Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Immunotoxicity

Administrative data

Endpoint:

immunotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23 February 2010 to 23 Mar 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Version / remarks:

Aug 1998

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

632-619-2

EC Number:

632-619-2

Cas Number:

881685-58-1

Molecular formula:

C20 H23 F2 N3 O

IUPAC Name:

632-619-2

Test animals

Species:

mouse

Strain:

other: Crl:CD1(ICR)

Sex:

female

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 consecutive days

Frequency of treatment:

Continuously in diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

Group 2. Dietary equivalent to 127 mg/kg bw/day.

Doses / concentrations 2

Dose / conc.:

2 500 ppm

Remarks:

Group 3. Dietary equivalent to 608 mg/kg bw/day.

Doses / concentrations 3

Dose / conc.:

5 000 ppm

Remarks:

Group 4. Dietary equivalent to 1356 mg/kg bw/day.

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

other: Positive control; cyclophosphamide in BPS via intraperitoneal injection (50 mg/kg/day)

Results and discussion

Effect levels

Dose descriptor:

NOEL

Remarks:

immune suppression

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no suppression of the humoral component of the immune system.

Remarks on result:

other: Dietary equivalent to 1356 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

ANALYTICAL CHEMISTRY

The analysed diet admix formulations were found to contain 94.3 % - 101 % of the test substance which was within the protocol-specified range (90 % to 110 % of target concentration), were homogeneous, and were stable after 15 days of room temperature storage. Based on these results, the protocol-specified doses of test substance were administered to the animals. The test substance was not detected in the vehicle formulation that was administered to the control group (Group 1)

 

SURVIVAL

There were no test substance-related deaths. One female (5000 ppm group) was found dead on study day 14. Clinical observations of decreased defecation and yellow material on the urogenital area and cumulative body weight loss were noted for this animal prior to its death. However, there were no macroscopic findings noted for this animal and no additional deaths were noted for any dose group. Therefore, this early death was not considered to be related to test substance administration.

 

CLINICAL OBSERVATIONS

There were no test substance-related clinical observations. All clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner and/or were common findings for laboratory mice of this age and strain.

 

BODY WEIGHTS

An test substance-related transient body weight loss was noted in the 5000 ppm group, resulting in slightly lower mean cumulative body weight gain and mean body weight when compared to the vehicle control group at the end of the study. A statistically significantly lower mean body weight gain, which constituted a slight body weight loss, was noted from study days 3 to 7 in the 5000 ppm animals. The mean cumulative body weight gain and mean body weight for the 5000 ppm group were slightly (but not statistically significantly) lower than the vehicle control group at the end of the study. This difference from the vehicle control group was attributed to the initial body weight loss during study days 3 through 7. There were no effects on body weight or body weight gain at 500 or 2000 ppm.

 

FOOD AND TEST SUBSTANCE CONSUMPTION

Food consumption was unaffected by test substance administration. There were no statistically significant differences when the control and test substance-treated groups were compared.

 

ANATOMIC PATHOLOGY

MACROSCOPIC EXAMINATION

There were no test substance-related macroscopic findings. All macroscopic findings were considered to be spontaneous and/or incidental in nature and unrelated to test substance administration.

 

ORGAN WEIGHTS

There were no test substance-related effects on absolute, adjusted, or relative spleen or thymus weights in the test substance-treated groups as compared to the vehicle control group. However, statistically significantly higher absolute and adjusted mean liver weights were noted in the 2500 and 5000 ppm group. The higher liver weight was attributed to test substance administration. There were no statistically significant organ weight differences noted for the 500 ppm group. As expected, statistically significant decreases were observed in the positive control group for the spleen and thymus organ weights. Specifically, as compared to the control group, the positive control group demonstrated 41 % and 40 % lower mean absolute and relative spleen weights, respectively; and 60 % and 56 % lower mean absolute and relative thymus weights, respectively. These effects are consistent with the known immunosuppressant effects of CPS and validate the appropriateness of the assay.

