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Short-term feeding study of lauric diethanolamide in rats
Fd Cosmet. Toxicol. Vol. 5, pp. 497-503. Pergamon Press 1967. Printed m Great Britain
Short-term Feeding Study of Lauric Diethanolamide in Rats I. F. GAUNT--Toxicology; MADGE FARMER a n d P. GRASSO---Pathology; a n d S. D. GANGOLLI--Analytical Chemistry British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey, England (Received 3 October 1966) Abstract A short-term feeding study has been earned out on laurie diethanolamlde (LDE) in the rat. Dietary levels of 0-0 (control),0 1,0-5,1-0 and 2 0% for 90 days evoked no adverse effect on the appearance or conditmn of the ammals. Growth retardation was associated w~th d~mmished food intake, at and above the 0"5 % level. Food refusal was demonstrably due to an effect of the test material on palatabihty of the d~et. Terminal haematologleal exammation revealed a reduction in the haemoglobin level, haematocrit and red cell count at the 1 and 2 % levels in females but less pronounced effects were seen in males. Serum levels of glutamic-oxaloaeetic transarninase were elevated at dietary levels of 0 5 % and above m females but only at 0"5 Yoin males. No untoward effect was observed in the renal functlon tests. The principal organ weight changes were: increases in the relative kidney weight m all test groups except at 0-1 Yom females and at 0-1 and 0"5 ~ in males, and increases in the relative hver weight in females on the two highest levels. The types and incidence of histological leslons were comparable m control and test groups. A no-effect level was established of0.1% LDE in the diet of rats for 90 days,'a level equivalent to 50 mg/kg/day. INTRODUCTION Laurie d i e t h a n o l a m i d e ( N , N ' - b i s ( h y d r o x y e t h y l ) l a u r a m i d e ; L D E ) finds a p p l i c a t i o n in f o o d - p a c k a g i n g m a t e r i a l s a n d a p p r o v a l has a l r e a d y been g r a n t e d in the U S A for specified uses in p a p e r , adhesive a n d textile f o r m u l a t i o n s (Title 21, C h a p t e r I o f the U S C o d e o f F e d e r a l Regulations, Sections 121.2526, 2519, 2520, 2535, 2536). F u r t h e r m o r e , a petition was filed on 11 A u g u s t 1965 with the F D A (Federal Register 1965, 30, 10211) to allow the use o f L D E as an antistatic agent in polyethylene containers for a n y foodstuffs except m i l k a n d b a b y food. A l t h o u g h in some cases the levels o f usage m a y be as high as 1% o f the finished p r o d u c t , in m a n y a p p l i c a t i o n s the c o n c e n t r a t i o n is m u c h lower. E x t r a c t a b i l i t y m a y be expected to be high since, to be an effective antistatic agent, the m a t e r i a l m u s t migrate freely to the surface. F r o m a toxicological viewpoint, a l t h o u g h the effects o f single a n d repeated doses o f L D E have been investigated, n o d a t a a p p e a r to be available on short- or l o n g - t e r m toxicity. The acute LDso in mice was estimated to be 9.65 a n d 0.287 g/kg b y the oral a n d intravenous routes respectively ( H o p p e r , H u l p i e u & Cole, 1949). I n rats, r a b b i t s a n d guinea-pigs no deaths o r p a t h o l o g i c a l changes were r e p o r t e d with single oral doses o f u p to 3 g/kg (Professor A. C. Frazer, u n p u b l i s h e d d a t a in 1962). H o w e v e r , a single oral dose o f 5 g/kg to rats i n d u c e d some thickening o f the collecting ducts o f the kidney with hyaline d e g e n e r a t i o n b u t g l o m e r u l a r d a m a g e was n o t observed. T h e livers o f these animals showed fatty change a n d nuclear abnormalities. I n further studies, i n t r a p e r i t o n e a l o r s u b c u t a n e o u s injection caused irritation a n d intense p e r i t o n e a l r e a c t i o n or sterile abscess f o r m a t i o n , thus preventing an estimate o f the intrinsic p a r e n t e r a l toxicity. 497
498
I F. GAUNT, MADGE FARMER, P. GRASSO
and
S. D. GANGOLL1
Limited reformation is available on the effects of repeated doses of LDE. Hopper et al. (1949) reported one death in a group of 10 mice given 25 daily oral doses, equivalent to onetenth of the LD50 (about 1 g/kg). According to Professor A. C. Frazer (unpublished data in 1962) dietary levels of 3.75 and 6"257oo but not 1-25% LDE when fed to rats for 10 days reduced body weight gain but this effect was attributed to an adverse effect on the palatability of the diet. The present study was initiated to investigate the effects of short-term feeding of LDE to rats. EXPERIMENTAL Materials. The sample of LDE used was supplied by Marchon Products Ltd., Whitehaven, Cumberland and details of the manufacturer's specification are as follows: LDE (92.36 ~ by difference); free diethanolamine (5.6 ~o); free alkali, as K O H (0-08 ~o); petroleum ether extractive (1.96 %). The petroleum ether extract was stated to consist mainly of fatty acids and esters thereof (52 and 30 %, respectively), fatty alcohols and sterols (10%); no nitrogen compounds were found present. Animals and diet. SPF rats of the Carworth Farm E strain were used and were maintained on powdered Spillers' Small Laboratory Animal Diet. Experimental design and conduct. Groups of 15 male and 15 female weanhng rats housed five/cage were fed diets containing 0.0 (control), 0.1, 0.5, 1.0 and 2.0 % LDE for 90 days. Food and water were supplied ad lib. Weekly records were made