3-anilino-5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione;famoxadone (ISO)

Immunotoxicity

Description of key information

28-day immunotox study in rats: NOAEL = 200 ppm, systemic toxicity observed, OPPTS 870.7800, Reliability = 1

28-day immunotox study in mice: NOAEL = 350 ppm, systemic toxicity observed, small decrease in primary humoural immune response to SRBC with equivocal biological significance, OPPTS 870.7800, Reliability = 1

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

immunotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Substance name: Famoxadone Technical
Lot #: DPX-JE874-221
Purity: 97.28% by analysis

Species:

mouse

Strain:

other: Crl:CD-1®(ICR)BR

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

7 000 ppm

Remarks:

Male: 1186; Female: 1664 mg/kg/day

Dose / conc.:

2 000 ppm

Remarks:

Male: 327; Female: 417 mg/kg/day

Dose / conc.:

100 ppm

Remarks:

Male: 55; Female: 72 mg/kg/day

Dose / conc.:

50 ppm

Remarks:

Male: 8; Female: 11 mg/kg/day

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Positive control:

The known immunosuppressive agent, cyclophosphamide monohydrate, was used to validate the SRBC-specific IgM ELISA by evaluating the potential of cyclophosphamide monohydrate to suppress the primary humoral immune response to SRBC in female mice.

Statistics:

Body weights, body weight gains, food consumption, food efficiency data, and SRBC-specific serum IgM antibody log2 titer data were initially checked for normality and homogeneity of variances using the Shapiro-Wilk and Levene’s tests, respectively. For parametric data, the Dunnett’s test was used to compare the treatment groups to the control. For non-parametric data, the Dunn’s test was used to compare the treatment groups to the control. Additionally, for monotone data, a 2-sided Jonckheere’s trend test was used in a step-down manner.
Clinical sign incidence data were evaluated by the Cochran Armitage test for trend.

Organ weights (mean absolute and relative [percent of final body weight]) were analyzed by a one-way analysis of variance (ANOVA). Pairwise comparisons among test and control groups were made with the Dunnett's test.

Separate analyses were performed on the data collected for each gender. The significance level was judged at p < 0.05.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related or statistically significant differences in the incidence of clinical signs of toxicity in either males or females at any dietary concentration.

Mortality:

no mortality observed

Description (incidence):

Test substance-related mortality did not occur.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse, test substance-related effects on body weight or body weight gain at any dietary concentration of test substance in either males or females. A slight, statistically significant increase in mean body weight compared to control was observed for the 7000 ppm female group on test day 14. The increase in body weight for the 7000 ppm females was considered spurious and not test substance-related as it did not consistently occur at other study intervals. A statistically significant increase and decrease in mean body weight gain was observed during the 7-14 and 14-21 day intervals, respectively, for the 7000 ppm female group. These alterations in body weight gain for the 7000 ppm females were not consistently observed at other study intervals and therefore were considered spurious and not test substance-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no adverse, test substance-related effects on food consumption at any dietary concentration of test substance in either males or females. Food consumption values for males and females fed a diet with the test substance were similar to their respective control groups.

The mean daily intake of test substance for male mice fed diets containing 0, 50, 350, 2000, or 7000 ppm was 0, 8, 55, 327, and 1186 mg/kg/day. The mean daily intake of test substance for female mice fed diets containing 0, 50, 350, 2000, or 7000 ppm was 0, 11, 72, 417, and 1664 mg/kg/day.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse, test substance-related effects on food efficiency at any dietary concentration of famoxadone in either males or females. There was a statistically significant increase and decrease compared to control in the food efficiency of the 7000 ppm female group for the 7-14 and 14-21 day intervals, respectively, which was attributed to similar alterations in mean body weight gain at these intervals. These alterations in the food efficiency values for the 7000 ppm females were not consistently observed at other study intervals and therefore were considered spurious and not test substance-related.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no adverse, test substance-related effects on either the absolute or relative spleen and thymus weights of the males or the thymus weights of the females at any dietary concentration. Test substance did not produce test substance-related effects on the spleen weights of female mice up to the 2000 ppm dietary concentration. Consistent with these data, dietary concentrations of test substance up to 2000 ppm in an eighteen-month feeding study in mice did not alter spleen weights or the histopathology of the spleen or thymus. In the 7000 ppm female group, test substance produced statistically significant increases in mean absolute and relative (% of body weight) spleen weights. Similarly, in a 90-day feeding study with test substance, 7000 ppm produced increased spleen weights as well as altered splenic histopathology (increased spleen red pulp and hemosiderin). Additionally, mice in the 7000 ppm group in the 90-day study had mild hemolytic anemia. The splenic alterations observed in the 90-day study were considered secondary to the hemolytic anemia. Therefore, the increase in spleen weight observed in this study for the 7000 ppm female group was considered test substance-related but secondary to test substance-induced hemolytic anemia and was not considered an immunotoxic effect.

