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Toxicological information

Immunotoxicity

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Administrative data

Description of key information

The following studies have been submitted to address the immunotoxicity endpoint:

Li, A. P., Dahl, A. R. and Hill, J. O. (1982). In Vitro Cytotoxicity and Genotoxicity of Dibutyltin Dichloride and Dibutylgermanium Dichloride. TOXICOLOGY AND APPLIED PHARMACOLOGY 64, 482-485.

Penninks, A. H. and Seinen, W. (1982). COMPARATIVE TOXICITY OF ALKYLTIN AND ESTERTIN STABILIZERS. Fd Chem. Toxic. Vol. 20. pp. 909 to 916.

Schobel, C. (1991). ZK 22.663: Local dermal tolerance test in rats after a single application and a subsequent rinsing. Report no.: IC 1/91. Report date: 1991-06-13.

Seinen, W. and Penninks, A. (1979). IMMUNE SUPPRESSION AS A CONSEQUENCE OF A SELECTIVE CYTOTOXIC ACTIVITY OF CERTAIN ORGANOMETALLIC COMPOUNDS ON THYMUS AND THYMUS-DEPENDENT LYMPHOCYTES. Ann. N. Y. Acad. Sci. USA. 320:499-517.

Seinen, W., Vos, J. G., van Kreiken, R., Penniks, A., Brands, R. and Hooykaas, H. (1977). Toxicity of Organotin Compounds. III. Suppression of Thymus-Dependent Immunity in Rats by Di-n-Butyltindichloride and Di-n-Octyltindichloride. TOXICOLOGY AND APPLIED PHARMACOLOGY 42, 213-224.

Snoeij, N. J., Penninks, A. H. and Seinen, W. (1988). Dibutyltin and tributyltin compounds induce thymus atrophy in rats due to a selective action on thymic lymphoblasts. Int. J. Immunopharmac. 10: 891-899.

DeWitt et al, 2005, Immune Responses in Sprague–Dawley Rats Exposed to Dibutyltin Dichloride in Drinking Water as Adults, Journal of Immunotoxicology, 2:151–160, 2005.

DeWitt et al, 2005, Developmental Exposure to 1.0 or 2.5 mg/kg of Dibutyltin Dichloride Does Not Impair Immune Function in Sprague-Dawley Rats, Journal of Immunotoxicology, 3:245–252, 2006.

Schobel (1991), Seinen et al (1977), Snoeij et al (1988), De Witt et al (2005) and De Witt et al (2006) have been allocated a Klimisch score of 2 with Seinen et al (1977) and Snoeij et al (1988) considered to be the key studies for this endpoint.

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
immunotoxicity: acute oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male Wistar-derived rats of 4-5 weeks of age, obtained from the Central Institute for Breeding of Laboratory Animals, TNO, Zeist, The Netherlands, were used. Rats were housed in plastic cages and kept at room temperature of 23 ± 2°C with 50-60% relative humidity and a constant 12h light/dark cycle. Di-n-butyltin dichloride (DBTC) was provided by Dr H. A. Meinema, Institute for Applied Chemistry, TNO, Utrecht, The Netherlands. The purity was >98%, as established by thin-layer chromatography.

Dose-effect relationships:
Randomized groups of four to five rats, weighing 50-60 g, were given single oral doses of DBTC (27 animals) ranging from 5 to 35 mg/kg. DBTC was dissolved in absolute ethanol and intubated as a 5% solution in corn oil (5 ml/kg body wt). Body at and weights of thymus, spleen, liver, kidneys and adrenals were recorded 4 days after intubation.

