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Immunotoxicity

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Description of key information

All studies are included as supporting information and are not considered for the CSA.

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
immunotoxicity: sub-chronic oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
15 Jun 2010 to 28 Jul 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.7800
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Dose descriptor:
NOAEL
Remarks:
immunotoxicity
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Based on the lack of effects on both the functional AFC assay for humoral immunity and the functional NKC assay for innate immunity. Dietary equivalent to 2025.8 mg/kg/day.
Critical effects observed:
no

Achieved dose levels

Concentration analysis results: The analyzed diet admix formulations contained 86.1%-104% of the test substance, except for the 100 ppm diet admix formulation prepared on 18 July 2010 (76% of target concentration), which was reformulated on 19 July 2010. This is within the research laboratory SOP range for acceptability (85% to 115% of target concentration), but not all diet admix formulations were within the protocol-specified range (90% to 110% of the target concentration). The test substance was not detected in the vehicle formulation that was administered to the vehicle control group (Group 1/1A).

Based on these results and the research laboratory SOP acceptability range for diet admix formulations, the protocol-specified doses 100, 1250, and 5000 ppm were offered to the animals.

Homogeneity results: The test diets were homogenous based on the protocol-specified acceptability limits (90% to 110% of the target concentration and had a relative standard deviation [RSD] ≤ 5%) and the research laboratory SOP acceptable range for diet admix homogeneity (85% to 115% of the target concentration and a RSD ≤ 10%). In addition, homogeneity assessments were conducted on diet admix formulations prepared for dosing, and the test diets were homogenous based on the research laboratory SOP acceptability range, but not homogenous based on the protocol-specified acceptability limits.

Stability results: All dietary formulations were stable for the duration of use.

 

Mortality: All animals survived to the scheduled necropsy.

 

Clinical observations: There were no test substance-related clinical observations.

Body weight and weight gain: Statistically significantly lower mean cumulative body weight gains were generally noted in the 5000 ppm AFC and NKC groups throughout the study compared to the vehicle control group. In addition, mean body weight losses were generally observed through study day 28, and at the end of the study, cumulative body weight gains were 69.6% and 83.3% lower than the control group in the 5000 ppm AFC and NKC groups, respectively. The lower body weight gains resulted in 7.8% and 10.2% lower mean body weights in the 5000 ppm AFC and NKC groups, respectively, at the end of the study compared to the vehicle control group.

There were notest substance-related effects on body weights or body weight gains in the 100 and 1250 ppm AFC and NKC groups compared to the vehicle control group. Sporadic incidences of higher or lower body weight gains were seen in these groups, but these were isolated and nottest substance-related.

 

Food consumption and compound intake: Food consumption was not affected by the test substance dietary formulations.

Dose rates (based on nominal dietary levels of the test substance) were calculated in terms of mg test substance/kg body weight. Mean values are shown below:

 

Table 1: Average dose received - average of AFC and NFC groups (mg/kg of body weight/day)

Test substance (ppm)

100

1250

5000

Females

37.2

447.8

2025.8

 

Sacrifice and pathology

Organ weights: There were notest substance-related effects on absolute liver weights in the AFC and NKC groups. Mean liver weights adjusted for final body weights were statistically significantly higher than the vehicle control group in the 5000 ppm AFC and NKC groups (22.4% and 15.8%, respectively) and in the 1250 AFC group (10.3%).

Absolute and adjusted spleen weights were statistically significantly lower than the vehicle control group (except for adjusted spleen weights in the 5000 ppm AFC group) in the 5000 ppm AFC and NKC groups, and this effect was considered test substance-related. Mean absolute spleen weights in the 5000 ppm AFC and NKC groups were 27.3% and 39.5% lower, respectively, and mean adjusted spleen weights were 12.3% and 29.4% lower, respectively, compared to the vehicle control group.

Mean absolute and adjusted thymus weights were lower than the vehicle control group in the 5000 ppm NKC group (24.7% and 20.5%, respectively); however, only the absolute value was statistically significant.

As expected, statistically significantly lower spleen weights were observed for the AFC positive control group, CPS. The positive control group had 39.9% and 38.9% lower absolute and relative mean spleen weights, respectively, compared to the vehicle control group. These effects were consistent with the known immunosuppressive effects of CPS and validate the appropriateness of the assay.

 

Macroscopic findings: There were notest substance-related macroscopic findings.

