cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Immunotoxicity

Description of key information

- Oral: No adverse effects on the immune system observed, female, mice, sub-acute, EPA 870.7800, Wasil 2012

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:

supporting study

Study period:

3 Jan 2012 to 13 Feb 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

mouse

Strain:

CD-1

Remarks:

Crl:CD1(ICR)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: were approximately 7 weeks old
- Weight at randomization: 20.5 to 26.7 g
- Housing: housed individually in clean, stainless steel, wire mesh cages suspended above cage board
- Diet: Certified Rodent LabDiet® 5002 (meal), ad libitum
- Water: Reverse osmosis-treated (on-site) drinking water, ad libitum
- Acclimation period: 13-day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean daily temperature ranged from 21.3 to 21.4 °C
- Humidity (%): mean daily relative humidity ranged from 34.8% to 38.9%
- Air changes (per hr): minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 Day

Frequency of treatment:

continuously

Dose / conc.:

10 ppm

Remarks:

Group 2. Dietary equivalent to 2.1 mg/kg of body weight/day.

Dose / conc.:

30 ppm

Remarks:

Group 3. Dietary equivalent to 7.0 mg/kg of body weight/day.

Dose / conc.:

100 ppm

Remarks:

Group 4. Dietary equivalent to 23.0 mg/kg of body weight/day.

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Positive control:

Cyclophosphamide (CPS) at a concentration of 5 mg/mL: Group 5.

Dose descriptor:

NOAEL

Remarks:

humoral immune response (AFC Assay)

Effect level:

100 ppm

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

30 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

100 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Analyses of Diet

Prior to dosing, test diet homogeneity was established at 8 and 120 ppm based on the protocol-specified acceptability limits (90% to 110% of the target concentration and had a relative standard deviation [RSD] ≤ 5%). In addition, the 8 and 120 ppm test diet admix formulations were found to be stable for up to 15 days of room temperature storage (≥90% of initial value). The analyzed diet admix formulations that were administered to the animals were found to contain 97.4% to 105% of the test substance, which were within the protocol-specified range (90% to 110% of target concentration and had an RSD ≤ 5%), except for the 10 ppm test diets prepared on 2 days which had RSD values of 9.1% and 5.9%, respectively, which were above the protocol-specified acceptability limits. However, these test diets were used for dose administration. Administration of the out-of-specification 10 ppm dietary admix formulations did not impact the integrity or interpretation of the study as the values were within the SOP range (RSD ≤ 10%). The test substance was not detected in the basal diet that was administered to the vehicle and positive control groups (Groups 1 and 5, respectively).

 

Survival

All animals survived to the scheduled necropsy.

 

Clinical Observations

There were no test item-related clinical observations. All clinical findings in the test item-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

Body weights were unaffected by administration of the test item in the diet. Mean body weight tended to be numerically lower than the vehicle control group values in the 100 ppm group after study day 10 of the study, but a clear relationship to treatment with statistical significance was not apparent. Occasional statistically significant differences in body weight gains were probably due to biological variability and were not considered related to test substance administration due to lack of a clear dose-response trend and/or the changes were in a direction not considered toxicologically relevant.

 

Food and Test Substance Consumption

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

 

Macroscopic examination

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

 

Organ weights

Test item-related higher liver weights were noted in the 100 ppm group. Higher mean absolute, relative (to terminal body weight), and adjusted mean liver weights were noted in the 100 ppm group when compared to the vehicle control group, and the adjusted values were statistically significant. Mean adjusted liver weights in the 100 ppm group were 20.5% higher than the vehicle control group. Based on the magnitude of the increase in liver weights and the lack of histopathological evaluation, this change was considered adverse. There were no treatment-related effects of the test item on spleen or thymus weights. In the positive control group (CPS), absolute and adjusted mean spleen and thymus weights were statistically significantly lower than the vehicle control group values.

 

AFC Assay

There were no test item-related effects on spleen cell numbers, specific activity, or total spleen activity at any dietary concentration evaluated. As expected, statistically significantly lower spleen cell numbers, specific activity, and total spleen activity were noted in the positive control (CPS) group when compared to the vehicle control group, which validated the functionality of the assay.

