Prometryn

Immunotoxicity

Description of key information

- Oral: NOEL = 2000 ppm (equivalent to 413.1 mg/kg bw/day), no adverse effects on the immune system observed, female, mice, sub-acute, EPA OPPTS 870.78000, 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:

key study

Study period:

Initiation: 11 August 2011, end: 6 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Version / remarks:

1998

Deviations:

no

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:

Source: Charles River Laboratories Inc., Portage, Michigan
Age: 38 days old at receipt, about 7 weeks at initiation of exposure
Weight: 20.8 to 26.1 g
Acclimation: 13 days
Housing: individually in stainless steel, wire mesh cages suspended above cardboard
Diet: Certified Rodent LabDiet 5002 (meal) basal diet supplied by PMI Nutrition International LLC ad libitum
Water: reverse-osmosis treated tap water
Temperature: 19.1 ºC to 22.2 ºC
Humidity: 41.1% to 58.9%
Lighting: 12 hours light to 12 hours darkness cycles
Air change: minimum 10 air changes per hour

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

The vehicle phosphate buffered saline, pH 7.4 in sterile water for injection was used for the positive control substance.

Details on exposure:

The test substances was mixed with an appropriate amount of basal diet to produce admixtures containing the appropriate dietary test concentrations. The basal and test diet admix formulations were offered ad libitum for 28 consecutive days (beginning on study day 0 and continuing until the time of the scheduled necropsy).
The Group 5 mice were administered the positive control substance, CPS, via intraperitoneal injection once daily on study days 24 through 27 at a dose level of 50 mg/kg/day and a dose volume of 10 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses to determine the homogeneity and stability of the test substance in the diet at 400 and 6000 ppm were conducted prior to the dosing phase of the study using test substance diets prepared solely for homogeneity and stability testing. All analyses were conducted using a validated gas chromatography method using flame ionization detection. The analyzed diet admix formulations that were administered to the animals were found to contain 91.5% to 94.8% of the test substance which were within the protocol-specified acceptability limits (90% to 110% of target concentration and RSD ≤5%).

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily, continuous administration via animal feed

Dose / conc.:

500 ppm

Dose / conc.:

2 000 ppm

Dose / conc.:

5 000 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

Based on the review of all appropriate pretest data animals judged suitable for assignment to the study were selected. Using a computerized program the females were randomized based on body weight stratification in a block design. A printout containing the animal numbers and individual group assignments was generated. Individual body weights at randomization were within ± 20% of the mean.
All mice were immunized via an intravenous lateral tail vein injection using an appropriately-sized syringe and needle with 0.2 mL of 1 x 108 sRBC (prepared in EBSS with HEPES) on study day 24. The Group 5 mice were administered the positive control substance, CPS, via intraperitoneal injection once daily on study days 24 through 27 at a dose level of 50 mg/kg/day and a dose volume of 10 mL/kg/day. Individual mouse positive control doses were based on the most recently recorded body weights (study day 24) to provide the correct mg/kg/day dose. The sRBC immunizations were administered prior to positive control (CPS) dose administration on study day 24.

Observations and clinical examinations performed and frequency:

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed once daily for all animals. Detailed physical examinations were conducted on all animals about weekly. Individual body weights were recorded approximately twice weekly. Food intake was calculated for each interval.

Sacrifice and pathology:

All surviving animals were sacrificed by inhalation of carbon dioxide after an exposure period of 28 days and subject to a detailed necropsy. Blood samples were collected at the time of euthanasia via the inferior vena cava. The necropsies included, but were not limited to, examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities, including viscera. Specific tissues were collected from all animals and placed in 10% neutral, buffered formalin, including bone marrow smear, liver, lymph node (mesenteric), Peyer's patches, spleen, thymus.

Cell viabilities:

Viability of splenocytes was determined using propidium iodide and a XL-MCL Flow Cytometer.

Humoral immunity examinations:

Spleen IgM Antibody-Forming Cell (AFC) Response to the T-cell Dependent Antigen, sRBC; Day 4 Response: The primary IgM response to sheep erythrocytes was measured using a modification of the original hemolytic plaque assay of (Jerne and Nordin, 1963; Jerne et al., 1974; White et al., 2010). After isolation and resuspension of the spleen cells, 1:30 and 1:120 dilutions were prepared. A 0.1 mL aliquot of spleen cells from the 1:30 and 1:120 suspensions was added to separate test tubes, each containing 25 μL of guinea pig complement, 25 μL of sRBC, and 0.5 mL of warm agar (0.5%). After
thoroughly mixing, each test tube mixture was plated onto a separate petri dish, covered with a microscope cover slip, and incubated at approximately 36-38 °C for 3 hours.

