The second international standard for chlortetracycline*

Bull. Org. mond. Sante' 1972, 47, 635-640 Bull. Wid Hith Org. The second international standard for chlortetracycline * J. W. LIGHTBOWN,1 JILLIAN M. BOND,1 MARJORIE V. MUSSETT,2 & LAURA L. TACKETT2 As supplies of the International Standard for Chlortetracycline were practically ex- hausted, it was replaced. The potency ofthe second international standard was defined on the basis ofan international collaborative assay comprising 157 individual assays performed in 9 laboratories in 6 countries. The mean potencies obtained in the participating laboratories, although they varied by only 7%, were heterogeneous. However, the mean potencyfor all the laboratories combined didnot differ significantlyfrom that ofthe first international standard. The International Unitfor chlortetracycline was therefore defined as the activity contained in 0.001 mg of the second international standard, corresponding to a potency of 1 000 IU/mg. The WHO Expert Committee on Biological Stan- dardization (1967) was informed that supplies of the first International Standard for Chlortetracycline were practically exhausted. Since active use was still being made of this standard and demand for it was considered likely to persist, material suitable for use as the second international standard was obtained and assayed against the first international standard. MATERIALS FOR THE STUDY The proposed second International Standardfor Chlor- tetracycline A sample of chlortetracycline hydrochloride, Batch No. 501-721B-36-2, weighing about 170 g was generously donated by Lederle Laboratories Division, American Cyanamid Company 3 through the good offices of Mr W. P. Jones of Cyanamid of Great Britain Ltd. The material, in two containers, each containing approximately 85 g, was received at the National Institute for Medical Research, London, in April 1966 and stored at -20°C, pro- tected from moisture. The following data were supplied by the manufacturer: Spectrophotometric assay (aver- age result for 2 laboratories) 99.3% * From the WHO International Laboratory for Biological Standards, National Institute for Medical Research, Mill Hill, London, England. I Division of Biological Standards. 2 Statistical Services Section. 3 Pearl River, N.Y., USA. Fluorometric assay (average result for 2 laboratories) 100.5% Microbial assay 4 1 090 " tg "/mg Tetracycline content 0 Colour value (E 1 %, 1 cm, 460 nm) 0.154 Volatile matter 0.34% Rabbit skin test Class 1 Heavy metals 26 mg/kg Specific rotation [a] 25 - 237.8' as is -238.6' anhydrous Chloride 6.92% ionic In August 1966 the material was distributed into approximately 2 300 neutral glass ampoules so that each contained approximately 75 mg. The ampoules were closed with vented polyethylene plugs and dried over phosphorus(V) oxide in vacuo to constant weight. After they had been filled with pure dry nitrogen the ampoules were sealed by fusion of the glass, tested for leaks, and stored in the dark at -200C. The residual moisture content of the material in the ampoules, measured as loss in weight on drying to constant weight over phosphorus(V) oxide at 560C and at a pressure of < 0.02 mmHg, was found to be 0.42% w/w (mean of 3 ampoules). When exposed to an atmosphere of 40% relative humidity at 200C, the material in the ampoules picked up <0.15% w/w in 60 minutes. The residual oxygen concentration in the ' When .g is placed between quotation marks (" ,ug "), it refers to a certain amount of activity (potency) and not to its accepted usage as a unit of mass. 2943 635 J. W. LIGHTBOWN ET AL. _% co U co~~~~ E 0.~~~~~~~ 0 0C 0EE E E oco.* 0 0 0W0 * * - C ;o - x 00~ 0 c05 0 .L C.2 0r0 0O - NC 0 NcN 00 0 0 0 0 00 0 C') 0~~~~~~~~~~~~~C C La r- 0 0e0T0 ')N W 0 a. V ~~~~~~00D 0 0m 0c 00OC-i ~~~~~~~0.0 co C >-E r--C 1:( Oco 0 .0 0.~~~~~~~~~~~~~~~~~~~~~-- (D)t EO co C0 N N00co L 10 0Z~~~co Co Z < 0E ZC')~~~~~O 0C-14 IU~-"( z z*~ M c m0U. " Z LO 0~ 00D E C-4 'lb I_b 14, CD 0 co .2 2 0 0 'Z -1EE E to S. 0co 0 ~ ~~ ~ ~ ~ ~ ~ ~ ~ ~ o0 2 cCu~~~~~2 0 Dr%O- c 2 636 CHLORTETRACYCLINE ampoules was found to be 0.065% v/v (mean of 6 determinations). The first International Standard for Chlortetracycline The proposed second international standard was assayed against the first international standard estab- lished in 1953 (Humphrey et al.). THE COLLABORATIVE ASSAY Five ampoules of the proposed second interna- tional standard, numbered 1 to 5, together with 4 ampoules of the first international standard, num- bered 1 to 4, were made available to each of the laboratories participating in the collaborative