TaIbi
Date: 13 December 1999
Doctor of Biochemistry
Head of Quality Assurance Unit
and Scientific Archives
(*) The dates indicated correspond to the dates of signature of audit reports by Study Director
and Management.
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SUMMARY
At the recmestof Elf Atochem S.A^_Paris-la.Defense. France, the acute oral toxicity of the test
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evaluated in rats according to OECD
(No. 401.24thFebruaryl987) and EC (92/69/EEC,"B.l, 31st July 1992) guidelines.
The study was conducted in compliance with the principles of Good Laboratory Practice
Regulations.
Methods
The test substance was administered by oral route (gavage) to one group of ten fasted Sprague-Dawley rats (five males and five females).
The test substance was administered undiluted at the dose of 2000mg/kg, taking into consideration that its specific gravity was 1.05 g/ml. Clinical signs, mortality and body weight gain were checked for a period of up to 14 days following the single administration of the test substance. All animals were subjected to necropsy.
Results
No deaths occurred at 2000 nig/kg.
The general behaviour and body weight gain of the animals were not affected by treatment with the test substance.
No apparent abnormalities were observed at necropsy in all animals.
Conclusion
Under our experimoital conditions, the oral LDo of the test substance)
^^^^^H^^^^BHS equal to or higher than 2000 mg/kg in rats. No signs of toxicity were
observed at this dose?
According to the classification criteria laid down in Commission Directive 93/2 I/EEC (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/EEC, the test substance does not present a significant acute toxic risk if swallowed.
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RESUME
B p r e s A la demande de Elf Atochem S.A., Paris-la-Defense, France, la toxicite aigue du produit
(92/69/EEC, B. 1,31 juillet 1992).
administration unique par voie orale chez Ie Rat a ete ictrices de 1'OCDE (n 401, 24 fevrier 1987) etde la CEE
L'etude a etc realisee confonnement aux regles de Bonnes Pratiques de Laboratoire.
Methode
Le produit a etc administre par voie orale (gavage) a un groupe de 10 rats Sprague-Dawley (5 males et 5 remelles) mis a la diete hydrique.
L'administration a ete effectuee avec le produit non dilue a la dose de 2000 mg/kg, en tenant compte de sa densite (d - 1,05 g/ml).
Les signes cliniques, la mortalite et revolution ponderale des animaux ont ete suivis pendant une periode de 14 jours apres 1'administration unique du produit.
Un examen anatomopathologique a etc effectue sur tous les animaux.
Resultats La mortalite est nulle a la dose de 2000 mg/kg. Aucun signe clinique n'est observe pendant 1'etude. devolution ponderale des animaux n'est pas influencee par le traitement. L'autopsie des animaux ne met en evidence aucune anomalie apparente.
Conclusion
Dans no_ conditions experimentales, la DL 0 orale du produit j
IH^^^^^Hest superieure ou egale a 2000 mg/kg chez le Rat. Aucun sighe de toxicite n'est
"observe a cette dose.
Selon les criteres de classification decrits dans la Directive 93/21/CEE (27 avril 1993) portant dix-huitieme adaptation au progres technique de la Directive 67/548/CEE, le produit ne presente pas de risque toxique aigu significatif en cas d'ingestion.
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1. INTRODUCTION
The objective of this study was to evaluate the toxicity of the test substance
following a single oral administration in rats.
In the assessment of the toxic characteristics of a test substance, determination of acute oral toxicity is an initial step. It provides information on health hazards likely to arise following a short-term exposure by the oral route in humans.
The study was conducted in compliance with: . OECD guideline No. 401, 24th February 1987, . EC Directive No. 92/69/EEC, B. 1, 31st July'1992.
2. MATERIALS AND METHODS 2.1 TEST SUBSTANCE
2.1.1 Identification
_n
The test substance|^^|BIBU^ised in the study was supplied by ElfAtochem S.A.
The test'substance was identified as follows:
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. ElfAtochemfilin^iumbe^BBBIIIJ . descriptionjiBBHUj^
. container: oneplasucuasK
. date of receipt: 5 May 1999
. storage conditions: at room temperature and protected from light . expiry date: May 2000.