of body weight and food consumption. Terminal haematological examination involved determinations of total erythrocyte count, haematocrit, haemoglobin concentration, reticulocyte count, total and differentlal leucocyte counts. Liver and kidney function tests were carried out terminally. Levels of serum glutamic-oxaloacetic and glutamic-pyruvic transaminases and of blood urea were determined. The urine was examined for colour, pH, microscopic constituents, protein, reducing substances, bile salts and blood and activity of glutamic-oxaloacetic transaminase. A concentration test was conducted in which measurements were made of the volume and specific grawty of the urine excreted during a 6-hr period of water deprivation and during a 4-hr period commencing 16 hr after a water load of 25 ml/kg. At autopsy, the gross appearance of the organs and the weights of brain, heart, hver, kidneys, adrenals and gonads were recorded. Paraffin wax sections of these organs together with a wide range of other organs were stained with haematoxylin and eosin for histological examination. Smears of femoral marrow were stained by the May Grunwald-Glemsa method. Palatability test. Pairs of male rats were allowed access to stock d~et and to d~et containing either one of the four d~etary test levels of LDE. The consumption of both diets was recorded for a period of 8 days. RESULTS In general, the rats remained m good health apart from two males on 1% LDE, which were killed on days 23 and 58 because of weight loss and respiratory distress. Extensive lung abscess formation was seen at autopsy and bronchopneumonia was confirmed histologically. Growth retardation occurred throughout the study at the two highest levels of 1.0 and 2-0 ~o in both sexes (Table 1). At 0.5 %, growth was inhibited throughout the test in males but only in the later stages in females, while at 0.1% no effect was seen in either sex. Food intake was reduced at all dietary levels except the lowest level of 0.1 ~o (Table 1). This effect
SHORT-TERM TOXICITY OF LAURIC DIETHANOLAM1DE
499
w a s m a m f e s t t h r o u g h o u t t h e s t u d y in b o t h sexes o n 2 % a n d m m a l e s o n 1 % L D E , b u t o t h e r w i s e a p p e a r e d in t h e r e m a i n i n g a f f e c t e d g r o u p s in t h e s e c o n d h a l f o f t h e s t u d y . T h e r e w a s a c o n s i d e r a b l e s c a t t e r i n g a n d w a s t a g e o f d i e t a t t h e 2.0 % level. I n t h e p a l a t a b i l i t y test, e x c l u s i v e p r e f e r e n c e w a s s h o w n t o t h e c o n t r o l diet, v i r t u a l l y n o t e s t . d i e t b e i n g c o n s u m e d a t a n y o f t h e d i e t a r y levels i n c o r p o r a t e d . Th~s o b s e r v a t i o n s u g g e s t s t h a t t o x i c a n o r e x i a w a s n o t the cause of reduced food intake. Table 1
Dietary level (%)
Mean values of body weight, food consumption and LDE retake of rats fed LDE at 0-2% of the dtet for 90 days Food consumption (g/rat/day) at wk
Body weight (g) at wk 02
4
8
13
0~
00 01 05 10 2'0
118 123 118 122 123
307 299 289** 273*** 233***
397 379 366** 342*** 282***
417 410 387** 391"** 302***
00 0 1 0'5 10 2-0
107 106 Ill 110 110
198 198 191 187"* 165"**
229 234 223 216"* 184"**
249 252 237* 232*** 198"**
Males 13 9 133 13 1 13 5 14 2
Intake of LDE (g/kg/day)t at wk
4
8
13
19-6 184 18 3 19 1 17 2
21 4 222 19'5 18 3 16"2
24 1 225 22 5 22'7 21'6
13'5 14 7 145 13-2 11'7
164 14"5 132 15 3 14 0
02
4
8
13
. . . 012 006 0 60 0 32 1 16 0"66 2 36 1 48
. 006 0-27 0 53 1 14
0-06 0-29 0-58 1"34
. . . 0 12 0 07 060 037 1 12 0 73 2 34 1 52
. 0-06 0-31 0 59 1 22
0 06 0"28 0'66 1'42
Females
11 3 11 9 124 11 6 12 7
13-9 14 7 141 13 7 12 7
"l-Calculated from data on body weight and food consumption. 2Day 1 of feeding. Values of body weight are the means for groups of 15 ammals. Values of food consumption are the means for 3 cages of 5 ammals. Values marked with asterisks differ s~gnificantly from those of controls: *P <0-05; **P <0-01 ; ***P <0.005. Table 2.
Termmal haematological response in rats fed LDE at 0-2 ~ of the dtet for 90 days Leucocytes Differential ( %)t
Dietary level (~)
Hb (g/lO0 ml)
HC (%)
RBC (lO°/mm a)
MCHbC (~)
00 0'1 05 1.0 20
161 16 0 158 15"3 150"
47 46 45 44 43
856 8'65 877 8 77 8'70
343 34'8 35"2 34"8 34"9
00 0 1 05 1"0 20
151 14 5 145 13'1"** 12-0"**
43 42 41 39 36
787 8"09 8-01 7 19"* 7"16"
352 34"6 354 33-6 33'3
MCV (/,3)
Ret,cs (% of RBC)
Total (lOa/mm a)
N
E
L
M
08 07 07 0'6 0"6
864 8"02 548 6 39 4'99
16 16 15 14 14
1 1 2 2 1
76 76 76 76 76
7 7 7 9 8
05 07 06 06 0'7
398 3-93 3'77 3-72 4-14
15 12 15 13 8
2 1 2 1 1
76 82 76 78 81
6 5 10 8 10
Males 55 53 51 50 50
Females
55 52 51 54 50
RBC.~-Red blood cell HC--Haematocnt Hb~-Haemoglobm M C H b C ~ - M e a n cell haemoglobin concn M C V - - M e a n cell volume N~Neutrophds E~Eosinophils ~Lymphocytes M~Monocytes "l'Basophils were absent except at 1% of the dlfferentml leucocyte count m the control male group. Values marked with asterisks differ significantly from those of controls: *P <0.05; **P < 0 01 ; ***P <0.001
500
1. F. GAUNT, MADGE FARMER, P. GRASSOa n d s. D. GANGOLLI
Haematological e x a m i n a t i o n revealed reductions in h a e m o g l o b i n levels, haematocrit values a n d red cell counts in females at the 1 a n d 2~0 levels b u t these effects were m u c h less p r o n o u n c e d in males (Table 2). E x a m i n a t i o n of the femoral m a r r o w smears showed n o deviation from normality.
