Document 6RZmykakRqR4D4dmvOxkZ53x6
SPONSOR Elf Atochem S.A. Direction Scurit Environnement - Produit La Dfense 10 - Cdex 42 92091 Paris-la-Dfense
France
AR226-3012
STUDY TITLE REVERSE MUTATION ASSAY ON BACTERIA SALMONELLA TYPHIMURIUM
STUDY DIRECTOR Brigitte Molinier
STUDY COMPLETION DA TE 24th March 1995
PERFORMING LA RORA TORY Centre International de Toxicologie (C.I.T.)
Miserey - 2705 Evreux - France
LABORATORY STUDY NTJMRER 12373 MMO
Company Sanitized. Does not contain TSCA C*?
CONTENTS
STATEMENT OF THE STUDY DIRECTOR
STATEMENT OF QUALITY ASSURANCE UNIT SUMMARY
1. INTRODUCTION
2. MATERLALS AND METHODS
2.1. TEST AND CONTROL SUBSTANCES 2.1.1 Identification 2.1.1.1 Test substance 2.1.1.2 Vehicle 2.1.2 Preparation 2.1.3 Positive controls 2.2. TEST SYSTEM 2.2.1 Bacterial strains 2.2.2 Metabolic activation system: S9 mix 2.3. EXPERIMENTAL DESIGN 2.3.1 Preliminary toxicity test 2.3.2 Mutagenicity tests 2.4. ASSESSMENT OF DATA 2.5. ARCHIVES
2.6. CHRONOLOGY OF THE STUDY
3. RESULTS
3.1. PRELIMINARY TOXICITY TEST (table 1) 3.2. MUTAGENICITY TESTS (tables 2 to 5)
4. CONCLUSION
5. REFERENCES Tables 1 to 5
`
.
6. TEST ARTICLE DESCRIPTION AND TEST ARTICLE ANALYSIS 7. HISTORICAL DATA
3
4 5 6
11 11 11 11 11 12 to 16 17 to 19 20 and 21
Company ^anWze'ij' Efeess n>>*
Company Sanitized. Does not contain TSCA CBI
STATEMENT OF THE STTTpy D T R F .rm p
Regulatiwis^aS
" CmpHanCe With PrfnciPles ^ Good Laboratory Practice
O.E.C.D. Principles of Good Laboratory Practice, C(81)30(final) Annex 2. May 12, 1981.
and the results
f ^ Procedures undertaken
27005 Evre^France.rmed * ^
IntemaiionaI de Toxicologie (C.I.T.), Miserey,
B. Molinier Study Director
Date: 24.3.95
Doctor of Toxicology
Head of Genetic Toxicology
Company Sanifizs'd. Doss not contain tr***
g 4 2 1 m ^ ^ m i J TY ASSURA IVrp TTxrrr 1. Specific study inspections
Type of inspections
Inspections
Dates (day/month/year) S tu d y S fo rQ
frequency defined in Q .A ^ p S e d u re s
f ^ Same tyPe ^cording to a
Inspected phase
_______
Treatment/test substance (incorporation) Treatment/test substance (preincubation)
Test substance/preparation
Dates (day/month/year)
Inspections
10.1.95 26.1.95 6.1.95
Report t0 Study Director ()
13.1.95 7.2.95
6.1.95
n (o Management (*)
16.1.95 7.2.95 10.1.95
pTr o ^
QuaJity Assurance Unit
( ) The dates mentioned corresDond tn thr
f
Director and Management. P
h d t&S of SISnature of audit reports by Study
Head of Quality Assurance Unit and Scientific Archives
C o m p a n y Sanitized. Does no! contain TSCA tua
CIT/StudyNo. 12373 MMO SUMMARY
If Atochem
5
Thaoobl jective of this study was to evaluate the potential o f the test s u b s ta n c e g a ^ iH i^ ^ n induce a ircevcerisce mutiatuion in boactiernia Salimonelula typhmimurniuwmn (Ames test). This test
enabless tthhee drieeftepc.rtfiionnn onffKboapsefl-poaiirr substitution or f-cr_a_m__eslh;i&ft._m_u_ta_g_e_n_s.
Methods
A preliminary toxicity test was performed to define the doses to be used for the mutageni
city study. The test substance was then tested in two independent tests, with or without a
metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9) of rats induced with Aroclor 1254.
The tests were performed according to the direct plate incorporation method except the second test with S9 mix, according to the preincubation method (1 hour, 37C).
Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to 5 doses of the test substance (3 plates/dose). After 48 to 72 hours of incubation at 37C, the revertant colonies were scored.
The test su b stan cejflH jj|H |H H H K y as dissolved in distilled water.
The doses of M m H ^ R p e r e : 312'5' 625' 1250* 2500 m d 5000 Pg/plate, DUuUfig/plate being the top dose recommended by international regulations.
The doses of the positive controls were as follows:
without S9 mix:
. 1 fig/plate of sodium azide (NaN3): TA 1535 and TA 100 strains, . 50 fig/plate of 9-Aminoacridine (9AA): TA 1537 strain, . 0.5 fig/plate of 2-NitrofIuorene (2NF): TA 98 strain, . 0.5 pg/plate of Mitomycin C (MMC): TA 102 strain.
with S9 mix:
. 2 pg/plate of 2-Antfaramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains
. 30 pg/plate of Danthron (DTH): TA 102 strain.
'
Results
For both tests, the control results were equivalent to those usually obtained in our
Laboratory. The number o f revertants induced by the positive controls was higher than the controls, indicating the sensitivity of the test system.
The test s u b s t a n c e g |^ ^ 9 |^ ^ 0 c i i d not induce any significant increase in the number of revertants, with or without S9 mix, in any of the 5 strains, for both tests.
Conclusion
Under our experimental conditions, the test s u b s t a n c ^ f l B H H H H d i d not show mutagenic activity in the reverse mutation assay on SalmoneU^yphirnunu}^
j&pittpany Sanitized. D oes not contain TSCA cTM
LT ruuLUCUl
6
1. INTRODUCTION
This study was perfonned at the request of elf Atochem S.A., Paris-la-Dfense, France.
Therjbjective of this study was to evaluate the potential of the test substance& B induce a reverse mutation in bacteria Salmonella typhimurium (A m esfS J.
sTM
m ^ n ani A? x s -.m 3 >
detection o f base-pair
hmited medium, while non-reverted strains cannot
& 1
T? p ! J was designed in accordance with the following guidelines-
" a hlStldlne-
2. MATERIALS AND METHODS
2.1. TEST AND CONTROL SUBSTANCES
2.1.1 Identification
2.1.1.1 Test substance
The test substance,! S.A. 1
--------- used in the stud'y was- -s-u-p--p--li-e--d--b- 7y -Elf Atochem
P d e n S a t i Qn:SUPPlied ^ ^ Sp nS r identified * e test substance as follows:
- protocol: - labelling^ . batch number; - protocol ~ - labelling . description!^^^^^^^^^^^^^^
. date o f ^ i p 2 ! n n94 5 g " a pIaStlC flask wraPPed in an aluminium foil . storage conditions: at room temperature, protected from light.
2.1.1.2 Vehicle The vehicle was distilled water.
2.1.2 Preparation The tea substance was dissolved in the vehicle at a concentration o f 50 mg/ml for both
The preparations were made immediately before use.
*
ICompany Sanitized. D ees not contain TSC
2.1.3 Positive controls
Without S9 mix . Sodium azide (NaN3) 9-Aminoacridine (9AA) . 2-Nitrofluorene (2NF) Mitomycin C (M M Q With S9 mix 2-Anthramine (2AM) Danthron (DTH)
ip u i Atocem
7
Dose (pg/plate)
& r c * Strains
1 50 0.5 0.5
2 30
TA 1535 - TA 100 TA 1537 TA 98 TA 102
TA 1535 TA 1537 - TA 98 - TA 100 TA 102
2.2. TEST SYSTEM 2.2.1 Bacterial strains
Berkeley, U.S.A ) Thev are
? 3): TA l535' TA > 7 , TA 98, ^ ' y^mes Laboratory (University of California
broth and 0.09 ml methylsulfoxide iHl l S M S ^ c o n S r . " " ^
` 011 m tri" `
2 r - " -- on in ,he sensitivity to mutagenic substances additional m m S f n f T ' a additio". increase their . the rfa mutation causes oartM In 5 i , m",atlons have been added:
TA to o p
R' factor 118 stodns TA 98'
' T A W ^ s S in ^ PAQI tetracyd " e " to plasmidicfector "h a s^ e e n added to the
Strains
TA 1535 TA 100 TA 102 TA 1537 TA 98
Histidine mutation
His G 46 His G 46 His G 428 (pAQl) His C 3076 His D 3052
Additional mutations
rfa uvrB rfa uvrB Factor R rfa - Factor R rfa uvrB rfa uvrB Factor R
and the TA 1537 and TA s/strain s b v ta r T 'h - f t'8118'1by base_Pair Substitution mutaens
detects oxidative mutagens
7
muta6" a-1" Edition, the TA 102 s
Com pany Sanitized. Does not contain Tftf^
M jru-uuacui
2.2.2 Metabolic activation system: S9 mix
afcrciot irdoiLng^t9o )A"mes eteal r i 97T> Th* qq w
mnction. b9 wwaassp'perefrofrrmeTd aTt C.If.rT.
