Dichlorodioctylstannane

Immunotoxicity

Description of key information

The studies which have been added to section 7.9.2 – Specific investigations -

Immunotoxicity of the dossier from Boyd, Henninghausen, Gruendel, Kishi, Miller, Penninks and Volsen are all demonstrating that the observed adverse effects can be produced by a single exposure with dioctyltin dichloride. The chronology of the different adverse effects after a single exposure are given in the following table:

0h           24h           48h          72h

Decrease thymus weight       -              -              (x)              X

Decrease body weight          -              -              -               -

Decrease Thymocyte           -              X              X              X

Decrease IL-2                    -              X              X              X

IgG₁(OX-18)                     no data    X              no data      X

CD4⁺CD8⁺                      -              X              X              X       

24 hours after a single administration of Dioctyltin dichloride to rats a decrease in thymocyte count, IL-2,  IgG1 and CD4+ CD8+ is reported.Almos all of the cited studies report a decrease of the thymus weight after 72 hours, only one study (Volsen et al., Int J Immunopharmacol. 1989;11(6):703-15 )reports this effect after 48 h. No effect on body weight was observed in the cited studies. 

The doses in the studies uses for oral administration varied between 6.3 and 100 mg DOTC /kg bw.

TheLOAEL for a single administration can be set to 6.3 mg DOTC /kg bw.  In absence of data, a NOAEL is not indicated.

 

The adverse effect caused by Dioctlytin after single administration relating to the immune system and the thymus were reversible within six weeks. The test systems recovered from lymphocytes inhibition  in less than eight days

 

 

According Regulation (EC) No.  1272/2008 Annex I: 3.8.2.1.1. a substance is to classify as STOT SE

-         

Substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following single exposure

Substances are classified in Category 1 for specific target organ toxicity (single exposure) on the basis of:

a.            reliable and good quality evidence from human cases or epidemiological studies; or

b.            observations from appropriate studies in experimental animals in which significant and/or severe toxic effects of relevance to human health were produced at generally low exposure concentrations. Guidance dose/concentration values are provided below to be used as part of weight-of-evidence evaluation.

 

-         

Substances that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following single exposure

Substances are classified in Category 2 for specific target organ toxicity (single exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations. Guidance dose/concentration values are provided below in order to help in classification.

In exceptional cases, human evidence can also be used to place a substance in Category 2 (see 3.8.2.1.6).

 

The cited studies demonstrate, that Dioctyltin dichloride causes an adverse effect to the immune system in guinea pig, mice and rats after a single administration. The LOAEL was set to 6.3 mg/kg bw.

 

Based on the finding in several species and in accordance  with (EC) No.  1272/2008 Annex I Table 3.8.2. (dose oral administrant below 300 mg/kg/bw). the criteria for STOT SE 1 are met.

 

A clsiification of Dioctyltin dichloride as STOT SE 1 – H370 (immune system, oral) is warranted.

 

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

6.3 ng/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

-

Effect on immunotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on immunotoxicity: via dermal route

Link to relevant study records

Reference

Reference 1

Endpoint:

immunotoxicity

Remarks:

acute

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no glp, no guideline, well documented

Qualifier:

no guideline required

Principles of method if other than guideline:

BN and LEW rats, two to three months old, and F1 hybrids form LEW females mated with BN males were injected intraperitoneally with DOTC (one time)

The antibody production (to SRBC) and the response to ConA was determined. Histopathology of the thymnus glands were performed.

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

other: LEW, BN, F1 Hybrids of LEW and BN

Sex:

male/female

Route of administration:

intraperitoneal

Vehicle:

other: ethanolglycerol

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

1 time

Frequency of treatment:

1 time, ip

Remarks:

Doses / Concentrations:
0
Basis:
other: mg DOTC / kg bw

Remarks:

Doses / Concentrations:
1
Basis:
other: mg DOTC / kg bw

Remarks:

Doses / Concentrations:
3
Basis:
other: mg DOTC / kg bw

Remarks:

Doses / Concentrations:
4
Basis:
other: mg DOTC / kg bw

No. of animals per sex per dose:

4 - 12

Control animals:

yes, concurrent vehicle

Dose descriptor:

other: thymus atrophy

Effect level:

ca. 2 other: mg DOTC / kg bw

Based on:

test mat.

Sex:

male/female

Dose descriptor:

other: immune parameters

Effect level:

ca. 2 other: mg DOTC / kg bw

Based on:

test mat.

Sex:

male/female

Dose descriptor:

other: LD100

Effect level:

ca. 4 other: mg DOTC / kg bw

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: all animals died within 9 days

Strain	DOTC mg/kg bw	Thymus wt (mg)	ConA resonse	PFC
BN	0	370 ± 20	106818 ± 16224	99 ± 22
BN	3	200 ± 41	16847 ± 4499	266 ± 45
LEW	0	325 ± 23	222024 ± 39966	136 ± 25
LEW	3	190 ± 19	20499 ± 8201	571 ± 184
F1	0	390 ± 40	Not done	113 ± 13
F1	3	250 ± 45	Not done	705 ± 213

Conclusions:

After a one time administration of DOTC to rats there are adverse effects in thymus and immune system

Reference 2

Endpoint:

immunotoxicity

Remarks:

acute

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no glp, no guideline study, well documented

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline required

GLP compliance:

no

Limit test:

no

Species:

other: rodent and human

Strain:

other: not relevant

Sex:

not specified

Route of administration:

other: not relevant

Vehicle:

other: not relevant

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

not relevant

Frequency of treatment:

not relevant

No. of animals per sex per dose:

not relevant

Publication describes the mechanism of apoptosi of thymocytes after activation of NR3C1.

It is shwon, that adverse effects beloning to immune suppressive substance are an acute effect , beginning after a few hours

Conclusions:

After activation of NR3C1 the DNA fragmentation (via duming of IL-6) of thymocytes tbeginns within a hour and cells lysis follows 3 top 5 hours later.
This results in thymus atrophy, depletion of lymphocytes

Executive summary:

Destruction of thymus cells was one of the earliest observed properties of adrenal glucocorticoids. The cells affected are primarily immature, CD4/CD8 double-positive lymphocytes. This process has been clearly shown in vivo and in vitro to be apoptosis, as characterized by cell shrinkage, membrane alterations, nuclear collapse and chromatin fragmentation into oligonucleosomes. Glucocorticoid-induced thymocyte death requires new mRNA and protein synthesis. A beginning has been made in identifying the genes involved in thymocyte apoptosis. A case is made for the death of unselected thymocytes in vivo being regulated by endogenous glucocorticoids.

Reference 3

Endpoint:

immunotoxicity

Remarks:

acute

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no glp, no guidelline, well documentaed, possible, vehicle Ethanol causes adverse effects to the immune system , but control group, so it is possible to compare the effects

Qualifier:

no guideline available

Principles of method if other than guideline:

Material:
The experimental animals used were female rats with a body mass of 150g beginning of the experiment. The animals received food and water ad libitum. Parker medium and calf serum were obtained from the Institut für Immunpräparate und Nährmedien Berlin-Weissensee. For i.v. application of DOTC was solved in 96% ethanol, followed by the 2 parts by volume the Ethanol solution with 3 volumes of glycerol were mixed.

Organ and bodyweight:
To the application, a total of 52 female rats (180g) initially every second day, then up to 35 days post-application every third and fifth day, at the 42nd Weighed day and 50 day (4 animals each) and sacrificed by cervical dislocation.
Spleen and thymus were removed and the wet weight determined. Equally procedure at d = 0 (4 animals) and d = +50 with the control group (solvents Ethanol/Glycerine only).

Number of Leucocytes:
It took place after homogenization of the organs in Parker medium. The number of cells was determined in Thoma-chamber.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Route of administration:

intravenous

Vehicle:

other: Ethanol / Glycerol

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

acute (1 time) by i.v.

Frequency of treatment:

1 time

Remarks:

Doses / Concentrations:
4 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
8 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
10 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
12 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

4 per day and dose

Control animals:

yes, concurrent vehicle

Dose descriptor:

other: adverse effet in thymus (immune system)

Effect level:

4 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see free text

Reversibe, dose related thymus atrophy and reduced number of cells in thymus after one time dose of DOTC

Conclusions:

Singe exposure to DOTC causes adverse effects in the immune system of rats. These effect is reversible

Executive summary:

One i.v. injection of Dioctyltin chloride in a dose of 4 mg/kg bw in female rats caused thymus atrophy, which was reversible within six weeks. Further the number of lymphocytes in the spleen was reduced. The periode of DOTC induced lymphocytes inhibition seems to cover less than eight days.

Reference 4

Endpoint:

immunotoxicity

Remarks:

other: gene expression, thymus, IL-6

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no glp, no guideline, well documentation

Qualifier:

no guideline required

Executive summary:

Thymopoiesis is essential for development and maintenance of a robust and healthy immune system. Acute thymic atrophy is a complication of many infections, environmental stressors, clinical preparative regimens, and cancer treatments used today. This undesirable sequela can decrease host ability to reconstitute the peripheral T cell repertoire and respond to new antigens. Currently, there are no treatments available to protect against acute thymic atrophy or accelerate recovery, thus leaving the immune system compromised during acute stress events. Several useful murine models are available for mechanistic studies of acute thymic atrophy, including a sepsis model of endotoxin-induced thymic involution. We have identified the IL-6 cytokine gene family members (i.e., leukemia inhibitory factor, IL-6, and oncostatin M) as thymosuppressive agents by the observation that they can acutely involute the thymus when injected into a young, healthy mouse. We have gone on to explore the role of thymosuppressive cytokines and specifically defined a corticosteroid-dependent mechanism of action for the leukemia inhibitory factor in acute thymic atrophy. We also have identified leptin as a novel, thymostimulatory agent that can protect against endotoxin-induced acute thymic atrophy. This review will highlight mechanisms of stress-induced thymic involution and focus on thymosuppressive agents involved in atrophy induction and thymostimulatory agents that may be exploited for therapeutic use.

Reference 5

Endpoint:

immunotoxicity

Remarks:

acute

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no glp, no guideline, well documented

Qualifier:

no guideline available

Principles of method if other than guideline:

Single Administration of DOTC to mice. Determination of thymus weight and number of thymocytes, antibody titer against SRBC

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

other: outbred AB

Sex:

male/female

Route of administration:

other: oral gavage, im, iv

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

1 time

Frequency of treatment:

1 time

Remarks:

Doses / Concentrations:
4 - 61 mg / kg i.v.
Basis:
other: nominal dosed

Remarks:

Doses / Concentrations:
30 - 120 mg / kg imn
Basis:
other: nominal dosed

Remarks:

Doses / Concentrations:
120 - 240 mg / kg oral
Basis:
other: nominal dosed

No. of animals per sex per dose:

5-10

Control animals:

yes, concurrent vehicle

Dose descriptor:

other: decrease thymus weight, number thymocytes, SRBC antibody titer

Effect level:

4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: i.v.