N o u n t o w a r d effect was seen in the renal f u n c t i o n tests (Table 3). Serum levels of glutamic--oxaloacetic t r a n s a m i n a s e were significantly elevated at dietary levels of 0.5 ~o a n d above in females b u t only at the 0.5 ~o level in males.
Table 3. Terminal serum and urine analysis o f rats f e d L DE at 0-2 % o f the diet for 90 days
Urine Concentratmntest Serum chemistry Dietary level (~o)
Urea (mg/100 ml)
GOT (ILl)
GPT (IU)
Specific gravityf pH
0"0 0"1 0"5 1"0 2"0
21"8 ~'8 22"7 23"2 19"4
63 80 100# 72 80
19 23 19 19 17
64 6"3 5"9 5"8 5"7
0"0 0"I 0"5 1"0 2"0
23"8 22"9 23"6 26"4 24"8
69 85 98# 93# 103#
18 17 11 11 10
5"8 6"4 5"9 6"0 6"1
GOT (IU)
Volume (ml)
Imtml
0-6 hr
16-20 hr
0--6 hr
16-20 hr
Males 4 7 2 4 6
4'7 3'4 4"5 4"9 4"8
5"3 5"4 4"2 4"9 6"2
8"4 7"5 7"8 6"0 8'5
5'2 4"3 5"4 8"0 5"3
1"8 2"8 2"1 2"8 1"7
Feamles 5 4 9 4 4
2"2 3"1 4"5 3"2 2"7
3"5 5"5 5"2 5"4 3"3
5"5 7"6 9"9 8"4 9"6
2"8 3"5 4"1 6"2 7"5
0"4 0"9 0"9 1"1 0"7
GOT-- Glutamic-oxaloacetic transaminase GPT--Glutamic-pyruvictransaminase ~-Expressed as (specific gravity x 100)--100. Results of serum chemistry are the means for groups of 15 animals and those of urine analysis are the means of two cages of five animals per group. Proteinurea was comparable in test and control groups. Tests for blood, bile salts and reducing substances were negative in all groups. Values marked with asterisks differ sJgnificantly from those of controls: *P <0"05.
A t autopsy, gross e x a m i n a t i o n revealed n o significant findings. The principal o r g a n weight changes were increases i n the relative kidney weight i n all test groups except at 0.1 ~o i n females a n d at 0.1 a n d 0.5 ~o in males a n d increases in the relative liver weight i n females o n the two highest levels (Table 4). O n histological e x a m i n a t i o n , the types a n d incidence o f pathological lesions were c o m p a r a b l e in c o n t r o l a n d test groups.
SHORT-TERM TOXICITYOF LAURIC DIETHANOLAMIDE
501
Table 4. Absolute and relative organ weights of rats fed LDE at 0-2 % of the diet for 90 days Organ weight Sex and dietary level (%)
Kidneys Brain
Heart
Liver
Spleen
Left
Right
Adrenals
Gonads
Terminal body weight (g)
Absolute organ weight (g) Male 0"0 0"1 0"5 1-0 2"0
1'94 1"97 1.95 2"04 1"90
1"44 1.45 1.35 1-33 1.07
14"54 14"15 13"60 14"85 I 1"46
0"80 0"73 0"67 0.72 0"57
1"29 1.31 1.32 1.36 1.07
1"33 1"31 1"34 1"45 I "08
0"055 0"067 0"063 0 066 0'054
3'56 3"45 3"48 3"57 3"56
4O9 408 391 388 301
Female 00 0"1 0"5 10 2-0
I "84 1"90 1"88 1"95 1 90
1"04 0"98 1"01 1"02 0"91
7"85 8"27 7"34 7"96 8"44
0'59 0'62 0'55 0"58 0"50
0"91 1"00. 1"02"* 1'08"** 0"92
0"91 0"99** 1 01"* 1"08"* 0"93
0 078 0'079 0"079 0'077 0'064
0'24 0'25 0"26 0'24 0"23
253 253 232 228 197
Relative organ weight (g/100 g body weight) Male 0.0 0.1 0.5 1.0 2"0
0.49 0.36 0.48 0.36 0.50 0.35 0.53 0.34 0"63j** 0"36
3.58 3-43 3.48 3.81 3"81
Female 0.0 0.1 0.5 1.0 2.0
0"73 0"76 0"82* 0"85*** 0"97***
3"11 0 23 3"28 0'24 3"18 0"24 3"49* 0"26 4"30*** 0"25
0"41 0"39 0"44 0"45 0"46
0.20 0.18 0.17 0"19 0'19
0.32 0.32 0.34 0-35* 0"36*
0.33 0.32 0.34 0.38* 0'36
0"36 0'36 0"39 0"39 0"44*** 0"44*** 0"48*** 0'47*** 0"47***0"47***
0.015 0.016 0.016 0.017 0'019"* 0"031 0 033 0"034 0"034 0'032
0.89 0.84 "0.89 0.84 1"19"** 0'10 0"11 0"11 0"11 0"12
Values are the means for groups of 15 animals and those marked with asterisks differ sigmfieantly from those of controls: *P <0.05; **P <0.01 ; ***P <0.001. DISCUSSION Feeding of L D E to rats at dietary levels of 0 - 2 ~o for 90 days produced n o observable adverse effect at the 0.1 ~o level or deaths at any level. G r o w t h r e t a r d a t i o n observed at a n d above 0.5 ~o appears to be due to a reduced food intake which o n the basis of the preference test a n d the scattering of food can be attributed to a n u n p a l a t a b l e diet. Sharratt (1961) showed that in general the degree of scattering was a n indication that the diet was unacceptable to the animals. Haematological e x a m i n a t i o n provided evidence of a n a n a e m i a b u t the n o r m a l appearance o f the marrow, n o r m a l retieulocyte c o u n t a n d other observations indicate that the cause was n o t m a r r o w depression or red cell haemolysis. It is possible that the reduced food intake m a y have led to some slight n u t r i t i o n a l deficiency, especially at the 2"0~o level where scattering of the diet would lead to a n even lower c o n s u m p t i o n t h a n that indicated in Table 1, since the m e t h o d of m e a s u r i n g food intake, i.e. by weighing food pots, does n o t take a c c o u n t of scattering.