mduced Wia Aroclor 1254 (S00 mgflcg) by m t r a S S a l m" , ? ' " ' i0m0genates frTM
s s9
The S9 mix contained per ml:
. 5 pmoles Glucose-6-Phosphate, . 4 pmoles NADP,
. 33 pmoles KC1,
. 8 pmoles MgCl2,
. 100 pmoles sodium phosphate pH 7.4,
. .
100 pi sterile
S9 (batch No. distilled water
38, protein q.s.p. 1 ml.
concentration:
27
SA),
2.3. EXPERIMENTAL DESIGN
. direct plate incorporation method (both tests without S9 mix first w
oQ s L
'5 1,11 f =9 45"C A ite rS L S h ^ COntainf * of Wstidine and biotin and m a in S L T a t
m0gem2atl n' 1,10
MS " - a p l a te ^ S n g
' S9 S k i d 'S r ^ , i f e" nd t e ` wifh
^ test suba" " solution, 0 5 ml of
counter (Aitek Counts,modeI 2.3.1 Preliminary toxicity test
2 t S t e i i 09jd ? A ^ a n ? T r ^ - `O ^ bacttria- 6 * 0 plate/dose) ,,ere
determined according to the international
W"
dumber of" o t e S
' " *PUX Cmpared to COntro1 ptotes and/or die
The sterility of the test substance was checked during this test and was found to be satisfac-
'Company Sanitized. Does not contain TSCA CBi
L-i/oiuuy jlnu. i z o / o M M l
Blf Atochem
2.3.2 Mutagenicity tests Rationale for rinse selection
^ c onned by s m iy Director m d wm be d ~ d " the following a iL b?.SeIected accordmB to the results of the preliminary toxicity test and to
' t o t a a u S r e S o n s " 6 teSt SUbS,anCeS' the top d0se to 5000 S ^ according to . ^ n o n - t o x ic , poorly soluble test substances, the top dose is the lowest precipitating
' 50% of the nnmtwr nf
* * * to based on the level o , , ^ sParse bacterial lawn and/or decrease by approximately
s" s ^ r i ^ s r dtofte"
s ^ ! 4 "i S w S o u l * * 0f t o SUbStm" (3 Plates/d" e> 1 on each
DS r h t St; the foUowinS controls were made using triplicate plates-
vehicle control: strain treated with the vehicle
P P S`
positive control: strain treated with the known mutagens mentioned in 2.1.3.
^ ,he
md * -
2.4. ASSESSMENT OF DATA
Treatment of results
TM p ttfw K " o S tS S m S ? ,* ' T h " P f mental Pint'
number of revenants
= = ^ 255"
Acceptance criteria
' w i t t i n g S i & S S " C "tt0lS TM Ugher to Evaluation criteria
f to . conois and
t l " C0"SMercd "
in this test system if the above two criteria
Biological and statistical significances were considered during the evaluation.
JrffimpaRy Sanltfeedf. D oes not contain TSCA f
2.5. ARCHIVES
The study archives: . protocol and possible amendments . raw data . correspondence . final report and possible amendments
end S S to the Sponsor.
! M
fT "'
5 ---
^ f penod' ** study * * e s will be returned
2.6. CHRONOLOGY O F TH E STUDY
The chronology of the study is summarized as follows:
Procedure
Protocol approved hv: . Study Director . . Sponsor
Preliminary toxicity test . treatment
First mutagenicity test . treatment
Second mutagenicity test . treatment
Date
12.10.94 3.10.94
17.1.95
20.1.95
26.1.95
Com pany Sanitized. Does not contain T S C * ^
3. RESULTS
ixvswueuLi
il
3.1. PRELIM INARY TOXICITY TEST (table 1)
The test su b stan ceiifli^ ^ H H Ilih JL .. ,, * , , , ,
50 mg/ml.
freely soluble m the vehicle (distilled water) at
When 0.1 ml o f this solution was addsH tn o mi of 5000Mg/p ,a ,e ,n o p r e c ip it a ^ ^ ^
Consequently, the doses were: 10,100,500,1000,2500 and 5000 pg/plate. The test substance was not toxic at the doses used.