Dose descriptor:

other: decrease thymus weight, number thymocytes, SRBC antibody titer

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: i.m.

Dose descriptor:

other: decrease thymus weight, number thymocytes, SRBC antibody titer

Effect level:

120 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: oral

decrease thymus weight, number thymocytes, SRBC antibody tiiter was reversible

Conclusions:

reversibe decrease thymus weight, number thymocytes, SRBC antibody titer after one time administration of DOTC

Executive summary:

After single administration of di-n-octyltin dichloride (DOTC) 4-61 mg/kg iv, 30-120 mg/kg im, and 120-240 mg/kg p.o.--a reversible, dose-dependent reduction of thymus weight and of the number of thymocytes was observed in mice. Di-n-butyltin dichloride (DBTC) induced similareffects. After iv administration of DBTC the level of total bilirubin in serum of mice was highly increased. DOCT did not elevate the bilirubin content of the serum. The level of bilirubin in serum did not correlate with the thymotoxiceffectsof DOTC and DBTC in mice

Reference 6

Endpoint:

immunotoxicity

Remarks:

acute

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no glp, no guideline, well documented

Qualifier:

no guideline available

GLP compliance:

no

Limit test:

no

Species:

other: rat and mice

Strain:

other: rat: wistar, mice: outbred AB

Sex:

male/female

Duration of treatment / exposure:

1 time

Frequency of treatment:

1 time

Remarks:

Doses / Concentrations:
mice, see attachment
Basis:
other: test nateruak

Remarks:

Doses / Concentrations:
rats, see attachment
Basis:
other: test material

No. of animals per sex per dose:

5-10

Control animals:

yes

Dose descriptor:

other: decrease thymus weight, number thymocytes

Effect level:

4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mice, iv

Dose descriptor:

other: decrease thymus weight, number thymocytes

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mice, i.m.

Dose descriptor:

other: decrease thymus weight, number thymocytes

Effect level:

60 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mice, p.o.

Dose descriptor:

other: decrease thymus weight, number thymocytes

Effect level:

4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: rat, i.v.

Dose descriptor:

other: decrease thymus weight, number thymocytes

Effect level:

15 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: rat, i.m.

Dose descriptor:

other: decrease thymus weight, number thymocytes

Effect level:

120 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: rat.p.o.

Species	Route of administration	Dose DOTC (ppm)	Relative thymus weight /% of control)	Thymocyte count (% of control)
Mouse	i.v.	8	41	47
	i.m.	30	74	76
		60	48	64
	p.o.	300	65	70
		500	70	50
Rat	i.v.	4	43	30
	i.m.	15	63	86
	p.o.	120	55	44

Conclusions:

A single DOTC cause a decrease thymus weight, number thymocytes in mice and rats after 4 days

Executive summary:

Theeffectsofdi-n-octyltin dichloride(DOTC) on thethymusinmicewere studied in dependence on the route of administration. Single administrations of 30, 60 and 120 mg/kg of DOTC i.m. induced a dose-related decrease of thethymusweight and of the number of nucleated cells in thethymusof malemice. 4 days after intravenous injection of 8 mg/kg of DOTC thethymusweight and the number of thymocytes were reduced to 50 percent of the control. In oral route of administration higher doses of DOTC (300 mg/kg) were necessary to provoke athymusatrophy. After pretreatment ofmicewith carbon tetrachloride thetoxiceffectsof orally administered DOTC were increased. The reduction of thethymusweight and of the number of thymocytes inmiceafter parenteral administration was accompanied by a depression ofthymus-dependent antibody formation against sheep red blood cells

Reference 7

Endpoint:

immunotoxicity

Remarks:

acute

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Not a recognised test method, no data on GLP or equivalent QA procedure, no purity reported.

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: BN and LEW

Sex:

male/female

Route of administration:

intraperitoneal

Vehicle:

other: 1:1 ethanol:glycerol

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

N/A

Frequency of treatment:

Single dose

Remarks:

Doses / Concentrations:
1, 3 or 4 mg DOTC/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

4 male, 4 female

Control animals:

yes, concurrent vehicle

Survival

All BN and LEW rats injected with 4 mg DOTC/kg body weight (using a concentration of 2 mg DOTC/ml solvent) died within 9 days but LEW survived significantly longer than BN. The percentage surviving also differed significantly on days 1 and 2 but not on days 3, 7 or 9. All animals injected with an equivalent volume of solvent without DOTC survived. It was found that, when the concentration of DOTC injected was increased to 4 mg/ml, both strains survived longer and this concentration was used for all studies described below.

Using the higher concentration, a difference between males and females was detected. The survival rates of BN males differed significantly (P<0.05) from that of females on all days except day 1 after injection of DOTC. LEW males differed significantly from females only on day 7. A significant difference between males of BN (17% survival at day 7) and LEW (58% survival at day 7) was alsoobserved. (LEW x BN) F₁ hybrids (not shown) behaved like the LEW strain with 56 % (5/9) of males surviving 7 days. There werenosignificant differences in survival of females among the 3 strains. All animals (groups of 4 to 6 for each strain) injected with 1 mg DOTC/kg survived.

Immunological parameters

No significant changes,in body weights or spleen weights were detected in any ofthe 3 strains whenratsinjected with DOTC were compared to those injected with solvent alone. The thymic weights and responses to ConA were decreased significantly when BN and LEW rats treated with DOTC were compared to controls. In contrast, the antibody responsesofthe 3 strains were increased, although the increase was not statistically significant. Responses of rats injected with 1 mg DOTC/kg body weight were comparable to those of controls. No significant differences between males andfemales were detected for any of these parameters.

Histopathology

There was a correlation between decreased thymic weightsand alterations detected histologically. BN rats and F₁ hybrids were similar and significant changes were detected only in females treated with DOTC. With LEWrats, significant changes were detected in both males and females. Tissues from ratstreated with solvent alone were all normal.

The major change observed in affected tissues was a marked depletion and thinning of the cortex. The junction between cortex and medulla was also somewhat obscured. With LEW rats, a significant increase in fibrous connective tissue was also observed in the thymus. Because some organotins are potent neurotoxins, the hippocampal formations of brains from the animals were also examined; all were normal. Other areas of the brain were not examined.

Conclusions:

Treatment with dioctyltin dichloride (DOTC) induced increased mortality, atrophy of the thymus and suppression of responses to concanavalin A. Brown Norway (BN) rats.were more susceptible than Lewis (LEW) to the lethal effects of DOTC and males were more susceptible than females. Increased mortality did not correlate with changes in the histological appearance of the thymus. LEW x BN F1 hybrids resembled the LEW parent in susceptibility to the lethal effects of DOTC and the BN parent in susceptibility to changes in the thymus.

Reference 8

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:

other: Single concentration used only without justification. No data on GLP or if test is performed to accepted method.

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Single dose. Animals observed for 12 days.

Frequency of treatment:

Single dose

Remarks:

Doses / Concentrations:
100 mg/kg BW
Basis:
actual ingested

No. of animals per sex per dose:

4 - 8 animals

Control animals:

yes

Changes in Body Weights

The body weights in animals given DOTC increased less in days 1 - 4 that the control. However, they soon recovered and no significant differences were observed at 8 days after treatment.

Organ Weights

For the thymus, a significantly low weight was observed 1 day after DOTC treatment, and the weight decreased until at least 7

days later. Thereafter, thymus weights of animals given DOTC were still significantly lower than those of control animals at the end of the experiment, although they tended to recover to control levels. Spleen weights were low in DOTC-treated animals in comparison to controls. Significantly low spleen weights in DOTC-treated animals were observed from 6 h to 3 days after treatment. However, these recovered to control levels 4 days after treatment. Although, low liver and kidney weights in DOTC-treated animals were also observed, these were temporary.

Changing pattern of tissue concentrations of dioctyltin and its major metabolite, monooctyltin

Most dioctyltin was accumulated in the liver after DOTC treatment. In the case of monooctyltin, a high accumulation was observed in the liver, but at lower levels than dioctyltin. Di- and monooctyltin concentrations in the liver and kidney increased peaked 2 days after DOTC treatment, and then decreased. Dioctyltin accumulation in the brain was very low, although the levels remained constant from 2 days after treatment. Monooctyltin accumulation in the brain was not detected during the experiment. Also, we could not detect di- or monooctyltin accumulation in the thymus or spleen.

Histological changes

In the histological study, no clear effects of DOTC administration were observed in all tissues examined. On the other hand, the number of TUNEL stained cells, apoptotic cells, in the thymus and spleen increased from 1 to 4 days after DOTC treatment, as compared with the controls. Thereafter, apoptotic cell numbers in the thymus and spleen in animals administered DOTC were similar to those in control animals until 8 days after treatment.

Expression of genes involved in apoptotic pathway in thymus

In the study, 154 probe sets of 97 genes involved in apoptosis were studied between the thymus of controls and DOTC-treated animals These genes were categorized as ones involved in apoptosis by Gene Ontology (GO) Data (Gene Ontology TM Consortium), Gene MicroArray Pathway Profiler or KEGG (Kyoto Encyclopedia of Genes and Genomes). Most of the studied probe sets did not show different expressions. Only three (1369146 a at, 1387690 at and 1368936 at) of the probes corresponding to Ahr, Casp3 and Txnll genes, respectively, showed significantly low signals. A significantly high signal was observed with the probe

for the Nr3cl gene.

Discussion

This is the first report to directly show the changing concentration patterns of dioctyltin and its major metabolite, monooctyltin, in each tissue of animals administered DOTC. Most accumulations of di- and monooctyltin were found in the liver. In other tissues, their accumulations were low or absent. In other tissues, their accumulations were low or absent. Penninks et al. (1987) also showed similar findings concerning the distribution of dioctyltin in tissues using 14C-labeled DOTC administration in rats. A decreased liver weight was observed when accumulations of di- and monooctyltin in the liver reached a peak. However, the liver toxicity of this compound would be low because liver weight recovered to control levels immediately, and no pathological changes were observed. Thymus and spleen weight losses were also found, although tissue accumulations of di- and monooctyltin were not detected. The reduced thymus and spleen weights were maintained longer than that observed in the liver. This suggests that this compound leads to immunotoxicity.

A higher number of apoptotic cells in the thymus was detected from 1 to at least 4 days after DOTC treatment as compared with controls, and this period roughly corresponds to the time of thymus weight decrease. Therefore, the increase in the number of apoptotic cells in these organs would provide an explanation for thymus weight loss, although DOTC also has an inhibitory effect of thymocyte proliferation. It supports the view that dibutyltin and tributyltin, organotins shown to be immunotoxic, similarly cause thymocyte apoptosis.

