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CONFIDENTIAL
DPT 429/984523/AC
ACUTE ORAL TOXICITY TO THE RAT (FIXED DOSE METHOD)
Sponsor
DuPont" Speciality Chemicals Jackson Laboratory Chambers Works Deepwater NJ 08023 USA
Page 1 of 1
Research Laboratory
Huntingdon Life Sciences Ltd P.O. Box 2
Huntingdon Cambridgeshire PE186ES
ENGLAND
Report issued 6 May 1999
Company Sanitized. Does not contain TSCA CBI
CONTENTS
DPT429/984523/AC
Page
COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ...................... 3
QUALITY ASSURANCE STATEMENT..................:............................................-..............
4
RESPONSIBLE PERSONNEL................................................................................................. 5
6 SUMMARY................................................................................................................................
7 INTRODUCTION......................................................................................................................
TEST SUBSTANCE.................................................................................................................. 8
EXPERIMENTAL PROCEDURE............................................................................................
9
12
RESULTS...........................................-...........................-.............................;..-.................-..
13 CONCLUSION..................................................--...--.........----.-........-...-....---..............
TABLES
1. Mortality
14
data.....------.......--.--.......................................--....................--..--..--.
2. Signs of reaction to treatment-prelimmary investigations........... --.......--.....----.......
15
3. Individual bodyweights & changes - preliminary investigations.------.------------...
15
4. Signs of reaction to treatment-main study..................----..--------.-----------..------
16
5. Individual and group mean bodyweights............................------ ----.............------ --
17
6. Individual bodyweight changes.--.....--------............--...............--------......--......
17
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DPT429/984523/AC COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and with the exception of that noted below I consider the data generated to be valid. The UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No 654).
OECD Principles of Good Laboratory Practice (as revised m 1997), ENV/MC/CHEM<98)T7: --------
EC Council Directive 87/18/EEC of 18 December 1986 (Official Journal No L 15/29), and from 1 May 1999 EC Commission Directive 1999/1 I/EC of 8 March 1999 (Official Journal No L 77/8). Chemical analysis of formulated test article for determination of stability, homogeneity and concentration was not undertaken for this study.
Lewis A. McRae, M.I.Sc.T., C.BioL, M.LBioL,
Study Director, Huntingdon Life Sciences Ltd.
Company SanstiEsd. Does not contain TSCA CS!
QUALITY ASSURANCE STATEMENT
DPT 429/984523/AC
The following have been inspected or audited in relation to this study
Study Phases Inspected
Standard Protocol Audit
Process Based Inspections Husbandry Housing/Environment Weighing of Animals Treatment Procedure Clinical Signs Post Mortem Records Audit Training Records
Report Audit
Date of Inspection
21 November 1997
14 September 1998 14 September 1998 14 September 1998 14 September 1998 14 September 1998 14 September 1998 14 September 1998 14 September 1998
4 January 1999
Date of Reporting
24 November 1997
17 September 1998 17 September 1998 17 September 1998 17 September 1998 17 September 1998 17 September 1998 17 September 1998 17 September 1998
6 January 1999
Standard Protocol Audit: An audit of the standard protocol generated for this type of study design was
conducted and reported to Company Management as indicated above.
Process based inspections: At or about the time this study was in progress inspections and audits of routine and repetitive procedures employed on this type of study were carried out. These were conducted and reported to appropriate Company Management as indicated above
Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above.
The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data.
K<o-)ro^cL-<y
6v '-"~s>
Margaret Blows, Quality Assurance Group Leader, Department of Quality Assurance, Huntingdon Life Sciences Ltd.
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RESPONSIBLE PERSONNEL
DPT429/984523/AC
Lewis A. McRae, M.I.Sc.T., C.Biol., M.I.Biol., Study Director,
Department of Acute Toxicology.