Time-effect relationships:
Three randomized groups of 21 rats, weighing 45-55 g were given 0, 15 mg DBTC per kg body wt by gastric intubation, resulting in an equimolar dose of 50 µmol/kg. At days 1, 2, 3, 4, 5, 7 and 9 after intubation, body wt and weights of thymus, spleen, liver, kidneys and adrenals of three animals per dose group were determined.
Moreover, on these days, except for day 5, cell suspensions were made of each isolated thymus gland separately. Thymocyte suspensions were prepared using phosphate-buffered saline supplemented with 2 mM D-glucose (PBS/glue) as isolation medium. Viability of cell suspensions was assessed with the trypan blue exclusion method and always exceeded 96% for thymocyte suspensions prepared from either untreated or organotin-exposed rats. Total cell yield per organ and the number of thymocytes with a small, intermediate or large volume were determined, using a Coulter Counter. When operating the counter with threshold values of 10, 40 or 70, cells with a mean volume larger than 30, 130 or 225 µmˆ3 respectively, could be counted within a single suspension. Threshold values were standardized using various batches of latex particles of known median size (Coulter Electronics). By subtracting the cell counts obtained at threshold values of 40 and 70 from cell counts determined at threshold values of 10 and 40, respectively, the number of small cells (mean volume <130 µmˆ3), intermediate cells (volume between 130 and 225 µmˆ3) and large cells (volume >225 µmˆ3) were determined.
The incorporation of DNA, RNA and protein precursors into acid-precipitable material of isolated thymocytes was measured. In short, thymocytes (2 x 10ˆ7) suspended in 1 ml of PBS/glue were incubated for 1 h at 37°C in a shaking bath. At 20, 40, and 60 min after the addition of 1 µCi ˆ3H-TdR (final concentration 25 nM), 1 µCi ˆ3H-Urd (final concentration 20 nM) or 50 nCi ˆ14-Leu (final concentration 145 µM), samples of 5 x 10ˆ5 cells were taken in four-fold and acid-precipitable material was harvested onto glass fiber filters with a 5% solution of trichloroacetie acid. Filters were processed for liquid scintillation counting in a Kontron MR 300.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Single oral dose.
Frequency of treatment:
Single oral dose.
Remarks:
Doses / Concentrations: Ranging from 5 - 35 mg/kg bw (total dose) Dose-effect relationship method
Dose / conc.:
5 mg/kg bw (total dose)
Remarks:
Time-effect relationship method
No. of animals per sex per dose:
Randomised groups of 4-5 rats per dose for dose-effect relationship method.
3 randomised groups of 21 rats for time-effect relationship method.
Control animals:
yes
Dose descriptor:
other: 50% reduction in thymus weight
Effect level:
18 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male

Dose-effect relationships:

Dosages of 5 mg DBTC per kg rat or more caused a statistically significant weight reduction of the thymus. Reduction was dose-related and appeared maximal 4 days after intubation. Regression analysis of the dose-effect relationships revealed that DBTC caused a decrease in relative thymus weight linear to the logarithm of the orally administered dose. The dose level calculated to give 50% reduction of relative thymus weight was 18 mg DBTC/kg.

On microscopic examination, a dose-related depletion of cortical lymphocytes was observed in thymus glands of DBTC-exposed rats. At dose levels higher than 100 µmol DBTC per kg rat, the lymphocyte depletion was associated with a pronounced reduction in the width of the thymic cortex. As a result of the thymic involution, the mast cells lying in the connective tissue between the thymic lobes were seen more closely together when compared to the controls.

Relative spleen weights were not affected and no erythrocyte rosettes were found in the mesenteric lymph nodes in the DBTC-exposed rats. The relative organ weights of kidneys and adrenals were not altered in any of the exposure groups. Growth reduction was noticed in the highest DBTC group only (35 mg/kg).

Time-effect relationships:

Thymus weight: During the relatively short test period of 9 days, the absolute thymus weights of control rats increased considerably, along with a rise in the number of cells that could be isolated from this gland. During the experimental period, the relative thymus weights remained at a constant level between 0.3 and 0.4% of body wt. From the second day after a single oral dose of 50 µmol DBTC per kg rat, the relative thymus weights were significantly decreased. Concurrently, the total cell count was diminished significantly at day 3, 4 and 7. These parameters, together indicative for thymus atrophy, were most severely reduced at day 4 after dosing of DBTC. Recovery was complete within 9 days for DBTC.

Relative cell counts: In thymocyte suspensions of control animals, the frequency of small cells (>130 µm³) increased gradually within the 9 days of the test period, while the intermediate (130-225 µm³) and large thymocytes (>225 µm³) progressively decreased in number. Already 1 day after intubation of DBTC, the frequency of the large thymocytes was markedly reduced to 45% of the control value. On the second day after dosing, the relative cell counts of both intermediate and large cells were significantly diminished to 75 and 51% of the control values, respectively. On the third day, the size distribution of thymocytes was normal again, while 4 days after intubation the frequency of the intermediate and large cells exceeded the controls. On this day the relative number of small cells was drastically decreased. Nine days after exposure, all populations were present with normal frequencies.