502
I.F. GAUNT, MADGE FARMER, P. GRASSO a n d s. D. GANGOLLI
T h e i n c r e a s e d level o f s e r u m g l u t a m l c - o x a l o a c e t i c t r a n s a m i n a s e was n o t a s s o c i a t e d w i t h d e t e c t a b l e o r g a n d a m a g e , a l t h o u g h liver w e i g h t was raised in t h o s e g r o u p s d i s p l a y i n g t h e g r e a t e s t increase in t h e a c t i w t y o f th~s e n z y m e . T h e i n c r e a s e d ratios o f liver a n d k i d n e y w e i g h t to b o d y w e i g h t were m a i n l y f o u n d in t h o s e g r o u p s d i s p l a y i n g significant g r o w t h r e t a r d a t l o n . W h e n e x p r e s s e d r e l a t i v e to b r a i n w e i g h t these increases were n o l o n g e r a p p a r e n t a n d h e n c e a p p e a r to be a reflection o f r e d u c e d b o d y w e i g h t gain. Finally, a no-effect level was e s t a b l i s h e d o f 0 . 1 % L D E in the dlet o f rats f o r 90 days, a level e q u i v a l e n t to 50 m g / k g / d a y .
REFERENCES Hopper, S. S., Hulpleu, H. R. & Cole, Vera V. (1949). Some toxicological properties of surface active agents. J. Am. pharm. Ass. 38, 428 Sharratt, M. (1961). The Aswsment of ToxwlO,--Studws m Body Weight Changes. Ph D. Thesis, University of Birmingham.
Etude de radministration fi court terme du di~thanolamide laurique dans l'alimentation du rat R&umg----Le dl6thanolam~de laurlque (DEL) a fait l'objet d'une 6tude dt6t6tique ~ court terme chez le rat. Admmlstr6 b. des doses respectives de 0 (pour le groupe t6moin), 0,1, 0,5, Iet 2% pendant 90 jours, le DEL n'a exerc6aucune influence d6favourable sur l'ast?ect ni sur l'6tat des rats. Dans les groupes de 0,5 ~ et plus, le retard de croissance a 6t6 associ6 ~t une d~mmutxon de la consommation d'ahments. Le refus de nourriture a 6t6 manifestement imputable ~ l'effet que le mat6nel utilis6 pour l'6tude a eu sur le goOt des aliments. L'examen h6matologlque final a fret apparaitre une &minutmn de la teneur en h6moglobine, des valeurs de l'h6matocnte et de la num6ratlon globulaire chez les rats femelles dans les groupes de 1 et 2%; ches les m~tles, les effets &aient morns prononc6s. Une 616vation de la transammase glutamlno--oxalac&tque dans le s6rum sanguin a 6t6 not6e chez les femelles des groupes de 0,5 % et plus; chez les mb.les, cette 616vatlon n'int6ressait que le seul groupe de 0,5 %. Aucune perturbation de la fonct~on r6nale n'a pu 6tre observ6e. Des changements de polds sensibles ont 6t6 relev6s au ntveau des organes smvants augmentation du polds relatif du rein chez les femelles dans tousles groupes sauf le groupe de 0,1%, et chez les mfiles darts les groupes de 0,1 et 0,5%; augmentation du poids relatff du foie chez les femelles dans les deux groupes aux teneurs les plus 616vees. Le type et la fr6quence des 16sions hlstologiques chez les rats t6moins 6taJent comparables aux observations faites dans les groupes trait6s. On a &abh que le mveau auquel les effets sont mexlstants est de 0,1% de DEL dans l'ahmentahon des rats durant 90 jours, ce qul 6quivaut ~. 50 mg par kg de polds par jour.
Kurzzeitiger Verfiitterungsversuch von Laurins~iuredi~ithanolamid an Ratten Zusammenfassung--Em
kurzzeltiger Verftitterungsversuch mit Laurlnsaure&athanolamid (LDA) wurde an Ratten durchgefuhrt. Die 90t~iglge Verabreichung von 0 (Kontrolle), 0,1, 0,5, 1,0 und 2 , 0 ~ LDA im Futter nef keme nachtelhge Wirkung auf das Aussehen oder den Zustand der Tlere hervor. Wachstumsverzogerung wurde mit vermmderter Futteraufnahme bet und uber der Konzentration von 0,5 % m Verbmdung gebracht. Die Futterverweigerung war nachwelslich auf die Geschmacksbeeintrachtigung des Futters durch die Versuchssubstanz zurtickzufuhren. Die termmale hamatologlsche Untersuchung ergab eine Vermmderung des Hamoglobmgehalts, des Hamatokritwertes und der Erythrozytenzahl bei den welblichen Tleren, an die I u n d 2% verabreicht worden waren; bei den m~mnlichen Tieren wurden weniger deuthche Wlrkungen beobachtet. Der Gehalt des Serums an Glutaminoxaloesslgsituretransaminase war bei der Verabreichung yon 0,5 % und dartiber bei weiblichen Tieren, aber nur bei Verabreichung von 0,5 % bel mannlichen Tleren erhoht. Kelne nachteilige Wirkung wurde bei den Nierenfunktionspriifungen beobachtet. Die haupts~chlichen Organgewichtsvera.nderungen waren: Zunahme des relativen Nierengewichts bei allen Versuchsgruppen,
SHORT-TERM TOXICITY OF LAURIC DIETHANOLAMIDE ausser bel 0,1 ~/o bel welblichen und bei 0,1 und 0,5 ~/o be1 mannhchen Tleren, und Zunahme des relatlven Lebergewlchts bel weiblichen Tieren nur bel den belden hochsten Konzentratlonen. Art und Haufigkeit der hlstologlschen Lasionen waren bei den Kontroll- und den Versuchsgruppen verglelchbar. Die wirkungsfreie Konzentration wurde mlt 0,1 ~, LDA lm Futter yon Ratten uber einen Zeitraum yon 90 Tagen festgestellt, eine Konzentratlon, die 50 mg/l~g/Tag entspricht.