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ce-rita'in TSCA CBi Company Sanii'ized. Doss "at
SUMMARY
DPT 429/984523/AC
This study was performed to assess the acute oral toxicity ^wU------B0we rat. The method followed
was based on that described in:
EEC Methods for the determination of toxicity, Annex to Directive 92/69/EEC (Official Journal No. L383A, 29.12.92), PartB, MethodB.l bis. Acute toxicity (oral) - fixed dose method.
OECD Guideline for Testing of Chemicals No.420 'Acute Oral Toxicity - Fixed Dose Method'
Adopted 17 July 1992.
A group of ten fasted rats (five males and five females) received a single oral gavage dose of the test
substance, formulated in distilled water. The administered dose after adjustment for water contenfU^
equated to 500 mg/kg bodyweight (all dosages referenced hereafter are expressed as dose concentrations
^dHII^HIBHUHlBiBIL after adjustment for
^ne niain study dose level selection was
supported by preliminary study fmdmgsanamcompuance wiuTthe test guidelines.
UnHIHt There were no deaths in. the main phase of the study in which a group of ten rats received a dosage of a dose level of 500 mg/kg bodyweight.
Clinical signs of reaction to treatment were characterised by piloerection, hunched posture, increased salivation and ungroomed appearance notable in all rats, with waddling/unsteady gait and thin appearance less commonly observed. All but piloerection (all rats) and hunched posture (males only) had resolved within 24 hours of dosing with piloerection (all rats) only evident from Day 4, resolving in all instances by Day 8.
All rats were considered to have achieved satisfactory bodyweight gains during the study.
No macroscopic abnormalities were observed in animals killed at study termination on Day 15.
ofHBHHH^^ The discriminating (non-lethal) oral dose to rats
bodyweight.
indicated to be 500 mg/kg
On the basis of findings in this study and in accord with EU hazard classification^BU|B|lwill not require labelling with the risk phrase R22 "Harmful if swallowed", in accordance with Commission Directive
93/2 I/EEC.
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INTRODUCTION
DPT429/984523/AC
The study was designed to assess the toxicity otdmU|bllowmg a single oral dose to the rat. The
rats were dosed by oral gavage as the test substance may be ingested accidentally.
fcr-- The study was conducted in compliance with:
EEC Methods for the determination of toxicity. Annex to Directive 92/69/EEC (Official Journal No. L383A, 29.12.92), Part B, Method B.I bis. Acute toxicity (oral) - fixed dose method.
OECD Guideline for Testing of Chemicals No.420 'Acute Oral Toxicity - Fixed Dose Method'
Adopted 17 July 1992.
The rat was chosen as it has been shown to be a suitable model for this type of study and is the animal recommended in the test guidelines.
The dose levels used in this study were chosen to help define the toxicity of the test substance and to be in > compliance with the test guidelines.
The protocol was approved by Huntingdon Life Sciences Management on 7 July 1998, by the Sponsor on 17 July 1998 and by the Study Director on 27 August 1998.
The experimental phase of the study was conducted between the 2 September and 13 November 1998.
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Company Sanitized. Does notconts'.n TSCA ASi
Identity: Chemical name:
Intended use:
I'.:
Appearance:
Storage conditions:
Lot number: Expiry:
Purity:-
I.
Sample received:
TEST SUBSTANCE
DPT 429/984523/AC
Room temperature
W
Two years from date of receipt
23 June 1998
^ : 8 :
SanSfesd. Ooes no"! c^ta.n TSCk
Company
EXPERIMENTAL PROCEDURE
DPT 429/984523/AC
ANIMAL MANAGEMENT
Animals chosen for this study were selected from a stock supply of healthy male and female CD rats of Sprague - Dawley origin (Hsd:Sprague-Dawley(CD)) obtained from Harlan UK Ltd, Bicester, Oxon,
England.
Animals in the main study were in the weight range of 78 to 97 g and approximately five to seven weeks of age prior to dosing (Day 1). All the rats in the main study were acclimatised to the experimental environment for a period of five days prior to the start of the study.
