Thiram

Immunotoxicity

Description of key information

Key, WIL-500003, U.S. EPA test guideline, mice, 28 days,

NOAEL (systemic and immunotoxicity): 150 ppm (corresponding to 25 mg/kg bw/day)

LOAEL (systemic and immunotoxicity): 450 ppm (corresponding to 74 mg/kg bw/day)

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:

key study

Study period:

17 Jun - 28 Oct 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: ca. 7 weeks
- Weight at study initiation: 24.3 - 31.8 g
- Fasting period before study: not applicable
- Housing: individually in clean, stainless steel, wire-mesh cages suspended above cage-board
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 - 21.5
- Humidity (%): 48.9 - 62.1
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 5 Jul 2011 To: 3 Aug 2011

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The basal and test substance diets were prepared approximately weekly, divided into aliquots for daily use, and stored frozen. On each day of diet administration, 1 aliquot from each group was removed from the freezer and allowed to thaw at room temperature prior to dispensation.

Positive control substance:
The cyclophosphamide (CPS) formulation administered to the positive control group (Group 5) was prepared on the day prior to the first dose (28 July 2011). The formulation was prepared in phosphate-buffered saline at a concentration of 5 mg/mL. The solution was prepared once, divided into 5 aliquots within a laminar flow hood using aseptic techniques, and stored frozen at approximately -20°C. On each day of dosing (study days 24 - 27), a vial was quickly thawed, stored on ice, and mixed thoroughly prior to dosing. Each aliquot was used within 3 hours of thawing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Various tests were performed to assess homogeneity and stability of the test material in the diet, before administration and during the testing period. Due to a decrease in the concentration noted in the 15 ppm (Group 2) samples following 1- and 2-day room temperature storage, the dosage concentration for Group 2 was changed to 17.5 ppm beginning on study day 3.

The analyzed dietary formulations were found to contain 93.4% to 106% of the test substance, were homogenous, and were stable at concentrations of 15 and 500 ppm following up to 2 days of room temperature storage or up to 9 days of frozen storage and at concentrations of 17.5 and 150 ppm following 1 day of room temperature storage, with the following exceptions. The analyzed concentration of the 15 ppm formulation following 1- or 2-days of room temperature storage was 84.5% and 80.5%, respectively, of the pre-storage values.

Duration of treatment / exposure:

28 days

Frequency of treatment:

continously via the diet

Dose / conc.:

15 ppm

Remarks:

From study Day 3 on, the dose was increased to 17.5 ppm due to limited stability of the test substance in the diet. The actual ingested dose was 3 mg/kg bw/day.

The actual digested dose was 3 mg/kg bw/day.

Dose / conc.:

150 ppm

Remarks:

The actual digested dose was 25 mg/kg bw/day.

Dose / conc.:

450 ppm

Remarks:

The actual digested dose was 74 mg/kg bw/day.

No. of animals per sex per dose:

10 males

Control animals:

yes, plain diet

Details on study design:

Sheep Red Blood Cell (SRBC) ADMINISTRATION
- Supplier: ImmunoTox®, Inc., Richmond, VA
- Storage: 2 - 8 °C
-Time schedule: Day 24 (For animals assigned to the positive control group, sRBC injections were administered prior to the first administration of CPS).
- Anesthesia: not reported
- number of cells injected: 1 * 10^8 SRBC/animal
- Procedure: All animals of all groups were injected (tail vein, 0.2 mL/animal) with SRBC preparation.
The spleens collected from these immunized mice at the scheduled necropsy were used for the Splenic Antibody-Forming Cell (AFC) assay to assess the TDAR (T-Dependent Antibody Response).

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon, for mortality and moribundity, clinical examinations were performed once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were conducted on all animals approximately weekly throughout the study, beginning 1 week prior to randomization, at the time of randomization, and prior to the scheduled necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily, beginning 1 week prior to randomization, at the time of randomization, on study day 0, and on the day of the scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

Organs weighed: Spleen, thymus

HISTOPATHOLOGY: Yes
- Tissues collected: Lymph nodes (left axillary, mandibular (2), mesenteric), Peyer’s patches, spleen (placed in EBSS/HEPES buffer), thymus
- fixative: 10% neutral-buffered formalin (except as noted)

Cell viabilities:

SPLEEN: Yes

THYMUS: No

BONE MARROW: No

Humoral immunity examinations:

SPLEENIC ANTIBODY FORMING CELLS (AFC) ASSAY: Yes
- Method: The spleens of all animals of all groups (treatment and control) were collected at the scheduled necropsy immediately following blood collection in EBSS solution, with 15 mM HEPES and supplemented with gentamicin. The spleen samples were processed into single-cell suspensions, centrifuged and resuspended in EBSS with HEPES. Spleen cell counts were performed using a Model Z1 Coulter Counter®. Viability of splenocytes was determined using propidium iodide and the Coulter® EPICS® XL-MCL™ Flow Cytometer.
- Dose groups: all, including the negative and positive control group
- No. of animals: all animals in all groups