assay. These laboratories, which represent 6 different coun- tries, are listed in Annex 1. Throughout the present article, each of them is referred to by a code number, which is not related to the order in which the laboratories are listed. The participants were asked to design assays in a way that made it possible to carry out tests of linearity and parallelism of the log-dose response lines and also to ensure that each assay should provide, from its own internal evidence, an estimate of potency of the proposed second standard in terms of the existing standard and fiducial limits to that estimate. The participants were also invited to com- pare the two materials by any physical or chemical method of analysis that was being used in their laboratory. STATISTICAL ANALYSIS OF RESULTS The 9 participating laboratories provided the re- sults of a total of 163 assays of the proposed second international standard in terms of the first interna- tional standard. These data were analysed statisti- cally by standard methods for parallel line assays, relating response (or transformed response) to the logarithm of the dose. Five assays were discarded as invalid because the departure from parallelism term, in the analysis of variance, was significant at the 1% level. Assays were not necessarily rejected on the grounds of significant deviations from linearity of the log-dose response lines, for reasons discussed pre- viously by Humphrey et al. (1953). However, a single assay from Laboratory 7 was omitted because not only was there significant curvature (P < 0.001) but also the estimated potency was discrepant from other values obtained by the same laboratory. In 6 of the 157 assays considered to be valid, the parallelism term was of borderline significance (0.01 <P < 0.05); thus the proportion of assays showing some degree of nonparallelism of the log-dose response lines was approximately the same as might have been expected to occur by chance. A breakdown of assays by laboratory, giving details of the assay methods, the response meta- meters used in the analysis, and the validity of the assays is given in Table 1. For most assays there was a satisfactory linear relationship between the re- sponse (as measured) and the log dose, but for Laboratory 7 the square of zone diameter was more satisfactory and for Laboratory 8 the square root of zone area was more linear with the log dose. Both these laboratories arranged dilutions of the prepara- tions under comparison in a Latin square design on large plates. Laboratory 3 also used large plates, whereas Laboratories 1 and 9 tested 3 dilutions of each preparation on Petri dishes. Some of the plate diffusion assays from Laboratory 2 were of a (3+3) design on Petri dishes; in others, 4 dilutions of each preparation were arranged in Latin squares on large plates. In the nephelometric assays from Labora- tory 2, 3-7 dilutions were used and analysis was done after rejection of a minimum amount of data from the ends of the log-dose response lines, in order to obtain parallelism. This was also improved by the use of a square root transformation, but 10 assays still showed significant curvature at the 1% level. These assays were not excluded, since the potencies did not materially differ from those obtained when the untransformed data were analysed or when the responses for a more limited range of doses were used. Further details of the assays from Laboratory 2 are given in Table 2. Laboratory 4 used two different designs on small plates, described here as methods 1 and 2. In me- thod 1, 3 replicates of a single dilution of the proposed standard were tested alongside 3 replicates of the corresponding dilution of the current stan- dard in each dish. In method 2, 3 positions on each plate were occupied by the same fixed dose of the standard, while one or other of the doses of either preparation was tested on the other half of the plate. In the latter assays, the deviation of the mean zone diameter for the dose being tested from the mean zone diameter for the fixed dose was used as the response metameter. For the basic analysis of the turbidimetric assays performed in Laboratories 5 and 6, optical density was related to the log dose. 637 Individual log potencies (MI) were tested for homogeneity within each laboratory and each assay method, using the formula: X2 = 2Wi (Mi-M)2 where the weights (Wi) are the reciprocals of the variances of the log potencies and M is the weighted mean potency. For Laboratories 1, 3, 6, 8, and 9, potency values were homogeneous-i.e., the probability of X2 was greater than 0.05 