Spleen weight also decreased after DOTC treatment. The number of apoptotic cells in the spleen as well as in the thymus increased. However, spleen weight reduction was relatively slight, and had recovered to control levels 4 days after treatment. Therefore, the toxicity of this compound to the spleen is relatively low compared to the thymus.

No other histopathological changes were noted in the brain, liver or kidney of animals given DOTC. The body weight of animals administered DOTC was significantly reduced 2 days after DOTC dosing as compared with the controls. However, it recovered immediately and was not significant 8 days after treatment. Therefore, it presented no serious toxicity.

In the study, genes in thymus involved in apoptosis were researched using a microarray technique. Most of the genes examined showed similar expressions between controls and DOTC-treated animals. Only three probes were found showing significantly low hybridization signals and one probe showing a significantly high signal: probes for the genes Ahr, Casp3, Txnll and Nr3cl, respectively. For expression analysis of these genes, not only single but also plural probes were determined in the present hybridization study. No significant difference was found using these plural probe sets. Therefore, it is not clear whether these genes are expressed differentially after DOTC treatment. However, the hybridization signal of these other kinds of probe sets against Ahr (1369147 at) and Txnll (139135 at, 1392058 at, 1388862 at, 1391789 at and 1368936 at) also tended to decrease, and that against Nr3cl (1368222 at and 1397004 at) tended to increase. So, Ahr and Txnll might be candidates of downregulated genes, and Nc3rl might be a candidate of upregulated genes after DOTC treatment. There is a report that nur77 is one of the candidate genes

for inducing apoptosis in the rat thymus after DBTC treatment (Gennari et al., 2002a). Nc3rl is one of nuclear receptor family

genes, like nur77. Therefore, Nc3rl may be a potential candidate gene for organotin-induced apoptosis. In conclusion, exposure

to DOTC causes immunotoxicity in developing rats, and is selectively toxic to thymus and spleen cells. One reason for the observed thymus weight loss may be the increased apoptosis of immune cells (possibly T-cells), as noted in cases of TBTC and DBTC treatment. To elucidate this further, Nc3rl gene expression in the thymus after DOTC treatment needs to be clarified.

Conclusions:

No significant differences in body weights of DOTC-treated rats relative to control animals. Di- and mono-octyltin accumulation detected mainly in the liver. Significantly low thymus and spleen weights. Increased apoptotic cell numbers in the thymus and spleen. No clearly different expressions of the 97 genes studied involved in apoptosis were detected in DOTC-treatment animals. Authors concluded that DOTC was selectively immunotoxic, and one mechanism for immunotoxicity is stimulation of immune cell apoptosis.

Reference 9

Endpoint:

immunotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No information on GLP. Study not performed to a recognised test method.

Qualifier:

no guideline followed

GLP compliance:

not specified

Species:

rat

Strain:

other: Inbred PVG rats

Sex:

male

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

8 or 12 weeks.

Frequency of treatment:

ad libitum in diet

Remarks:

Doses / Concentrations:
0, 75 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

minimum of 8 animals for each parameter investigated

Control animals:

yes

Previous studies had shown a slight drop in growth rate in rats fed 150 ppm DOTC-containing diets for longer than 2 weeks, and animals in this investigation were therefore fed diets containing 75 ppm DOTC to avoid any confounding factors.

Thymic weight was markedly reduced in all treated groups whereas no effect on body weight gain or feed uptake was apparent. No histological changes attributable to DOTC treatment were observed in tissue sections of spleen, liver, prostate, seminal vesicles, and testes compared with tissue from control rats by light microscopy. Thymus sections from DOTC-fed rats showed a gross depletion of small thymocytes, septal thickening, and loss of corticomedullary boundaries in all cases. The thymic atrophy was accompanied by some loss of circulating leukocytes (WBC) which was first apparent 4 weeks after commencement of treatment, but showed little further decrease over time of treatment. After 8 weeks treated rats had a mean WBC count of 9.9 X 10²/ mm³ compared to 13.27 X 103/mm³ in control rats (p<0.001) and at 12 weeks the mean WBC count was 10.4 X 10²/mm³ and 13.8 X 10³/mm, respectively (p<0.001). is decrease was not accompanied by a decrease in cell viability which was always greater than 95% as determined by trypan blue exclusion. Analysis of the T-cell subpopulations present in the circulating lymphocytes demonstrated a significant (p<0.01) preferential loss of cells expressing W3/25 phenotype in samples obtained from treated rats. The absolute numbers of W3/25-positive lymphocytes had decreased from a mean of 6.8 X 10³/mm³ in control rats to 4.35 X 10³/ mm³ in DOTC-fed animals after 12 weeks feeding.. No significant difference was found in cells expressing the MCR OX8 phenotype between treated and control rats.

The ability of lymphocytes from treated rats to respond to PHA-induced blastogenesis was reduced at all three concentrations of PHA used in the assay system compared to untreated animals. A decrease of [³H]thymidine incorporation into unstimulated lymphocytes obtained from treated rats was also observed. Thymidine incorporation was 27, 42, and 63% of control values at 1.25, 2.5, and 3.75 µg/ml PHA, respectively, after 8 weeks of DOTC exposure. The level of decreased responsiveness compared to concurrent controls was the same after the 8- and 12-week feeding regimen, and was always very much greater at the suboptimal dose of PHA present during the assay (p<0.01) These assays were unaffected by. the reduction in total lymphocytes obtained from treated compared to control animals, since all tests were performed at a concentration of 1 X 10⁶ lymphocytes per ml.

Both DOTC-treated and control animals were able to mount specific antibody responses against SRBC, a heterologous T-cell-dependent antigen. Hemagglutinin assays showed little difference in responsiveness between the treated and untreated groups after either 8 or 12 weeks of treatment. The slight reduction in antibody production seen after 12 weeks exposure from 4.5 to 4.0 units expressed as loge dilutions are in marked contrast to experiments with nude rats which demonstrated antibody responses of less than 0.5 units.

As little difference was found in the alteration of thymus-dependent immune responses between 8 and 12 weeks DOTC exposure, investigation into allogenic responsiveness and natural cytotoxicity were carried out only in 8-week exposed animals. Experiments measuring GvH reactions showed slight differences in the weights of draining lymph nodes from F₁ rats injected with cells obtained from DOTC-treated rats compared with those injected with cells from control animals. The reduction in lymph node weight, more discernable when expressed as proliferation index (PI), was not indicative of an impaired allogeneic response. The mean PI was 18.87 and 17.61 for control and DOTC-treated respectively.

A marked reduction in allogenic recognition in in vitro MLR assay. Lymphocytes isolated from treated PVG rats showed a significantly reduced proliferative response when cultured with either syngeneic (p<0.05) or allogeneics (p<0.01) stimulator cells compared to control rats. Natural cytotoxicity, a measure of early killing of allogeneic cells by unsensitized hosts, was also determined after 8 weeks on DOTC-containing diets. Syngeneic recipients (DA rats) were used as negative controls. Whereas the radioactivity recovered from lymph nodes was in excess of that recovered from kidneys in syngeneic recipients (lymph node/kidney = 14.2), both control and DOTC-fed allogeneic PVG rats were able to destroy a substantial proportion of ⁵¹Cr-labeled injected cells within 24 hr. The lymph node to kidney ratio was equal to 0.75 for control and 0.71 for treated animals, demonstrating no significant difference in activity between the two groups.

Conclusions:

Dioctyltin accumulations were mainly detected in the liver, and peaked 48 h later. A similar pattern was also found in the kidney, but the levels were low or trace amounts. Significantly low thymus and spleen weights were detected in DOTC-treated animals. Increased apoptotic cell numbers in the thymus and spleen were observed in DOTC-treated animals also. Although the expression of 97 genes involved in apoptosis was studied in the thymus, at least 24 h after treatment, we could not detect clearly different expressions between DOTC- and vehicle-treated animals. The present results suggest that DOTC was selectively immunotoxic. One of the mechanisms for its immunotoxicity would be via its stimulation of immune cell apoptosis.

Reference 10

Endpoint:

immunotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported.

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No information on GLP. Study not performed to a recognised test method.

Qualifier:

no guideline followed

GLP compliance:

not specified

Species:

mouse

Strain:

Balb/c

Sex:

female

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

8 weeks

Frequency of treatment:

Once a week

Remarks:

Doses / Concentrations:
20, 100 and 500 mg/kg bw
Basis:
nominal conc.
in corn oil vehicle

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Statistics:

Data were analyzed using analysis of variance followed by Student's t test where appropriate, values of P < 0.05 being considered statistically significant.

Antibodies with specificity for self erythrocytes were first detected in 80% of control animals 7 days after the second rat erythrocyte injection. The antibody titer increased rapidly after the third injection and at this stage all control mice were producing a high-titer antibody response. The weekly administration of 20 or 100 mg/kg body wt DOTC prior to each RRBC injection had no significant influence on this response, but 500 mg/kg body wt DOTC significantly depressed the response from Week 3 onwards.

The parallel anti-rat erythrocyte antibody response occurred rapidly, with anti-body being detectable in the serum 7 days after the first injection of RRBCs. The antibody titer was seen to increase following subsequent injections of rat erythrocytes. The administration of 500 mg/kg body wt DOTC caused a statistically significant depression of the response at Weeks 1 (p < 0.05), 4 (p < 0.01), and 5 (p < 0.05); the administration of lower doses of DOTC was without effect. None of the DOTC doses employed affected the 48-hr delayed-type hypersensitivity response against oxazolone assessed during Week 8 of the treatment regime. Hematological analysis performed at the end of the experiment revealed a reduced hemoglobin level in those mice treated with 500 mg/kg body wt DOTC, though RBC and WBC counts remained within the control range. The administration of the lower concentrations of DOTC had no effect on these parameters. Compared with control, relative thymus weight was decreased (p < 0.01) liver weight was increased, but spleen weight was normal in animals receiving 500 mg/kg body wt DOTC weekly. The administration of 20 or 100 mg/kg body weight DOTC, did not influence the relative organ weights.

Conclusions:

Oral weekly administration of DOTC caused a suppression of the anti-self erythrocyte antibody response. The humoral response against rat erythrocytes was also diminished. Although humoral responsiveness was affected only at a dose level which caused thymic atrophy (500 mg/kg body wt) the delayed type hypersensitivity reaction to oxazolone, a measure of cell-mediated immunity, was not affected. These data indicate that DOTC can suppress specific aspects of murine immunocompetence and may be relevant in designing studies for assessing possible hazards associated with environmental chemicals.

Reference 11

Endpoint:

immunotoxicity

Remarks:

subacute

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No information on GLP. Study not performed to a recognised test method.

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: PVG

Sex:

male

Route of administration:

other: Oral food and also intramuscular in some rats

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

maximum of 4 weeks

Frequency of treatment:

ad libitum in food

Remarks:

Doses / Concentrations:
0, 150 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

6 male

Control animals:

yes, plain diet

Statistics:

Differences between the parameters measured in treated and control animals were compared using Student's t test. The level of significance chosen for all studies was P<0.01.

