Teflubenzuron

Immunotoxicity

Administrative data

Endpoint:

immunotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-28 to 2011-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-(3,5-dichloro-2,4-difluorophenyl)-3-(2,6-difluorobenzoyl)urea

EC Number:

617-441-5

Cas Number:

83121-18-0

Molecular formula:

C14 H6 Cl2 F4 N2 O2

IUPAC Name:

1-(3,5-dichloro-2,4-difluorophenyl)-3-(2,6-difluorobenzoyl)urea

Test material form:

solid

Test animals

Species:

mouse

Strain:

C57BL

Remarks:

(6 J Rj)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Raison sociale: JANVIER S.A.S., Route des Chênes Secs - C.S. 4105 - Le Genest-St-Isle, 53941 St. Berthevin Cedex, France
- Age at study initiation: 48-50 days
- Weight at study initiation: ca. 22 g
- Fasting period before study: no
- Housing: housed individually in Polycarbonate cages, type M II with wire cover
- Diet: ad libitum, ground Kliba maintenance diet mouse-rat “GLP”
- Water: ad libitum, tap water
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: On the basis of the analytical findings the drinking water and food was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: For each concentration, the test substance was weighed out and mixed with a small amount of food. Then corresponding amounts of food, depending on the test group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Details of the mixers used are retained with the raw data. The test-substance preparations were mixed once before the start of the administration period.
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse-rat “GLP”
- Storage temperature of food: room temperature

POSITIVE CONTROL:
Cyclophosphamide monohydrate (positive control substance) was applied as a solution via gavage. To prepare the solution, the appropriate amount of Cyclophosphamide monohydrate was weighed out depending on the desired concentration. Then the vehicle (drinking water) was filled up to the desired volume, subsequently mixed using a magnetic stirrer. The positive control substance preparations was prepared once, split in daily aliquots and deep frozen at -18°C. The mixtures were applied when reaching room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the diet was demonstrated over a period of 31 days at room temperature. The concentration control analyses revealed that the mean values of the test substance in feed and the positive control substance (Cyclophosphamide monohydrate) in drinking water were in the expected range of the target concentrations, i.e. were always in a range of 92.9-108.1% for the test-substance preparations and 97% for the positive control substance.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

continuous

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 ppm (nominal)

Remarks:

approximately 44 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 ppm (nominal)

Remarks:

approximately 218 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

5 000 ppm (nominal)

Remarks:

approximately 1059 mg/kg bw/day

No. of animals per sex per dose:

8 males per dose

Control animals:

yes, plain diet

Examinations

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. All animals were checked daily for any clinically abnormal signs. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50×37.5 cm with sides of 25 cm high). The following parameters were examined:
1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on study day 0 was calculated as body weight change.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Individual food consumption was determined once a week and calculated as mean food consumption in grams per animal and day. The mean daily intake of test substance (test group means) was calculated based upon individual values for body weight and food consumption.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Drinking water consumption was observed by daily visual inspection of the water bottles for any overt changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

HISTOPATHOLOGY: No

Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined:
1. Anesthetized animals
2. Spleen
3. Thymus

Cell viabilities:

SPLEEN: No

THYMUS: No

BONE MARROW: No

Humoral immunity examinations:

ANTIBODY PLAQUE FORMING CELLS (PFC) ASSAY: No

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes
Immunization
- Sterile, heparinized sheep blood (Acila SARL, Weiterstadt, Germany) was washed with sterile 0.9% NaCl solution and adjusted to 4×10^8 RBC/mL.
- On study day 23, each mouse was immunized with 0.5 mL of the SRBC solution injected intraperitoneally.
- On study day 29, blood was taken from each animal for the SRBC ELISA.