Incorporation of macromolecule precursors: In association with the frequency of intermediate and large cells, the incorporation of Urd and TdR in thymocytes of control animals showed a tendency to decline during the 9 day period. Intubation of DBTC markedly reduced the incorporation of the RNA precursor during the first 2 days. The time-effect relationships for the incorporation of Urd were strikingly similar to the kinetics of the intermediate and large cells. The effects of DBTC on the incorporation of TdR, however, were more severe and more persistant. Recovery occurred 1 day later (day 4) and a rebound may have taken place between day 4 and 7. Although the incorporation of Leu was less affected by DBTC, the time-effect curves for this amino acid resembled the ones for the incorporation of Urd. Incorporation rates were normalized after 7-9 days after organotin intubation. In rats given DBTC, the incorporation of TdR, Urd, and Leu was already reduced significantly after 1 day to 48, 50 and 63% of control values, respectively. Absolute cell counts: Using the frequency and the total cell count data, the absolute numbers of small, intermediate and large cells were determined. These absolute cell counts also indicate that DBTC caused a selective reduction of the large cells in the first 2 days after dosing. At day 4, the small, intermediate and total cell counts were drastically diminished. In thymocyte suspensions prepared 4 days after organatin exposure, the frequency of the large cells.was found to be markedly increased. However, the absolute number of large cells did not exceed the respective control value. As with the relative cell counts, the absolute numbers were normal again at day 9.

Conclusions:
A dose-related reduction of thymus weight was observed to achieve a maximum 4 days after dosing. It was calculated that a 50% reduction in thymus weight would occur at a dose level of 18 mg/kg bw of DBTC.
A dose-related depletion of cortical thymus lymphocytes was observed.
Executive summary:

In this immunotoxicity study, atrophy of the thymus was found in rats given a single oral dose of DBTC. Doses as low as 5 mg DBTC or 10 mg TBTC per kg body wt decreased relative thymus weight. It was calculated that a 50% reduction in thymus weight would occur at a dose level of 18 mg/kg bw of DBTC.

Thymus atrophy caused by DBTC is due to a selective effect on the population of large-sized, rapidly proliferating lymphoblasts. These cells, which are present in a limited number, generate the large population of small, non-dividing cells that populate the thymic cortex. As a result of this selective action on the lymphoblasts, a marked depletion of cortical lymphocytes develops several days after exposure to DBTC.

Endpoint:
immunotoxicity: acute oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read across material
Justification for type of information:
Study conducted on structural analogue dibutyltin chloride
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
other: 50% reduction in thymus weight
Effect level:
18 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1 mg/kg bw/day
Study duration:
subacute
Species:
rat

Effect on immunotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

From SPCFC (2004) specific experimental data for Dibutyltin (chloride) indicate immunotoxic effects at doses as low as 1 mg/kg bw in preweaning exposure studies (source unclear). This value is then apparently disregarded, and a LOAEL for DBT is cited as 2.5 mg/kg bw/day. SPCFC also indicate read-across from tributyltin to dibutyltin to be appropriate since the mechanism of action is similar and the immunotoxicity of TBT may be attributable to DBT as a metabolite; an immunotoxicity NOAEL for TBT is identified at 0.025 mg/kg bw/day by repeated dietary exposure. It must be noted that dose spacing in the critical study of TBTO (Wester et al 1988, 1990) is large and the NOAEL correspondingly imprecise. Further, if toxicity of TBTO is even in part due to formation of DBT as the ultimate toxicant, then a NOAEL based on an appropriate study of DBT-Cl is the preferable endpoint.

Modern and robust studies of the immunotoxicity of DBTC in adult rats and in pups exposed during gestation and lactation (DeWitt et al, 2005, 2006) found no repeatable effect on immune function at doses up to 2.5 mg/kg bw/day, although at this dose an effect on bodyweight was reported (possibly due to palatability); 1 mg/kg bw/day was a NOAEL.In a series of other appropriate studies of DBTC (Gaunt et al 1968; Pennincks et al 1982; Osterburg 1993; Waalkens et al 2003) dose levels of approximately 2-2.5 mg/kg/bw/day are an inconsistent NOAEL/LEL; but no effects are detected at 1 mg/kg bw/day or below. EFSA/SPCFC review did not include the significant studies by Osterburg (1993), Waalkens (2003), and DeWitt et al (2005, 2006). Use of a NOAEL at 0.3 mg/kg bw/day form Waalkens (2003) is therefore a robust and precautionary endpoint.

Justification for classification or non-classification

According to Regulation (EC) no 1272/2008 the test substance would be classified as a STOT Single Exp. Category 1 based on results obtained in Snoeij, N.J., Penninks, A.H. and Seinen, W. (1988) which indicate 50% reduction of thymus weight following a single oral dose of 18 mg/kg. Signal word: Danger; Hazard statement: H370 Causes damage to thymus.