diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

Immunotoxicity

Description of key information

Immunotoxicity (EPA OPPTS 870.7800), rat: NOEL immunotoxicity = 591 mg/kg bw/day (females), corresponding to 7500 ppm (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:

18 Aug - 06 Oct 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 Aug 1998

Deviations:

yes

Remarks:

female rats only, no testing in both sexes as required by the guideline

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Rj:WI (IOPS HAN)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: 170 – 216 g
- Fasting period before study: no
- 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: filtered and softened water from the municipal water supply, ad libitum
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY:

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: 18 Aug 2010 To: 28 Sep 2010

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was incorporated into the diet to provide the required dietary concentrations. The test substance was ground to a fine powder before being incorporated into the diet by dry mixing.

DIET PREPARATION
- Rate of preparation of diet (frequency): There was one preparation for each concentration.
- Storage temperature of food: When not in use, the diet formulations were stored at approximately -18°C.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of test substance in diet was verified before the study for the lowest and highest concentrations to demonstrate adequate formulation procedures. Dietary levels of the test substance were verified for each concentration. The mean value obtained from the homogeneity check was taken as measured concentration.
Homogeneity and concentration results ranged from 93 to 100% of the nominal concentration and were within the in-house target range of 85-115% of the nominal concentration. Stability in rodent diet was analysed in a previous study (refer to study M-136542-01-1-01 under “Repeated dose toxicity”). The stability of the test substance in the dietary formulation was checked under the study conditions. The test substance was found to be stable at 7500 ppm in rodent diet when stored frozen for a period of 32 days, followed by 11 days at room temperature (95% of the nominal concentration). At 500 ppm, the recovery after identical storage conditions was found to be marginally outside the in-house target range (84% instead of 85% of the nominal concentration). Nevertheless, the formulations were considered to be acceptable for the study.

Duration of treatment / exposure:

30 days

Frequency of treatment:

continuously (via diet)

Dose / conc.:

500 ppm (nominal)

Remarks:

equivalent to 40 mg/kg bw/day

Dose / conc.:

2 500 ppm (nominal)

Remarks:

equivalent to 199 mg/kg bw/day

Dose / conc.:

7 500 ppm (nominal)

Remarks:

equivalent to 591 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were chosen on the basis of the results of subacute and subchronic toxicity studies.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily in weekends or public holidays)
- Cage side observations included: morbidity, mortality, clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly
- Detailed physical examinations included: nature, onset, severity, reversibility and duration of clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the start of the study (study Day 1), then at weekly intervals throughout the treatment period and before terminal 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: No. The food consumption for each group was determined and the group mean diet consumption (g/day) was calculated per week. The weight of food supplied and of that remaining at the end of the food consumption period was recorded weekly for all animals during the treatment period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. The weekly main achieved dosage intake in mg/kg bw/day for each week and for weeks 1 to 4 was calculated. For details please refer to “Any other information in materials and methods incl. tables”.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

IMMUNOLOGICAL ANALYSIS: Yes
- Time schedule for collection of blood: Blood samples were taken by puncture of the retro-orbital venous plexus 4 days after sheep red blood cell (SRBC) sensitisation. Animals were anesthetised by inhalation of Isoflurane.
- Animals fasted: No
- How many animals: all animals
- Parameters checked: SRBC-specific immunoglobulin M in response to antigen administration.

Sacrifice and pathology:

SACRIFICE: On study Day 30 all animals from all groups were sacrificed by exsanguination while under deep Isoflurane anesthesia.
GROSS PATHOLOGY: Necropsy included the examination of all major organs, tissues and body cavities. Significant macroscopic abnormalities were recorded but not sampled. At final sacrifice, the following organs were weighed: spleen and thymus.
HISTOPATHOLOGY: No

Cell viabilities:

SPLEEN: No

THYMUS: No

BONE MARROW: No

Humoral immunity examinations:

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes
- Method: Female rats were treated continuously with the test item, vehicle control or positive control via dietary administration for a period of 28 days. 4 days before necropsy, all animals were immunized with Sheep Red Blood Cell (SRBC) antigen by intravenous injection. At scheduled necropsy, the immunological IgM response following immunization with SRBC was quantified for all animals.
- Dose groups: all dose groups
- No. of animals: all animals

Positive control:

Cyclophosphamide 3.5 mg/kg bw/day, administered by oral gavage at a constant dosage volume of 5 mL/kg bw.