SRBC ELISA
-The ELISA was performed according to TEMPLE et al. (1995).
- In deviation to the mentioned reference, a standard curve (7 standards in a two-fold dilution) was established using anti-SRBC IgM positive serum pool, for comparison to subsequent test runs (arbitrary lab units/mL; stock standard aliquots stored at -80°C).
- Each serum sample was applied to the ELISA in two dilutions:
1.negative control and dosed animals: 1:256 and 1:512;
2. positive-control animals: 1:64 and 1:128
-Generally, the 1:256 dilution for the negative control and dosed animals and the 1:64 dilution for the positive-control animals was reported.
- Result deviations (lab units/mL) in both dilutions of more than 25% were confirmed
- OD values of the sample dilutions outside of the linear range of the standard curve were repeated with a couple of two-fold lower or higher dilutions.
- Generally, two in house controls were measured with each test run.
- The ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
- Dose groups: all
- No. of animals: 8 per group

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

Positive control:

Cyclophosphamide monohydrate (10 mg/kg bw/day, oral route) was selected to be sufficient to cause immunosuppressive activity as positive control substance.

Statistics:

1. food consumption, body weight, and body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means
2. Clinical pathology parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians.
3. Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each test group with the control group was performed using the WILCOXON test for the hypothesis of equal medians

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One animal (test group 1 [200 ppm]) showed a teeth anomaly at the upper jaw on study days 28 and 29 of the administration period. One animal (test group 2 [1000 ppm]) showed alopecia at the abdominal region from study day 21 and at the hindlimb from study day 23 onwards until the end of the administration period. Due to the single occurrence and the absence of a dose-response relationship the findings were assessed as spontaneous in nature and not test substance-related.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight parameters of all animals treated with the substance were not significantly affected by test substance administration. Body weight and body weight change of mice treated with Cyclophosphamide monohydrate (positive control group) were significantly decreased from study day 7 onwards, throughout the whole administration period, reaching a maximum of -8.4% in mean body weight on study day 28 and a body weight loss between on study days 0 to 7. These findings were assessed as being treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of animals receiving test item and animals receiving the positive control substance (Cyclophosphamide monohydrate) was not significantly reduced during the administration period. On study day 7, food consumption values of two animals receiving the positive control substance were out of the biological range. However, the changes were without any influence on clinical signs and body weight and, therefore, not related to treatment. The approximate, mean daily test-substance intake in mg/kg body weight/day over the entire study period was 44 mg/kg bw/day (200 ppm group), 218 mg/kg bw/day (1000 ppm group) and 1059 mg/kg bw/day (5000 ppm group).

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test substance-related findings were observed.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Description (incidence and severity):

Six days after immunization, no changes in the SRBC IgM titers were found in male mice dosed with the test substance (up to 5000 ppm [approximately 1059 mg/kg bw/d]; highest dose tested [limit dose]), whereas the SRBC titers were significantly lower in mice of the positive control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No weight deviations were recorded for animals of test groups 1-3 (200, 1000 and 5000 ppm). The positive control group (test group 4: Cyclophosphamide Monohydrate) revealed a significant decrease of terminal body weight, spleen and thymus weights, which was the expected result.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Both observed gross lesions occurring in 2 different organs in 2 separate animals (sparse hair [alopecia] in 1 of 8 animals in test group 2 [1000 ppm]; enlarged kidneys in 1 of 8 animals in test group 3 [5000 ppm]) were considered to be incidental and not related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Specific immunotoxic examinations

Cell viabilities:

not examined

Humoral immunity examinations:

no effects observed

Description (incidence and severity):

The SRBC IgM antibody titers in male mice dosed with the test substance were not changed (up to 5000 ppm [approximately 1059 mg/kg bw/d]; highest dose tested [limit dose]). In contrast, the mice dosed with the positive control compound had significantly reduced SRBC IgM antibody titers.

Specific cell-mediated immunity:

not examined

Non-specific cell-mediated immunity:

not examined

Other functional activity assays:

not examined

Description (incidence and severity):

The results obtained with the positive control substance Cyclophosphamide monohydrate
verified the sensitivity of the present immunotoxicity study (with TDAR method). The
administration of Cyclophosphamide monohydrate (10 mg/kg bw/d) led to effects indicative of
immunotoxicity as indicated by the reduced SRBC IgM antibody titers and reduced absolute
and relative spleen and thymus weights.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

5 000 ppm (nominal)

Based on:

test mat.

Remarks:

corresponding to ca. 1059 mg/kg bw/d

Sex:

male

Basis for effect level:

other: No adverse effects observed up to highest dose tested.

Target system / organ toxicity

Key result

Critical effects observed:

no