and the weighted means (expressed as potency ratios, on a weight-to-weight basis) are given in Table 3, together with their confidence limits, based on the total weights. Where the X2 test indicated heterogeneity, un- weighted geometric mean potencies were evaluated and confidence limits were estimated by the " direct " method-i.e., by calculating the variances direct from the distributions of individual log potencies. For Laboratory 2, all 50 potencies obtained by plate diffusion methods were homogeneous, and the weighted means are given in Tables 2 and 3. For the nephelometric assays the potencies were hetero- geneous for each test organism, but the two direct estimates were homogeneous with each other and with the results for the plate diffusion assays and so were combined to give an overall value for the laboratory. For Laboratory 4, the assays done by method 1 were homogeneous. With method 2, however, there was heterogeneity within the set and with the potency for method 1. For this reason no overall estimate for the laboratory was derived, the values in Table 3 re- presenting the weighted mean for method 1 and the unweighted mean for method 2. Laboratory 5 carried out 2 assays per day for 4 days. As there was homogeneity within days, but not between days, the overall mean was calculated direct from the 4 daily means. There was some heterogeneity among the Bacillus pumilus assays provided by Laboratory 7, but when assays were grouped according to the days on which they had been carried out the x2 between days was only of borderline significance and the weighted mean for the 4 days was homogeneous with the results obtained from the Bacillus cereus assays. The log potencies for each laboratory, weighted either from the internal evidence of the assays or from the variation between assays (as described above) were found to be heterogeneous (P < 0.001). This did not appear to stem from the results of any particular laboratory or assay method, so the final value for the study of 1.004 with confidence limits 638 J. W. LIGHTBOWN ET AL. Da) 'O N.o0) 0) 0) 0)° °l a) '- ' 0O~ O1 O~ O~ 0) oo 00 a) a) N _ o -- Nn o o 0 0 O ) X W C' C' C' C' o C 9 0 o 0 0 0) 0) o o C'4 00 N D 0) 0) o o r- ( C) N N o * oE 00C 0 S o o X Co) a0) a, cI 0 0 L 00 CO 05 -00 CD 0 0)- 0 a.0 C *n : 0 S oo C I- =OQ 0 S (i 0 a)00C' 0 0 o 0 0 Co C') Co 0 a, C 0 0. C C 0) co C_ C v O. to a) 0 V 0C E o 0 0 o 0 a, ._S C 0 -C 0 N 0 .0 -J -o .0 'a, U1) CA 0 - la 0 n cu E oW a)(a 0 MA 40 a) a) I-4 L) 0 0 0 0 0 0 on en 0 07 (00 I- Z X 0 O @ 0Q 0 Q *QC<i t ii m x x '- O, ~ Ce0M 0 9 0 0 .C .C 0. 0.0 0 0 c 0. 0. 0 0O N P' q(_ L to0 0 .o Na .2 E - ) 0 0 c 0.CL CHLORTETRACYCLINE Table 3. Potencies obtained by different laboratories Laboratory N.oasys Potency Confidence No. No. of assays ratio limits (P = 0.95)w/w 1 13 1.049 1.038-1.060 2 plate diffusion (50) 0.978 0.968-0.988 nephelometric (14) 0.971 a 0.943-1.001 64 0.977 0.968-0.987 3 14 0.999 0.986-1.011 4 method 1 (4) 0.984 0.972-0.996 method 2 (4) 1.054 1.015-1.095 5 8 1.013 b 0.995-1.032 6 8 1.027 1.020-1.034 7 B. cereus (6) 1.025 1.012-1.038 B. pumilus (14) 1.042 c 1.034-1.050 20 1.037 1.030-1.044 8 B. cereus (8) 1.007 0.998-1.016 B. pumilus (7) 0.992 0.979-1.005 15 1.004 0.997-1.012 9 7 1.005 0.993-1.017 a Based on weighted means for 2 test organisms. b Unweighted mean of 4 daily weighted means. c Based on 4 daily weighted means. (P = 0.95) of 0.998-1.011 was obtained as a direct estimate from the distribution of the 157 log poten- cies considered to be valid. As the International Standard for Chlortetra- cycline had a potency of 1 000 IU/mg, the proposed second international standard was estimated to con- tain 1 004 (998-1 011) IU/mg. DISCUSSION Considering the putative purity of the two mate- rials being compared in this collaborative assay, the variations in potency obtained by the different labo- ratories is surprising. A much greater range of assay conditions was used than is usual in a collaborative assay, but it is difficult to see any relationship between the variations in potency and the variations in assay conditions. Laboratory 2 made an extensive comparison of the two materials by a variety of methods and techniques encompassing most of those used by the other participating laboratories. The results showed good agreement between the different Table 4. Summary of results obtained by chemical and physical methods for the comparison of purity of the International Standard for Chlortetracycline (IS) and the Proposed Second International Standard (PSIS) Laboratory Ratio of purity a Method used 3 1.00 Chiccarelli