imidacloprid (ISO); 1-(6-chloropyridin-3-ylmethyl)-N-nitroimidazolidin-2-ylidenamine

Immunotoxicity

Description of key information

Key, M-394980-01-2, U.S. EPA test guideline, rat, 28 days,

NOAEL (systemic): 600 ppm (corresponding to 47.1 mg/kg bw/day)
LOAEL (systemic): 2400 ppm (corresponding to 186 mg/kg bw/day)
NOAEL (immunotoxicity): 2400 ppm (corresponding to 186 mg/kg bw/day, 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:

5 May - 16 Jul 2010

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:

Wistar

Remarks:

Rj:WI (IOPS HAN)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France.
- Age at study initiation: 7 weeks
- Weight at study initiation: 218 - 258 g
- Fasting period before study: not applicable
- Housing: individually in suspended, stainless steel, wire-mesh cages
- Diet: Certified rodent powdered and irradiated diet A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: tap water, filtered and softened, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 5 May To: 15 Jun 2010

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST ITEM:
DIET PREPARATION
- Rate of preparation of diet (frequency): once for each concentration
- Mixing appropriate amounts with: The test substance was ground to a fine powder before being incorporated into the diet by dry mixing with Certified rodent powdered and irradiated diet A04CP1-10
- Storage temperature of food: -18 °C

CYCLOPHOSPHAMIDE
PREPARATION OF DOSING SOLUTIONS:
- Lot/Batch Nr: 097K1311
- Appearance: white powder
- Purity: 100.2% (analysis by HPLC)
- Storage: 2 - 8 °C
- Date of Analysis: 08. Mar 2004

Route of exposure:
- gavage
-Time schedule: daily
- Concentration: 3.5 mg/kg bw/day
- Volume: 5 mL/kg body weigh

Preparation:
- Rate of preparation (frequency): twice
Procedure: The dosing formulation of cyclophosphamide was prepared by suspending the
substance in sterilized water to produce the required dosing concentration.
Storage: +5°C in the dark when not in use

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of test substance in diet was verified for the lowest and highest concentrations to demonstrate adequate formulation procedures. The mean value obtained from the homogeneity check was taken as measured concentration. Dietary levels of the test substance were verified for each concentration.

To determine the stability of the test substance in the frozen diet, samples from the highest and lowest concentrations were analyzed after having been frozen for 28 days then thawed and kept at room temperature for two weeks.

For the positive control, the homogenity cyclophosphamide in the vehicle sterilized water was verified on the first formulation to demonstrate adequate formulation procedures. The concentration of cyclophosphamide in the vehicle was verified for each preparation. Stability of of cyclophosphamide in vehicle has been demonstrated in a previous study (ODIN-FEURTET M. (SA 09023, 2009): Cyclophosphamide: 28-Day oral immunotoxicity study in Wistar rats by gavage, Bayer CropScience, Sophia Antipolis, France.) at concentrations of 0.1 and 1 g/L for a time period which covers the period of storage and usage for the current study.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

150 ppm (nominal)

Remarks:

corresponding to an actual dose digested of 11.7 mg/kg bw/day

Dose / conc.:

600 ppm (nominal)

Remarks:

corresponding to an actual dose digested of 47.1mg/kg bw/day

Dose / conc.:

2 400 ppm (nominal)

Remarks:

corresponding to an actual dose digested of 186 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Gender selection rational: There is no evidence of immunotoxicity with imidacloprid in rats or mice. Therefore, the selection of species and gender for the immunotoxicity study is based on which one is more sensitive to the compound based on other toxicologic endpoints.
- Specied selection rationale: selection rationale: The test substance was previously tested in rat and mice. A subchronic dietary study in Wistar rats showed higher effects in males than females. Body weight was more effected in males (LOAEL = 61 vs 422 mg/kg bw/day in females) as well as the liver (LOAEL = 300 vs 422 mg/kg bw/day in females). A chronic dietary study showed that the test substance had no influence on body weight at 300 ppm in males and females (corresponding to 16.9 mg/kg bw/day for males and 24.9 mg/kg bw/day for females) but males were more sensitive to thyroid mineralization (LOAEL = 17 mg/kg bw/day vs 73 mg/kg bw/day for females). In B6C3F1 mice, a subchronic study showed higher sensitivity in males (NOAEL = 391 mg/kg bw/day vs 3087 mg/kg bw/day in females). Also in the oncogenicity study in mice the overall NOAEL was based on decreased body weight at 1000 ppm in males (208 mg/kg bw/day) and females (274 mg/kg bw/day).
- Dose selection rationale: Based on the previously described studies, dose levels were set to 0, 150, 600 and 2400 ppm