In all treated groups thymic weight (gram wet weight) was markedly reduced. In the animals fed DOTC-containing diets for 4 weeks, return-to-normal diets resulted in increased cellularity of the thymus, although thymic weight remained significantly reduced. The low thymic weight after treatment was accompanied by a decrease in circulating lymphocytes. Surface marker analysis did not, however, demonstrate a decrease in the proportion of T cells present (80 and 77% for control and treated rats, respectively). Viability, as determined by trypan blue exclusion, was greater than 95% at all times. The PHA-induced lymphocyte blastogenesis was significantly reduced: after 4 weeks of DOTC-containing diet compared to control animals and remained reduced after cessation of treatment.

Histopathological and Ultrastructural Observations

Control rats. Thymuses from untreated rats showed normal thymic morphology and a distinct corticomedullary junction was always apparent at the light microscope level. Ultrastructurally cortical thymocytes were characterised by round nuclei, dense heterochromatin, little cytoplasm, and few mitochondria. The reticular epithelial cells (REC) exhibited slender cytoplasmic processes and large, pale nuclei. The abundant cytoplasm contained tonofilaments and some rough endoplasmic reticulum and mitochondria. Small numbers of electron dense granules were also observed in the cytoplasm along with occassiorial multivesicular bodies. Few macrophages were present in the cortical area.

Adrenalectomized rats. Both at light and electron microscope levels thymuses from operated animals exhibited normal morphological features.

DOTC-treated rats. Thymuses from rats fed DOTC-containing diets for 2 weeks showed a partial loss of cortical thymocytes and indistinct corticomedullary junctions. A number of vacuolated REC was observed together with normal appearing REC. There were few macrophages present and very few pyknotic thymocytes. Ultrastructurally the vacuolated REC showed an increase in both nuclear and cytoplasmic densities with vesiculation of the rough endoplasmic reticulum and an accumulation of intracellular vacuoles, some containing amorphous material. Many of the otherwise normal appearing REC showed an increase in the number of secretory droplets present in the distal cytoplasm. The majority of remaining thymocytes had a normal appearance.

With increasing length of exposure to DOTC there was a continued loss of cortical thymocytes. Positive identification of the corticomedullary junction was not possible and the number of vacuolated REC observed was greatly increased. When the animals were returned to a normal diet a weekly examination of the thymuses showed a considerable repopulation with, however, persistance of hypocellular areas and of numerous vacuolated epithelial cells.

Adrenalectoinized and DOTC-treated rats. Morphological changes following oral DOTC treatment were as marked in adrenalectomized rats as in nonoperated animals.

Hydrocortisone treated rats. Animals treated with hydrocortisone showed severe depletion of cortical thymocytes and loss of the corticomedullary junction. No alteration in the appearance and distribution of REC was observed, but macrophages were present in greatly increased numbers. Ultrastructural ex-amination showed that the macrophages contained large lipid droplets within secondary lysosomes.

Conclusions:

Thymus weight was decreased. Histopathological observations of the thymus included loss of cortical thymocytes and vacuolation of reticular epithelial cells. PHA-induced lymphocyte blastogenesis was decreased. Circulating lymphocytes were decreased.

Reference 12

Endpoint:

immunotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No information on GLP. Study not performed to a recognised test method.

Qualifier:

no guideline followed

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: PVG

Sex:

male

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Maximum of 6 days.

Frequency of treatment:

Groups gavaged on alternative days.

Remarks:

Doses / Concentrations:
0, 75 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Statistics:

Differences between the parameters measured were compared using a small sample t-test. The level of significance chosen for all experiments was p<0.05.

Reduction of thymic weight was first apparent 48 h after DOTC administration. Alterations in thymocyte activity was apparent prior to the decline in thymic weight loss. Studies on thymocytes obtained from untreated rats and rats 24, 48 and 72 h after commencement of treatment demonstrated a marked decline in both the basal level of DNA synthesizing cells present in the thymus, and the degree of spontaneous in vitro proliferative activity.

When thymocytes were incubated together with tritiated thymidine for 30 min, a progressive decrease in thymidine incorporation was evident. Decrease in thymidine uptake occurred before thymic involution was apparent, and significant difference were apparent 24 h after the first oral intake of DOTC when compared to thymocytes obtained from untreated rats (p<0.05). Mitotic activity was almost entirely absent in thymocytes obtained after two administrations of DOTC (P<0.001), although considerable thymic tissue was still present in the treated rats.

Proliferation of thymocytes obtained from untreated rats continued during a 3 h incubation period, reflecting the spontaneous proliferative capacity of thymocytes when maintained in in vitro cultures. In contrast, DNA synthesis was significantly decreased in cell populations obtained from rats 72 h after commencement of treatment (P<0.001) indicating a marked decline in mitotic activity.

Sera obtained from DOTC-treated rats inhibited the mitogen responsiveness of thymocytes obtained from control rats. The ability of thymocytes to respond to PHA stimulation was significantly reduced when the cells were cultured in the presence of either 2.5 or 5% rat sera obtained from treated animals. The inhibitory effect of serum samples was evident shortly after commencement of DOTC administration, and was greater when sub-optimal concentrations of PHA were used . At this PHA level (1.0 µg/ml), sera obtained 24 h after commencement of DOTC treatment significantly reduced PHA-induced thymocyte blastogenesis (P<0.05) when added to the culture medium at 50%. Sera obtained 48 and 72 h after commencement of treatment caused a highly significant depression of thymocyte responsiveness at both PHA levels. Similar results were obtained with 2.5% sera although reduction in thymocyte proliferation was less marked than at the higher concentration of rat sera. Addition of rat sera obtained from untreated control rats had no inhibitory effect on the thymocyte proliferative response and the level of thymidine incorporation was similar to that obtained under standard culture conditions. Addition of DOTC (7.5 µg/ml) to control sera did not cause any depression of thymocyte proliferation.

Conclusions:

Oral administration of dioctyltin dichloride (DOTC) results in the production or release of factors which appear to prevent thymocyte maturation at an early stage. Sera obtained from rats gavaged with 75 mg DOTC/kg on alternate days, significantly inhibited PHA-induced blastogenesis of normal thymocytes. In vitro uptake of tritiated thymidine by thymocytes obtained from DOTC-gavaged rats was markedly reduced. These events occurred 24 to 72 h after commencement of DOTC treatment and preceded overt thymic weight loss. The effects of DOTC may therefore be a consequence of factor(s) capable of blocking the intrathymic differentiation of T cell precursors.

Reference 13

Endpoint:

immunotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported.

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No information on GLP. Study not performed to a recognised test method. Very little information on methods.

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: PVG

Sex:

not specified

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

8 weeks

Frequency of treatment:

Not stated, but test substance was incorporated into rats feed.

Remarks:

Doses / Concentrations:
75 ppm
Basis:
nominal conc.

Control animals:

not specified

Test 1

At post mortem, thymuses from pregnant rats maintained on control diets showed slight thymic involution; however the thymuses from both virgin and pregnant DOTC treated rats were markedly atrophied. Tissue sections of thymuses from post partum control animals apeared normal, with a clear distinction between cortex and medulla. Reticulo-epithelial cells (RECs) were present in both the cortex and medulla and often contained large numbers of non-osmiophillic vacuoles. Virgin animals exposed to DOTC for the same period showed massive thymocyte depletion in the cortical region, the number of thymocytes was also slightly reduced in the medullary region. There was no marked increase in the degree of vacuolation in the RECs. Pregnant animals given DOTC during pregnancy and killed immediately post partum presented a similar picture in terms of thymocyte depletion, however, the degree of vacuolation in the RECs was greatly increased. It would therefore appear that the changes in the thymus occurring during pregnancy are qualitatively, but not quantitatively similar to those observed during DOTC-exposure. In both instances there was evidence of vacuolation in RECs however, the degree of vacuolation observed as well as the number of vacuolated cells was greater in rats exposed during pregnancy. It therefore appears that the changes in endocrine control that occur during pregnancy may exacerbate the effects of DOTC.

Test 2

It was apparent that almost all of the radioactivity was excreted in the faeces by 48 hrs. Nevertheless the presence of radioactivity in the liver and urine indicated that a small proportion of the administered compound was absorbed and possibly metabolised. Furthermore although only a minute amount of label was found to be present in the thymus significantly more was found in the pituitary.

Despite the very specific action DOTC has on rat thymic tissues only a trace of radioactivity was found associated with the organ, it therefore seems unlikely that thymic atrophy is the result of a local DOTC-mediated toxic event. The observation that label was also present in the pituitary lends further support to the postulate that endocrine factors may play a role in DOTC induced thymic atrophy.

Conclusions:

The changes in endocrine control that occur during pregnancy may exacerbate the effects of DOTC.

Despite the very specific action DOTC has on rat thymic tissues only a trace of radioactivity was found associated with the organ, it therefore seems unlikely that thymic atrophy is the result of a local DOTC-mediated toxic event. The observation that label was also present in the pituitary lends further support to the postulate that endocrine factors may play a role in DOTC induced thymic atrophy.

Reference 14

Endpoint:

immunotoxicity

Remarks:

subchronic

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No information on GLP. Studies not performed to recognised test methods. Very little information on methods.

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Atrophy of Thymus and Thymus-Dependent Lymphoid Tissues in Rats Fed Di-n-Octyltindichloride

In rats fed di-n-octyltindichloride (DOTC) at dietary levels of 0, 50 or 150 mg/kg diet (ppm) a prominent and dose-related decrease of thymus and popliteal lymph node weight was noted.Thymic atrophy progressively increased Already after a 4-day feeding period thymus weights were significantly lower as compared to control animals. Thymus weights of rats fed 50 or 150 ppm DOTC for 4 weeks were only 48% and 16% of the control weights. Histologically, lymphocyte depletion was observed in the thymus and in thymus-dependent areas of the spleen and lymph nodes, whereas in other organs no treatment-related histopathologic changes were noted. Although most of the lymphoid tissue of the thymus had disappeared, signs of cell destruction (karyorrhexis and "starry sky" formation) as seen after treatment with corticosteroids antimetabolites, alkylating agents, and other antitumor compounds were never observed after DOTC treatment. This does not, however, exclude lymphocytotoxicity, as the viability of nucleated cells isolated from thymus and spleen of rats fed 50 or 150 ppm DOTC for 4 weeks was significantly decreased. However, the effect on thymocyte number was much more pronounced than the effect on all viability. Already, after 2 days feeding of DOTC the total number of thymocytcs showed a dose-related decrease but cell viability was unaltered (as measured with the dye exclusion test). Total thymocyte counts progressively diminished to 33% and 6% of the control value at week 4 in animals fed 50 or 150 ppm DOTC, respectively. These data indicate that DOTC primarily induces an inhibition of the division of thymic lymphocytes and secondarily causes cell death.