The rats were allocated without conscious bias to cages within the treatment groups. They were housed in groups of up to'five rats of the same sex-in metal cages (RS Biotech Sub-Dividable Rodent Cages polished stainless steel (20cm high x 39cm wide x 39cm long)). The cages were fitted with grid floors to ensure rapid removal of waste material to undertrays. The cages were suspended in mobile stainless steel racks in Room 6 of Building R14.
A standard laboratory rodent diet (Special Diet Services RM1(E) SQC expanded pellet) and drinking water were provided ad libitian. Access to food only was prevented overnight prior to and for approximately 4 hours after dosing. The batch of diet used for the study was analysed for certain nutrients, possible contaminants and micro-organisms. Results of routine physical and chemical examination of drinking water, as conducted by the supplier, are made available to Huntingdon Life
Sciences Ltd. as quarterly summaries.
Thermostatic controls were set to maintain a temperature of223C. Relative humidity was not fully
controlled but was anticipated to be in the range 30 - 70%. Temperature and humidity were recorded continuously using a seven day recorder. Actual measurement of these parameters revealed that animal room temperature was in the range 21 to 24C and relative humidity was in the range 38 - 61%. Permanent daily recordings of these parameters were made and these are archived with other Departmental raw data. Lighting was controlled by means of a time switch to provide 12 hours of artificial light (0700 -1900 hours) in each 24-hour period.
Each animal was identified by cage number and ear punching. Each cage was identified by a coloured label displaying the dose level, study schedule number, animal mark and the initials of the Study Director and Home Office licensee.
TEST SUBSTANCE PREPARATION
concentration of 20% v/v in distilled waterandadmuusteredat^dose volume of TO ml/kg bodyweight
in order to achieve the desired dosage.
The test substance was prepared on the day of dosing.
The absorption and characterisation of the homogeneity, stability in vehicle and purity of the test substance was not undertaken in this study and remains the responsibility of the Sponsor.
9 :
Company Sanitksd, Does not contasn 7SCA C
DPT429/984523/AC
TREATMENT PROCEDURE
Preliminary investigations
f
In the absence of precise toxicological information a preliminary study was conducted to help define the
toxic potential of the test substance and aid in selection/validation of the dosage selected for use in the
o^H^HBIInd main study. Initially, one female rat was treated with the test substance as su]mlidatl25mg/kg, but as
aresult of a marked toxic response thought to be associated with the pH
in the
interests of animal welfare, a further female was treated at 125 mg/kg with theiest substanceformulated
in distilled water and for further clarification of oral fexicrfy a female raE was'dosed at 500 mg/kg^
bodyweight and finally one male and one female (per dosage) treated at 1000 and 2000 mg/kg.
Main study
A group often rats (five males and five females) received a single oral gavage dose of the test substance at' a dose level of 500 mg/kg bodyweight; This dose level was chosen in compliance with the test
guidelines.
No control animals were included in this study.
ADMINISTRATION OF THE TEST SUBSTANCE
The appropriate dose volume of the test substance was administered to each rat by oral gavage using a syringe and cannula of the appropriate gauge.
The day of dosing was designated Day 1.
OBSERVATIONS
Mortality Cages of rats. were checked at least twice daily for mortalities. Clinical signs
Animals were observed soon after dosing and at frequent intervals for the remainder of Day 1. On
^.subsequent days, animals were observed once in the morning and again at the end of the experimental
day (with the exception of the last day of observation - morning only). The nature and severity of the
clinical signs and time were recorded at each observation.
' '*
Animals in the preliminary and main study were observed for up to 7 or 14 days respectively. after dosing.
Bodyweight
The bodyweight of each rat in the preliminary study was recorded on Days 1 (prior to dosing) and 8 (or at death) and in the main study on Days 1 (prior to dosing), 2,3,4, 8 and 15.