 

Natural Killer Cell (NKC) Assay

Based on the lack of indications for immunotoxicity, there was no need to conduct an NKC assay with the test item.

Conclusions:

The administration of the test item in the diet for 28 consecutive days to female Crl:CD1(ICR) mice at dose levels of 10, 30, and 100 ppm resulted in a no-observed-effect-level (NOEL) for the AFC assay (humoral immune response) of 100 ppm (equivalent to 23.0 mg/kg of body weight/day), the highest dose level evaluated. The no-observed-adverse-effect level (NOAEL) for this study, based on the increased liver weights observed in the 100 ppm dose group, was 30 ppm (equivalent to 7.0 mg/kg of body weight/day).

Executive summary:

The test substance, was offered ad libitum in the diet for 28 consecutive days to female Crl:CD1(ICR) mice in Groups 2, 3, and 4 at dietary concentrations of 10, 30, and 100 ppm, respectively. The concurrent control group (Group 1) and the positive control group (Group 5) were offered the basal diet on a comparable regimen to the test item-treated groups. All mice (Groups 1-5) were immunized with an intravenous injection of sheep red blood cells (sRBC) on study day 24. Mice in the positive control group (Group 5) were administered the positive control substance, cyclophosphamide (CPS), via intraperitoneal injection (50 mg/kg/day) once daily for 4 consecutive days (study days 24 through 27). Each group consisted of 10 mice/group. 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 approximately weekly and on the day of the scheduled necropsy. Individual body weights and food consumption were recorded approximately twice weekly. Complete necropsies were conducted on all animals. The liver, mesenteric lymph node, Peyer’s patches, spleen, and thymus were collected at the scheduled necropsy, and the liver, spleen, and thymus were weighed. Spleens were placed in Earle’s Balanced Salt Solution (EBSS)/HEPES buffer, spleen cell suspensions were prepared, spleen cell counts were performed, and the number of specific IgM antibody-forming cells directed towards the sRBC antigen were determined to measure the humoral immune response using the splenic Antibody-Forming Cell (AFC) assay.

All animals survived to the scheduled necropsy. There were no test item-related clinical observations, macroscopic findings, or effects on body weights or food consumption. There were no test item-related effects on spleen cell numbers and the test item did not significantly suppress the humoral immune response when evaluated as either specific activity (AFC/10⁶ spleen cells) or as total activity (AFC/spleen) of splenic IgM to the T-cell dependent antigen sRBC. Adverse, test item-related higher liver weights were noted in the 100 ppm group. For the positive control group, CPS, statistically significantly lower spleen and thymus weights, spleen cell numbers, specific activity, and total spleen activity of IgM antibody-forming cells were noted when compared to the vehicle control group. These effects were consistent with the known immunosuppressant effects of CPS and validated the functionality of the assay.

Based upon the results of this study, the test item administered ad libitum in the diet for 28 consecutive days to female Crl:CD1(ICR) mice at dose levels of 10, 30, and 100 ppm resulted in a no-observed-effect-level (NOEL) for the AFC assay (humoral immune response) of 100 ppm (equivalent to 23.0 mg/kg of body weight/day), the highest dose level evaluated. The no-observed-adverse-effect level (NOAEL) for this study, based on the increased liver weights observed in the 100 ppm dose group, was 30 ppm (equivalent to 7.0 mg/kg of body weight/day).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

7 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

Study performed under GLP and in accordance with EPA 870.7800 Guideline

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 substance was administered in the diet for 28 consecutive days to female mice at dose levels of 0, 10, 30 and 100 ppm (equivalent to 0, 2.1, 7.0 and 23.0 mg/kg bw/day) resulted in and increased absolute and relative liver weight at 100 ppm and higher. No effects on the immune system were found. The NOEL for the AFC assay (humoral immune response) of 100 ppm (equivalent to 23.0 mg/kg bw/day), the highest dose level evaluated. The no-observed-adverse-effect level (NOAEL) for this study, based on the increased liver weights observed in the 100 ppm dose group, was 30 ppm (equivalent to 7.0 mg/kg bw/day)

Justification for classification or non-classification

Based on the available information, classification for effects on the immune system upon repeated exposure is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC)1272/2008.