PKThree groups of pregnant rats were exposed to 1% nitrous oxide 6 hours a day, 5 days per week for the first, first and second, and all 3 weeks of gestation. The offspring were weighed and measured at weekly intervals. The following variables were evaluated in control and experimental groups: litter size, body weight, tail length and body length. Litter sizes in animals exposed for the first and second weeks of gestation were significantly smaller than the controls. A two-way nested statistical analysis of the data revealed that over an 8-week period the experimental rats were significantly smaller than the control rats of the same age. Analysis of the 95% confidence intervals revealed no particular pattern related to the timing of the exposure to nitrous oxide

Austin, J.

Cleaton-Jones, P. E.

et. al

Viera, E

English

Wits Institutional Repository on DSPACE

106 S A M |; I )  I I: S 1; T  Y n S K K I  I 21 Januarie 1978
Intermittent Exposure of Gravid Rats to 1% Nitrous Oxide 
and the Effect on the Postnatal Growth of their Offspring
E. VIEIRA. P. H. CLP.ATON-JONES. J. AUSTIN. P. L. PATTI
SUMMARY
Three groups of pregnant rats were exposed to 1% nitrous  
oxide 6 hours a day, 5 days per week fo r the firs t, firs t 
and second, and all 3 weeks of gestation. The o ffsp ring  
were w eighed and measured at weekly intervals. The fo l­
low ing variables were evaluated in contro l and experim en­
tal groups: litte r size, body weight, tail length and body 
length. L itte r sizes in animals exposed fo r the firs t and 
second weeks of gestation were s ign ifican tly  smaller than 
the con tro ls . A tw o-w ay nested s ta tis tica l analysis of the 
data revealed that over an 8-week period the experimental 
rats were s ign ifican tly  smaller than the control rats of the 
same age. A nalysis of the 95%  confidence intervals re ­
vealed no particu la r pattern related to the timing o f the 
exposure to n itrous oxide.
■V. Afr. nicil. J.. 53, 106 (1978).
'There is a growing concern today that anaesthetic gases 
which escape into the operating theatre may be hazardous 
to operating room personnel. Nitrous oxide (N.-O) may be 
one of the anaesthetic agents responsible for spontaneous 
abortion in female anaesthetists and theatre nurses,1 pos­
sibly owing to the teratogenic effect of the gas.2,3 The 
purpose o f this study was to determine the effect on the 
litter size and growth of the offspring when gravid rats 
had been intermittently exposed to a TV, N.O atmosphere 
during various stages of gestation.
MATERIALS AND METHODS
Twelve-week-old female Wistar strain albino rats were 
obtained from the South African Institute for Medical 
Research. Rats were mated by placing three female rats 
with one male rat in a cage in the evening. The following 
morning the females were examined for copulation plugs 
and mating was confirmed by examining vaginal smears 
for the presence of spermatozoa. In this study the time 
o f conception was taken to be midnight on the night of 
mating. The day o f the positive smear was designated day 
0, the next day 1, and so on. Pregnant rats were divided 
into 6 groups o f 8 each, of which 3 groups were subjected
MUG Dental Research Unit, University of (lie VVitwatersrand,
Johannesburg
I V I E J H A .  nil1. Mioiumioi..
i’. K. CUKATON-IONKS. ii.n.s.. m .ii. n.oii.. i-ii.n.. i>..\.
J. AUSTIN, n.v.so.
Department of Applied Mathematics, University of tile Wil- 
walersrand, Johannesburg
P. I’ATTf. si.se.
Date received: 26 July 1977.
to N :0 exposure during gestation and 3 groups served as 
controls. Of the experimental groups, the first group was 
exposed to N ;0  during their entire gestation (i.e. 3 weeks), 
for 6 hours per day (from lOhOO to 16h00), Monday to 
Friday. The second group was exposed for the first and 