The total number and viability of spleen and peripheral lymph node cells was less severely decreased by DOTC, than the number and viability of thymus cells. After 4 weeks feeding of 150 ppm DOTC, the numbers of nucleated spleen and peripheral lymph node cells were 75% and 70%, respectively, of the control counts. The total number of peripheral blood lymphocytes was generally unaltered by organoun treatment. Also, the number and viability of bone marrow cells was not decreased by DOTC feeding. Effects of organotin compounds on bone marrow stem cells are further excluded by the spleen colony assay technique with balb/c mice. This experiment was carried out with di-n-butyltin dichloride (DBTC), a compound with effects on lymphoid tissue similar to those observed with DOTC. The numbers of 10-day spleen colonies produced in lethally irradiated recipient mice injected with bone marrow cells of donor mice treated 1 or 3 days before cell transplantation with 0, 2, or 10 mg DBTC/kg intravenously, were essentially the same. In addition, spleen weights of mice reconstituted with bone marrow cells from DBTC-treated and -untreated animals were comparable. Thus, neither the number and viability of bone marrow cells nor the number of colony-forming stem cells was decreased by organotin treatment. These results further support the selective cytotoxicity of DOTC for thymus and thymus-dependent lymphocytes.

A selective lymphocyte depletion in the thymus can also be induced by a stress-related release of glucocorticoids. The selective effects of DOTC on the thymus are not related to stress since:

(1) Thymus weights were as severely decreased in adrenalectomized as in sham-adrenalectomized DOTC-fed rats;

(2) relative adrenal weights were the same in DOTC-fed and control animals;

(3) histologically, no signs of hyperactivity of the adrenal cortex, nor destruction of lymphocytes in the thymic cortex were observed; and

(4) in vitro, DOTC is cytotoxic for rat thymocytes.

Reversibility of Thymus Atrophy

Although DOTC-induced thymus atrophy occurs rapidly and its severity mimics a "chemical thymectomy," the atrophy was completely reversible. A 78% reduction of thymus weight induced by feeding 150 ppm DOTC for 4 weeks was followed by a fast recovery, when the animals were returned to the stock diet. After a rehabilitation period of 2 weeks, thymus weights of DOTC treated animals did not differ significantly from the controls. After 4 weeks thymus weights of treated animals were even higher than the controls, which is considered to be a compensatory reaction as is seen in many healing processes. Eight weeks after treatment thymus weights were the same as the controls. By restriction of food intake in weanling rats for 28 days, they induced a thymus reduction of 81%. But on rehabilitation the thymus weight returned to control values within 16 days. The similarity of the recovery of the thymus after DOTC feeding and nutritional deprivation suggests that the half-life of DOTC may be short.

Intravenous Treatment Study in Rats

After repeated intravenous injections of DOTC a marked thymus atrophy was observed. This atrophy occurred already at doses of 1 and 2 mg/kg, which did not affect other organ systems nor the body weight of the animals. Even a single injection of 1 mg/kg DOTC caused thymus atrophy, which was most pronounced 4 days after treatment. Given by the intravenous route DOTC was severely toxic. Doses of 10 mg/kg body weight produced death in about 50% of the animals (both mice and rats).

Species Differences

Lymphoid atrophy did not occur in guinea pigs, Japanese quail, or chickens. However, after intravenous administration of DOTC in rats and mice, comparable thymus effects were found. So there may be differences in uptake and/or distribution between rats and mice after oral administration. Although a selective organotin-induced lymphoid atrophy could not be established in guinea pigs, weanling animals fed 150 ppm DOTC died within 3 weeks. These animals appeared to be emaciated, showed abdominal edema and enlarged caecums filled with a fluid. Because of the antibacterial activity of dialkyltin compounds, caecal fermentation was disturbed, by which the production of free fatty acids in the caecum was almost completely inhibited Morphologically a severe villous atrophy was observed in the gastrointestinal tract of these animals, which may be related to the cytotoxic activity of DOTC. Japanese quail appeared to be very insensitive to the toxic effects of dialkyltin compounds, since feeding levels up to 600 ppm did not induce any sign of toxicity.

IMMUNE FUNCTION STUDIES AFTER IN VIVO EXPOSURE TO ORGANOTIN COMPOUNDS

Since the thymus and thymus-derived lymphocytes play a central role in the immune system, a number of immune function studies were performed in rats, rnieq and guinea pigs. In the thymus (blood-borne) stem cells migrate into the cortex where, under the stimulatory and perhaps regulatory influence of thymic epithelial cells, cell division and differentiation begin, giving rise to the forming of various subclasses of (thymus-derived) T-lymphocytes. This process starts at an early stage of ontogeny. So, thymectomy of neonates results in a reduced number of lymphocytes a diminished capacity to produce serum antibodies, and failure to reject skin grafts from foreign strains. These animals often die within 3 or 4 months from runting disease,a. Thymic ablation of adult animals is not accompanied by severe disturbances in lymphopoiesis or immune functions. As DOTC causes a severe thymic atrophy suggestive of a "chemical thymectomy," the immune function studies presented here are carried out with animals treated after weaning age as well as exposed during the developmental phase of the immune system (pre and/or postnatal exposure).

It appears that all immune reactions in which T-lymphocytes participate are compromised in the rat. Cell-mediated immunity was depressed in all test systems (delayed type hypersensitivity to tuberculin, skin graft rejection, graft-versus-host reaction, resistance to Listeria monocytogenes infection, lymphocyte transformation by T-cell mitogens). However, the humoral response was impaired with sheep red blood cells (SRBC) a thymus-dependent antigen, but the antibody response to E.coli ( lipopolysacharide (LPS) a thymus-independent antigen was unaffected. The response of lymphocytes from thymus and spleen to the T-cell mitogens phytoheamagglutinin (PHA) and concanavalin A (Con A) was severely depressed, in particular in pups that were exposed postnatally by oral intubation during 3 weeks following birth, whereas the response to the B-cell mitogen LPS was not impaired. From these data, it seems likely that DOTC suppresses various subpopulations of T-lymphocytes without affecting B lymphocytes. This selectivity may be explained by a selective toxic action on lymphocytes in the thymus, resulting in a reduced number and reduced responsiveness of peripheral T-lymphocytes. Also macrophages do not seem to be compromised, since blood monocyte numbers (as precursors of macrophages) and the in vivo phagocytizing capacity of macrophages as measured by the clearance of carbon particles were the same for treated and control animals.

Immune functions of DOTC-fed mice and guinea pigs were not impaired, which is in agreement with the absence of lymphoid atrophy observed in these species. No function studies were performed in mice that were exposed to these compounds by the intravenous route.

A remarkable effect of DOTC, probably not related to the immune system is the increase of endotoxin sensitivity. Weanling rats fed 150 ppm of the organotin compounds for 4 weeks died from an i.v. injection of 100 µg/E.coli LPS whereas the controls died at concentrations of 2500 µg LPS.

IN VITRO STUDIES

A cytotoxic effect of DOTC is demonstrated in suspension cultures of rat thymocytes.

In contrast to the effect on rat thymocytes, DOTC did not reduce rat bone marrow cell survival under the same culture conditions. Number and viability of rat bone marrow cells remained the same when cultured with 0, 5, or 50 µg of DOTC/ml for periods up to 40 hr.

Besides this difference in sensitivity between rat thymus and bone marrow cells, also a difference was found between thymocytes from various species. The viability of human thymocytes was not distinctly decreased, but these cells aggregated in a dose-dependent fashion in the presence of 0.5 and more DOTC per ml medium. Agglutination did not occur when the cells were cultured at low concentrations in a shaking waterbath. But, the ability of these thymocytes to form so-called E-rosettes with SRBC was rapidly reduced indicating that DOTC interferes with lymphocytic receptors for SRBC that are specific for human T-lymphocytes. The number of E-rosettes decreased in a direct proportion to the dose of organotin compounds in the incubation medium. Concentrations as low as 0.5 µg DOTC/ml already decreased the formation of E-rosettes.

Besides the dye exclusion test, which measures cell death, also the effects of various compounds on cell division in mitogen-stimulated as well as unstimulated lymphocyte cultures were studied. In the presence of DOTC, the division of thymus and spleen lymphocytes was dose-relatedly inhibited. At a concentration of 0.5 µg/ml medium DOTC, as well as the other dialkyl compounds tested, induced a complete inhibition of DNA synthesis of rat thymocytes. The viability of spleen and thymus cells was not impaired at these concentrations by the dialkyltin compounds. Therefore we conclude that dialkyltin compounds primarily induce an inhibition of cell proliferation and secondarily cause cell death.

In the lymphocyte transformation test no distinct differences in antiproliferatire activity of the various dialkyltins were observed. So, the differences in lymphocytotoxicity observed after in vivo exposure between the water soluble and water insoluble dialkyltins can not be explained by differences in in vitro activity of these compounds.

Species differences as observed after in vivo exposure to organotin compounds, did not occur in the lymphocyte transformation test, since comparable effects were found on DNA-replication of rat and mouse as well as human thymocytes. This indicates an absence of species differences in organotin sensitivity in vitro. Although, with viability testing, species differences in vitro were noted. This discrepancy can not be explained yet, but indicates that the absence of organotin-induced cell death, as measured with a dye exclusion test, will not exclude a cytotoxic action of these compounds.

The estertin compound bis-ß-carbobutoxyethyltin dichloride, which did not cause lymphoid atrophy in vivo, actively decreased the thymidine uptake of lymphocytes in vitro. However, its hydrolysis product only slightly decreases thymidine incorporation at the maximum concentration of 360 µg per ml medium. Probably, bis-13-carboxyethyltin does not enter lymphocytes. Also other dicarboxylic acids, such as succinic acid and fumaric acid are not able to penetrate cells.

A remarkable although not large difference was noted in the blast transformation test between thymus and bone marrow cells. Complete inhibition of DNA-replication of bone marrow cells occurred at concentrations 5 times higher than with thymus and spleen cells. Also with tumor cell lines inhibition of DNA replication was obtained, but often at higher concentrations than with thymocytes.

Conclusions:

The effect of DOTC on lymphoid tissues in vivo was selective. It induces thymus atrophy with signs of myelotoxicity and compromising other organs. The number and viablilty of lymphoid cells from thymus, spleen and lymph nodes decreased in a dose related fashion, whereas the viability of bone marrow was not changed.

DOTC can be referred to as immunosuppressive.

Reference 15

Endpoint:

immunotoxicity

Remarks:

subchronic

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No information on GLP. Studies not performed to recognised test methods.

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Species:

other: Rats, mice, guinea pigs and quail

Route of administration:

other: Oral-in feed and intravenous

Statistics:

The data were analysed for significance of differences by Student's t-test

Oral Route

DOTC caused a significant decrease in thymus weight at both feeding levels in male and female rats. This was seen as a dose related effect.