10
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TERMINAL STUDIES
DPT429/984523/AC
^
Termination
The animals (that survived treatment) in the preliminary study were killed on Day 8 (study termination) and all animals in the main study were killed on Day 15 by carbon dioxide asphyxiation.
Macroscopic pathology
All animals were subjected to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities. The macroscopic appearance of all examined organs was recorded.
ARCHIVES
All raw data and study related documents generated during the course of the study at Huntingdon, together with a copy of the final report will be lodged in the Huntingdon Life Sciences Archives,
Huntingdon.
Such records will be retained for a minimum period of five years from the date of issue of the final report. At the end of the five year retention period the Sponsor will be contacted and advice sought on the future requirements. Under no circumstances will any item be discarded without the Sponsor's
knowledge.
DEVIATIONS FROM PROTOCOL
There were no deviations from the protocol that were considered to have affected either the validity or the integrity of this study. However, the following deviation did occur; to assist in further defining the
.toxicity of the test substance one male and one female (per dosage) were treated at 1000 and
2000 mg/kg each dosage. These animals were also slightly above the upper weight limit specified in the study protocol.
: 11 : Company Sanifeed. Dees not c.Q-Aaw TSCA C',
^
RESULTS
OPT 429/984523/AC
PRELIMINARY STUDY (Tables 1,2 and 3)
Two females receivec|Um|BRit a dosage of 125 nig/kg (administered either as supplied or as an
'; :aqueous formulation). A further female was treated at 500 mg/kg and fmally one male and one female
^~(perdosage) was treated at 1000 and 2000 mg/kg bodyweight. Results were as follows:
I- Deaths were recorded between Day 3 and Day 4 for one female dosed at 1000 mg/kg and one male" ana'
'-one female treated at 2000 mg/kg.
yg^-;' (Day 8).
All other animals were killed as scheduled at study termination
l||s. .
Clinical signs comprised piloerection, hunched posture, waddling/unsteady gait and dark colouring to eyes observed in rats at all dosages. In addition, ungroomed/thin appearance, lethargy, abnormal faeces, protruding eyes, abnormal respiration, pallid extremities, walking on toes, blue/cold extremities, increased salivation, increased lacrimation and body tremors were observed in rats at one or more
dosages.
Bodyweight gains were considered to be within acceptable limits for a study of this nature and duration.
Macroscopic examination of animals treated at 125 and 500 mg/kg killed at study termination revealed no abnormalities. Examination of the one rat that survived treatment at 1000 mg/kg revealed pallor of the brain and kidneys, slightly enlarged liver, kidneys and darkened spleen. Findings in decedents were primarily congestive in nature (characterised by darkened tissues, prominent blood vessels and enlarged
tissues/organs) in subcutaneous tissue, brain, heart, lungs, liver, spleen and along the alimentary tract. Also observed in the latter was fluid/food retention.
Findings from this phase of the study demonstrated that administration oilJBHHH^ dosages of
1000 and 2000 mg/kg resulted in ^50% mortality.
MAIN STUDY
o^MBB----ft There were no deaths in a group of ten rats (five males and five females) following a single oral
administration
a dose level of 500 mg/kg bodyweight.
CLINICAL SIGNS (Table 4)
Piloerection increased salivation and ungroomed appearance were observed in all rats within seven minutes of dosing and accompanied later on Day 1 or thereafter by hunched posture notable in all rats with waddling/unsteady gait and thin appearance less commonly observed. All but piloerection (all rats) and hunched posture (males only) had resolved within 24 hours of dosing with hunched posture resolving in all instances by Day. 4 and piloerection resolving in all instances by Day 8.
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Company Sanitized. Ccas not conTsjn TSCA CBi
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DPT429/984523/AC
1="?'''-?' BODYWEIGHT (Tables 5 and 6)
^0 '
t?"
All rats were considered to have achieved satisfactory bodyweight gains during the study.
1^. MACROSCOPIC EXAMINATION
^s----N&niacroscopic abnormalities were observed for animals killed at study termination on Day 15.