second weeks of gestation, while the third group was 
exposed for the first week of gestation only.
The rats were exposed to N :0  by placing their cages in 
a specially constructed perspex environmental chamber,* 
into which a mixture of compressed air (9.9 I min) and 
NiO (0,11/min) was introduced. This llow rah- provided 
for It) changes of atmosphere per hour. 'To enstr an even 
distribution of the gas mixture it was passed ' ougli a 
centrifugal fan for mixing before entering th chamber. 
The gas mixture was vented through an exhaust duct out 
of the experimental room. T he positions of the cages in 
the chamber were alternated on a daily rotational basis to 
provide for any variations in exposure to the gas. The 
humidity in the chamber ranged between 50% and 60% 
and no CO.- build-up was delected. Standard mouse diet 
(Epol, Vcrecniging Consolidated Mills) and water were 
provided ail libitum to all the rats. T he control rats were 
exposed to atmospheric air in the same room as the ex­
perimental groups.
Each gravid female was isolated shortly before giving 
birth. A t birth all litter mates were examined for gross 
defects and the number in each litter was recorded.
A t weekly intervals the young rats were weighed, and 
tail and body length were measured with an apparatus 
described by Jcfferys/'
Statistical Analysis
The experimental format was a mixed two-way nested 
design. T he statistical analysis consisted of a nested two- 
way analysis of variance for a fixed main group effect 
(control and N :0  exposed groups) and random subgroup 
effects (rat litters). The former analysis was carried out by 
means of a modified formula according to Johnson and 
Leone." The probability values of the calculations were 
obtained from standard F-valuc tables.
Finally, the 95% confidence intervals of all the results 
o f all the groups at the various time intervals were 
examined to see which experimental group was significantly 
different from the controls.
A one-way analysis o f variance and Student’s / test using 
the within-groups mean square for pooled variance was 
applied to test for differences in litter size.
RESULTS
Litler Size
A total of 410 rals was born, of which 248 were control 
rats and 162 were experimental rats. The mean number
21 Jaiuiarv 1978 S A M t: n i < a i . .1 o u it n a i. 107
TABLE I. LITTER SIZE FOR VARIOUS RAT GROUPS
P eriod  o f S tu d e n t's  t test
e xp osure  to N u m b e r o f T o ta l Rats pe r lit te r — .
G ro u p N 20 /a ir l it te rs o ffs p r in g (m ean dz S D ) f P va lue
C ontro l None 8 248 10,4 ztz 2,3 — —
t 3 weeks 8 66 8,3 ziz 3,2 6,4 <0,001
2 t st and 2nd weeks 8 50 6,3 ziz 2,6 11,2 <0,001
3 1 st week 8 46 5,7 ztz 1,6 12,4 <0,001
of young rats per litter is shown in 'table I. There were 
significantly fewer rats per litter in all females exposed to 
l°f. N.O during gestation.
In Table II the mean body weights of both the N.O- 
exposed and the control animals are listed, as well as the 
values obtained through pooling of all the results o f all 
the experimental animals. Experimental animals were 
significantly lighter than the control rats throughout the 
8-week period. A t each of the weekly intervals there was 
a significant difference between the three experimental 
and the control groups. There were also significant 
differences between litters at all intervals except 7 weeks.
Table I I I  shows the results obtained in the tail length 
measurements. A t I and 2 weeks there were no statisti-
werc significant differences between the body lengths of 
rats in the N.O-exposed and the control groups for the 
first G weeks of the 8-week period.
Examination of the 95°,'. confidence intervals of the 
data at the various periods revealed no particular pattern 
of effects related to the timing of the exposure to N :0. 