Data relating to the characterization of the test substance are documented in a test article description and in an analytical certificate (presented in appendix 1) provided by the Sponsor.
2.1.2 Formulation procedure The test substance was used undiluted.
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2.2 TEST SYSTEM
2.2.1 Animals Species, strain: rat, Sprague-Dawley ICO: OFA-SD (IOPS Caw). Reason for this choice: rodent species generally accepted by regulatory authorities for this type
of study. Breeder Hfa Credo, 69210 L'Arbresle, France. Number and sex: one group of ten animals (five males and five females). Age/weight: on the day of treatment, the animals were approximately 6 weeks old, and had a mean body weight standard deviation of 177 4 g for the males and 145 7 g for the females. Acclimatization: at least 5 days before the beginning of the study. Identification of the animals: the animals were identified individually by earmarks or eamotches.
2.2.2 Environmental conditions During the acclimatization period and throughout the study, the conditions in the animal room were set as follows:
.temperature: 212C . relative humidity: 30 to 70% . light/dark cycle: 12h/12h
. ventilation: approximately 12 cycles/hour of filtered, non-recycled air. The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals. The animals were housed in polycarbonate cages (48 cm x 27 cm x 20 cm). Each cage contained one to seven animals of the same sex during the acclimatization period and five rats of the same sex during the treatment period. Each cage contained dust-free sawdust (SICSA, 94142 Alfortville, France). Bacteriological and chemical analyses of the sawdust, including the detection of possible contaminants (pesticides, heavy metals), are performed regularly by external laboratories. The results of these analyses are archived at CIT.
2.2.3 Food and water
All the animals had free access to A04C pelleted diet (UAR, 91360 Villemoisson-sur-Orge,
France), except as noted in "2.3.1 Fasting of the animals". Each batch of food was analysed by the supplier for'composition and contaminant levels. The diet formula is presented in appendix 2.
Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum. Bacteriological and chemical analyses of the water and diet, including the detection of possible contaminants (pesticides, heavy metals and nitrosamines), are performed regularly by external
laboratories.
The results of these analyses are archived at CIT.
No contaminants were known to have been present in the diet, drinking water or bedding material at levels which may be expected to have interfered with or prejudiced the outcome of
the study.
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23 TREATMENT
2.3.1 Fasting of the animals The animals were fasted for an overnight period of approximately 18 hours before dosing, but had free access to water. Food was given back approximately 4 hours after administration of the test substance.
2.3.2 Administration of the test substance As the test substance was anticipated to be non-toxic at 2000 mg/kg, a limit test was performed by administering 2000 mg/kg of the test substance to one group of ten animals (five males and five females).
The test substance was administered undiluted, taking into consideration its specific gravity (1.05g/ml).
The administration was performed in a single dose by oral route using a metal gavage tube fitted to a 1 ml plastic syringe (0.01 ml graduations).
The volume administered to each animal was adjusted according to body weight determined on the day of treatment.
2.3.3 Chronology of the study The single administration was performed on 13 July 1999 in the morning (day 1) and was followed by a 14-day observation period until 27 July 1999 (day 15).
2.4 CLINICAL EXAMINATIONS
2.4.1 Clinical signs and mortality The animals were observed frequently during the hours following administration of the test substance, for detection of possible treatment-related clinical signs. Thereafter, observation of the animals was made at least once a day until day 15. Type, time of onset and duration of clinical signs were recorded for each animal individually.
Time of death was recorded individually, in terms of the number of hours or days after dosing.
2.4.2 Body weight The animals were weighed individually just before administration of the test substance on day 1 and then on days 8 and 15.
The body weight gain of the treated animals was compared to that of CIT control animals with the same initial body weight.
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2.5 NECROPSY
On day 15, all animals were killed by carbon dioxide asphyxiation and a macroscopic examination was performed.
After opening the thoracic and abdominal cavities, a macroscopic examination of the main organs (digestive tract, heart, kidneys, liver, lungs, pancreas, spleen and any other organs with obvious abnormalities) was performed. In case of macroscopic lesions, organ samples were taken and preserved in 10% buffered formalin.
No microscopic examination was performed.