At necropsy, a pronounced reduction in size of the thymus was found in all animals fed DOTC.

The most prominent effect found was lymphocyte depletion in lymphoid organs, and it was most pronounced in the thymic cortex of animals fed DOTC. At the 150-ppm level, the cortex was almost completely depleted. Signs of cell destruction, such as karyorrhexis or "starry sky" formation was never observed. Also, lymphocyte depletion was present in the thymus-dependent areas of the spleen and popliteal lymph node (respectively, periarteriolar lymphocyte sheets and paracortical areas). Other treatment-related histopathological changes were not noted.

Other animal species

Growth of guinea pigs on the DOTC diet showed a dose-related decrease. At the 100-ppm level, growth was severely inhibited. One guinea pig of the highest dose group died and the remaining animals were in very poor condition. The general appearance of quail fed DOTC did not differ from that of the controls; body weights were the same in the various groups.

In contrast to rats, weights of lymphoid organ were not affected by DOTC in quail. In guinea pigs, however, thymus weight was significantly decreased at the 100-ppm level and was associated with a strongly decreased body weight. In addition, the relative adrenal weights of these animals were markedly increased from 0.41 ± 0.09 mg/g body weight in the control group to 0.61 ± 0.06 mg/g in the 100-ppm group (p < 0.001). The weights of the liver and kidney did not differ between the test and control groups.

At pathological examination, the guinea pigs fed 100 ppm of DOTC appeared to he emaciated. Two animals showed abdominal edema. The content of the gastrointestinal tract contained fluid. A reduction in size of the thymus was found in six guinea pigs al 100 ppm of DOTC. Histologically, there was marked depletion of lymphocytes in the thymic cortex. At variance with rats, in the cortex "starry sky" appearance was noted; ie. phagocytosis of pycnotic lymphocytes by macrophages. Also, the Hassal corpuscles were conspicuous.

Rats: Recovery experiment

Body weights of DOTC-treated animals were significantly reduced up to 2 weeks after treatment and remained lower than the controls during the whole period. During Week 3, three animals of the DOTC group died. They showed thickened and dilated bile ducts and abdominal edema.

The severe reduction of thymus weight (78%) induced by 150 ppm of DOTC for 4 weeks was followed by fast recovery. After 2 weeks feeding of the stock diet, thymus weights of DOTC-treated animals did not differ significantly from the controls. After 4 weeks, thymus weights of treated animals were higher than the controls. This is considered to be a compensatory reaction as is seen in many healing processes. Eight weeks after treatment thymus weights were the same as the controls.

Intravenous Route

Rats

DOTC was observed to have a strong effect on thymus weight. This is in agreement with the feeding experiment. Thymus atrophy was present at doses of 1 and 2 mg/kg, which did not affect growth or liver and kidney weights. At the 4-mg/kg dose, thymus weight was severely depressed. However, at this dose ncreased kidney weight was noticed. The weights of spleen and adrenals were not affected by this route of administration of DOTC.

At microscopic examination, thymus atrophy, associated with lymphocyte depletion. was observed in animals repeatedly dosed with 1, 2, or 4 mg/kg. Other treatment-related histopathological changes were not noted.

A dose-related reduction of thymus weight. cell number, and cell viability was present in all treatment groups (1, 2, 4, and 8 mg/kg).

Only at the highest dose level was body weight reduced; thymus weight was 24% of the control value, whereas cell number was only 7% of that of the controls.

in Vitro Experiments

The effect of DOTC on rat thymocyte cultures showed decreased cell count and viability at concentrations of 0.012 and 0.12 mM.

In contrast DOTC did not reduce rat bone marrow cell survival under the same culture conditions. Number and viability of rat bone marrow cells were the same when cultured with 0, 5, or 50 µg of DOTC/ml for periods up to 40 hr.

Conclusions:

DOTC caused a dose related reduction of thymus weight, cell count and cell viability in rats. The effect was completely reversible. These effects were not seen in mice, guinea pigs or japanese quail.

In vitro tests supported in vivo studies in terms of selectivity of cell type. A dose related decrease in rat thymocytes was observed, whereas bone marrow cells were unaffected.

Reference 16

Endpoint:

immunotoxicity

Remarks:

subchronic

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported.

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No information on GLP. Studies not performed to recognised test methods.

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Species:

other: Rats, mice and guinea pigs.

Strain:

other: Specific pathogen-free Wistar-derived (WU), inbred Wistar (WAG) and (WAG x B) F1 hybrid rats, inbred Swiss mice and Hartley strain guinea pigs

Sex:

male/female

Route of administration:

other: Oral: feed and gavage.

Vehicle:

other: arachis oil used for gavage

Analytical verification of doses or concentrations:

no

Statistics:

Student's t test or the distribution-free Wilcoxon test were used to calculate one-sided significances of differences between values of treated and control animals.

Delayed-Type Hypersensitivity Reaction to Tuberculin Rats sensitized with mycobacterium tuberculosis M 37 Ra revealed a well-developed skin reaction after intradermal tuberculin injections. The reaction was most pronounced when measured 14 days after sensitization. DOTC produced a dose-related reduction of the delayed-type hyper-sensitivity reaction to tuberculin. At the 50- and 150-ppm levels, the diameter as well as the thickness of the reaction site was significantly decreased. Histopathological examination of the skin lesions revealed a reduced infiltration of mononuclear cells (lymphocytes and monocytes) in the treated animals. Thymus and popliteal lymph node weights were decreased by DOTC. Histopathologically, it was associated with a lymphocyte depletion of the thymus and thymus-dependent peripheral lymphoid organs. There was also a dose-related decrease in absolute adrenal weight, but the relative weight of this organ did not differ significantly from the control weight. Body weights were slightly decreased only at the highest dose level. In contrast to rats, in guinea pigs the delayed-type hypersensitivity reaction to tuberculin was not distinctly affected. After 5 weeks at 50 ppm of DOTC, skin reactions of test and control groups were the same. Seven weeks after the start of the experiment, the thickness of the skin reaction was slightly decreased, but the diameter of the reaction was not affected. Guinea pigs at 100 ppm were not tested, because general systemic toxicity supervened. In Weeks 4 and 5, respectively, two and eight animals died. These animals were emaciated and displayed a clear edema in the pleural and peritoneal cavity. The remaining animals were in poor condition, had rough coats, and weighed only 64% of the controls at Week 5. Body weights of guinea pigs at 50 ppm after termination of the experiment did not differ significantly from the controls. In comparison with rats at 50 ppm, thymus weights of guinea pigs were slightly but significantly decreased. Adrenal weights were significantly increased. Allograft Rejection Rats were fed DOTC at levels of 0, 50, or 150 ppm in the diet for 9 weeks.The rejection time of tail skin grafts from (WAG x B) F₁ hybrids to WAG rats was significantly prolonged by DOTC at the 150-ppm level. Antibody Synthesis (Plaque-Forming Cell, Hemagglutination, and Hemolysin Assay) The hemolysin response showed the same effects as the agglutinin response. Ten days after a primary SRBC sensitization the titers of rats fed 150 ppm of DOTC were significantly decreased. However, after secondary immunization no differences in antibody response were found. Carbon Clearance Carbon clearance, was not affected in rats dosed three times a week from birth to Week 7 via gastric intubation with 0, 5, or 15 mg of DOTC/kg. The corrected phagocytic index was the same in the control and test groups.

Conclusions:

DOTC induces immunosuppression in rats by a selective inhibition of T-lymphocyte activity. Immune suppression was most pronounced in animals exposed to the chemicals during the developmental phase of the lymphoid system.

Reference 17

Endpoint:

immunotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not mentioned.

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No information on GLP. Studies not performed to recognised test methods.

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

peanut oil

Analytical verification of doses or concentrations:

not specified

Statistics:

The data, from those experiments in which the dams were dosed and pups from both sexes were evaluated, were analyzed by two-way analysis of variance with sex, dose, and dose by sex interaction effects examined using the general linear models procedure of the Statistical Analysis System. The purpose of this analysis was to determine whether or not the primary analysis should be done on males and females combined or separately. Williams's test was used as the primary analysis to determine the lowest dose at which a significant (p < .05) effect occurred. The data from rats dosed with DOTC as adults or pups were analysed using Dunnett's t-test

Acute oral dosing of adult female rats with DOTC on 10 consecutive days (Experiment I) resulted in thymic atrophy at all doses and significant suppression of the LP response to both T- and B-cell mitogens at 20 mg/kg/d. The most dramatic suppression was a 66% decrease observed for the response to PHA. Splenic NK cell activity against both YAC-1 and W/Fu targets was unaffected by DOTC at the doses examined. Since no changes in NK activity were observed at any E:T ratio, only the 50:1 data are shown. The primary antibody response to SRBC as measured by the IgM PFC assay was also unaffected at the doses examined.

Exposure to DOTC during d 10-20 of gestation (Experiment II) caused significant increases in the body, spleen, and thymus weights of 6-wk-old offspring born to dams dosed at 50 mg/kg/d. This increase in body and organ weights was observed for both male and female offspring. The LP response of splenocytes from female offspring was significantly (p < .01) lower compared with controls for Con A, PWM, and STM. No decrease in the LP response was observed for male offspring. NK cell activity was unaffected by this treatment.

The offspring in Experiment III were evaluated at 4, 8, and 16 weeks of age. No consistent, significant changes in body or lymphoid organ weights were observed at the 3 ages examined, although differences were observed in female body weights and male spleen weights of 8-week-old offspring nursed by dams exposed to 40 mg/kg/d. No consistent alterations in the LP response were observed for rats at 4 and 16 wk of age, although a significant decrease in the LP response to Con A, PWM, and STM was observed for 8-weeks-old male rats nursed by darns exposed to 40 mg/kg/d. Similarly, no significant differences were observed between control and dosed groups in NK activity or in the IgM PFC response to SRBC, although there was an increase, albeit not significant, in the PFC response for 8-week-old male rats nursed by dams exposed to 40 mg/kg/d.

Maternal pre- and postnatal exposure to DOTC (Experiment IV) caused no observable effects on the immune parameters measured in, their offspring. No significant differences were observed for body and lymphoid organ weights, LP responses, or NK activity in 8-week-old rats born to dams dosed on d 11-20 of gestation and d 2-11 postpartum with DOTC at 20 or 30 mg/kg/d.

In contrast to those experiments in which the darns were dosed with DOTC, exposure of newborn pups to DOTC during the first 3 weeks of life, 3 times per week, caused significant decreases in the LP responses. In Experiment V both male and female littermates were exposed to DOTC at 10 mg/kg-d. At 8 week of age, no differences were observed in body or lymphoid organ weights or in NK activity. However, the LP responses to Con A, PHA, and PWM were significantly (p < .01) lower in both males and females dosed with DOTC compared with controls. The reduction in the PHA response of DOTC-treated rats was more pronounced than Con A and PWM responses (i.e., 75% vs. 64% and 63%, respectively). At 12 weeks of age the LP responses of treated rats were comparable to controls.