3'2` M UTAGENICITY TESTS (tables 2 to 5)
indicating the sensitivity o f the test system.
^ o r a t o i y . The controls was higher than the controls,
The selected doses w e r e -312 5 6os n ^ n n<nn , the top dose recommended by 'intemationS^eguktiSs.
5 Pg/plate beinS
The test s u b s ta n c e fr flB H H |M M l a-.j nnt .
.
number of
in any*of th e ^ str ^ n s!^ ^ fareaSe "
4. CONCLUSION
Under our experimental conditions, the test s u b s t a n c J H U H i ^ M ) ,., mutagenic activity in the reverse mutation assay on S a l M S y S H S ^ M Sh "
5. REFERENCES
simple test system combining'J iv ^ h o jg e n ^ Y &' R D `- CarcinoSens are mutagens: a Proc. Nat. Acad. Sci. U .S.A .,70,2281-2285 (1 9 7 3 ^ aCVaon m d bacteria for detection.
mutagens with^the ^ a lm o n d h Research, 31, 347-364 (1975).
.Methods for detecting carcinogens and -microsome mutagenicity test. Mutation
Mutation Research, X ^ l 73-215 f l 9 ^ methods for the Salmonella mutagenicity test.
5aTiifizeli 13ms Hoi contain TSCA CW
' / Turivi Table 1: Preliminary test o f toxicity
F jc ii Arocnem
12
T : toxicity : - : no + : slight
+ + : moderate + + + : considerable to total
P : precipitate of the test substance :
- : no + : slight 1 + + : moderate + + + : strong
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Table 2: First m utagenicity test without m etabolic activation
JSfRnpaify Satilffxecf, Does Bet con*a5n TSO* fy*'
U -rJ_L r -V l.\ JV llW X X J.
Table 3: Second m utagenicity test without metabolic activation
14
number o f revertants with the vehicle
J
`' "'Bi
/J.X X T .f c W V l I W l l X
Table 4: First mutagenicity test with metabolic activation
15
ratio :
number of,revertants with the test substance number o f revertants with the vehicle
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J.T x a v x v
iu m ucueui
16
Table 5: Second mutagenicity test with metabolic activation : Preincubation m ethod
strains
doses
TP
(/g/plate)
0 --
312.5
--
TA 1535
625 1250
---
2500
-+
5000
+ ++
2AM(2)
--
0 --
312.5
--
TA 1537
625 1250
- -' --
2500
++
5000
+ ++
2AM(2)
--
0 --
312.5
--
625 - -
TA 98 1250 - -
2500
-+
5000
+ ++
2AM{2)
--
0 --
312.5
--
625 - -
TA 100 1250 - -
2500
-+
5000
- ++
2AM(2)
--
0 --
312.5
--
625 - -
TA 102
1250
--
2500
-+
5000 . - + + '
DTH(30)
--
0 : vehicle control (sterile distilled water)
reyertante per plate
mean standard ratio
10 8 7 8 17 7 198 10 5 8 6 7 6 225 21 16 21 18 25 20 2449 114 95 105 102 106 98 1862 263 193 329 301 372 297 765
8 10 9 12 10 14 285 6 6 5 4 12 5 334 32 19 22 20 21 24 2385 111 86 89 100 102 104 2540 315 249 376 230 320 307 830
deviation
99
1
68
2
11 9
2
89
2
11 13
4
12 11
4
253 245
44
10 9
2
45
1
66
2
65
1
79
3
97
2
323 294
60
22 25
6
22 19
3
17 " 20
3
23 20
3
20 22
3
25 23
3
2629 2488
127
84 103
17
90 90
5
85 93
11
92 98
5
96 101
5
95 99
5
2297
2233
344
207 262
54
216 219
28
296 334
40
276 269
36
343 345
26
279 294
14-
987 861
114
T : toxicity (cf.Table 1)
_
0.9 1.0 1.0 1.4 1.2 27.3
_
0.6 0.7 0.6 1.0 0.8 33.9
0.8 0.8 0.8 0.9 0.9 99.5
_
0.9 0.9 1.0 1.0 1.0 21.7
0.8 1.3 1.0 1.3 1.1 3.3
P : precipitate (cf. Table 1)
ratio :
number o f revertants with the test substance number o f revertants with the vehicle
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u t u u j r n u . j / j lYJLLvr
'U Aiocnem
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17
6. TEST A R TIC LE DESCRIPTION AND TEST ARTICLE ANALYSIS
v-ii/jLuuy inu. 1ZJ/JJVUVH.
