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Immunotoxicity

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Description of key information

The evaluated immune functions were not affected by the mixture or by MMT alone.

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
immunotoxicity: short-term oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not reported
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
This study was a part of the original dossier for MMTE. It remains in the dossier to provide continuity to reviewers and an historical perspective of the changes which are being made to the dossier. New data on in vitro metabolism, cited in section 7.1.1 of this dossier, caused a change in the interpretation of the data cited. The new in vitro metabolism data support the conclusion that MMTE does not metabolise to MMTC under simulated mammalian gastric conditions [pH ~2 and 37 °C as was formerly believed. It then follows that the toxicology of MMTE via the oral route in mammalian species cannot be accurately predicted based on studies conducted with MMTC via the oral route in mammalian species. The implications are clear: (a) dietary feeding and oral gavage studies conducted with MMTC cannot be read-across to MMTE, and (b) for studies conducted with MMTC, their relevance for hazard classification of MMTE must be reduced or eliminated. Therefore, the Klimisch score of this study has been reduced to Klimisch 4 because the read-across strategy from this study to MMTE is no longer valid (though the study itself is still considered to be reliable) but in this case as the reference is considered to concern methyltin and organotin compounds in general, it can be considered to provide useful information on the lack of immunotoxicity of organotin substances.
Justification for type of information:
This paper is included as supporting information for completeness and in order to provide further detail on how a mixture of organotin compounds is understood to affect immune functions and to indicate that thymus effects do not indicate immunotoxicity.
Reason / purpose for cross-reference:
other: read across target
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to determine whether immune function was affected by organotins given as a mixture. An evaluation of immune functions in adult male Sprague-Dawley rats, a model sensitive to the immunomodulatory effects of organotin compounds, was carried out. Rats were exposed to a mixture of organotins, or to MMT alone, via drinking water. Immune function was evaluated by monitoring antibody responses to sheep red blood cells (SRBCs), delayed-type hypersensitivity (DTH) responses to bovine serum albumin (BSA), and natural killer (NK) cell cytotoxicity.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Dibutyltin dichloride (DBTC), dimethyltin dichloride (DMTC), monobutyltin trichloride (MBT) and monomethyltin trichloride (MMT)
- Purity: DBTC 96 %, DMTC 97 %, MBT 95 % and MMT, 97 %
- Lot/batch No.: DBTC 02211AO, DMTC BA14015HS, MBT 05427LU and MMT 12609CD
- Source: Aldrich Chemical Company (Milwaukee, WI)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 to 7 weeks old
- Weight at study initiation: Approximately 275 g
- Fasting period before study: No
- Housing: Animals were housed individually in polycarbonate cages with pine shavings
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22.3 ± 1.1 °C
- Humidity: 50 ± 10 %
- Photoperiod: A 12-h light (06:00 h) to dark (18:00 h) cycle
Route of administration:
oral: drinking water
Vehicle:
other: Alkamuls EL-620
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared twice weekly in 25-L polycarbonate carboys and then transferred into individually labelled plastic drinking water bottles topped with double ball bearing sipper tubes.
The proportions of the dosing solutions were 2:2:1:1 (DBTC:DMTC:MBT:MMT), at concentrations of 20:20:10:10 (high mix), 10:10:5:5 (medium mix), or 5:5:2.5:2.5 (low mix) mg organotins/L of water. The solutions of MMT alone contained 20 or 40 mg of MMT/L water. Based on average body weight and average water consumption throughout the study, the calculated dosage (mg/kg body weight/d) of each of the chemicals were as follows: 20 mg MMT/L, 2.2 mg/kg/d; 40 mg MMT/L, 4.1 mg/kg/d; low mix, 1.3 mg/kg/d; medium mix, 2.5mg/kg/d; and high mix, 4.7 mg/kg/d.

VEHICLE
- Name: Alkamuls EL-620 (Alkamuls; ethoxylated castor oil)
- Justification for use and choice of vehicle (if other than water): Although only DBTC and MBT are not miscible in water, all organotins were solubilised in Alkamuls prior to mixing with water.
- Concentration in vehicle: The final concentration of Alkamuls in all dosing preparations was 0.5 % v/v.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
Continuous in water
Remarks:
5:5:2.5:2.5 mg/L for DBTC:DMTC:MBT:MMT
Remarks:
10:10:5:5 mg/L for DBTC:DMTC:MBT:MMT
Remarks:
20:20:10:10 for DBTC:DMTC:MBT:MMT
Dose / conc.:
20 mg/L drinking water
Remarks:
MMT
Dose / conc.:
40 mg/L drinking water
Remarks:
MMT
No. of animals per sex per dose:
8 males per dose group
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were chosen based on water palatability determined from previous studies, potential toxicity and the proportion of each compound expected to occur in drinking water. In sampled PVC pipes, organotin concentration was MMT > DMTC = DBTC > MBT and frequency was MMT = DMTC > DBTC = MBT. MMT doses were also chosen to minimise any potential complications from neurotoxicity. The proportions of the dosing solutions were therefore 2:2:1:1 DBTC:DMTC:MBT:MMT.
- Schedule: Dosing was initiated on day 0; DTH sensitisation took place on day 22; primary immunisation took place on day 24; exposure ended on day 28; DTH challenge, IgM serum collection and natural killer cell activity took place on day 29; booster immunisation took place on day 43; and IgG serum collection took place on day 48.
Observations and clinical examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed twice weekly during the dosing period and also just prior to sacrifice.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Dosing water was changed and water consumption was monitored twice weekly.