Statistics:

Please refer to the attached document "Statistical analysis_M-402229-01-1.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed throughout the study. An increase in salivation was noted in 4, 3 and 1 animals at 500, 2500 and 7500 ppm, respectively, however, in the absence of a dose-effect relationship, this observation was considered not to be treatment-related. The few other clinical signs observed were considered to be chance findings.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in any group throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean absolute body weight gain during the first week of treatment at 7500 ppm was 33% lower than in the controls (not statistically significant). Thereafter, mean body weight gain in this group was comparable to controls, however, at the end of the study the absolute body weight gain remained 13% lower compared to the controls (not statistically significant).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Description (incidence and severity):

Please refer to "Specific immunotoxic examinations" for further details.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no relevant changes in organ weights (spleen and thymus) in treated animals when compared to the controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic changes in treated animals when compared to the controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Cell viabilities:

not examined

Humoral immunity examinations:

no effects observed

Description (incidence and severity):

The high mean anti-SRBC IgM concentration observed in the control group confirmed the sensitization of the animals. A high inter-individual variability was noted in all groups as is usually observed with SRBC sensitization in the rat. No statistically significant difference was noted in anti-SRBC IgM concentrations up to 7500 ppm. Treatment with the positive control resulted in a markedly lower anti-SRBC IgM concentration when compared to untreated controls, thus demonstrating the functionality of the test system.

Specific cell-mediated immunity:

not examined

Non-specific cell-mediated immunity:

not examined

Other functional activity assays:

not examined

Other findings:

not examined

Key result

Dose descriptor:

NOEL

Remarks:

immunotox

Effect level:

7 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: no immunotoxic potential identified

Remarks:

highest dose tested

Key result

Critical effects observed:

no

Table 1: SRBC-specific IgM response

SRBC-specific IgM concentration (u/mL) mean ± standard deviation
Dose (ppm)	0	500	2500	7500	CPA
Study Day 30	12972 ± 10031	13164 ± 9123	16798 ± 14767	17498 ± 18135	1690** ± 1051
CPA: Cyclophosphamide, 3.5 mg/kg bw/day; SRBC: Sheep Red Blood Cell ** p < 0.01

Conclusions:

Treatment of female Wistar rats for 30 days did not impair the immunological IgM resonse following immunization with SRBC antigen up to the highest tested dose of 7500 ppm (corresponding to 591 mg/kg bw/day).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

591 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to assess the endpoint immunotoxicity under Regulation (EC) No. 1907/2006.

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

The test item was investigated for immunotoxicity in a subacute oral toxicity study according to EPA OPPTS 870.7800 and in compliance with GLP. The test item was incorporated into the ground diet and administered daily via the diet for a period of 30 days. Groups of 10 female rats per dose received doses of 500, 2500 and 7500 ppm, corresponding to 40, 199 and 591 mg/kg bw/day. Similar constituted groups of animals received the plain diet (negative control) or 3.5 mg/kg bw/day cyclophosphamide (positive control). Four days before necropsy, all animals were immunized with Sheep Red Blood Cell (SRBC) antigen by intravenous injection.

The animals were observed twice daily for mortality and clinical signs; a detailed physical examination was performed prior to and once weekly during the study. In addition, individual body weights and food consumption were recorded once weekly. Just before necropsy, blood samples were collected from each animal for specific anti-SRBC immunoglobulin M (IgM) analysis. After 30 days of treatment, all animals underwent necropsy, gross pathological examination and selected organs were weighed for each animal.

There was no mortality observed during the study period. Clinical signs of toxicity comprised salivation at all dose groups, but a clear dose-response relationship was lacking, thus the findings were considered to be unrelated to treatment. The mean absolute body weight gain of the high dose group animals was reduced during the first week of the study, but comparable to those of control animals thereafter. At the end of the study, the absolute body weight gain remained 13% lower to those of controls. The effect was not statistically significant and therefore considered toxicologically not relevant. At gross pathological examination there were no treatment-related macroscopical changes and no differences in organ weight.

Analysis of the SRBC-induced IgM response revealed no differences of treated animals and control animals up to the highest dose tested. Under the experimental conditions, the test item has no immunotoxic potential. The NOAEL for systemic and the NOEL for immunologic toxicity were 7500 ppm, corresponding to 591 mg/kg bw/day.

Justification for classification or non-classification

The available data on immunotoxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.