SERVICE DE TOXICOLOGIE CONFIDENTIEL 3 octobre 1994
lf Atochem
18
elf atochem sa
La Dfense 10, cdex 42
92091 Paris-La Dfense France
FICHE D'INFORMA FIONS
IDENTIFICATION
Nom commun Nom chimique
Origine; n de lot N d'archivage (CAL)
Elf Atochem, CRRA, Opration Industrielle 88/94
PROPRIETES PHYSICO-CHIMIQUES
Apparence Viscosit Temprature bullition PH Densit Point d'clair Solubilit
INFORMATIONS TOXICOLOGIQUES ET PRECAUTION D'EMPLOI DLQ1rat 1oral > 2000 mSfog- Non irritant pour la peau et les yeux. Non sensibilisant.
CONDITIONS DE CONSERVATION ET DE DESTRUCTION
Conservation Stabilit
Destruction
: A l'obscurit et l'abri de la chaleur.
. Stable jusqu en Septembre 1995 dans ces conditions de stockage
: Incinration.
S a oe d .D ^n o tco ^fS C ^ f Company
v^xx/ututijr n u . / j iVilViwi
eiF atochem
Ijc ii Aiocnem
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Centre de Recherche Rhone-Alpes
V .W V .V W .V A ^ 'A W S '.W /M V
Date : 3 --)o S /3 t
Rfrence de l'Opration Date de fabrication...........
ij o/sh
IJSLi
r
RESISTANCE l'EAU... RESISTAN CE ('HUILE. KIT V A LU E......................
$ m C 0 ip L u s\o ^\
M I
Destinataires :
MM. CHAIZE / PIED
LAIGNEL MONTAGNON GROGNET
'1
. ------
Signatura :
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/ i Im
L- J
M M O /M M J30.1.95
controls
tmoins
NaN3 2NF 9AA 2AM 2AM(P) 0-S9mix 0+S9mix
TA 1535
mean (SD) mini-maxi
moyenne (cart-type) 371(101)
149-564
Nb values
Nombre de valeurs
105
TA 1537
mean (SD) mini-maxi Nb values
moyenne (cart-type)
Nombre de valeurs
TA 98
mean (SD) mini-maxi
moyenne (cart-type)
Nb values
Nombre de valeurs
276(52) 253(43) 10(3) 12(3)
131-409 111-333
4-20 4-20
66 54 132 135
189(83) 174(65) 336(71) 9(4) 9(3)
60 - 499 81-439 189-457 4-21 4-20
111
72 1495(345) 936-2975
75
54 2633(327) 1685-3250 60
138
29(9)
11-55
135
150
26(7)
14-47
159
controls
tmoins
NaN3 MMC ENNG 2AM 2AM(P) DTH DTH(P) 0 - S9mix 0 + S9mix
TA 100
mean (SD) mini-maxi Nb values
moyenne (cart-type)
517(115)
237-751
Nombre de valeurs
111
1500(371) 823-2328 2512(350) 1691-3093
72 57
96(16) 104(19)
61-142 60-156
135 150
TA 102
WP2 uvrA
mean (SD) mini-maxi Nb values mean (SD) mini-maxi Nb values
moyenne (cart-type)
Nombre " moyenne de valeurs (cart-type)
Nombre de valeurs
614(280) 230-1395
824(122) 609-990
896(228) 602-1483
802(107) 603-1066
60
846(138) 568-1191
36
292(45) 152-414 105 26(11) 11-57
358(62) 224-509
105
30(9) 14-63
45 21 27
48 51
NaN3: Ipg/plate/bote
2NF: 0.5/ig/plate/bote
9AA: 50pg/plate/bote
MMC: 0.5/ig/plate/bote
ENNG: 2/rg/plate/bote
.
2AM: 2 or 10pg/plate for Salmonella typhimurium or for WP2uvrA, respectively.
2AM: 2 ou 10/ig/bote pour Salmonella typhimurium ou pour WP2uvrA, respectivement.
DTH: 30fig/plate/bote
(P): preincubation method/mthode par princubation.
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