CONCLUSION
IBfc.- .-^_The acute lethal oral dose to rats o'
bodyweight.
demonstrated to be greater than 500 mg/kg
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TABLE 1 Mortality data
DPT 429/984523/AC
-: Not applicable
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DPT 429/984523/AC TABLE 2 Signs of reaction to treatment - preliminary investigations
Signs of reaction
Piloerection Hunched posture Waddling/unsteady gait Increased salivation Ungroomed appearance1' Thinappearance Lethargy Abnormal respiration' Pallid extremities Walking on toes Abnormal faeces'" Protruding eyes Blue/cold extremities Dark colouring to eyes Increased lacrimation Body tremors Prostration
No. of rats in group of 1' or 22 showing signs
Dose (mg/kg)
125'+ 125 500
- -- ...-- --JL ,1
F
F
F
10002 M F
20002 M----F~
1#
1
1
1 1
1
1#
1
.
1
1 1
1
1
1
0
1 1
1
0
0
0
0
1
1
1
0
0
11 1
1
0
0
11 0
1
0
0
0 0
1
1#
1
0
0 0
1
1
0
0
0 0
1
1
0
0
0 0
1
1
0
0
1 1
1
1
0
0
11 1
1
0
0
0 0
1
1
0
0
1
1
1
1
0
0
0
00 1
1
0
0
0
0 0
1
0
0
0
0
0 0
1
0
1.
Preliminary study.
2.
Main study
+:
vas administered as supplied
#: ""Still evident at study termination
a:
Characterised by increased, decreased or gasping/noisy breathing
b:
Characterised by soiled/stained fur around the face/muzzle or ano/genital region or wet far
c:
Characterised by soft to liquid faeces/few
TABLE 3 Individual bodyweighte and gains (g) - preliminary investigations
Dose
(mg/kg) 125+ 125 500
1000
2000
Animal No.
&Sex
IF
2F . 3F
23 M
24 F
: 25 M 26 F
Bodyweight (g) at Day
I*
8
Death
116
124
[8]
120
165
[45]
122
161
[39].
197
206
[9]
185
-
235
-
[-]
173
[--]
186
199
-
[-]
191
Lto dosing
HiUp +:
administered as supplied
[1-- Weight gain shown in parenthesis
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TABLE 4 Signs of reaction to treatment - main study
DPT429/984523/AC
Signs of reaction
Piloerection Hunched posture Waddling/unsteady gait Increased salivation Ungroomed appearance" Thin appearance
No. of rats in group of 5 per sex showing signs
Dose ( 500 mg/kg)
-Kr-----
5
-F------
5
5
5
5
0
5
5
5
5
1
1
a:
Characterised by soiled/stained fur around the
face/muzzle or ano/genital region or wet fur M: Male rats
F:
Female rats
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DPT429/984523/AC TABLES Individual and group mean bodyweights (g) - main study
Dose
Animal No.
(mg/kg)
&Sex
I*
11M
86
12 M
..89
13 M
78
14 M
86
15 M
85
500
Mean
85
16F
95
17F
88
18F
84
19 F
97
20 F
94
Mean
92
*: Prior to dosing
Bodyweight (g) at Day
2
3
4
8
15
86
88
98
127
187
'94"
102
TT6"
142----1-93--
87
94
99
122
174
95
103
113
146
210
91
101
112
144
204
91
98
108
136
194
109
118
117
139
154
99
108
117
136
156
93
102
109
124
145
108
120
123
142
172
100
112
118
136
160
102
112
117
135
157
TABLE 6
Individual and group mean bodyweight changes (g) - main study
Dose (mg/kg)
500
Animal No. &Sex 11M
12 M 13 M 14 M 15 M
Mean 16F 17F 18F 19 F 20 F
Mean
Bodyweight'gains (g)
Day 1-8
41
Day 8-15 60
53
51
44
52
60
64
59
60
51
57
44
15
48
20
40
21
45
30
42
24
44
22
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