Registration Dossier

Administrative data

Endpoint:
biotransformation and kinetics
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Exposure period is one our and therefore no equilibrium is achieved but due to the readily biodegradability of both Chloramine T and p-TSA it is very unlikely that this would have been easy to obtain. From the uptake and depuration rates a reliable worst-case BCF was calculated. This test was performed in order to establish residues of Chloramine-T trihydrate in fish that are meant for consumption and not according standard guidelines like OECD 305. Despite of the different setup of the test a reliable kinetic BCF for p-TSA equivalent residues could be derived.
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
No reference is made to a standard guideline, test performed to establish residue concentrations in exposed animals
GLP compliance:
yes
Remarks:
according to US FDA principles of GLP
Radiolabelling:
yes
Vehicle:
no
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Rainbow trout (Oncorhynchus mykiss) obtained as eyed eggs were reared in the laboratory. Fingerlings (1.82-4.50 g) and juvenile trouts (36.1-88.7 g) were used in this study. The test animals were selected for the study without regard to sex. The animals were acclimated for at least 7 days in a 495L fibreglass tank under flow through conditions at a temperature of 12° C and photoperiod of 12:12>.

Diet: Animals were fed commercial trout food (Nelson’s Sterling Silver Cup Salmon Food, Murray Elevators, Murray, UT)( the feed was analysed for environmental contaminants and these were below those known to interfere with similar studies in mammals). Fingerlings received 3.9% of bw and juvenile fish received 2.0% of bw. The animals were not fed 1 day before the study commenced and was provided again during the recovery phase.
Route of exposure:
aqueous
Test type:
static
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
60 min
Test temperature:
11 -7°C (11.3-12.0) during the depuration phase for fingerling fish
11.8°C (11.6-12.2) during the depuration phase for juvenile fish
pH:
8.09 (7.87-8.16) during the depuration phase for fingerling fish
7.95 (7.63-8.07) during the depuration phase for juvenile fish
Dissolved oxygen:
10.0 mg/L (9.74-10.2) ; percent saturation of oxygen, 93.7% (90.8-95.0) during the depuration phase for fingerling fish
9.21 mg/L (7.75-9.62) ; percent saturation of oxygen, 87.3% (73.8-91.6) during the depuration phase for juvenile fish
Details on test conditions:
Exposure groups:
Separate groups of fingerling and juvenile rainbow trout were allowed to acclimate to conditions in the environmental chamber for at least 1 week before testing. Accumulation and elimination of Chloramine-T from the two-size classes of fish was determined in four separate experiments. The first two documented accumulation and elimination in fingerlings , the second two documented accumulation and elimination in juveniles. Exposures to solutions of [14C]Chloramine-T were made in large (60 L) stainless steel exposure tanks for up to 1 hour. Chloramine-T accumulation was assessed by exposing 48 finger1ings in two tanks (four groups of 12 fish each) and 40 juveniles (10 fish in each of four tanks; four groups of 10 fish) by static bath to solutions of [14C]Chloramine-T.

Four groups (12 fish per group for fingerlings; 10 fish per group for juveniles) were removed from the exposure tanks, one group at each 15-min interval through the hour of exposure.
Control groups for each fish size were sampled before the exposure. The rates of residue elimination were assessed by exposing 108 fingerlings in
one tank (12 fish per group x nine groups) and 72 juvenile fish in three tanks (24 fish/ tank) by static bath to a solution of [14C]Chloramine-T for
1 hour. After the 1-hour exposure, fish were removed from the exposure, tanks, placed in a fresh water rinse, and then randomly transferred to one of six 48-L glass-holding aquaria that were supplied with fresh, flowing (1 L/min; >one tank exchange/hour) water and held until the animals were sampled. The calculated concentration of [14C]Chloramine-T in each exposure tank was 20 mg/L, twice the treatment concentration proven to be effective in controlling disease agents responsible for bacterial gill disease (BGD). The loading rate for any single exposure did not exceed 15 g of fish per liter of test solution. Previous studies indicated that this loading rate was proper to maintain adequate concentrations of dissolved oxygen (≥ 7.0 mg/L) in the test water during a 1-hour exposure. Test water was aerated for at least 1 hour before addition of the chemical; tanks were not aerated during the exposures. Holding aquaria were cleaned and observations of activity and food consumption were made daily for all test groups. Temperature, pH, conductivity, and dissolved oxygen concentration were monitored in the recovery water throughout the depuration phase of the study using a Hydrolab Scout water quality analysis system.

