Dioctyltin dilaurate

Immunotoxicity

Description of key information

The immunotoxicity caused by Dioctyltin is an acute effect, The value acute is missing in selection possibilities for time frame of effect

 

LOAEL Dioctyltin oxide 11.8 mg/kg/day => LOAEL DOTL 3.9 mg/kg/day

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

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 2

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 3

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 4

Endpoint:

immunotoxicity: acute oral

Remarks:

Part of the OECD 414 study on the read across substance Dioctyltin oxide.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

READ ACROSS
Hydrolysis under stomach condition (pH 1.2) of Diotyltin dilaurate into Dioctyltin oxide and lauric acid (cp. Section basic toxicokinetics: Nasshan, In vitro metabolism study)
Study for justification of read across is link via cross reference.

DIOCTYLTIN DILAURATE
Furthermore another study with dioctyltin dilaurate shows additional information regarding the immune toxicity.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

other: Hydrolysis under stomach condition (pH 1.2)

Reason / purpose for cross-reference:

other: Main study (OECD 414, rat)

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 85-7 (Immunotoxicity)

Deviations:

not applicable

Principles of method if other than guideline:

see attached report: study performed as port of an OECD 414 study

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

female

Route of administration:

oral: feed

Vehicle:

other: in diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

72 h

Frequency of treatment:

in diet

Dose / conc.:

0 mg/kg diet

Remarks:

0 mg/kg bw/day

Dose / conc.:

5 mg/kg diet

Remarks:

0-4 mg/kg bw/day

Dose / conc.:

25 mg/kg diet

Remarks:

1.8 mg/kg bw/day

Dose / conc.:

200 mg/kg diet

Remarks:

11.8 mg/kg bw/day

No. of animals per sex per dose:

5 in control, low and mid dise, 4 in high dose group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Subpart of OECD 414 study in rat, test substance Dioctyltin oxide. So rationale for dose selection is the main study. Dose selection form outcome of former studies with Dioctyltin oxide ((Waalkens-Berendsen, OECD 422, DOTO)

- Rationale for selecting satellite groups:
Determination of immunotoxicity as maternal toxicity

- Post-exposure recovery period in satellite groups:
Not planed, but time of last exposure to necropsy showed recovery of the immune system of test animals

- Rationale for animal assignment (if not random):
Subpart of OECD 414 study in rat, test substance Dioctyltin oxide

- Other:
Immunophenotyping:
Immunophenotyping was performed on Immunotoxicity animals from blood samples (0.5mL in EDTA) during the predose phase and on Gestation Days 7 (24 hours after first dose), 8 (48 hours after first dose), 9 (72 hours after first dose) and 10 (prior to necropsy)

Cytokine Measurement:
Blood samples (0.5 mL in serum separator tubes) were collected from Immunotoxicity cohort animals during the predose phase and on Gestation Days 7 (24 hours after first dose), 8 (48 hours after first dose), 9 (72 hours after first dose) and 10 (prior to necropsy)

Observations and clinical examinations performed and frequency:

Immunophenotyping:
Immunophenotyping was performed on Immunotoxicity animals from blood samples (0.5mL in EDTA) during the predose phase and on Gestation Days 7 (24 hours after first dose), 8 (48 hours after first dose), 9 (72 hours after first dose) and 10 (prior to necropsy)

Cytokine Measurement:
Blood samples (0.5 mL in serum separator tubes) were collected from Immunotoxicity cohort animals during the predose phase and on Gestation Days 7 (24 hours after first dose), 8 (48 hours after first dose), 9 (72 hours after first dose) and 10 (prior to necropsy)

Positive control:

not required

Clinical signs:

not specified

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In the moin OECD 414 study no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In the moin OECD 414 study no effects observed

Food efficiency:

no effects observed

Description (incidence and severity):

In the moin OECD 414 study no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Description (incidence and severity):

not reported at the moment, if findings, report in endpoint of main OECD study

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not reported at the moment, if findings, report in endpoint of main OECD study

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

see section specific immunotoxic examinations

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

4ß percent of thymus decrease in high dose group

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not reported at the moment, if findings, report in endpoint of main OECD study

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not reported at the moment, if findings, report in endpoint of main OECD study

Other effects:

not examined

Description (incidence and severity):

not reported at the moment, if findings, report in endpoint of main OECD study

Details on results:

not reported at the moment, if findings, report in endpoint of main OECD study

Cell viabilities:

effects observed, treatment-related

Description (incidence and severity):

Blood
At gestation Day 9, activated B lymphocyte numbers (CD3-/CD45RA+/CD25+) were markedly higher than the control group animals (approximately 3 to 4 times for the majority of the animals) at the 11.8mg/kg/day dose level.
Other observed differences, were limited to several lymphocyte sub-populations and tended to be slight to moderate in nature. On gestation Day 9, moderate increases were noted for CD3+/CD8+/CD25+, CD3+/CD25+ and CD3+/CD4+/CD25+ cell counts in animals dosed 11.8mg/kg/day. When compared to the control group results, the aforementioned populations were +85%, +60% and +50% greater, respectively.
At gestation Days 8 and 10, all groups administered Dioctyltin Oxide, displayed slightly lower numbers and relative percentages of NK cells (CD3-/CD161a+) when compared to the control group, although results within the treated groups were relatively unchanged when compared to other sampling days. No other changes of note were observed on Days 8 and 10.
Cytokines
Results for serum concentrations of IL-2 varied from day to day, with almost all animals, across all groups including controls, displaying quantifiable results on Days 8 and 10, but no animals recording results above the lower limit of quantitation (LLOQ) on Day 10. No test article-related effect could be determined, as the frequency and magnitude of quantifiable IL-2 were similar across all groups, and comparable to the results observed during the pre-dose phase. Quantifiable results, where observed were either slightly above the LLOQ value, or at least within 3 times the LLOQ value.

Humoral immunity examinations:

not examined

Specific cell-mediated immunity:

not specified

Non-specific cell-mediated immunity:

not specified

Other functional activity assays:

not examined

Other findings:

not specified

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 11.8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

immunology

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1.8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

immunology

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

11.8 mg/kg bw/day (nominal)

System:

immune system

Organ:

thymus

other: immune system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

The LOAEL for acute immune toxicity for Dioctyltin oxide was determined to be 11.8 mg / kg bw7day thus a NOAEL of 1.8 mg/kg bw7day results.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

25.46 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

REMARK: Study duration: acute !; fIn the fiield study duration the value "subacute" was chosen, in due the filed has ti be filled in for TCC. Value "acute" in the choice box not included.

Effect on immunotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Several studies demonstrate clearly the immunotoxicity of Dioctyltin is an acute effect. These adverse effects related to the immune system take place hours after administration of the test substance to the animals. A few hours later there are changes in the thymus, too.

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

Justification for classification or non-classification

In due to mechanism DOT is to classify as STOT Single 2