503
Short-term Feeding Study of Lauric Diethanolamide in Rats I. F. GAUNT--Toxicology; MADGE FARMER a n d P. GRASSO---Pathology; a n d S. D. GANGOLLI--Analytical Chemistry British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey, England (Received 3 October 1966) Abstract A short-term feeding study has been earned out on laurie diethanolamlde (LDE) in the rat. Dietary levels of 0-0 (control),0 1,0-5,1-0 and 2 0% for 90 days evoked no adverse effect on the appearance or conditmn of the ammals. Growth retardation was associated w~th d~mmished food intake, at and above the 0"5 % level. Food refusal was demonstrably due to an effect of the test material on palatabihty of the d~et. Terminal haematologleal exammation revealed a reduction in the haemoglobin level, haematocrit and red cell count at the 1 and 2 % levels in females but less pronounced effects were seen in males. Serum levels of glutamic-oxaloaeetic transarninase were elevated at dietary levels of 0 5 % and above m females but only at 0"5 Yoin males. No untoward effect was observed in the renal functlon tests. The principal organ weight changes were: increases in the relative kidney weight m all test groups except at 0-1 Yom females and at 0-1 and 0"5 ~ in males, and increases in the relative hver weight in females on the two highest levels. The types and incidence of histological leslons were comparable m control and test groups. A no-effect level was established of0.1% LDE in the diet of rats for 90 days,'a level equivalent to 50 mg/kg/day. INTRODUCTION Laurie d i e t h a n o l a m i d e ( N , N ' - b i s ( h y d r o x y e t h y l ) l a u r a m i d e ; L D E ) finds a p p l i c a t i o n in f o o d - p a c k a g i n g m a t e r i a l s a n d a p p r o v a l has a l r e a d y been g r a n t e d in the U S A for specified uses in p a p e r , adhesive a n d textile f o r m u l a t i o n s (Title 21, C h a p t e r I o f the U S C o d e o f F e d e r a l Regulations, Sections 121.2526, 2519, 2520, 2535, 2536). F u r t h e r m o r e , a petition was filed on 11 A u g u s t 1965 with the F D A (Federal Register 1965, 30, 10211) to allow the use o f L D E as an antistatic agent in polyethylene containers for a n y foodstuffs except m i l k a n d b a b y food. A l t h o u g h in some cases the levels o f usage m a y be as high as 1% o f the finished p r o d u c t , in m a n y a p p l i c a t i o n s the c o n c e n t r a t i o n is m u c h lower. E x t r a c t a b i l i t y m a y be expected to be high since, to be an effective antistatic agent, the m a t e r i a l m u s t migrate freely to the surface. F r o m a toxicological viewpoint, a l t h o u g h the effects o f single a n d repeated doses o f L D E have been investigated, n o d a t a a p p e a r to be available on short- or l o n g - t e r m toxicity. The acute LDso in mice was estimated to be 9.65 a n d 0.287 g/kg b y the oral a n d intravenous routes respectively ( H o p p e r , H u l p i e u & Cole, 1949). I n rats, r a b b i t s a n d guinea-pigs no deaths o r p a t h o l o g i c a l changes were r e p o r t e d with single oral doses o f u p to 3 g/kg (Professor A. C. Frazer, u n p u b l i s h e d d a t a in 1962). H o w e v e r , a single oral dose o f 5 g/kg to rats i n d u c e d some thickening o f the collecting ducts o f the kidney with hyaline d e g e n e r a t i o n b u t g l o m e r u l a r d a m a g e was n o t observed. T h e livers o f these animals showed fatty change a n d nuclear abnormalities. I n further studies, i n t r a p e r i t o n e a l o r s u b c u t a n e o u s injection caused irritation a n d intense p e r i t o n e a l r e a c t i o n or sterile abscess f o r m a t i o n , thus preventing an estimate o f the intrinsic p a r e n t e r a l toxicity. 497
498
I F. GAUNT, MADGE FARMER, P. GRASSO
and
S. D. GANGOLL1
Limited reformation is available on the effects of repeated doses of LDE. Hopper et al. (1949) reported one death in a group of 10 mice given 25 daily oral doses, equivalent to onetenth of the LD50 (about 1 g/kg). According to Professor A. C. Frazer (unpublished data in 1962) dietary levels of 3.75 and 6"257oo but not 1-25% LDE when fed to rats for 10 days reduced body weight gain but this effect was attributed to an adverse effect on the palatability of the diet. The present study was initiated to investigate the effects of short-term feeding of LDE to rats. EXPERIMENTAL Materials. The sample of LDE used was supplied by Marchon Products Ltd., Whitehaven, Cumberland and details of the manufacturer's specification are as follows: LDE (92.36 ~ by difference); free diethanolamine (5.6 ~o); free alkali, as K O H (0-08 ~o); petroleum ether extractive (1.96 %). The petroleum ether extract was stated to consist mainly of fatty acids and esters thereof (52 and 30 %, respectively), fatty alcohols and sterols (10%); no nitrogen compounds were found present. Animals and diet. SPF rats of the Carworth Farm E strain were used and were maintained on powdered Spillers' Small Laboratory Animal Diet. Experimental design and conduct. Groups of 15 male and 15 female weanhng rats housed five/cage were fed diets containing 0.0 (control), 0.1, 0.5, 1.0 and 2.0 % LDE for 90 days. Food and water were supplied ad lib. Weekly records were made of body weight and food consumption. Terminal haematological examination involved determinations