The remaining fish from each group were pooled and homogenized. These samples were stored at -20 °C until LSC and HPLC analysis.
Samples of exposure eater were collected every 10 minutes during each 1 hour exposure period and analysed for radioactivity by LSC and for the concentrations of Chloramine-T and PTSA by HPLC.
Nominal and measured concentrations:
The exposure concentration found by radiochemical analysis in the water samples from all test baths (n=10) was initially 17 mg/L after 60 min the concentration remained above 16 mg/L. HPLC analysis gave a concentration of 19 mg/L which decrease to 18 mg/L after 60 minutes.
Simultaneous analysis of the exposure water for Chloramine-T and its primary decomposition product, p-TSA, indicated that p-TSA was present in a1l exposure waters immediately following the addition of [14C]Chloramine-T (average concentration was 0.76 ± 0.08 mg/L) . Its concentration gradually increased during the 60-min exposures to an average of 1.30 ± 0.25 mg/L. The sum of the Chloramine-T concentration and p-TSA concentration converted to Chloramine-T equivalents in the test water was approximately 20 mg/L.

Residues of Chloramine-T were not observed in any of the fish tissues analyzed for this study. Chloramine-T was apparently rapidly reduced to the primary decomposition product, p-TSA. Therefore, all tissue residues determined either by radiometric or by HPLC methods were reported on the basis of equivalent concentrations of p-TSA.
Reference substance (positive control):
no
Key result
Conc. / dose:
20 mg/L
Temp.:
11.8 °C
pH:
8
Type:
BCF
Value:
2.2 dimensionless
Basis:
whole body w.w.
Calculation basis:
other: K1/K2 ratio
Remarks:
based on LSC concentrations
Metabolites:
Findings, disparity between radiometric and HPLC analysis, suggested that other metabolites besides PTSA are present in the tissues.
Data were evaluated as a percent of the HPLC to LSC concentrations; percentages less than 100% were assumed to indicate the presence of an additional metabolite(s) of Chloramine-T in the tissues.

The values of HPLC-concentration calculated as a percent of LSC concentration were least during the accumulation phase of the study, suggesting that a metabolite was formed almost immediately after the initial exposure. The percentage decreased slightly during the accumulation phase of the study in both fingerling and juvenile fish. Percentages increased toward 100% in all tissues during the elimination phase of the study. It was attempted to identify the other metabolites and one peak was found. Since Chloramine-T is a mild oxidizing agent, the metabolite may be a complex or conjugate with a tissue antioxidant such as glutathione. Such a metabolite would be expected to occur in highest concentrations during and immediately after exposure and decrease there after.

1994 study:
In a follow up study rainbow trout were exposed to 20 mg/L Chloramine-T for 60 ± 3 minutes. Samples of fillet tissue, residual carcass, and gall bladder bile were homogenized and analyzed for radioactive residues by gradient high performance liquid chromatography (HPLC) equipped with in-1ine radio chromatography detection. As expected residues of Chloramine-T were not observed in any of the fish tissues analyzed. However in this second study, there was no evidence of any residues other than PTSA in any of the tissues analyzed, even when multiple extracts were pooled and analyzed to increase the sensitivity. Samples of archived tissue from the previous residue study were extracted and analyzed using procedures from the present study and the unidentified residue was confirmed. Concurrent purity analyses of the test articles from the previous and the present studies indicated that the test article from the previous study contained a radioactive contaminant that accounted for approximately 5% of the total radioactivity of the sample. We feel that the unidentified residue from the previous study was an anomaly that resulted from the presence of an impurity in the original test article.

Chloramine T was not found in any of the fish tissues analyzed in this study, rather the reduction product p-TSA was present.

No Steady-state p-TSA equivalent residue concentration was achieved within the accumulation period considered (i.e. 60 minutes). The tissue-equivalent concentrations of p-TSA in whole-body homogenates were significantly greater (P<0.05) in fingerlings than in juveniles.

The p-TSA equivalent concentration in composite whole body homogenate samples from fingerlings was 0.98µg/g after the 60 minutes exposure, a value that was only about 5% of the concentration in the exposure water. From this 0.98 µg/g p-TSA equivalent only 0.36 µg/g was p-TSA.

The kinetic whole body BCF as calculated from the accumulation and depuration from fingerling data using the p-TSA equivalent concentration (worst-case) is 2.19. It should be noted that this ratio must be considered as a worst-case value because it is calculated using the p-TSA equivalent concentration for fingerlings, using the average depuration rate during the whole curve expressed in relation to the whole body. Lower ratios would be obtained if the data for juvenile rainbow trout would have been used. Based on the measured log Pow values for Chloramine T and p-TSA, Chloramine T will have a lower BCF than pTSA.

