clothianidin (ISO); (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine

Immunotoxicity

Description of key information

Key, M-394980-01-2, U.S. EPA test guideline, rat, 28 days,
NOAEL (systemic): 500 ppm (corresponding to 45.8 mg/kg bw/day in males and 46.2 mg/kg bw/day in females)
LOAEL (systemic): 3000 ppm (corresponding to 252.8 mg/kg bw/day in males and 245.0 mg/kg bw/day in females)
NOAEL (immunotoxicity): 3000 ppm (corresponding to 252.8 mg/kg bw/day in males and 245.0 mg/kg bw/day in females, highest dose tested)

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:

29 Jun - 05 Aug 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Version / remarks:

adopted 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD®(SD)IGS BR VAF/Plus®

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, USA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ca. 6-7 weeks
- Weight at study initiation: 161-216 g (males), 155-183 g (females)
- Fasting period before study: not applicable
- Housing: individually in stainless steel, wire-bottom cages
- Diet: Certified Rodent Diet® #5002 (PMI Nutrition International), ad libitum
- Water: filtered tap water, ad libitum
- Acclimation period: approximately 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Jun 2004 To: 05 Aug 2004

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks on MMAD:

Not applicable

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): diets were prepared by the sponsor and shipped to the testing facility. Each diet ration was used for one week.
- Mixing appropriate amounts with: Certified Rodent Diet® #5002 (PMI Nutrition International)
- Storage temperature of food: Frozen (-15 °C to -25 °C), stored at room temperature during each week of use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogenity and stability of the test substance in the diet was tested previously conducted studies with the test substance in the diet and proven for concentrations of 50 and 5000 ppm. Stability was verified for 7 days at room temperature and for 28 days in a refrigerator. Analysis of the test item concentration in the diet determined concentrations of 154, 524 and 3120 ppm.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily, continously, via the diet

Dose / conc.:

150 ppm (nominal)

Remarks:

corresponding to 13.8 mg/kg bw/day for males and 14.0 mg/kg bw/day for females

Dose / conc.:

500 ppm (nominal)

Remarks:

corresponding to 45.8 mg/kg bw/day for males and 46.2 mg/kg bw/day for females

Dose / conc.:

3 000 ppm (nominal)

Remarks:

corresponding to 252.8 mg/kg bw/day for males and 245.0 mg/kg bw/day for females

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses were based on a previous subchronic study in rats with similar concentrations. Based on this results, the highest dose was expected to produce signs of toxicity but no mortality, the intermediate concentration was chosen in order to cause minimal toxic effects and the lowest dose should not have any impact on the rats.

Treatment with Sheep Red Blood Cells (sRBCs)
Four days before sacrifice, all rats were administered a single intravenous injection 0.5 mL of sRBCs via the tail vein.

Treatment of the positive control:
The ten male and ten female rats in the positive control group were administered 50 mg/kg bw (10 mg/mL) cyclophosphamide via intraperitoneal injection for four consecutive days before sacrifice. This injection of cyclophosphamide was administered after the administration of the sRBCs.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly during the acclimatisation period, during administration twice daily on day 1, 2, and 3 of treatment and after that once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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 AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

OTHER: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Rats were sacrificed by CO2 asphyxiation. Necropsy included the examination of external surfaces, orifices, as well as an internal examination of tissues and organs in situ. Gross necropsy of the thoracic, abdominal and pelvic viscera was performed.

The spleen was harvested, transfered to Earle's Balanced Salt Solution (BBSS) in HEPES medium with gentamicin added as a bacteriostat and refrigerated for further analysis. Single-cell suspensions were prepared from each spleen using a Stomacher® 80 Lab Blender. Cell suspensions were then centrifuged and resuspended in Earle's Balanced Salt Solution with HEPES.

Organ weight was recorded for: Spleen

HISTOPATHOLOGY: No.