 

AFC Assay: Test substance-related effects on spleen cellularity were noted in the 5000 ppm AFC group. Statistically significantly lower (34%) spleen cell numbers were noted in the 5000 ppm AFC group compared to the vehicle control group. As expected, statistically significantly lower (59%) spleen cell numbers were observed in the positive control group (CPS) when compared to the vehicle control group.

In the functional evaluation of the IgM AFC response, treatment with the test substance did not suppress the humoral immune response when evaluated as either specific activity (AFC/106 Spleen Cells) or total spleen activity (AFC/Spleen). As anticipated, statistically significant lower specific activity (100%) and total spleen activity (100%) was observed in the positive control group (CPS) animals when compared to the vehicle control group animals.

 

NKC Assay: There were notest substance-related effects on NKC activity. A statistically significantly higher (38%) NKC activity was noted in the 1250 ppm NKC group at the effector: target ratio of 200:1; however, in the absence of a dose‑response trend this was not considered to betest substance-related. Spleen cell numbers in the 5000 ppm NKC group were statistically significantly lower (40%) compared to the vehicle control group, which was similar to the effects observed on spleen cellularity in the 5000 ppm AFC group. As expected, statistically significantly lower NKC activity was observed in the positive control group (anti asialo GM1) when compared to the vehicle control group.

 

DISCUSSION: Lower spleen weights observed for the 5000 ppm AFC and NKC groups, and lower thymus weights observed for the 5000 ppm NKC group were seen only in the presence of a large effect on body weight. In addition, lower spleen cell numbers in the 5000 ppm AFC and NKC groups mirrored the effects on spleen weight. Therefore, in the absence oftest substance-related effects on the humoral (AFC) or innate (NKC) functional immune response, these effects were considered an indication of general toxicity and not immunosuppression.

Conclusions:
Based on the results of this study, treatment of female B6C3F1 mice with the test substance on a continuous basis in the diet for a minimum of 28 consecutive days resulted in no suppression of the humoral (AFC) or innate (NKC) components of the immune system. Based on lower spleen and thymus weights and lower spleen cell numbers at 5000 ppm, the no observed effect level (NOEL) for the non-functional immune parameters (i.e., spleen and thymus weights and spleen cell numbers from the NKC group animals) was 1250 ppm (equivalent to approximately 447.8 mg/kg/day); however, the changes were considered to be secondary to signs of general toxicity including markedly lower body weights at 5000 ppm and higher liver weights at 1250 and 5000 ppm. Based on the lack of effects on both the functional AFC assay for humoral immunity and the functional NKC assay for innate immunity the overall NOEL would be 5000 ppm, the highest dose examined. Further signs of general toxicity included lower body weights at 5000 ppm and higher liver weights at 1250 and 5000 ppm.
Executive summary:

The test substance was offered ad libitum in the diet for a minimum of 28 consecutive days to female B6C3F1 mice in Groups 2, 3, and 4 at dietary concentrations of 100, 1250, and 5000 ppm, respectively. Ten mice/group (Subset A) were used for the splenic Antibody-Forming Cell (AFC) assay and 10 mice/group (Subset B) were used for the Natural Killer Cell (NKC) assay. Mice in AFC positive control group (Group 5A) were administered the positive control substance, cyclophosphamide (CPS) via intraperitoneal injection (50 mg/kg/day) once daily for 4 consecutive days (study days 24 through 27). All AFC group mice (Groups 1A-5A) were immunized with an intravenous injection of sheep red blood cells (sRBC) on study day 24, approximately 96 hours prior to the scheduled necropsy. Mice in the NKC positive control group (Group 6B) were administered the positive control substance (anti asialo GM1) via a single intravenous injection (0.2 mL/animal) on study day 27, approximately 24 hours prior to the scheduled necropsy. The concurrent control groups (Groups 1A and 1B) and the positive control groups (Group 5A and 6B) were offered the basal diet on a comparable regimen as the test substance-treated groups. All animals were euthanized on study day 28. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed once daily for all animals. Detailed physical examinations were performed once weekly and on the day of the scheduled necropsy. Individual body weights were recorded twice weekly and food consumption was recorded approximately weekly. Complete necropsies were conducted on all animals. The liver and spleen were collected from all AFC- and NKC-designated animals and weighed at the scheduled necropsy. In addition, the thymus was collected and weighed from all NKC-designated animals. Spleens were placed in Earle’s balanced salt solution/HEPES buffer and shipped to an external party. After arrival at the external party, spleen cell suspensions were prepared, spleen cell counts were performed, and the number of specific IgM antibody-forming cells directed towards the sRBC antigen were determined for the AFC-designated animals to measure the humoral immune response. In addition, external party’s personnel prepared the spleens from the NKC-designated animals to measure innate immunity using the NKC assay.