et al. (1957) 4 0.998 (0.994-1.001) Extinction in N. H2S04 A = 274 nm 0.999 (0.993-1.005) Extinction in N. H2S04 A = 274 nm after heating 9 1.005 (0.998-1.012) Grove & Randall (1955) a Ratio PSIS: IS with 95 % confidence limits. methods, again suggesting that the recorded varia- tions in assay conditions did not influence the poten- cy ratios obtained. Although mean potency ratio values for the differ- ent laboratories were not statistically consistent, they varied by only about 7%, whereas potency ratios from the 157 individual assays extended over a range of approximately 30%. All the laboratories except one assayed solutions on the day they were pre- pared, but it is possible that there were differences in the time that elapsed between the solution of the samples and their use in the assay, which might account for some of the variations in potency. No significant difference between the activity of the proposed second standard and that of the first standard was demonstrated, and it was suggested to the participating laboratories that the new standard should be assigned a potency of 1 000 IU/mg. The participating laboratories were invited to com- pare the existing and proposed standards by any chemical and physical methods they were currently using. Such comparisons were made by 3 labora- tories and their results are summarized in Table 4. The ratios obtained agreed closely between the labo- ratories and supported a conclusion of equal activity for the materials compared. In accordance with the authorization of the WHO Expert Committee on Biological Standardization (1967), the proposed material was established as the Second International Standard for Chlortetracycline and, with the agreement of the participants in the study, the International Unit for chlortetracycline was defined as the activity contained in 0.001 mg of the Second International Standard for Chlortetra- cycline. 639 640 J. W. LIGHTBOWN ET AL. R1TSUMJR DEUXIEME tTALON INTERNATIONAL DE CHLORTSTRACYCLINE Le Comite OMS d'experts de la Standardisation biolo- gique (1967) ayant not6 que les stocks de l'etalon inter- national de chlortetracycline etaient en voie d'epuisement, avait demande au National Institute for Medical Research, de Londres, d'etablir un deuxieme etalon international. Du materiel approprie en vue du remplacement du premier etalon a ete obtenu et soumis 'a un titrage compa- ratif dans neuf laboratoires de six pays. Au total, 157 6preuves ont e pratiquees, par diffusion et turbidime- trie, sur une serie de micro-organismes. L'analyse statis- tique a fait ressortir l'heterog6neite des estimations de l'activite dans quatre laboratoires, de meme que des variations de I'activite moyenne de la pr6paration pro- posee suivant les laboratoires, mais dans une proportion ne depassant pas 7 %. La cause de ces variations intra- et inter-laboratoires n'a pas ete determinee. L'examen des resultats d'ensemble a montre que F'activite de la prepa- ration proposee ne differait pas sensiblement de celle du premier 6talon. En consequence, les participants ont decide de cons- tituer le materiel propose en deuxieme etalon international de chlortetracycline, dont l'activit6 a e fixee a 1000 UI par milligramme. L'unite internationale de chlortetracy- cline a e definie comme l'activit6 de 0,001 mg du deuxieme etalon international de chlortetracycline. REFERENCES Chiccarelli, F. S. et al. (1957) J. Ass off. agric. Chem., 40, 922 Grove, D. C. & Randall, W. A. (1955) Assay methods of antibiotics, New York, Medical Encyclopedia Humphrey, J. H. et al. (1953) Bull. Wld Hlth Org., 9, 851 WHO Expert Committee on Biological Standardization (1967) Wld Hlth Org. techn. Rep. Ser., No. 361, p. 10 Annex 1 PARTICIPATING LABORATORIES National Biological Standards Laboratory Department of Health Canberra, Australia (Mr N. M. Semple) Institute of Hygiene and Epidemiology Brussels, Belgium (Dr A. Lafontaine & Dr A. Vanden Bulcke) Drug Control Department Brussels, Belgium (Dr J. Dony, Miss I. Boudru, & Mr A. De Roeck) Cyanamid of Great Britain Ltd Gosport, Hants., England (Mr W. P. Jones) National Institute for Medical Research London, England (Dr J. Bond & Mr J. W. Lightbown) Research Institute for Pharmaceutical Chemistry Budapest, Hungary (Dr I. Horvath, Dr I. Koczka, & Dr A. Szab6) Cyanamid International Wayne, N.J., USA (Dr S. H. Babcock, Jr) Food and Drug Administration Department of Health, Education, and Welfare Washington, D.C., USA (Dr W. W. Wright) State Control Institute for Medical Biological Prepara- tions Moscow, USSR (Professor A. T. Kravcenko & Professor L. M. Jacobson)