of total erythrocyte count, haematocrit, haemoglobin concentration, reticulocyte count, total and differentlal leucocyte counts. Liver and kidney function tests were carried out terminally. Levels of serum glutamic-oxaloacetic and glutamic-pyruvic transaminases and of blood urea were determined. The urine was examined for colour, pH, microscopic constituents, protein, reducing substances, bile salts and blood and activity of glutamic-oxaloacetic transaminase. A concentration test was conducted in which measurements were made of the volume and specific grawty of the urine excreted during a 6-hr period of water deprivation and during a 4-hr period commencing 16 hr after a water load of 25 ml/kg. At autopsy, the gross appearance of the organs and the weights of brain, heart, hver, kidneys, adrenals and gonads were recorded. Paraffin wax sections of these organs together with a wide range of other organs were stained with haematoxylin and eosin for histological examination. Smears of femoral marrow were stained by the May Grunwald-Glemsa method. Palatability test. Pairs of male rats were allowed access to stock d~et and to d~et containing either one of the four d~etary test levels of LDE. The consumption of both diets was recorded for a period of 8 days. RESULTS In general, the rats remained m good health apart from two males on 1% LDE, which were killed on days 23 and 58 because of weight loss and respiratory distress. Extensive lung abscess formation was seen at autopsy and bronchopneumonia was confirmed histologically. Growth retardation occurred throughout the study at the two highest levels of 1.0 and 2-0 ~o in both sexes (Table 1). At 0.5 %, growth was inhibited throughout the test in males but only in the later stages in females, while at 0.1% no effect was seen in either sex. Food intake was reduced at all dietary levels except the lowest level of 0.1 ~o (Table 1). This effect
SHORT-TERM TOXICITY OF LAURIC DIETHANOLAM1DE
499
w a s m a m f e s t t h r o u g h o u t t h e s t u d y in b o t h sexes o n 2 % a n d m m a l e s o n 1 % L D E , b u t o t h e r w i s e a p p e a r e d in t h e r e m a i n i n g a f f e c t e d g r o u p s in t h e s e c o n d h a l f o f t h e s t u d y . T h e r e w a s a c o n s i d e r a b l e s c a t t e r i n g a n d w a s t a g e o f d i e t a t t h e 2.0 % level. I n t h e p a l a t a b i l i t y test, e x c l u s i v e p r e f e r e n c e w a s s h o w n t o t h e c o n t r o l diet, v i r t u a l l y n o t e s t . d i e t b e i n g c o n s u m e d a t a n y o f t h e d i e t a r y levels i n c o r p o r a t e d . Th~s o b s e r v a t i o n s u g g e s t s t h a t t o x i c a n o r e x i a w a s n o t the cause of reduced food intake. Table 1
Dietary level (%)
Mean values of body weight, food consumption and LDE retake of rats fed LDE at 0-2% of the dtet for 90 days Food consumption (g/rat/day) at wk
Body weight (g) at wk 02
4
8
13
0~
00 01 05 10 2'0
118 123 118 122 123
307 299 289** 273*** 233***
397 379 366** 342*** 282***
417 410 387** 391"** 302***
00 0 1 0'5 10 2-0
107 106 Ill 110 110
198 198 191 187"* 165"**
229 234 223 216"* 184"**
249 252 237* 232*** 198"**
Males 13 9 133 13 1 13 5 14 2
Intake of LDE (g/kg/day)t at wk
4
8
13
19-6 184 18 3 19 1 17 2
21 4 222 19'5 18 3 16"2
24 1 225 22 5 22'7 21'6
13'5 14 7 145 13-2 11'7
164 14"5 132 15 3 14 0
02
4
8
13
. . . 012 006 0 60 0 32 1 16 0"66 2 36 1 48
. 006 0-27 0 53 1 14
0-06 0-29 0-58 1"34
. . . 0 12 0 07 060 037 1 12 0 73 2 34 1 52
. 0-06 0-31 0 59 1 22
0 06 0"28 0'66 1'42
Females
11 3 11 9 124 11 6 12 7
13-9 14 7 141 13 7 12 7
"l-Calculated from data on body weight and food consumption. 2Day 1 of feeding. Values of body weight are the means for groups of 15 ammals. Values of food consumption are the means for 3 cages of 5 ammals. Values marked with asterisks differ s~gnificantly from those of controls: *P <0-05; **P <0-01 ; ***P <0.005. Table 2.
Termmal haematological response in rats fed LDE at 0-2 ~ of the dtet for 90 days Leucocytes Differential ( %)t
Dietary level (~)
Hb (g/lO0 ml)
HC (%)
RBC (lO°/mm a)
MCHbC (~)
00 0'1 05 1.0 20
161 16 0 158 15"3 150"
47 46 45 44 43
856 8'65 877 8 77 8'70
343 34'8 35"2 34"8 34"9
00 0 1 05 1"0 20
151 14 5 145 13'1"** 12-0"**
43 42 41 39 36
787 8"09 8-01 7 19"* 7"16"
352 34"6 354 33-6 33'3
MCV (/,3)
Ret,cs (% of RBC)
Total (lOa/mm a)
N
E
L
M
08 07 07 0'6 0"6
864 8"02 548 6 39 4'99
16 16 15 14 14
1 1 2 2 1
76 76 76 76 76
7 7 7 9 8
05 07 06 06 0'7
398 3-93 3'77 3-72 4-14
15 12 15 13 8
2 1 2 1 1
76 82 76 78 81
6 5 10 8 10
Males 55 53 51 50 50
Females
55 52 51 54 50
RBC.~-Red blood cell HC--Haematocnt Hb~-Haemoglobm M C H b C ~ - M e a n cell haemoglobin concn M C V - - M e a n cell volume N~Neutrophds E~Eosinophils ~Lymphocytes M~Monocytes "l'Basophils were absent except at 1% of the dlfferentml leucocyte count m the control male group. Values marked with asterisks differ significantly from those of controls: *P <0.05; **P < 0 01 ; ***P <0.001
500
1. F. GAUNT, MADGE FARMER, P. GRASSOa n d s. D. GANGOLLI
Haematological e x a m i n a t i o n revealed reductions in h a e m o g l o b i n levels, haematocrit values a n d red cell counts in females at the 1 a n d 2~0 levels b u t these effects were m u c h less p r o n o u n c e d in males (Table 2). E x a m i n a t i o n of the femoral m a r r o w smears showed n o deviation from normality.