Positive control:

Cyclophospamide

Statistics:

See any other information on materials and methods

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical findings in the treated groups were noted with similar incidence in the vehicle 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.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly lower mean body weight gain was noted in the 5000 ppm group on study days 0 to 3 compared to the vehicle control group, and was attributed to treatment. Mean body weight gains were generally similar to the vehicle control group for the remainder of the study. Although mean body weights for the 5000 ppm group remained slightly lower throughout the study due to the initial lower body weight gain, values were not
statistically significant compared to the vehicle control group, and mean body weights were only 3.7% lower on study day 28.

Food consumption and compound intake (if feeding study):

no effects observed

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant effects on spleen cell number at any dose level of prometryn. In the functional evaluation of the IgM AFC response, treatment with prometryn did not result in statistically significant effects on the humoral immune response when evaluated as either specific activity (AFC/106 spleen cells) or as total spleen activity (AFC/spleen) at 500 and 2000 ppm; however, there were statistically significant decreases in specific activity and total spleen activity in the 5000 ppm group compared to the vehicle control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All macroscopic findings noted were considered to be spontaneous and/or incidental in nature and unrelated to test substance administration.

Cell viabilities:

no effects observed

Humoral immunity examinations:

effects observed, treatment-related

Description (incidence and severity):

Exposure of mice to the 500 ppm or 2000 ppm in the diet did not result in any statistically significant effects when evaluated as either Specific Activity (left panel) or Total Spleen Activity (right panel). At 5000 ppm a statistically significant decrease was observed in both Specific Activity (45%) and in Total Spleen Activity (46%) as compared to the vehicle control animals.

Key result

Dose descriptor:

NOEL

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

immunology

Remarks on result:

other: corresponding to 413.1 mg/kg bw/day

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

5 000 ppm

System:

immune system

Organ:

spleen

Results with positive control: As expected, a statistically significant decreases in spleen cell numbers, specific activity, and total spleen activity were noted in the positive control (CPS) group when compared to the vehicle control group.

Conclusions:

Administration of the substance to female mice in the diet for 28 consecutive days resulted in a reduced humoral immune response at a dose of 5000 ppm, based on lower specific activity and total spleen activity.

Executive summary:

The potential immunotoxic effects of the substance when administered orally via the diet to female CD-1 for 28 consecutive days were studied under GLP to EPA OPPTS guideline 870.7800. The test substance was mixed into basal animal feed at concentrations of 500, 2000 or 5000 ppm, and admixtures were offered ad libitum to groups of ten test animals per test concentration over the course of the study period. The concurrent negative control group of ten female mice and the positive control group of ten female mice received plain diet. All mice were immunised with an intravenous injection of sheep red blood cells (sRBC) on study day 24. Mice in the positive control group were administered cyclophosphamide via intraperitoneal injection (at 50 mg/kg bw/day) once daily for four consecutive days from day 24 through day 27. All animals were euthanised on study day 28 by inhalation of carbon dioxide. The concentrations of test substance in feed admixtures were analysed by a validated gas chromatography method and representative samples taken over the course of the study were confirmed to contain 91.5% to 94.8% of the test substance which were within the protocol-specified acceptability limits (90% to 110% of target concentration and RSD ≤5%). Mean achieved doses in the treatment groups were 105.8, 413.1 and 1044.5 mg/kg bw/day. All animals survived to the scheduled necropsy. There were no substance related clinical observations, macroscopic findings or effects on food consumption or organ weights. The body weight gain at 5000 ppm was lower from study days 0 to 3. Body weights at 5000 ppm were generally similar to those in the negative control group from study day 7 to the end of the study. There was no significant effect on spleen cell number. However, the substance did significantly suppress the humoral immune response at 5000 ppm when evaluated as specific activity (AFC/10^6 spleen cells) and total activity (AFC/spleen) of splenic IgM to the T-cell dependent antigen sRBC, which was overall considered to be a non-adverse effect. The positive control substance cyclophosphamide caused statistically significant decreases in spleen cell numbers, specific activity, and total spleen activity of IgM antibody-forming cells, validating the functionality of the assay. Based on the reduced humoral immune response at the highest dose of 5000 ppm (corresponding to a mean dose of 1044.5 mg/kg bw/day), the NOEL for the humoral immune response was 2000 ppm (equivalent to 413.1 mg/kg bw/day).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 044.5 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

One guideline-compliant GLP study on immunotoxicity available

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

The test substance was administered in the diet for 28 consecutive days to female mice at dose levels of 0, 500, 2000, and 5000 ppm. The exposure resulted in a transient (day 1 to 3) decrease in body weight gain and reduced humoral immune response based on lower specific activity and total spleen activity at 5000 ppm. The NOEL was 2000 ppm (dietary equivalent to 413.1 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.