2.6 DATA EVALUATION
Evaluation of the toxicity of the test substance following a single oral administration in rats should include the relationship, if any, between the animals' exposure to the test substance and the incidence and severity of all abnormalities including behavioural and clinical abnormalities, macroscopic lesions, body weight changes, mortality and any other toxic effects.
2.7 PROTOCOL ADHERENCE
The study was performed in accordance with Study Protocol No. 18745 TAR and subsequent
amendments, with the following deviations from the agreed Study Protocol: . the temperature and relative humidity recorded in the animal room were sometimes outside of
the target ranges specified in the protocol.
These minor deviations were not considered to compromise the validity or integrity of the study.
2.8 ARCHIVING
The study documentation and specimens generated during the course of the study are archived at CIT, 27005 Evreux, France, for 10 years after the end of the in vivo phase of the study.
The archived study materials include: . protocol and possible amendments, . raw data,
. correspondence, . final report and possible amendments.
On completion of this period, the archived study materials will be returned to the Sponsor, or may be archived at CIT for a further period.
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3. RESULTS 3.1 CLINICAL EXAMINATIONS 3.1.1 Clinical signs and mortality (table 1) No clinical signs and no deaths were observed during the study. 3.1.2 Body weight (figures 1 and 2, tables 2 and 3) The body weight gain of the treated animals was similar to that of CIT historical control animals. 3.2 PATHOLOGY (table 4) Macroscopic examination of the main organs of the animals revealed no apparent abnormalities. 4. CONCLUSION
m^^Wi^Sts Under our experimental conditions, the oral LDo of the test substanceU^^----HH----| equal to or higher than 2000 mg/kg in rats. No signs of toxicity were "observed at this dose? According to the classification criteria laid down in Commission Directive 93/2 I/EEC (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/EEC, the test substance does not present a significant acute toxic risk if swallowed.
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Figure 1: Body weight of treated rats
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Figure 2: Body weight ofCIT historical control rats
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
----Mde -a--Femde
e.g.: CIT Historical data of animals dosed by the oral route: results of control animals fynicQACtf
October 1995 to December 1997.
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Table 1: Individual clinical signs and mortality
Dose
(mg/kg)
Time
Animals
Males
Females
Mortality
2000
lh-2h 1
-4h
[ 01-02-03-04-05 06-07-08-09-10 No
D2toD15 J
mm: minutes h :hour
D : day
Clinical signs None
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Table 2: Individual and mean body weight and weekly body weight change (g)
Dose ing/kg
Volume Sex
ml/kg
Animals 1
Days
(1)
8
(1)
2000
1.91
Male
01
174
64
238
62
02
173
80
253
69
03
177
73
250
56
04
180
74
254
58
05
182
72
254
48
M
177
73
250
59
SD
4
6
7
8
2000
1.91
Female
06
144
39
183
8
07
136
51
187
14
08
151
43
194
37
09
142
32
174
46
10
154
44
198
33
M
145
42
187
28
SD
7
7
9
16
(1) - Body weight gain
M -Mean SD - Standard Deviation
15
300 322 306 312 302
308 9
191 201 231
220 231
215 18
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Table 3: Mean body weight and weekly body weight change of CIT historical control rats
BODY WEIGHT OF CONTROL RATS
(g)
Dose nig/kg
0
Volume ml/kg
10
Sex Male
0
10
Female
M : mean SD : standard deviation
n : number of animals
Days
------------------------------------------.........
1
8
15
M
182
261
315
SD
10
12
21
n
45
45
45
M
147
190
215
SD
10
15
17
n
45
45
45
BODY WEIGHT CHANGE OF CONTROL RATS
(g)
Dose nig/kg
0
Volume ml/kg
10
Sex
Male
Days
.,,.,,..,,.,,----------.-.--.--------.----.---------
1 to 8
8 to 15
M
79
54
SD
7
13
0
10
Female
M
SD
43
25
8
7
M : mean SD : standard deviation
e.g.:
CIT Historical data of animals dosed by the oral route: results of control animals from
October 1995 to December 1997.