Mean test substance consumption in the 0, 100, 1250, and 5000 ppm groups were 0, 37.2, 447.8, and 2025.8 mg/kg/day, respectively, over the entire 28-day study. All animals survived to the scheduled necropsy. There were no test substance-related clinical observations or effects on food consumption or macroscopic findings for either the AFC or NKC assay animals. There weretest substance-related lower mean body weights and body weight gains in the 5000 ppm AFC and NKC groups. There were test substance-related higher mean adjusted liver weights in the 5000 ppm AFC and NKC groups and the 1250 ppm AFC group. In addition, lower mean absolute and adjusted spleen weights were noted in the 5000 ppm AFC and NKC groups, and lower mean absolute and adjusted relative thymus weights were noted in the 5000 ppm NKC group. In the AFC assay, there were no effects attributed to thetest substanceon the specific activity or total activity of splenic IgM AFC to the T cell‑dependent antigen sRBC. Test substance-related lower spleen cell numbers were observed in the 5000 ppm AFC group. For the AFC positive control group (CPS), statistically significantly lower spleen cell numbers (59%), specific activity (100%), and total spleen activity (100%) of IgM antibody‑forming cells were noted when compared to the vehicle control group. These effects on spleen weights, spleen cell numbers, and spleen activity (i.e., number of specific IgM antibody-forming cells directed towards the sRBC antigen) were consistent with the known immunosuppressant effects of CPS and validated the functionality of the AFC assay. In the NKC assay, there were no effects attributed to thetest substanceon NKC activity. For the NKC positive control group (anti asialo GM1), statistically significantly lower NKC activity at the effector:target ratio of 200:1 was noted when compared to the vehicle control group. This effect was consistent with the known effects of anti asialo GM1 on NKC activity, which validated the functionality of the NKC assay.

In conclusion, based on the results of this study, treatment of female B6C3F1 mice with the test substance on a continuous basis in the diet for a minimum of 28 consecutive days resulted in no suppression of the humoral (AFC) or innate (NKC) components of the immune system. Based on lower spleen and thymus weights and lower spleen cell numbers at 5000 ppm, the no-observed-effect level (NOEL) for the non-functional immune parameters (i.e., spleen and thymus weights and spleen cell numbers from the NKC group animals) was 1250 ppm (equivalent to approximately 447.8 mg/kg/day); however, the changes were considered to be secondary to signs of general toxicity including markedly lower body weights at 5000 ppm and higher liver weights at 1250 and 5000 ppm. Based on the lack of effects on both the functional AFC assay for humoral immunity and the functional NKC assay for innate immunity the overall NOEL would be 5000 ppm, the highest dose examined. Further signs of general toxicity included lower body weights at 5000 ppm and higher liver weights at 1250 and 5000 ppm.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
447.8 mg/kg bw/day
Study duration:
subacute
Species:
mouse

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

28-Day Dietary Immunotoxicity Study (Crittenden 2011)

Treatment of female B6C3F1 mice with the test substance on a continuous basis in the diet for a minimum of 28 consecutive days resulted in no suppression of the humoral (AFC) or innate (NKC) components of the immune system. Based on lower spleen and thymus weights and lower spleen cell numbers at 5000 ppm, the no observed effect level (NOEL) for the non-functional immune parameters (i.e., spleen and thymus weights and spleen cell numbers from the NKC group animals) was 1250 ppm (equivalent to approximately 447.8 mg/kg/day); however, the changes were considered to be secondary to signs of general toxicity including markedly lower body weights at 5000 ppm and higher liver weights at 1250 and 5000 ppm. Based on the lack of effects on both the functional AFC assay for humoral immunity and the functional NKC assay for innate immunity the overall NOEL would be 5000 ppm, the highest dose examined. Further signs of general toxicity included lower body weights at 5000 ppm and higher liver weights at 1250 and 5000 ppm.

Justification for classification or non-classification

Immunotoxic potential of thiamethoxam has been investigated in a guideline 28-day immunotoxicity study in mice in which thiamethoxam did not impact humoral immunity or innate immunity (lack of effects on both AFC assay and NKC assay) up to a dose level of 2025.8 mg/kg bw/d. No clear evidence on a potential immunotoxicity of the substance is available from the other toxicity studies. A weight of the evidence evaluation of the available data does not indicate consistent evidence of adverse effects of thiamethoxam on the immune system. Classification for STOR RE is not warranted.