On comparison of the N.-O-exposed groups, it appeared 
that N.O hail a greater effect on the growth of rats ex­
posed for only the first week o f the gestation. This observa­
tion was not constant throughout the 8-week period of the 
study. For body weight this was found during the first, 
fourth anil sixth weeks of growth, for body length during 
the first, third and fourth weeks o f growth, and for tail 
length during the third, fifth  and eighth weeks o f growth.
cally s ign ificant differences between the ta il lengths o f rats 
in the con tro l and the N^O-exposed groups, but thereafter 
the differences were h ig h ly  significant.
D I S C U S S I O N
The cond itions o f exposure to m ixtures o f N :0  a
The g ro w th  in body length is listed in Table  IV . There reporte il thus fa r vary  considerably. F ink c l  olC  e;
W e e k s C o n tro l
TABLE II. BODY WEIGHT (g)
P ooled va lu e s  fo r a ll ------------------
N 20 -e x p o s e d  g ro u p s  3 w e e ks
P eriod  e xp o se d  to  N 20  
1 st and 2nd w e e ks 1 st w eek
p o s tp a rtu m (m ean ziz S D ) (m ean 'iz  S D ) (m ean dz S D ) (m ean ziz S D ) (m ean d z  S D )
1 10,0 ziz 1,2 7 ,0 -dz 1,2* 8,1 ziz 1,6 8,3 ziz 1,4 6,5 ziz 0,53
2 17,6 4,7 15,5 dz 4,83* 17,2 ziz 2,7 16,1 d z  4,0 13,4 ziz 7,8
3 31,6 dz 4,5 24,5 d z  6,13* 27,7 d :  2,8 24,8 dz 4,8 20,9 dz 10,8
4 42,3 ztz 3,9 39,1 zL: 4 ,2 7 t 39,4 d: 3,5 41,9 ziz 5,6 31,2 d z  13,7
5 54,0 ziz 10,3 52,8 • 10,27* 50,6 ztz 5,6 58,0 ziz 14,3 50,0 ziz 10,9
6 67,0 d r  7,7 65,0 d z  11,23* 64,7 ziz 8,7 68,4 dz 10,7 6 2 ,3 -zt 14,3
7 85,6 ziz 9,3 80,2 ztz 14,20t 81,5 dz: 17,7 85,0 d z  18,3 74,2 d z  6,6
8 107,8 ziz 8,10 90,7 ztz 15,80* 91,3 d r  21,0 94,3 ziz 21,2 86,7 ziz 5,2
1* values for m ain group effects: • /><0.001, t 
W e e k s  C o n tro l
P< 0.05.
TABLE III. TAIL LENGTH (mm)
Pooled va lu e s  fo r a ll -------------  —
N 20 -e x p o s e d  g ro u p s  3 w e e ks
P eriod e xp o se d  to  N 20  
1st and 2nd w e e ks 1 st w eek
p o s tp a rtu m (m ean d :  S D ) (m ean i  S D ) (m ean 1 S D ) (m ean ziz S D ) (m ean dz S D )
1 54,0 4,1 23,0 ±. 4,6 24,6 -L 4,0 25,4 ziz 5,8 20,6 dz 4,1
2 71,0 dr. 3,7 36,2 d : 4,6 38,6 dz: 2,2 38,6 ziz 6,3 28,5 zi. 5,3
3 78,1 ' 5,9 43,0 t. 6,6* 48,7 iz  5,8 42,8 iz 6,4 40,1 dz 7,7
4 95,1 dz 14,2 62,0 i :  6 ,4 * 66,9 d - 11,8 68,6 d z  3,5 50,5 -iz 3,9
5 118,7 zt: 7,0 80,0 d . 10,3* 82,3 z t 13,8 88,1 dz 5,3 67,1 dz 12,8
6 132,4 ziz 8,8 89,1 dz 5 ,8* 80,8 ±  2,5 95,6 dz 13,1 90,8 dz 2,0
7 147,7 dz: 9,8 103,9 L- 7 ,9 1 105,6 ' 10,5 108,6 ztz 10,7 97,5 iz 2,7
8 159,2 d :  6,9 110,9 1 10,63* 1 1 1,9 1 12,8 115,0 d 12,5 105,8 • 6,6
/' values for main group effects: /'<0.001. t /»<0.02.
108 S A M i: i )  i i; s i: I v d s k r i  i 21 .laiuianc 1 ‘>78
TABLE IV. BO DY LENGTH (mm)
Period exposed to N^O
Weeks Control N,0-exposed qroups 3 weeks 1st and 2nd weeks 1 st week
postpartum (mean :-i: SD) (mean "  SD) (mean 1 SD) (mean : 1 SD) (mean 1 SD)
1 59,5 6,9 54,0 1 4 ,9 * 59,0 1 4,4 56,3 5,2 48,1 1 5,3
2 79,5 -■ 8,2 71,1 1 6 ,5 * 77,8 5,9 76,7 4,9 60,0 8,7
3 88,2 -±z 7,3 78,1 iz  16.0* 88,0 4,2 77,8 -iz 21,3 68,6 —  9,2
4 108,8 ziz: 9,9 95,1 -iz. 9 ,8 1 101,3 r± : 8,3 107,0 z±z 13,8 77,0 -iz: 7,4
5 138,7 ziz: 7,9 1 18,7 ::L: 18,7* 124,8 -iz 16,6 131,5 ziz 13,1 100,5 ziz 26,4
6 148,6 ± 10,5 132,4 -i :  15,0* 128,8 r i : 11,8 142,6 ± 15,4 125,8 z t. 17,8
7 1 56.7 6,7 147,7 iz  8,0 150,6 L: 8,6 152,4 ziz 9,1 140,0 6,3
8 167,3 ziz 5,6 159,2 i -  7,0 160,6 1 8,2 162,0 ziz 7,5 155,0 -iz  5,5
/ ’ values for main group effects: * / ’ •110.001. t P<  0.05.
Sprague-Dawling rats continuously to 4 5  - 5 0 " .'. N.O in air to 0.01 N.O. Kamazolto c / al.' repor led a signilic;
from clay 8 of the gestation period, for 2. 4. and 6 days, 
i.e. during the second week o f pregnancy, at an unknown 