N o u n t o w a r d effect was seen in the renal f u n c t i o n tests (Table 3). Serum levels of glutamic--oxaloacetic t r a n s a m i n a s e were significantly elevated at dietary levels of 0.5 ~o a n d above in females b u t only at the 0.5 ~o level in males.
Table 3. Terminal serum and urine analysis o f rats f e d L DE at 0-2 % o f the diet for 90 days
Urine Concentratmntest Serum chemistry Dietary level (~o)
Urea (mg/100 ml)
GOT (ILl)
GPT (IU)
Specific gravityf pH
0"0 0"1 0"5 1"0 2"0
21"8 ~'8 22"7 23"2 19"4
63 80 100# 72 80
19 23 19 19 17
64 6"3 5"9 5"8 5"7
0"0 0"I 0"5 1"0 2"0
23"8 22"9 23"6 26"4 24"8
69 85 98# 93# 103#
18 17 11 11 10
5"8 6"4 5"9 6"0 6"1
GOT (IU)
Volume (ml)
Imtml
0-6 hr
16-20 hr
0--6 hr
16-20 hr
Males 4 7 2 4 6
4'7 3'4 4"5 4"9 4"8
5"3 5"4 4"2 4"9 6"2
8"4 7"5 7"8 6"0 8'5
5'2 4"3 5"4 8"0 5"3
1"8 2"8 2"1 2"8 1"7
Feamles 5 4 9 4 4
2"2 3"1 4"5 3"2 2"7
3"5 5"5 5"2 5"4 3"3
5"5 7"6 9"9 8"4 9"6
2"8 3"5 4"1 6"2 7"5
0"4 0"9 0"9 1"1 0"7
GOT-- Glutamic-oxaloacetic transaminase GPT--Glutamic-pyruvictransaminase ~-Expressed as (specific gravity x 100)--100. Results of serum chemistry are the means for groups of 15 animals and those of urine analysis are the means of two cages of five animals per group. Proteinurea was comparable in test and control groups. Tests for blood, bile salts and reducing substances were negative in all groups. Values marked with asterisks differ sJgnificantly from those of controls: *P <0"05.
A t autopsy, gross e x a m i n a t i o n revealed n o significant findings. The principal o r g a n weight changes were increases i n the relative kidney weight i n all test groups except at 0.1 ~o i n females a n d at 0.1 a n d 0.5 ~o in males a n d increases in the relative liver weight i n females o n the two highest levels (Table 4). O n histological e x a m i n a t i o n , the types a n d incidence o f pathological lesions were c o m p a r a b l e in c o n t r o l a n d test groups.
SHORT-TERM TOXICITYOF LAURIC DIETHANOLAMIDE
501
Table 4. Absolute and relative organ weights of rats fed LDE at 0-2 % of the diet for 90 days Organ weight Sex and dietary level (%)
Kidneys Brain
Heart
Liver
Spleen
Left
Right
Adrenals
Gonads
Terminal body weight (g)
Absolute organ weight (g) Male 0"0 0"1 0"5 1-0 2"0
1'94 1"97 1.95 2"04 1"90
1"44 1.45 1.35 1-33 1.07
14"54 14"15 13"60 14"85 I 1"46
0"80 0"73 0"67 0.72 0"57
1"29 1.31 1.32 1.36 1.07
1"33 1"31 1"34 1"45 I "08
0"055 0"067 0"063 0 066 0'054
3'56 3"45 3"48 3"57 3"56
4O9 408 391 388 301
Female 00 0"1 0"5 10 2-0
I "84 1"90 1"88 1"95 1 90
1"04 0"98 1"01 1"02 0"91
7"85 8"27 7"34 7"96 8"44
0'59 0'62 0'55 0"58 0"50
0"91 1"00. 1"02"* 1'08"** 0"92
0"91 0"99** 1 01"* 1"08"* 0"93
0 078 0'079 0"079 0'077 0'064
0'24 0'25 0"26 0'24 0"23
253 253 232 228 197
Relative organ weight (g/100 g body weight) Male 0.0 0.1 0.5 1.0 2"0
0.49 0.36 0.48 0.36 0.50 0.35 0.53 0.34 0"63j** 0"36
3.58 3-43 3.48 3.81 3"81
Female 0.0 0.1 0.5 1.0 2.0
0"73 0"76 0"82* 0"85*** 0"97***
3"11 0 23 3"28 0'24 3"18 0"24 3"49* 0"26 4"30*** 0"25
0"41 0"39 0"44 0"45 0"46
0.20 0.18 0.17 0"19 0'19
0.32 0.32 0.34 0-35* 0"36*
0.33 0.32 0.34 0.38* 0'36
0"36 0'36 0"39 0"39 0"44*** 0"44*** 0"48*** 0'47*** 0"47***0"47***
0.015 0.016 0.016 0.017 0'019"* 0"031 0 033 0"034 0"034 0'032
0.89 0.84 "0.89 0.84 1"19"** 0'10 0"11 0"11 0"11 0"12
Values are the means for groups of 15 animals and those marked with asterisks differ sigmfieantly from those of controls: *P <0.05; **P <0.01 ; ***P <0.001. DISCUSSION Feeding of L D E to rats at dietary levels of 0 - 2 ~o for 90 days produced n o observable adverse effect at the 0.1 ~o level or deaths at any level. G r o w t h r e t a r d a t i o n observed at a n d above 0.5 ~o appears to be due to a reduced food intake which o n the basis of the preference test a n d the scattering of food can be attributed to a n u n p a l a t a b l e diet. Sharratt (1961) showed that in general the degree of scattering was a n indication that the diet was unacceptable to the animals. Haematological e x a m i n a t i o n provided evidence of a n a n a e m i a b u t the n o r m a l appearance o f the marrow, n o r m a l retieulocyte c o u n t a n d other observations indicate that the cause was n o t m a r r o w depression or red cell haemolysis. It is possible that the reduced food intake m a y have led to some slight n u t r i t i o n a l deficiency, especially at the 2"0~o level where scattering of the diet would lead to a n even lower c o n s u m p t i o n t h a n that indicated in Table 1, since the m e t h o d of m e a s u r i n g food intake, i.e. by weighing food pots, does n o t take a c c o u n t of scattering.