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): No

Specific cell-mediated immunity:

ONE-WAY MIXED LYMPHOCYTE CULTURE (MLC) ASSAY: No

DELAYED-TYPE HYPERSENSITIVITY (DTH) REACTION: No

CYTOTOXIC T-LYMPHOCYTE (CTL) ASSAY: No

Non-specific cell-mediated immunity:

NATURAL KILLER (NK) CELL ACTIVITY: No

MACROPHAGE NUMBER AND FUNCTION: No

Other functional activity assays:

SPLEEN CELL PROLIFERATION ASSAY (ANTI-CD3 MEDIATED T CELL PROLIFERATION): No

ENUMERATION TOTAL B CELLS, TOTAL T CELLS AND T CELL SUBPOPULATIONS: No

Positive control:

Yes, Cyclophosphamide monohydrate (CPS)
The positive control substance, CPS, was administered to Group 5 via intraperitoneal injection once daily on study Days 24 through 27. The positive control substance dosage level was 50 mg/kg bw/day and the dosage volume was 10 mL/kg bw/day.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean.

Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Body weight, body weight change, and food consumption data were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group.
The positive control data were evaluated using the Student’s t-Test and compared to the vehicle control group.

The AFC data was expressed as both specific activity (IgM antibody forming cells per million spleen cells [AFC/106 spleen cells]) and as IgM total spleen activity (AFC/spleen). Data were first tested for homogeneity of variances using the Bartlett’s Chi Square test. Homogenous data were evaluated using a parametric one-way ANOVA. When significant differences occurred, the treatment groups were compared to the vehicle control group using Dunnett’s test. Non-homogenous data was evaluated using a non-parametric ANOVA. When significant differences occurred, the treatment groups were compared to the vehicle control group using the Gehan-Wilcoxon test, when appropriate. The Jonckheere’s test was used to test for dose-related trends across the vehicle control and treatment groups. The positive control data was evaluated using the Student’s t-Test and compared to the vehicle control group. The criteria for accepting the results of the positive control group included a statistically significant (p≤0.05) decrease in the response compared to that of the vehicle control group.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical observations.

Summarized results can be found in Attachment 1 in the attached background material.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy.

Summarized results can be found in Attachment 1 in the attached background material.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on body weights. Any statistically significant differences between the control and test substance-treated groups were of a small magnitude, were most likely due to biological variability, and were not considered related to test substance administration due to the lack of a dose-response trend.

Summarized results can be found in Attachment 1 (Summary of all data) and 2 (Overview oif relevant data) in the attached background material.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance-related effects on food consumption. Any statistically significant differences between the control and test substance-treated groups were of a small magnitude, were most likely due to biological variability, and were not considered related to test substance administration due to the lack of a dose-response trend.

Summarized results can be found in Attachment 1 in the attached background material.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

no effects observed

Description (incidence and severity):

Please refer to the detailed section below.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 15 ppm: No treatment related differences were observed.
- 150 ppm: relative spleen weights were statistically significantly increased (24%) compared to the control group.
- 450 ppm: absolute (36%) and relative (38%) spleen weights were statistically significantly increased compared to the control group.

These higher spleen weights were not indicative of immunotoxicity. When animals have been immunized with sheep erythrocytes, the spleens of the animals undergo proliferation in response to the sRBC injection and this can alter the determination of the actual effect of the compound on spleen weight.

As expected, statistically significant decreases were observed in the positive control (CPS) group for both absolute (53%) and relative (50%) spleen weights.

No treatment related differences were observed regarding absolute and relative thymus weights.

Summarized results can be found in Attachment 1 (Summary of all data) and 2 (Overview oif relevant data) in the attached background material.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related macroscopic findings at the scheduled necropsy.
All macroscopic findings noted were considered to be spontaneous and/or incidental in nature and unrelated to test substance administration.

Summarized results can be found in Attachment 1 (Summary of all data) and 2 (Overview oif relevant data) in the attached background material.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Cell viabilities:

no effects observed

Description (incidence and severity):

There were no statistically significant effects in spleen cell number for the animals treated with any of the three dose levels of the test substance, as compared to the control group. As expected, a statistically significant decrease (68%) was observed in the positive control (CPS) group for the spleen cell number, as compared to the vehicle control group.

Summarized results can be found in Attachment 1 (Summary of all data) and 2 (Overview oif relevant data) in the attached background material.