 

AFC ASSAY

There were no test substance-related effects on spleen cell numbers. However, statistically significantly higher (25 %) spleen cell numbers were observed in the mid-dose group, but no effect in spleen cell numbers was observed for the low- and high-dose groups. Therefore, in the absence of other immune-related findings, the slightly higher spleen cell numbers observed at 2500 ppm were considered not treatment-related. As expected, statistically significantly lower (48 %) spleen cell numbers were observed in the positive control group when compared to the vehicle control group. In the functional evaluation of the IgM antibody-forming cell response, treatment with test substance did not suppress the humoral immune response when evaluated as either specific activity (AFC/106spleen cells) or total spleen activity (AFC/spleen). As anticipated, statistically significantly lower specific activity (100 %) and total spleen activity (100 %) was observed in the positive control group animals when compared to the vehicle control group animals.

Applicant's summary and conclusion

Conclusions:

The no-observed-effect level (NOEL) for suppression of the immune response in female CD-1 mice exposed to the test substance on a continuous basis in the diet for 28 days was 5000 ppm (equivalent to 1356 mg/kg/day), thus the NOEL for suppression indicates that the test substance is not immunotoxic.

Executive summary:

In an EPA OPPTS 870.7800 study in compliance with GLP, groups of Crl:CD1(ICR) female mice were exposed to test substance given via in the diet ad libitum for a minimum of 28 consecutive days. Three groups of mice (Groups 2-4) were administered dietary concentrations of 500, 2500, and 5000 ppm, corresponding with 0, 127, 608, and 1356 mg/kg/day, respectively, over and mice in group 5 were given the positive control substance, cyclophosphamide (CPS), via intraperitoneal injection (50 mg/kg/day) for 4 consecutive days (study days 24 through 27). The vehicle and positive control groups (Groups 1 and 5, respectively) were offered the basal diet on a comparable regimen as test substance-treated groups. Additionally, all mice were immunised with an intravenous injection of sheep red blood cells (sRBC) on study day 24, approximately 96 hours prior to the scheduled necropsy. Each group consisted of 10 females. 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, spleen, and thymus were collected from all animals and weighed at the scheduled necropsy. Spleens were placed in EBSS/HEPES buffer, spleen cell suspensions were prepared. Spleen cell counts were performed, and the number of specific IgM antibody-forming cells directed toward the sRBC was determined.

There were no treatment-related deaths. There were no test substance-related effects on clinical observations or macroscopic findings. There were no test substance-related effects on absolute, adjusted, or relative spleen or thymus weights. There were no effects attributed to test substance on the specific activity or total activity of splenic IgM antibody-forming cells to the T cell-dependent antigen sRBC. A transient body weight loss was noted at 5000 ppm. Higher mean absolute and adjusted liver weights noted at 2500 and 5000 ppm test substance when compared to the vehicle control group were consistent with previous studies assessing test substance at similar exposure levels. Lower mean absolute and adjusted spleen and thymus weights were noted for the CPS positive control group when compared to the vehicle control group. Additionally, CPS administration produced statistically significantly lower spleen cell numbers (48% lower), specific activity (100% lower), and total spleen activity (100% lower) of IgM antibody-forming cells when compared to the vehicle control group. These effects are appropriateness of the assay.

Based on the results of this study, treatment of female Crl:CD-1(ICR) mice with test substance on a continuous basis in the diet for 28 days resulted in no suppression of the humoral component of the immune system. The no-observed-effect level (NOEL) for suppression of the immune response was 5000 ppm (equivalent to 1356 mg/kg/day). The only effects attributed to test substance were high liver weights noted at 2500 and 5000 ppm, and a transient body weight loss at 5000 ppm. There were no indications in this study that test substance was immunotoxic. Similarly, data from subchronic, chronic, and reproductive toxicity studies of test substance in the rat, mouse and dog (as appropriate) did not provide any evidence of an effect of test substance on the immune system, as indicated in the Minutes and Memorandum of Understanding from the 17 Dec 2009 Meeting with the US EPA Dose Adequacy and Review Team (DART) and the sponsor (dated 12 Jan2010).

Hence, the NOEL for suppression of the immune response in female CD-1 mice was 5000 ppm (equivalent to 1356 mg/kg/day) and it was concluded that there was no evidence of immunotoxicity associated with test substance, and therefore, the need to conduct additional immunotoxicity assessments, specifically a natural killer cell (NKC) activity assay, was not identified at this time.