IMMUNE FUNCTIONS: YES
- Time schedule for examinations: Different immune functions were evaluated in separate groups of animals 1 d after dosing ended.
Humoral immunity examinations:
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes
- Method: Animals were immunised on d 24 of dosing by intravenous injection of 2 × 10^8 SRBCs in 0.5 mL sterile saline and were bled 5 d later by tail vein transection. Blood was held at room temperature for 30 min and centrifuged at 4 °C to separate serum; serum was frozen at –80 °C until analysis of SRBC-specific IgM. Two weeks after primary immunisation, a booster immunisation of SRBCs (2 × 10^8) was administered intravenously to the same animals. Blood from decapitated animals was collected 5 d after the booster immunisation and processed as described above for later analysis of SRBC-specific IgG. The relative serum titres of SRBC-specific IgM and IgG antibodies were measured by enzyme-linked immunosorbent assay (ELISA). All ELISA procedures were optimised for differences in SRBC membrane preparations and each new lot of secondary antibody.
- Dose groups: All
- No. of animals: Eight animals per dose were used to measure T-cell-dependent antibody responses (immunoglobulin [Ig] M and IgG) to SRBC.
Specific cell-mediated immunity:
DELAYED-TYPE HYPERSENSITIVITY (DTH) REACTION: Yes
- Method: BSA (1 mg/mL in sterile saline) was emulsified in Freund’s complete adjuvant at a 1:1 ratio. Animals anaesthetised with isoflurane were sensitised on d 22 of dosing by injecting 0.1 mL BSA-CFA subcutaneously into the caudal tail fold. Seven days later, animals were anaesthetised with isoflurane and challenged by injecting 0.1 mL heat-aggregated BSA into the right rear footpad. The left rear footpad was injected with the same volume of saline and served as the injection control. BSA was aggregated by heating 10 mg BSA/mL of sterile saline to 75 °C for 1 h and removing excess saline by centrifuging for 10 min at 650 × g. After 24 h, footpad thickness (triplicate measurements) was determined in anaesthetised animals with an electronic calliper designed and built in the model shop at the U.S. EPA (Research Triangle Park, NC). The device applies very light, even, and reproducible pressure on the footpad for each measurement, thus increasing the accuracy of measurements. Standards of known thickness were measured before and after experimental measurements. Swelling was calculated by subtracting the mean saline-injected, left footpad thickness from the mean BSA-injected right footpad thickness.
- Dose groups: All
- No. of animals: Eight animals per dose were used to measure the DTH responses to purified (Fraction V) bovine serum albumin.
Non-specific cell-mediated immunity:
NATURAL KILLER (NK) CELL ACTIVITY: Yes
- Method: Terminal body weight (BW) was recorded, the spleen and thymus were removed, and the weights of both organs were recorded. Splenocyte single cell suspensions were prepared and cultured with 51Cr-labelled murine YAC-1 lymphoma target cells (100 μCi/1 × 10^6 cells) in round-bottomed microtitre plates at effector to target cell ratios of 25:1, 50:1, or 100:1. After a 4-h incubation at 37 °C, microtitre plates were centrifuged (290 × g) and 25 μL of supernatant was transferred to a 96-well LumaPlate, covered, and read on a TopCount NXT microplate scintillation counter. The data are presented as the average adjusted percent cytotoxicity for three replicates [(mean counts per min in supernatant – spontaneous release of 51Cr) / (mean releasable counts from target cells – spontaneous release of 51Cr) × 100].
- Dose groups: All
- No. of animals: Eight animals per dose were used to measure NK cell activity the day after dosing ended (d 29).
Statistics:
Statistical analyses were performed with the SAS System (SAS Institute, Inc., Cary, NC). Analysis of variance (ANOVA) was used to analyse immune responses by dose and, when appropriate, linear regressions were used to determine dose response. When ANOVA indicated a statistically significant treatment effect, individual post hoc comparisons were made using Tukey’s test and the least square means t-test with a Tukey’s adjustment for controlling the familywise error rate. A repeated-measures ANOVA was used to analyse BW changes over time and dose. Statistical significance was determined using an alpha of .05.
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
no effects observed
Description (incidence and severity):
For the organotin mixture groups, body weight did not differ between controls and dosed animals during any part of the dosing period nor when terminal weights were collected. Exposure to MMT had no statistical effect on body weight.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was reduced by approximately 20 - 30 % in all organotin mixture groups relative to controls. Exposure to MMT had no statistical effect on water consumption.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Cell viabilities:
not examined
Humoral immunity examinations:
no effects observed
Description (incidence and severity):
No statistical differences between control and organotin-exposed animals were detected for primary and recall antibody responses to sheep red blood cells (SRBCs).
Specific cell-mediated immunity:
no effects observed
Description (incidence and severity):
No statistical differences between control and organotin-exposed animals were detected for delayed-type hypersensitivity responses to bovine serum albumin..
Non-specific cell-mediated immunity:
no effects observed
Description (incidence and severity):
No statistical differences between control and organotin-exposed animals were detected for natural killer cell cytotoxicity.
Spleen and thymus weights of animals used to evaluate natural killer cell activity did not differ between controls and dosed animals following exposure to the organotin mixture or to MMT alone.
Other functional activity assays:
not examined
Other findings:
not examined