In a subsequent experiment, in which neonatal male rats were dosed with DOTC (Experiment VI), significant reductions in the LP response to Con A and PHA at all exposure doses, STM at 10 and 15 mg/kg/d and PWM at 15 mg/kg/d were observed at 7 weeks of age. The PHA response was significantly lower at 10 weeks of age at all exposure doses. NK cell activity was not affected at any age. In both Experiments V and VI, significant reductions in the LP response of splenocytes were observed from 4 to 7 weeks following the last exposure to DOTC. These alterations were observed in the absence of decreases in body or lymphoid organ weights. Analysis of the splenic T-lymphocyte subsets in 7-week old rats in Experiment VI revealed no alteration in the percentage or absolute number of W3/25- and OX8-positive cells between control and 15 mg/kg/d DOTC-dosed rats. (Control rats had 28.5 ± 0.9% W3/25+ and 30.5 ± 1.8% OX8+ cells, and rats dosed at 15 mg/kg/d had 25.6 ± 2.2% W3/25+ and 33.7 ± 1.8% OX8+ cells.) This lack of difference between control and DOTC-treated rats for these two lymphocyte subsets occurred despite significant reduction in the LP response of splenocytes to Con A (67%) and PHA (61%) for DOTC-treated rats. It is interesting to note that, in addition to those observations regarding T-lymphocytes of splenic origin, no consistent change was observed in the LP response to Con A or PHA of thymocytes from 7- and 10-week-old rats exposed to DOTC in Experiment VI.

In order to test the hypothesis that the developing immune system is potentially more susceptible to perturbation by an immunotoxicant than the fully developed immune system, an experiment was conducted in which young adult (8 weeks old) functionally mature rats were dosed with DOTC on a schedule identical to that used in Experiments V and VI. Three days after the last dosing a significant reduction was observed in the thymus weights of rats dosed at 10 and 20 mg/kg/d. No changes in body or spleen weights or in adrenal weights were observed between control and DOTC-dosed rats. The LP response to Con A was reduced at 20 mg/kg/d and to PHA at 10 and 20 mg/kg/d. The LP response to PWM was reduced at 10 and 20 mg/kg/d, albeit significantly only at 10 mg/kg/d. At 4 weeks after the last dosing, however, all parameters tested had returned to control levels. Once again, NK cell activity was not affected by this dosing scheme.

Conclusions:

Decreased thymus weight and thymic atrophy were observed at all dose levels. Suppression of lymphoproliferation to T- and B-cell mitogens was observed. Spleenic natural killer (NK) cell activity and primary antibody response to sheep red blood cells (SRBC) were not affected.

Decreased lymphoproliferation response; no effects on lymphoid organ weights or NK cell activity.

Lymphoproliferation response decreased; NK cell activity unaffected.

Both doses resulted in decreased thymus weight. Lymphoproliferation response was reduced. Body, spleen, and adrenal weights were unchanged. NK cell activity was unaffected.

Offspring body, spleen, and thymus weights increased. Female offspring had decreased lymphoproliferation response. NK cell activity was unaffected.

Offspring immune function unaffected.

Reference 18

Endpoint:

immunotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no glp information, no guideline study, well documentation,

Qualifier:

no guideline available

Principles of method if other than guideline:

Oral administration of 100 mg/kg bw test substance to male rats.
Determination of:
- Distribution of the substance and its metabolite in organs (six timeframes)
- Differential gene expression (gene chip)
For further details see publication.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

1 time

Frequency of treatment:

1 time

Remarks:

Doses / Concentrations:
100 mgkg bw
Basis:
actual ingested

No. of animals per sex per dose:

a) gene expression: 5 animals, 1 time frame
b) organ distribution: 8 animals per time frame, 7 time frames

Control animals:

yes, concurrent vehicle

Dose descriptor:

other: change in immune system, effect in thymus

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

The changes in thymus realted to the changes in gene expression caused by DOTC.

The activation of Nr3c1, also known as GR (glucorticoide receptor), by dioctyltin dichloride causes can explain the adverse effects in the immune system.

Conclusions:

A one time dose to dioctyltin dichloride causes adverse effects ine the immune system and causes changes in the thymus.

Reference 19

Endpoint:

immunotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no glp, no guuideline, good documantation

Qualifier:

no guideline required

Principles of method if other than guideline:

Animals:
Male PVG rats aged 4 -5 weeks were maintained on a stock laboratory diet and given water ad libitum throughout the experiment. Groups of animals ware garaged on alternative days 75 mg Dioctyltin dichloride in corn oil /kg bw. Control animals received corn oil alone. Five animals were scarified immediately prior forst gavage and on the subsequent 1, 2 , 3 and 6 days following commencement of treatment, and body, thymus and liver weights were recorded. Blood samples and thymocyte cell suspension were obtained on days 0, 1, 2 and 3. Thymuses were obtained separate control PVG rats.

Preparation of serum
Blood samples were collected by exsanguinations under ether anesthesia. Serum was removed aseptically form each blood sample and stored in aliquots at -20 °C. Samples form each treatment group was pooled prior to use.

Preparation of thymocyte suspension
Thymuses were teased apart in RPMI 1640 culture medium, filtered through wire gauze, washed 3 times in RPMI and resuspended.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: PVG

Sex:

male

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

1 time (necorpsy after 1,2,3 and 6 days)=

Frequency of treatment:

1 time

Remarks:

Doses / Concentrations:
75 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5 animals

Control animals:

yes, concurrent vehicle

Dose descriptor:

other: adverse effect relating to thymus, thymus cells

Effect level:

75 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Thymic attrophy after 48h, a lterations in thymocyte activity were apparent prior to decline in thymic weight loss

Conclusions:

Thymic attrophy after 48h, a lterations in thymocyte activity were apparent prior to decline in thymic weight loss

Executive summary:

Oral administration ofdioctyltin dichloride(DOTC) results in the production or release of factors which appear to preventthymocytematuration at an early stage. Sera obtained from rats gavaged with 75 mg DOTC/kg on alternate days, significantly inhibited PHA-inducedblastogenesis of normalthymocytes.In vitrouptake of tritiated thymidine bythymocytesobtained from DOTC-gavaged rats was markedly reduced. These events occurred 24 to 72 h after commencement of DOTC treatment and preceded overt thymic weight loss. The effects of DOTC may therefore be a consequence offactor(s) capable of blocking the intrathymic differentiation of T cell precursors.

Reference 20

Endpoint:

immunotoxicity

Remarks:

acute

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no guideline, no glp information, well documentated study

Qualifier:

no guideline required

Principles of method if other than guideline:

Tissue distribution studies
The tissue distribution of radioactivity was determined in male rats
weighing 80--100 g following a single i.v. or oral (by gavage) administration
of 1.2 and 6.3 mg [~C]DOTC/kg body weight, respectively. Groups of 3
animals were killed by decapitation at 1, 2, 4 and 7 days after the administration,
and blood was collected in heparinized tubes. The animals were
dissected immediately to sample and weigh the organs and tissues. Duplicate
or triplicate 50--100-mg samples of most organs and tissues were cut,
weighed, and transferred to liquid scintillation vials to examine their content
of radioactivity. For the smaller organs, e.g. adrenal, pituitary glands etc. the
radioactivity of the total organ was measured. To each vial 1 ml of the
tissue solubilizer Soluene 350 (Packard Instruments, Packard Dekker
B.V., The Netherlands) was added and allowed to stand for one night at
50--60°C. After solubilization, samples of liver, kidney, heart, lung and
blood were decolourized by adding 0.5 ml of isopropanol followed by 0.5
ml of a 30% solution of H202, to reduce colour quenching. After adding
10 ml scintillation fluid to each vial the content of radioactivity was determined
in a Mark II (Nuclear Chicago) liquid scintillation counter and corrected
for background. Counting efficiency was determined by the external
standard technique. The tissue radioactivity was expressed in dpm/mg tissue.
From these data the relative accumulation index of radioactivity in each of
the organs and tissues was calculated according to the equation: R t = (Ct/Ht).
In this equation R t stands for the relative accumulation of radioactivity at
time t (days); C t for radioactivity in dpm/mg tissue at time t, and H t for the
radioactivity in dpm/mg tissue which was calculated by dividing the total
radioactivity in the sampled organs and tissues by their total weight at time
t (see Table I and III). The weights of blood, muscle and fat tissues were
estimated to contribute to 8, 45 and 8% of the total body weight, respectively
[19].
Excretion studies
Groups of 3 male rats weighing 100--120 g were given a single i.v. or a
single p.o. dose (by gavage) of 1.2 mg and 2 mg [14C] DOTC/kg body weight,
respectively. Then for 25 days urine and feces of each animal were dally
collected separately and their r a d i o a c t i v i t i e s were measured. Triplicate
50-pl aliquots of urine from the individual animals were transferred to
s c i n t i l l a t i o n vials and 1 ml Soluene-350 was added. The fecal samples of the
individual animals were dried at 50--60°C, weighed, and ground to a fine
powder with a mortar and pestle. Then t r i p l i c a t e 10--20-mg samples from
the individual animals were transferred to s c i n t i l l a t i o n vials and rehydrated
with 0.1 ml water (1 h) before adding 1 ml Soluene-350. The vials of urine
and fecal samples were closed and incubated overnight at 50--60°C. To reduce

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

other: single i.v. or oral

Vehicle:

ethanol

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

1 time

Frequency of treatment:

1 time

Remarks:

Doses / Concentrations:
1.2 mg/kg bw
Basis:
other: concentration nominal, i.v.

Remarks:

Doses / Concentrations:
6.3 mg/mg bw
Basis:
other: concentration nominal, oral

No. of animals per sex per dose:

3 per necropsy day and way (necropsy on 1, 2, 4 and 7 days)

Control animals:

yes, concurrent vehicle

Dose descriptor:

other: decrease in thymus weight

Effect level:

1.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: way: i.v.