Humoral immunity examinations:

effects observed, treatment-related

Description (incidence and severity):

- 15 and 150 ppm: No treatment related differences were observed.
- 450 ppm: In the functional evaluation of the IgM AFC response, exposure to the test substance resulted in lower Specific Activity and Total Spleen Activity in the 450 ppm group, when compared to the vehicle controls. Only the difference in the Specific Activity (51%) was statistically significant.

As anticipated, the positive control, CPS, produced statistically significant decreases in both Specific Activity and Total Spleen Activity.

Summarized results can be found in Attachment 1 (Summary of all data) and 2 (Overview oif relevant data) in the attached background material.

Specific cell-mediated immunity:

not examined

Description (incidence and severity):

not applicable

Non-specific cell-mediated immunity:

not examined

Description (incidence and severity):

not applicable

Other functional activity assays:

not examined

Description (incidence and severity):

not applicable

Other findings:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

systemic and immunological toxicity

Effect level:

150 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects were observed at this dose level.

Remarks on result:

other: corresponding to an actual ingested dose of 25 mg/kg bw/day.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic and immunological toxicity

Effect level:

450 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

immunology

Remarks on result:

other: corresponding to an actual ingested dose of 74 mg/kg bw/day.

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day

System:

hepatobiliary

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

No indications for an immunotoxic potential were seen in the toxicity data set comprising short- and long-term toxicity studies, reproductive toxicity and neurotoxicity studies, i.e. effects on spleen or thymus weight, spleen, thymus or lymph nodes histopathology or effects on clinical pathology parameters relevant for immunotoxicity.

Conclusions:

The study was conducted in compliance with GLP and OCSPP Guideline 870.7800, and is therefore considered as valide.
In the conducted study, the NOAEL for T-cell dependent antigen response (TDAR-NOAEL) was 25 mg/kg bw/day (150 ppm). Increased spleen weighs were noted at 25 mg/kg bw/day and 74 mg/kg bw/day (450 ppm). No effects on spleen cellularity and thymus were observed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

The available study was conducted according to U.S. EPA test guideline and GLP, thereby fulfilling the criteria of suitability as key study.

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

Mode of Action Analysis / Human Relevance Framework

Please refer to the information given in the endpoint summary.

Additional information

The potential to interfere with the immune system was assessed in male CD-1 mice under subacute test conditions (feed, 28 days), according to an U.S. EPA test guideline and GLP. This study is considered suitable as key study (Report number WIL-500003).

Groups of 10 Wistar rats/per dose were exposed daily via the diet to 15/17.5, 150 and 450 ppm test item, corresponding to 3, 25 and 74 mg/kg bw/day of the test substance for at least 28 days according to OCSPP Guideline 870.7800 and in compliance to GLP. In the low dose group, from study Day 3 on, the dose was increased to 17.5 ppm due to limited stability of the test substance in the diet. One group of mice received 50 mg/kg bw Cyclophosphamide as a positive control for immunosuppression. Cyclophosphamide was administered via intraperitoneal injection from Day 24 - 27.

On Day 24, rats were injected in the tail vein with 0.2 mL preparation of Sheep Red Blood Cells (SRBC), containing 1 x 10^8 cells/mL. 4 days after SRBC immunization, animals were sacrificed and the spleen was analysed with the spleenic antibody forming cells (AFC) assay. When animals were sacrificed, the gross necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded but not sampled. Organ weight was recorded for spleen and thymus.

 

No mortality, clinical signs or changes in body weight or food consumption were observed in the study. Animals of the mid- and high dose group showed increased mean absolute and/or relative spleen weight compared to controls but these higher spleen weights were not indicative of immunotoxicity. Treatment-related decreases in both specific activity and total spleen activity in the 450 ppm group were observed. No indications for an immunotoxic potential of the test substance were seen in the toxicity data set comprising short- and long-term toxicity studies, reproductive toxicity and neurotoxicity studies, i.e. effects on spleen or thymus weight, spleen, thymus or lymph nodes histopathology or effects on clinical pathology parameters relevant for immunotoxicity.

The SRBC response was unchanged in treatment groups compared to control groups.

In the positive control group, no treatment-related clinical signs or influence on food consumption or body weight occurred. The positive control substance produced statistically significant decreases in both specific activity and total spleen activity when compared to the vehicle control

Group and statistically significantly reduced absolute and relative spleen and thymus weights. With these results, the positive control cyclophosphamide confirmed the sensitivity of the test system.

 

Under the conditions of the study, the NOAEL for systemic and T-cell dependent antigen response (TDAR-NOAEL) was 25 mg/kg bw/day (150 ppm). Increased spleen weighs were noted at 25 mg/kg bw/day and 74 mg/kg bw/day (450 ppm). No effects on spleen cellularity and thymus were observed.