Exposure to these organotin mixtures or to MMT alone did not statistically alter the adaptive (i.e., delayed-type hypersensitivity or T-cell-dependent antibody responses to SRBC) or the innate immune functions (i.e., natural killer cell cytotoxicity) examined.

The data therefore suggest that immunotoxicity is unlikely to result from the concentration of organotins present in drinking water delivered via PVC pipe, as the concentrations used here were several orders of magnitude higher than those expected to leach from PVC pipe.

A lack of effects on thymus weight in the study is likely attributable to several factors, such as the dose, strain of rat used and route of exposure. The high mix dose contained 60 mg organotins/L, which resulted in an approximate dosage to the rats of 4.7 mg organotins/kg/d. However, as serum organotin concentrations were not measured, it is not possible to know how the relative contribution of parent compounds, metabolites and potential interactions among the organotins may have affected the thymotoxicity of one or more of the organotins within the mixture.

The data provide evidence that even when organotin concentrations in drinking water exceed expected human exposures from drinking water alone by nearly 300 000 times, negative impacts on functional immunity are unlikely.

Conclusions:
No statistical differences between control and organotin-exposed animals were detected for primary and recall antibody responses to sheep red blood cells, delayed-type hypersensitivity responses to bovine serum albumin or natural killer cell cytotoxicity.
Executive summary:

Immune function was assessed in male Sprague-Dawley rats exposed to the mixture of organotins used in PVC pipe production.

Adult male rats were given drinking water for 28 d containing a mixture of dibutyltin dichloride (DBTC), dimethyltin dichloride (DMTC), monobutyltin trichloride (MBT), and monomethyltin trichloride (MMT) in a 2:2:1:1 ratio, respectively, at 3 different concentrations (5:5:2.5:2.5, 10:10:5:5, or 20:20:10:10 mg organotin/L). Rats were also exposed to MMT alone (20 or 40 mg MMT/L) or plain water as a control. Delayed-type hypersensitivity, antibody synthesis, and natural killer cell cytotoxicity were evaluated in separate endpoint groups immediately after exposure ended.

The evaluated immune functions were not affected by the mixture or by MMT alone. The data suggest that immunotoxicity is unlikely to result from the concentration of organotins present in drinking water delivered via PVC pipes, as the concentrations used were several orders of magnitude higher than those expected to leach from PVC pipes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Effect on immunotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

As part of the original dossier for MMTE a study was included which utilized read-across from MMTC. This study remains in the dossier to provide continuity to reviewers and an historical perspective of the changes which are being made to the dossier. New data on in vitro metabolism, cited in section 7.1.1 of this dossier, caused a change in the interpretation of the data cited. The new in vitro metabolism data support the conclusion that MMTE does not metabolise to MMTC under simulated mammalian gastric conditions [pH ~2 and 37 °C] as was formerly believed. It then follows that the toxicology of MMTE via the oral route in mammalian species cannot be accurately predicted based on studies conducted with MMTC via the oral route in mammalian species. The implications are clear: (a) dietary feeding and oral gavage studies conducted with MMTC cannot be read-across to MMTE, and (b) for studies conducted with MMTC, their relevance for hazard classification of MMTE must be reduced or eliminated. Therefore, the Klimisch score of the study has been reduced to Klimisch 3 because the read-across strategy from this study to MMTE is no longer valid (though the study itself is still considered to be reliable).

A paper is included as supporting information for completeness and in order to provide further detail on how a mixture of organotin compounds is understood to affect immune functions.

Immune function was assessed in male Sprague-Dawley rats exposed to the mixture of organotins used in PVC pipe production.

Adult male rats were given drinking water for 28 d containing a mixture of dibutyltin dichloride (DBTC), dimethyltin dichloride (DMTC), monobutyltin trichloride (MBT), and monomethyltin trichloride (MMT) in a 2:2:1:1 ratio, respectively, at 3 different concentrations (5:5:2.5:2.5, 10:10:5:5, or 20:20:10:10 mg organotin/L). Rats were also exposed to MMT alone (20 or 40 mg MMT/L) or plain water as a control. Delayed-type hypersensitivity, antibody synthesis, and natural killer cell cytotoxicity were evaluated in separate endpoint groups immediately after exposure ended.

The evaluated immune functions were not affected by the mixture or by MMT alone. The data suggest that immunotoxicity is unlikely to result from the concentration of organotins present in drinking water delivered via PVC pipes, as the concentrations used were several orders of magnitude higher than those expected to leach from PVC pipes.

Justification for classification or non-classification