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Table 4: Individual macroscopic examinations at necropsy
Dose mg/kg
Time
Males
Animals Females
2000 D:day
D 15
01-02-03-04-05 06-07-08-09-10
18
Macroscopic abnormalities No apparent abnormalities
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APPENDICES
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20
1. Test article description and analytical certificate
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TOXICOLOGY DEPARTMENT
CONFIDENTIAL April 99
elf atochem s.a. La defense 10, cours Michelet
92091 Paris-la-Defense, France
TEST ARTICLE DESCRIPTION
PHYSICAL AND CHEMICAL PROPERTIES
Appearance Melting point Flash point
Solubility__
TOXICOLOGICAL INFORMATIONS AND USE SAFETY
See safety data
sheet_______________________
STORAGE AND DISPOSAL
Storage Expiry date
Disposal______
: in dark and at room temperature : may 2000 : incineration
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ZA Vilon Sani Paul Rloui B.P. 20 C0670 Ricw (FiBnce) T61: A<.7<.4-1.7< Fax : <.74..l;.07 . Titci : H0429ATQVSP
Villcr* Salnl Paul, Ic 9 Novem
CERT1F1CAT D'ANALYSJg
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Point eclair
pH
Taillc dcs Partic ulcs Test d'application Cuir oldophobic ct hydrophobie
Uoit<S R6sultat Specification
% W
dc fibrication
t----1
c 1----1
[r) flT [--]
nm 1
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2. Diet formula
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Ref: A04
COMPLETE DIET RAT AND MOUSE MAINTENANCE DIET
Appearance: 15 mm diameter pellets or powder Conditioning: 25 kg double paper bag with aluminium on the outside
Daily portion: Rat 18-25 g. Mouse 5-10 g, water ad libitiim.
FORMULA %
M1NERAILS (calculaited in mg/kg
Nat
CMV
Cereals
and
cereal
byproducts
s......
88
Vegetable protein (soya bean
val.
val.
Total
meal, yeast).......................... Animal protein (fish)............. Vitamin and mineral mixture.
7 P............ 5900 0
2
Ca
3 300
5 000
3
K
6 700
0
5 900 8 300 6 700
Na
300
1 600
1900
AVERAGE ANALYSIS %
Mg.....,.,, 1900 100 2 000
Mn
50
40
90
Calorific value (KCal/kg)
2900
Fe
90
150
240
Moisture............................... 12 Cu 15 15 30
Proteins................................ 17 Zn 40 45 85
Lipids...................................
3
Co
T
1.5
1.5
Carbohydrates (N.F.E.)........
58.7
I............. 0.3
0
0.3
4
Fibre.....................................
Minerals (ash).......................
5
AMINO ACID VALUES (calculated in mg/kg)
VITAMINS (calculated per kg)
Nat
CMV
val.
val.
Total
Arginine................................ Cystine.................................. Lysine.................................... Methionine............................ Tryptophan............................ Glycine..................................
FATTY ACID VALUES
(calculated in mg/kg)
9800 2300 8500 3200 1900 8100
Palmitic acid.......................... Palmitoleic acid.................. . Stearic acid........................... . Oleic acid......................... ..... Linoleic acid........................... Linolenic acid.........................
2600
Traces 500
8000 14500
Traces
Vitamin A Vitamin D3 Vitamin Bl Vitamin B2 Vitamin B3 Vitamin B6 Vitamin B 12 Vitamin E Vitamin K3 Vitamin PP Folic acid
Biotin
Choline
Traces
Traces
6mg 2mg
10 mg 1 3 mg 0.01 mg 15 mg
0.25 mg 60 mg
0.5 mg 0.04 mg 1200mg
Available under quality "Control Ref: A04 C "
7500 IU 1500RJ
1 mg
4.5 mg 6.5 mg 1.3 mg 0.01 mg 15 mg
2.25 mg 15 mg Omg Omg
400 mg
7500 IU
1500RJ
7mg
6.5 mg 16.5 mg 2.6 mg 0.02 mg
30 mg
2.5 mg 75 mg 0.5 mg 0.04 mg 1600mg
UAR, 7 rue Gallieni, 91360 Villemoisson - Tel: 01.69.04.03.57 - Fax : 01.69.04.81.97 (Ref. Doc. UAR: 1992)
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