Cell viabilities:

SPLEEN: YES
- Method: Viability of splenocytes was determined using propidium iodide (PI) and the Coulter EPICS XL-MCL Flow Cytometer
- Dose groups: All
- No. of animals: all

THYMUS: No

BONE MARROW: No

Humoral immunity examinations:

ANTIBODY FORMING CELLS (AFC) ASSAY: Yes
- Method: modified hemolytic plaque assay of Jerne (1974). The cell suspension obtained from the spleen was centrifuged and resuspended in 6 mL and 1:50 and 1:150 dilutions were prepared. Form these dilutions, 0.1 mL was added to 25 µL guinea pig complement, 25 µL sRBC, and 0.5 mL of warm agar (0.5%). The mixture was plated on a petri dish, covered with a microscope cover slip and incubated for 3 h (36-38 °C). The plaques, which developed, were counted using a Bellco plaque viewer. A plaque, occurring from the lysis of sRBC, is elicited as a result of the interaction of complement and antibodies (produced in response to the i.v. sensitization) directed against sRBC. Each plaque is generated from a single IgM antibody-producing B cell, permitting the number of AFC present in the whole spleen to be calculated.
- Dose groups: all
- parameters measured: specific activity (AFC/10^6 spleen cells) and total spleen activity (AFC/spleen)
- No. of animals: all

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

Other examinations:

None

Positive control:

Cyclophosphamide monohydrate, ISOPAC® (CPS) - a white powder, diluted in Phosphate Buffered Saline, pH 7.2 (PBS, without calcium chlorite and magnesium chlorite)

Statistics:

Significance was set to p < 0.05 with the exception of the Bartlett's test, were significance was defined as p < 0.001.

For parametric data such as body weight, feed consumption etc, a Bartlett's test was used to assess homogenity of the variance. If this was significant (meaning variances were not homogeneous), a nonparametric test is performed (see below). If variances were homogeneous, data were compared using the Analysis of Variance Test (ANOVA) followed by Dunnett's Test if differences were found. Nonparametric data were analyzed with the Kruskal-Wallis Test, when 75% or fewer of the scores in all the groups were tied, folllowed by a Dunn's Test if differences were found. When more than 75% of the scores in any dosage group were tied, Fisher's Exact Test was used to compare the proportion of ties in the dosage groups.

Clinical observation incidence data, as well as other proportion data, were analyzed as contingency tables using the Variance Test for Homogeneity of the Binomial Distribution.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical signs that were observed were non-treatment related because they occurred sporadically and evenly throughout dose groups. These were red perioral substance, slight excess salivation, chromorhinorrhea, red substance in the cage pan, chromodacryorrhea, misaligned and/or missing or broken incisors, a scab on the neck, head or nose, swollen digit, abrasion on the neck or dehydration.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

not applicable

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 3000 ppm: decreased mean body weight for males (-12.8 to -14.8%) and females (-8.8 to -12.6%) on study Day 8, 15, 22, 29; decreased mean body weight gain in study intervals 1 to 8 (-60%), and 1 to 29 (-25.2%) for males and 1 to 8 (-72.3%), 15 to 22 (-48.1%) and 1 to 29 (44.6%) for females, decreased terminal mean body weight for males (-12.8%) and females (-12.6%).

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

Positive control: reduced mean body weight on DS 29, reduced mean body weight gain on days 22 to 29 and 1 to 29 in males and females, reduced terminal mean body weight.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 3000 ppm: reduced absolute mean feed consumption throughout the study in males (-15.1 to -32.5%) and females (-14.6 to -27.1%), reduced relative mean feed consumption on days 1 to 8 and 1 to 29 in males (-26.4 and -10.3%, respectively) and females (-23.0 and -8.7%, respectively)

Summarized data can be found in Attachment 2 in the attached background material.

Positive control: reduced absolute and relative feed consumption in males on study days 22 to 29 and 1 to 29.

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):

For the test substance, no effects were observed. Non-treatment related effects were seen in the 150 and 500 ppm treatment group (only for males): total spleen activity was increased in 150 and 500 ppm animals and specific activity in the 500 ppm exposure group. This enhancement was not dose-dependent, it did not occur in female rats and it was related to a low response in control animals. Therefore, it was considered incidental.

Positive control
The positive control produced a decrease in specific activity (96%) and total spleen cell activity (99%).

Summarized data can be found in Attachment 3 in the attached background material.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute and relative spleen weight was not affected by treatment with the test substance.

Positive control: reduced absolute (-55 to -62%) and relative weight (-50 to -54%) of the spleen in males and females was observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Only incidental findings were observed. These were a urinary bladder with thick walls and tan areas present on the left lateral lobe of the liver (1 male in the positive control group)
and a mass (1.8 x 1.4 x 0.8 cm) in the left lateral lobe of the liver with abdominal adipose and pancreatic tissue adhered to the mass (1 female, positive control group).