502
I.F. GAUNT, MADGE FARMER, P. GRASSO a n d s. D. GANGOLLI
T h e i n c r e a s e d level o f s e r u m g l u t a m l c - o x a l o a c e t i c t r a n s a m i n a s e was n o t a s s o c i a t e d w i t h d e t e c t a b l e o r g a n d a m a g e , a l t h o u g h liver w e i g h t was raised in t h o s e g r o u p s d i s p l a y i n g t h e g r e a t e s t increase in t h e a c t i w t y o f th~s e n z y m e . T h e i n c r e a s e d ratios o f liver a n d k i d n e y w e i g h t to b o d y w e i g h t were m a i n l y f o u n d in t h o s e g r o u p s d i s p l a y i n g significant g r o w t h r e t a r d a t l o n . W h e n e x p r e s s e d r e l a t i v e to b r a i n w e i g h t these increases were n o l o n g e r a p p a r e n t a n d h e n c e a p p e a r to be a reflection o f r e d u c e d b o d y w e i g h t gain. Finally, a no-effect level was e s t a b l i s h e d o f 0 . 1 % L D E in the dlet o f rats f o r 90 days, a level e q u i v a l e n t to 50 m g / k g / d a y .
REFERENCES Hopper, S. S., Hulpleu, H. R. & Cole, Vera V. (1949). Some toxicological properties of surface active agents. J. Am. pharm. Ass. 38, 428 Sharratt, M. (1961). The Aswsment of ToxwlO,--Studws m Body Weight Changes. Ph D. Thesis, University of Birmingham.
Etude de radministration fi court terme du di~thanolamide laurique dans l'alimentation du rat R&umg----Le dl6thanolam~de laurlque (DEL) a fait l'objet d'une 6tude dt6t6tique ~ court terme chez le rat. Admmlstr6 b. des doses respectives de 0 (pour le groupe t6moin), 0,1, 0,5, Iet 2% pendant 90 jours, le DEL n'a exerc6aucune influence d6favourable sur l'ast?ect ni sur l'6tat des rats. Dans les groupes de 0,5 ~ et plus, le retard de croissance a 6t6 associ6 ~t une d~mmutxon de la consommation d'ahments. Le refus de nourriture a 6t6 manifestement imputable ~ l'effet que le mat6nel utilis6 pour l'6tude a eu sur le goOt des aliments. L'examen h6matologlque final a fret apparaitre une &minutmn de la teneur en h6moglobine, des valeurs de l'h6matocnte et de la num6ratlon globulaire chez les rats femelles dans les groupes de 1 et 2%; ches les m~tles, les effets &aient morns prononc6s. Une 616vation de la transammase glutamlno--oxalac&tque dans le s6rum sanguin a 6t6 not6e chez les femelles des groupes de 0,5 % et plus; chez les mb.les, cette 616vatlon n'int6ressait que le seul groupe de 0,5 %. Aucune perturbation de la fonct~on r6nale n'a pu 6tre observ6e. Des changements de polds sensibles ont 6t6 relev6s au ntveau des organes smvants augmentation du polds relatif du rein chez les femelles dans tousles groupes sauf le groupe de 0,1%, et chez les mfiles darts les groupes de 0,1 et 0,5%; augmentation du poids relatff du foie chez les femelles dans les deux groupes aux teneurs les plus 616vees. Le type et la fr6quence des 16sions hlstologiques chez les rats t6moins 6taJent comparables aux observations faites dans les groupes trait6s. On a &abh que le mveau auquel les effets sont mexlstants est de 0,1% de DEL dans l'ahmentahon des rats durant 90 jours, ce qul 6quivaut ~. 50 mg par kg de polds par jour.
Kurzzeitiger Verfiitterungsversuch von Laurins~iuredi~ithanolamid an Ratten Zusammenfassung--Em
kurzzeltiger Verftitterungsversuch mit Laurlnsaure&athanolamid (LDA) wurde an Ratten durchgefuhrt. Die 90t~iglge Verabreichung von 0 (Kontrolle), 0,1, 0,5, 1,0 und 2 , 0 ~ LDA im Futter nef keme nachtelhge Wirkung auf das Aussehen oder den Zustand der Tlere hervor. Wachstumsverzogerung wurde mit vermmderter Futteraufnahme bet und uber der Konzentration von 0,5 % m Verbmdung gebracht. Die Futterverweigerung war nachwelslich auf die Geschmacksbeeintrachtigung des Futters durch die Versuchssubstanz zurtickzufuhren. Die termmale hamatologlsche Untersuchung ergab eine Vermmderung des Hamoglobmgehalts, des Hamatokritwertes und der Erythrozytenzahl bei den welblichen Tleren, an die I u n d 2% verabreicht worden waren; bei den m~mnlichen Tieren wurden weniger deuthche Wlrkungen beobachtet. Der Gehalt des Serums an Glutaminoxaloesslgsituretransaminase war bei der Verabreichung yon 0,5 % und dartiber bei weiblichen Tieren, aber nur bei Verabreichung von 0,5 % bel mannlichen Tleren erhoht. Kelne nachteilige Wirkung wurde bei den Nierenfunktionspriifungen beobachtet. Die haupts~chlichen Organgewichtsvera.nderungen waren: Zunahme des relativen Nierengewichts bei allen Versuchsgruppen,
SHORT-TERM TOXICITY OF LAURIC DIETHANOLAMIDE ausser bel 0,1 ~/o bel welblichen und bei 0,1 und 0,5 ~/o be1 mannhchen Tleren, und Zunahme des relatlven Lebergewlchts bel weiblichen Tieren nur bel den belden hochsten Konzentratlonen. Art und Haufigkeit der hlstologlschen Lasionen waren bei den Kontroll- und den Versuchsgruppen verglelchbar. Die wirkungsfreie Konzentration wurde mlt 0,1 ~, LDA lm Futter yon Ratten uber einen Zeitraum yon 90 Tagen festgestellt, eine Konzentratlon, die 50 mg/l~g/Tag entspricht.
503