Sheep Red Blood Cell (SRBC) ADMINISTRATION

SRBC
- Supplier: BioMérieux
- Reference Number: 72141
- Storage: 2 - 8 °C

-Time schedule: Day 26
- Anesthesia: YES if necessary (Isoflurane (Baxter, Maurepas, France))
- Procedure: On the day of injection, Sheep Red Blood Cells were washed in PBS (Phosphate Buffered Saline), counted using a cell counting instrument (Siemens Advia 120) and diluted in PBS in order to obtain a 5 x 10^8 cells/mL preparation. SRBC preparation was kept on ice until use. All animals of all groups were injected (tail vein, 05 mL/animal) with SRBC preparation.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily on weekends and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

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
- time schedule: weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 4 days after SRBC immunization (terminal sacrifice)
- Animals fasted: No
- How many animals: all survived animals
- Parameters examined: SRBC-specific IgM

HAEMATOLOGY: No

URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
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, thymus

HISTOPATHOLOGY: No

Cell viabilities:

No data

Humoral immunity examinations:

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes
- Method: Rat Anti-Sheep Red Blood Cell IgM ELISA kits from Life Diagnostics (West Chester, PA 19380 – USA)
- Dose groups: all
- No. of animals: 10/dose group (all animals)

Specific cell-mediated immunity:

No data

Non-specific cell-mediated immunity:

No data

Other functional activity assays:

None

Other examinations:

None

Positive control:

Yes

Statistics:

Mean and standard deviation were calculated for each group and time period.

Body weight change, terminal body weight, absolute and relative organ weight parameters
To analyse the differences between the control and the positive group cyclophosphamide, an F-test was performed. if this showed significant differences, a 2-sided modified t-test followed. If no significance was found, a 2-sided 2-test was performed.

To compare control and test substance groups, group variances were analyzed with the Bartlett test followed with an Analysis of Variance (ANOVA) and a 2-sided Dunnett's test (if needed) when variance was equal and a Kruskal-Wallis and 2-sided Dunn test (if needed) when variance differed.

Body weight and average food consumption/day parameters
The comparison between the control and positive group was similar to the above described, only that the data was transformed using the log transformation if the F test showed a significant difference. Then, the procedure was repeated on the transformed data.

The comparison between the control and test substance groups was also similar to the above described, only that the data was transformed using the log transformation if the Bartlett test showed a significant difference. Then, the procedure was repeated on the transformed data and followed as described above.

If one or more group variance (s) equaled 0, means were compared using non-parametric procedures.

Immunological parameter
The control and positive group were compared using the 2-sided Mann-Whitney-test

The control and test substance group were compared with the Kruskal-Wallis-test. is a significant difference was found, the 2-sided Dunn Test was used.

Significance in all of the tests was defined as p ≤ 0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 2400 ppm: piloerection on Day 8, 15 and 22 (1/10), wasted appearance Day 8 (1/10)

Cyclophosphamide:
No treatment-related signs observed

Summarized data can be found in Table 1 in Attachment 1.

Mortality:

no mortality observed

Description (incidence):

not applicable

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 2400 ppm: mean body weight reduced throughout the study (19-21%), mean body weight gain was reduced especially during the first two weeks of the study (Day 0 to 8: weight loss of -4 g compared to weight gain +54 g in the controls; Day 8-15: weight gain of 3.5 g vs 5.5 g in the controls)

Cyclophosphamide:
mean body weight was reduced by between 3 and 5% throughout the study (this was statistically significant on study Days 15 and 22).

Summarized data can be found in Table 2-4 in Attachments 2-4.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 2400 ppm: reduced mean food consumption by 46, 24, 20 and 17% on study Days 8, 15, 22 and 29, respectively (p≤0.01)

Cyclophosphamide:
no effects observed

Summarized data can be found in Table 5 in Attachment 5.

Compund intake
The mean achieved doses of the test substance were 11.7, 47.1 and 186 mg/kg bw/day at 150, 600 and 2400 ppm, respectively

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. One control animal showed unusally high anti-SRBC IgM concentrations.

Cyclophosphamide
anti-SRBC IgM mean concentration was 95% lower than the controls, proving the sensitivity of the assay to identify an immunosuppressive agent.

Data is summarized in Table AB1 and AB2 in Attachment 6.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Terminal body weight
- 2400 ppm: reduced mean terminal body weight (19%)
Summarized data can be found in Table AB3 in Attachment 7.