Dose descriptor:

other: decrease in thymus weight

Effect level:

6.3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: way: oral gavage

thymic atrophy and weight decrease in thymus

Conclusions:

Immunotoxicity and the decrease of thymus weight are acute effects of oral and i.v. exposure to dioctyltin dichloride

Reference 21

Endpoint:

immunotoxicity

Remarks:

subacute

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

1976-2004

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: no method, no glp information, reviewy

Justification for type of information:

General mechanism of immune toxcity of dioctyltin compounds

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

GLP compliance:

not specified

"Thymic atrophy produced by certain organotins, such as triphenyltin, tributyltin, dibutyltin, and dioctyltin compounds, involves a decrease in the number of cortical thymocytes, resulting in reduced thymus weight. With prolonged exposure, T-cell-mediated immune responses are. Loss of thymocytes appears to involve suppression of proliferation of immature thymocytes and, at higher dosages, apoptosis of mature thymocytes. These appear to be direct effects on the thymus as both cytotoxicity and apoptosis have been observed in thymocyte cell cultures exposed to di- or tributyltin . Cytotoxicity of butyltin compounds in thymocyte cultures involves suppression of DNA and protein synthesis (), and also induction of the expression of genes involved in apoptosis, such asnur77, a transcription factor member of the steroid/thyroid hormone receptor superfamily (Gennari et al. 2002b). An early and, possibly, the initiating event of apoptosis is a rise in cytosolic ionized calcium (Ca2+) concentration, caused both by intracellular calcium stores as well as by disruption of calcium transport at the cell membrane. Disruption of the regulation of intracellular calcium levels and, possibly, direct effects on energy metabolism of mitochondria either contributes to or gives rise to the uncontrolled production of reactive oxygen species, release of cytochromecto the cytosol, and the proteolytic and nucleolytic cascade of apoptosis. Modification of the cytoskeleton through Ca2+ -independent disruption of F-actin may also contribute to DNA fragmentation.

Alkyltin compounds, in particular butyltin compounds, suppress T-cell-mediated immune responses,

Including antibody formation against foreign antigens, delayed hypersensitivity reactions, and allograft rejection. These effects appear to result from suppression of proliferation of immature thymocytes (CD4-CD8+) which would, otherwise, differentiate into mature T-cells possessing

the complete antigen-recognizing T-cell receptor complex, resulting in lower numbers of circulating

functional T-cells. Suppression of lymphoproliferative responses to T- and B-cell mitogens also has been demonstrated for triphenyltin. Direct effects on lymphocyte function may also contribute certain aspects of immune suppression observed in animals exposed to butyltin compounds, including decreased natural killer cell activity.In vitro, butyltin compounds suppress cytotoxic activity of human natural killer cells that function in the immune response to tumors and virally-infected cells. Mechanisms for suppression of killer cells appear to involve loss of cell surface receptors important for binding to target cells, possibly secondary to a loss of regulation of intracellular cAMP, and disruption of the transcription of genes for the cytotoxic proteins granzyme B and perforin, which are proteins contained in granules released by NK cells."

Conclusions:

Dioctyltin compounds have immune suppresion properties.

Reference 22

Endpoint:

immunotoxicity

Remarks:

other: gene expression

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no glp, no guideline study, well documanted

Qualifier:

no guideline required

Principles of method if other than guideline:

Reagents
Mouse monoclonal antibodies specific for cytokeratin and isotype
IgG were purchased from Dako (Carpinteria, CA). Neutralizing
anti-IL-6 was from R&D Systems (Minneapolis, MN). Alexa Fluor
488-conjugated goat anti-mouse IgG was from Invitrogen Corp.
(Carlsbad, CA). Recombinant mouse cytokine IL-6 was from Pepro-
Tech, Inc. (Rocky Hill, NJ). Jak2 inhibitor AG490 was from Sigma–
Aldrich Co. (St. Louis, MO) and dissolved in dimethylsulfoxide
(DMSO).

Cell culture
Fresh, discarded thymus tissue was taken from healthy children
during corrective cardiovascular surgery and from thymo-thymectomies
of patients with thymoma. The study protocol was approved
by Human Experiment and Ethics Committee of National
Cheng Kung University Hospital of Taiwan since 02/25/2008. The
certification number is ER-96-157. Briefly, thymus tissue was
minced and filtered, and then digested by collagenase and deoxyribonuclease
(DNase). After they had been centrifuged several
times and then washed with phosphate-buffered saline (PBS), thymus
cell pellets (7.5 107) were resuspended in a T-75 plastic
flask containing 2 mL of RPMI medium 1640 with 10% fetal bovine
serum, 1 mmol/L of L-glutamine (25030081; Invitrogen, Carlsbad,
CA, USA), 10 IU/mL of penicillin (15140122; Invitrogen), and
10 lg/mL of streptomycin (15140122; Invitrogen) and then cultured
in a humidified atmosphere of 5% CO2 and 95% air at 37 C.
The culture medium was changed every 2–3 days, and fibroblast
inhibitor (H1637; Sigma–Aldrich) was added. The cells were collected
for further study when 80–90% of the flash was occupied
by cells in a confluent monolayer, usually after 2–3 weeks of incubation.
The supernatant was collected before every change of medium
and before the cells were harvested for a cytokine antibody
array. Some of the collected cells were for flow cytometric analysis,
and others were subsequently cultured with or without serum for
further study. For morphological observation, the cells were visualized
under a microscope (IX71; Olympus, Tokyo, Japan).

Flow cytometric analysis
The cells were detached using 1000 U/mL of trypsin and 0.5 mM
of ethylene diamine tetra acetate (EDTA). Suspended cells were
fixed and permeabilized using a kit (BD PharMingen Cytofix/Cytoperm;
BD Biosciences, San Jose, CA). Antibodies specific for cytokeratin
(Dako) or isotype IgG were added to the cells and
incubated at 4 C for 1 h. After they had been washed with PBS,
the cells were incubated with Alexa Fluor 488-conjugated secondary
antibodies at 4 C for 1 h. The incubated cells were then
washed again with PBS and analyzed using flow cytometry with
excitation set at 488 nm (FACSCalibur; BD Biosciences).

Cytokine antibody array analysis
The cytokine production profile was determined using a cytokine
antibody array (Human Cytokine Antibody Array III AAHCYT-
3-8; RayBiotech, Inc., Norcross, GA) according to the manufacturer’s
instructions. Briefly, 100 lg/mL of protein from cell-conditioned
culture medium was incubated with the array membrane
at 4 C for 1 h. After they had been washed with PBS-T (PBS plus
0.05% Tween-20), the cell membranes were incubated with primary
biotin-conjugated antibodies and HRP-conjugated streptavidin.
Signals were detected using enhanced chemiluminescence
(ECL) (Pierce Biotechnology Inc., Rockford, IL), and the relative signal
intensity was quantified using densitometry with bio-imaging
software (LabWorks; UVP, Inc., Upland, CA) with reference to the
positive controls on the membrane

ELISA
Cells were seeded in 96-well plates at a density of 1 104 cells/
well. After the treatment, we used commercial ELISA kits (88-
7066-77; eBioscience, San Diego, CA) and (DY275; R&D Systems,
Minneapolis, MN), according to the manufacturer’s instructions,
to detect the concentrations of mouse IL-6 and GRO-a, respectively,
in cell-conditioned culture medium. Cell-conditioned culture
medium was regularly diluted, and the concentrations were
calculated so that they did not exceed detection limits. IL-6 and
GRO-a concentrations were measured using spectrophotometry
(Spectra MAX 340PC; Molecular Devices Corporation, Sunnyvale,
CA) at 540 nm, and the concentration was calculated using a standard
curve with ELISA software (Softmax Pro; Molecular Devices).

Viability assay
Cell viability was assayed using a colorimetric assay (Cell
Counting kit-8 (CK04); Dojindo Laboratories, Gaithersburg, MD)
according to the manufacturer’s instructions. Aliquots of the culture
media were transferred to 96-well microplates. A microplate
reader (Spectra MAX 340PC; Molecular Devices) was used to
measure the absorbance at 620 nm with a reference wavelength
of 450 nm, and the data were analyzed with ELISA software (Softmax
Pro; Molecular Devices). The relative optical density was
calculated.

Statistical analysis
Data were analyzed using Student’s t test with commercially
available statistical software (SigmaPlot 8.0 for Windows; Systat
Software, Inc., San Jose, CA). Statistical significance was set at Pvalues
< 0.05.

Limit test:

no

Species:

mouse

Strain:

other: not relevant

Sex:

not specified

Route of administration:

other: not relevant

Vehicle:

other: not relevant

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

see priciples of methode if other tahn guideline

Frequency of treatment:

see priciples of methode if other tahn guideline

No. of animals per sex per dose:

see priciples of methode if other tahn guideline

Conclusions:

IL-6 is a critical cytokine in the
thymus and is of paramount importance for the differentiation,
proliferation, and survival of both thymic epithelial cells and thymocytes.

IL-6 is an immunosuppressive cytokine that induces
acute thymus atrophy

Reference 23

Endpoint:

immunotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no glp, no guuideline, good documantation

Qualifier:

no guideline available

Principles of method if other than guideline:

Thymocytes from DOTC trated and control rats were seperated into low and high proliferative fractions and characterised as a function of both their spontaneous DNA synthesis, antigenic phenotype and specific mRNA transscrips

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: PVG (Olac)

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

a) 1x, 100 mg/kg bw
b) 1x, 75 mg/kg bw
c) 2x (0, 48h), 75 mg/kg bw

Frequency of treatment:

a) 1
b) 1
c) 2 (0,48 h)

Remarks:

Doses / Concentrations:
0, 75, 100 mg/kg/bw
Basis:
actual ingested

No. of animals per sex per dose:

4 dosed +4 contaol per dose and timeframe

Control animals:

yes, concurrent vehicle

Dose descriptor:

other: addverse effect for immune system

Effect level:

75 other: mg /kg bw

Based on:

test mat.

Sex:

male

see attachmentn (nwner ship protection)

Conclusions:

Dioctyltin dicoloride causes addverse effects in immune system (e.g. thymus atrophie) after a single dose

Executive summary:

Experiments have been performed to characterise early changes in the proliferative capacity and phenotypic makeup of thymocytes obtained from DOTC-treated PVG rats. The analysis of density gradient-separated thymocytes demonstrated that within 72 h of oral dosing, spontaneous in vitro thymocyte proliferation was markedly suppressed. A concomitant depletion of an MRC OX18 positive thymocyte population was also observed. These events occurred prior to both overt thymic weight loss and characteristic, treatment-induced histopathological changes. Since recent evidences suggest that the growth factor interleukin-2 (IL-2) may play an important role in thymocyte proliferation, additional molecular biological studies were performed to evaluate whether DOTC may exert its anti-proliferative effects by compromising IL-2 production. Using a mRNA cytoplasmic dot blot technique, the examination of thymocyte lysates from treated and control animals revealed that DOTC markedly down regulated and at high doses abolished, the normal expression of the IL-2 gene. The high turnover gene alpha-actin was, however, unaffected by its action, thus demonstrating the selective effects of DOTC.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Several demonstrate clearly, the the immunotoxicity of Dioctyltin is a acute effect. The adverese effects related to the immune system takes place hours after administration of the test substance to the animals. A few hours later there is to changes in the thymus, too.

Justification for selection of effect on immunotoxicity via oral route endpoint:
use of an acuty study, to effect, itest type subacute is chosen in due to default selection criteria

Justification for classification or non-classification

In due to mechanism DOTC is to classify as STOT Single 1