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):

Spleen: The number of viable cells in all treatment groups were comparable to the control group. The positive control decreased the number of viable spleen cells (-87%).

Humoral immunity examinations:

no effects observed

Description (incidence and severity):

see above (immunological findings)

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

Effect level:

500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 524 ppm (analytical concentration), 45.8 mg/kg bw/ day (males) and 46.2 mg/kg bw/day (females)

Conclusions:

The potential immunotoxicity of the test substance was assessed in male Wistar rats under subacute test conditions (feed, 28 days), according to an U.S. EPA test guideline, under GLP conditions. Under the conditions of the test, the dietary NOAEL for the test substance was 45.8 and 46.2 mg/kg bw/day for males and females, respectively. At ca 250 mg/kg bw/day (252.8 and 245.0 for males and females, respectively) reduced body weight and feed consumption was observed. No effect on IgM response was found. The NOAEL for immunotoxicity was therefore set to ca 250 mg/kg bw (the highest administered dose). The positive control cyclophosphamide confirmed the sensitivity of the test system.
The study is considered valid and in compliance with GLP and the guideline US-EPA-OPPTS Series
870, Health Effects Testing Guidelines, N°870.6300.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

252.8 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

U.S. EPA test guideline and GLP study, 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

Additional information

To evaluate the effects of the test substance on the immune system, one key study was conducted. Groups of 10 Crl:CD®(SD)IGS BR VAF/Plus® rats/sex/dose were exposed daily via the diet to 150, 500 and 3000 ppm of the test substance for at least 28 days according to US-EPA-OPPTS Series 870, Health Effects Testing Guidelines, N°870.6300 and in compliance with GLP (M-123395-01-1).  The analytical concentration of the test substance in the feed was 154, 524 and 3120 ppm. Males ingested on average 13.8, 45.8 and 252.8 mg/kg bw/day and females 14.0, 46.2 and 245.0 mg/kg bw/day. The dosing solutions were prepared by mixing the appropriate amounts of the test substance with the diet. One group of rats received Cyclophosphamide as a positive control for immunosuppression. 50 mg/kg bw (10 mg/mL) cyclophosphamide was administered via intraperitoneal injection for four consecutive days before sacrifice.

Both the concentration and homogeneity of the test substance in the diet and the positive control in the vehicle were analyzed and considered valid. Clinical signs were recorded daily and body weight and food consumption were measured weekly. Four days before sacrifice, rats were injected in the tail vein with 0.5 mL of a preparation of Sheep Red Blood Cells (SRBC). When animals were sacrificed, the gross necropsy included the examination of external surfaces, orifices, as well as an internal examination of tissues and organs in situ. Gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The spleen was harvested and a single cell suspension was prepared. This was then analyzed for plaque formation using the modified hemolytic plaque assay of Jerne (1974). Also, the cell viability of spleenocytes was assessed.

No mortality or treatment-related clinical signs were observed in the study. At 500 ppm of the test substance, systemic toxicity was evident. The animals showed reduced body weight (-8.8 to -14.8%), reduced body weight gain (up to -72.3%) and reduced feed consumption (absolute and relative) compared with the vehicle control. The SRBC response was unchanged in treatment groups compared to the vehicle control group and no difference was found in terms of spleenocyte viability between the treated and vehicle control animals. In the positive control group, no treatment-related clinical signs or mortality was observed but the mean body weight and feed consumption (absolute and relative) were reduced, compared with the vehicle control. No immunotoxic effects were observed in rats at doses of test item up to and including 3120 ppm, corresponding to 250 mg/kg bw/day. In the positive control immunotoxicity was noted as a decreased number of viable spleen cells (-87%), the specific activity (96%) and total spleen cell activity (99%). With these results, the positive control cyclophosphamide confirmed the sensitivity of the test system.

Under the conditions of the test, the dietary NOAEL for the test substance was 45.8 and 46.2 mg/kg bw/day for males and females, respectively. At 252.8 and 245.0 mg/kg bw/day for males and females, respectively, reduced body weight and feed consumption was observed. No effect on IgM response was found. The NOAEL immunotoxicity was therefore set to 252.8 and 245.0 mg/kg bw/day for males and females, respectively (the highest administered dose).

Justification for classification or non-classification

The available data on immunotoxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive and do not warrant classification