Cyclophosphamide
no effects observed

Thymus weight
- 2400 ppm: reduced mean absolute thymus weight (28%), increased mean spleen to body ratio (19%)
Both of these findings were attributed to the lower terminal body weight and therefore considered related to systemic toxicity rather than caused by immunotoxicity. The apparent decrease in relative thymus weight in the top dose group (-11%), was not considered to be a treatment-related effect, since the difference from controls was not statistically significant and as the mean value still remained within the range of historical control data as available for Wistar rats from 28-day toxicity studies.

Summarized data as well as Historical control data can be found in Table AB4, AB5, and AB6 in Attachment 8.

Cyclophosphamide:
Reduced mean absolute and relative thymus and spleen weight (25- 31%) were reported.

Summarized data can be found in Table AB 6 and AB7 in Attachment 8.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

-2400 ppm: atrophic/small thymus (3/10), congested/red intestines (3/10)
The atropic/small thymus is considered related to systemic toxicity (see above), it is unclear whether the intestinal appearance was incidental or also a sign of systemic toxicity.

Cyclophosphamide
- Atrophic/small spleen (7/19), atrophic/small thymus (3/10)

Summarized data can be found in Table 9 Attachment 9.

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:

not examined

Description (incidence and severity):

not applicable

Humoral immunity examinations:

not examined

Description (incidence and severity):

not applicable

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

Effect level:

47.1 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

186 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

Immunology

Effect level:

186 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

immunology

Remarks on result:

other: No Observed Effect Level for the immunotoxicological parameters

Key result

Critical effects observed:

no

Conclusions:

The potential immunotoxicity of present 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 NOEL for the test substance was 47.1 mg/kg bw/day. At 186 mg/kg bw/day, the Maximum Tolerated Dose was exceeded, resulting in systemic toxicity. This was shown by reduced body weight and food consumption. Reduced absolute thymus and increased relative spleen weight were considered to be due to systemic toxicity rather than immunotoxicity. The NOEL for immunotoxicity was therefore set at 186 mg/kg bw/day, which was the highest dose level tested. 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

186 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

Mode of Action Analysis / Human Relevance Framework

No data available.

Additional information

The potential immunotoxicity of present 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. This study is considered suitable as key study (M-394980-01-2).

Groups of 10 Wistar rats/per dose were exposed daily via the diet to 150, 600 and 2400 ppm test item, corresponding to 11.7, 47.1 and 186 mg/kg bw/day (mean ratios) 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 to GLP (M-394980-01-2).

The dosing solutions were prepared by mixing the appropriate amounts of the test substance with the diet. One group of rats received 3.5 mg/kg bw Cyclophosphamide as a positive control for immunosuppression. Cyclophosphamide was administered solubilized in deionized water via gavage. 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. Detailed physical examination was performed weekly throughout the study. On Day 26, rats were injected in the tail vein with 0.5 mL preparation of Sheep Red Blood Cells (SRBC), containing 5 x 10^8 cells/mL. 4 days after SRBC immunization, blood was taken from all animals (non-fasted) and analyzed for anti SRBC- IgM using an ELISA kit. 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 was observed in the study. At 186 mg/kg bw/day of the test substance, systemic toxicity was evident, observed as reduced body weight (19-21%), reduced body weight change and reduced food consumption compared to the control group. Clinical signs observed were piloerection on Day 8, 15, and 22 and wasted apearance in one animal in the high-dose group on Day 8. Animals of this test group also showed a smaller thymus (in appearance and weight) and an increased spleen:body weight ratio. However, these findings cannot be specifically attributed to immunotoxicity but are rather related to the systemic toxicity. The SRBC response was unchanged in treatment groups compared to control groups. In 3 of 10 animals receiving 186 mg/kg bw/day of the test substance, the intestine was red, it was not determinable whether this finding was incidental or a sign of systemic toxicity. 

In the positive control group, no treatment-related clinical signs or influence on food consumption were observed but the mean body weight was reduced throughout the study (3-5%) as compared to controls. The concentration of anti-SRBC IgM was 95% lower than the controls. Spleen and thymus were smaller and lighter than in control animals. With these results, the positive control cyclophosphamide confirmed the sensitivity of the test system. Thus, no immunotoxic properties could be evidenced in male rats at doses of test item up to and including 2400 ppm, corresponding to 186 mg/kg bw/day.

Under the conditions of the study, the NOEL for immunotoxicity was set to 186 mg/kg bw (the highest administered dose), whereas the overall NOAEL with respect to systemic toxicity was set at 47.1 mg/kg bw/day. The observations described showed, that at 186 mg/kg be/day, the Maximum Tolerated Dose was exceeded, resulting in systemic toxicity.

Justification for classification or non-classification

No classification is required since no immunotoxicity was observed.