HPLC

Depuration:

LSC: Log C = -0.011 * time – 0.3091 (r = 0.9794)

K2 = LN(10)* slope = 0.0253hours -1

T½ = LN(2)/ K = 27.4 hours

HPLC: Log C = -0.0081 * time – 0.6277 (r = 0.9840)

K2 = LN(10)* slope = 0.0187 hours -1

T½ = LN(2)/ K = 37.0 hours

DT90:

The initial mean p-TSA equivalent concentration in fingerlings after 1 hour of uptake = 0.26 µg/g. After 120 hours of depuration this concentration has dropped to 0.03 µg/g

Uptake:

K1 = (Cf * K2)/Cw * (1 – e^(-K2*t)))

Cw = 19 µg/L (on average taken from appendix 9)

Cf LSC = 0.549 µg/g based on graph after 0.525 hours

Cf HPLC = 0.176 µg/g based on graph after 0.525 hours

K1 LSC = 0.0554 hours-1

K1HPLC = 0.0177 hours-1

BCF LSC = K1/K2 = 2.19

BCF HPLC = 0.95

The depuration was observed during 240 hours. No residues remained after this depuration time. About 90% removal was observed after 120 hours of depuration.

Validity criteria fulfilled:
not applicable
Conclusions:
The test data show that for p-TSA equivalent residues a worst-case bioconcentration factor of 2.2 was derived using fingerling fish and the p-TSA equivalent concentration. Based on the rapid transformation and low log Pow, Chloramine T has a low bioaccumulation potential.
Executive summary:

Chloramine T was not found in any of the fish tissues analyzed in this study, rather the reduction product p-TSA was present.

No Steady-state p-TSA equivalent residue concentration was achieved within the accumulation period considered (i.e. 60 minutes). The tissue-equivalent concentrations of p-TSA in whole-body homogenates were significantly greater (P<0.05) in fingerlings than in juveniles.

The p-TSA equivalent concentration in composite whole body homogenate samples from fingerlings was 0.98 µg/g after the 60 minutes exposure, a value that was only about 5% of the concentration in the exposure water. From this 0.98 µg/g p-TSA equivalent only 0.36 µg/g was p-TSA.

The kinetic whole body BCF as calculated from the accumulation and depuration from fingerling half-lives using the p-TSA equivalent concentration (worst-case). The k1/k2 ratio as calculated according to OECD guideline 305 is 2.2 based on LSC. It should be noted that this ratio must be considered as a worst-case value because it is calculated using the p-TSA equivalent concentration for fingerlings, using the average depuration rate during the whole curve expressed in relation to the whole body. Lower ratios would be obtained if the data for juvenile rainbow trout would have been used. Based on the measured log Pow values for Chloramine T and p-TSA, Chloramine T will have a lower BCF than pTSA.

Data source

Referenceopen allclose all

Title:
No information
Author:
Dawson, V.K. and Gingerich, W.H., 1991, Accumulation and clearance of chloramine-T residues in rainbow trout after use pattern treatment with [ring UL-14C] chloramine-T, National fisheries Research Center, La Crosse, Wisconsin, USA Report No.: LNFRC-CLT-90-01, April 1, 1991,
Title:
No information
Author:
Dawson, V.K., et al., 1994, Isolation and characterization of Chloramine-T metabolites in rainbow trout after use pattern treatment with [ring UL-14C] Chloramine-T, National Biological Survey Upper Mississippi Science Center, La Crosse, Wisconsin, USA, Report No.: CAP-93-00057, December 1, 1994,

Materials and methods

Type of medium:
animal

Test material

Reference
Name:
Unnamed
Type:
Constituent

Results and discussion

Transformation products:
yes

Any other information on results incl. tables

RS-Freetext:
Chloramine T was poorly absorbed. After 30, 45 and 60 
minutes of exposure fingerling trout has greater 
concentrations of total residues than juveniles. Chloramine

was not found in any of the fish tissues analysed in this 
study, rather the reduction product p-toluenesulfonamide 
(ptsa) was present. Based on radiometric analysis, the 
concentrations of ptsa-equivalent in whole body homogenates
of fingerlings and juveniles after one hour were
respectively 0.98 µg/g (5% of that in exposed water) and
0.57 µg/g (3% of exposed water). Based on HPLC analysis
however the concentrations were respectively 0.36 µg/g and
0.17 µg/g: a concentration of about one third of those
expected from the radiochemical analysis. 
The differences in these concentrations are explained in the
second study of December 1994, where it was found that the
radio-active material from the first study contained 5%
contamination which accounted for the difference in 
radiometric versus HLPC analysis. In this study it was found

that Chloramine T is rapidly reduced to the primary 
decomposition product ptsa, and that there were no other
residues than ptsa. Furthermore elimination rate of
Chloramine T and ptsa 14C was characterized by a 
rapid loss of radioactivity over the first 24 hours after 
withdrawal and a further loss until 240 hours or until they
fell under the detection limit. The estimated half-lifes in 
fingerlings and juveniles were 27.3 hours and 32.6 hours.

Applicant's summary and conclusion

Conclusions:
CL-Freetext:
It is concluded that immediately after exposure no
Chloramine T is present. Only minimal residual amounts
of the decomposition product ptsa is present in the fish,
and
with a maximumof 5% of that in the exposed water. Half-life
time is 27 to 33 hours. At the end of the study (at 240
hours
after withdrawal) the residual concentration had fallen
below the detection limit of 0.0074 µg/g ptsa equivalent.