number of air changes per hour. Corbett cl a ll in one 
experiment used a continuous exposure, during the second 
and third weeks, to 0.1%, and 1.5"', mixtures of N.O and 
air. In a second experiment the exposure was for 8 hours 
per day to 0.1% and 0.01".', N=0 and air. No details were 
given o f the number of air changes per hour. Ramazotto 
< 7  al.' used an exposure of 25 minutes per day to 50% 
N.O and air during each o f the first, second, third and 
all 5 weeks of pregnancy but gave no further details of 
their technique.
Tn this study 1% was chosen as the experimental con­
centration because this approximates the highest N.O 
pollution level reported in a dental surgery by Millard and 
Corbett." The exposure time o f 6 hours a day. 5 days per 
week, was selected to stimulate N O levels which may be 
encountered during a dentist’s working day when N.O 
sedation is being used.
The mean litter size of the control animals exposed to 
atmospheric air in our study, namely 10.4. closely approxi­
mates the mean litter size o f 11.1 and 10.2 reported in 
two groups of gravid control rats that breathed compressed 
air in an environmental chamber and atmospheric air, 
respectively/ In a more recent unpublished study by us. 
the mean litter size of gravid rats that were exposed to 
compressed air in an environmental chamber was 1 1.3.
The reduction in litter size found in our study is in 
agreement with the results obtained in the other N.O 
exposure studies. Fink ct al.3 reported a 28% fetal resorp­
tion rate, while Corbett ct alI noted signilicar t reductions 
in litter size in rats exposed to 1.5% and 0.1%. N.O. but not
crease in fetal death in those animals exposed to the an.tes- 
tlictic during the first, third and all 3 weeks of pregnancy. 
This confirms that N.O is toxic to the developing rat 
Ictus.
I hc present study is the first study o f postnatal growth 
of rats exposed to N.O during the prenatal period. It has 
shown that the 1% N.O and air mixture decreased the 
postnatal growth of the rats. T he offspring that had been 
exposed during the first week of pregnancy were sig­
nificantly smaller than those in the other two groups. The 
reason for this is not clear but may be related to exposure 
of the fertilized ovum before, during or immediately after 
implantation on day 5 of pregnancy.
CONCLUSIONS
It is not possible to extrapolate directly the results of this 
study to man. However, the fact that intermittent ex­
posure to a low concentration of N.O resulted in retarded 
postnatal growth suggests that growth studies need to he 
undertaken in the offspring of female anaesthetists and 
operating room personnel.
Ri-r-Tiu-Ncrs
1. Corbet I. T . I f .  (19721: r c r l i l .  and S lcril., 23, 864.
2. Corbett. T . IT.. Cornell, R . G., End res, J. L. ct al. (1973): Ancs* 
flicsiolnuv. 39. 299.
\. F ink. It. U .. Shepard. T. H . and Itlnndau, K. J. (1967): Nature. 214. 
146.
4. Cleaton-Jones. I*.. Austin. J. C ., Ranks, D. ct al. (1977): Hr it. J. 
Anaeslh., 49 (in press).
5. Jcflerys. .1. I; . (1969): 'G row th  pattern and environment*. M .I) .  thesis. 
University o f Nijmegen. The Netherlands.
6. Johnston, N. L. and Leone, I7. C. (1964): Statistics and Experimental 
Dc.siyn, vol. 11. New Y o rk : John W iley .Sons.
7. Ramazotto. /.. J., Katz. A. and Cupiola, R. (1975): J. dent. Res., 54, 
special issue A , IA IJR  abstract L31.
S. M illa rd , R. I. and Corbett, T . H . (1974): J. oral S lirg., 32. 593.


Intermittent exsposure of gravid rats to 1% nitrous oxide and the effect on the postnatal growth of their offspring

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Intermittent exsposure of gravid rats to 1% nitrous oxide and the effect on the postnatal growth of their offspring

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