Humoral immunity examinations:

effects observed, treatment-related

Description (incidence and severity):

The dietary exposure of male mice to 7000 ppm resulted in a statistically significant decrease of 12% compared to control in the primary humoral immune response to SRBC. The biological significance of the decreased humoral immune response in the 7000 ppm male group is equivocal. The lower humoral immune response occurred only at the highest dietary concentration (>1000 mg/kg/day). The 1000 mg/kg/day dose is the limit dose for oral studies as specified by the EPA (870.3100 90-Day Oral Toxicity). Additionally, no alterations in the humoral immune response occurred in female mice nor in male and female rats in a separate 28-day feeding study with famoxadone. Furthermore, the magnitude of the altered humoral immune response was small (i.e., a 12% decrease relative to control). One can argue that the immune system has a “functional reserve capacity” that must be overcome before altered host resistance to tumors or infectious agents becomes detectable. For example, Dean et al. reported that the humoral immune response had to be decreased by approximately 40% before an increased susceptibility to influenza virus was observed. On the other hand, investigators examining cyclophosphamide found that the majority of the immune test-host resistance relationships approached a linear parameter model. Based on these data, the investigators suggested that small changes in an immune test may be associated with a change in host resistance. Nevertheless, the lower immune response in the male 7000 ppm group was considered to be indicative of minimal immunotoxicity. There were no adverse, test substance-related effects on the primary humoral immune response to SRBC at any dietary concentration of test substance in females.

A statistically significant decrease of 53% compared to control in the primary humoral immune response to SRBC was observed in female mice dosed with 90 mg/kg/day cyclophosphamide monohydrate for 5 days. Therefore, the SRBC-specific ELISA test system was valid for this feeding study with the test substance.

Reference: Dean, J.H., Thurmond, L.M., Lauer, L.D (1986). Comparative toxicology and correlative immunotoxicology in rodents. In: Burger E.J., Tardiff, R.G., Bellanti, J.A., eds. Environmental Chemical Exposures and Immune System Integrity. 85, 102. Princeton, NJ: Princeton Scientific.

Dose descriptor:

NOEL

Remarks:

for systemic toxicity

Effect level:

7 000 ppm

Based on:

test mat.

Sex:

male

Remarks on result:

other: highest dose tested

Dose descriptor:

NOEL

Remarks:

for systemic toxicity

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: increased spleen weight

Key result

Dose descriptor:

NOEL

Remarks:

for immunotoxicity

Effect level:

7 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

immunology

Remarks on result:

other: highest dose tested

Key result

Dose descriptor:

NOEL

Remarks:

for immunotoxicity

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: slightly lower primary humoural immune response to SRBC

Conclusions:

NOEL for immunotoxicity (mouse): 2000 ppm for males (327 mg/kg/day); 7000 ppm for females (1664 mg/kg/day), highest dose tested

Executive summary:

This study was conducted following US EPA guideline OPPTS 870.7800. The objective of this study was to evaluate the potential of the test substance to suppress the primary humoral immune response to sheep red blood cells (SRBC) when incorporated into nutritionally adequate diet and fed to male and female Crl:CD-1®(ICR)BR mice for at least 28 days. Five groups of 10 male mice and five groups of 10 female mice were fed either 0, 50, 350, 2000, or 7000 ppm test substance. Body weights were measured once per week and clinical signs were recorded weekly. Food consumption was determined weekly. To evaluate the primary humoral immune response, all animals were injected on test day 23 with SRBC and sacrificed on test day 28. Following sacrifice, the spleen and thymus were removed from each animal and weighed and serum was collected and analyzed for SRBC-specific IgM antibody. Sera previously collected from mice injected with SRBC and dosed with the known immunosuppressive agent cyclophosphamide monohydrate were analyzed for SRBC-specific IgM antibody concurrently with the study samples as a positive control.

The mean daily intake of test substance for male mice fed diets containing 0, 50, 350, 2000, or 7000 ppm was 0, 8, 55, 327, or 1186 mg/kg/day. The mean daily intake of test substance for female mice fed diets containing 0, 50, 350, 2000, or 7000 ppm was 0, 11, 72, 417, or 1664 mg/kg/day.

There were no test substance-related effects on body weight, body weight gain, food consumption, food efficiency, clinical signs of toxicity, or mortality at any dosage of test substance in either males or females.

Test substance did not produce test substance-related effects on the spleen weights of male mice or the thymus weights of male and female mice. The test substance did not produce test substance-related effects on the spleen weights of female mice up to the 2000 ppm dietary concentration. A test substance-related increase in spleen weight for female mice fed 7000 ppm test substance in the diet was observed but was considered secondary to test substance-induced hemolytic anemia, based on findings from hematology evaluations in a previous study. The dietary exposure of male mice to 7000 ppm test substance resulted in a statistically significant lower primary humoral immune response to SRBC. Although the biological significance of this lower humoral immune response was equivocal, this effect was considered indicative of minimal immunotoxicity. The test substance did not suppress the primary humoral immune response to SRBC of female mice at any dietary concentration.

The no-observed-effect level (NOEL) in this study for systemic toxicity, other than immunotoxicity, for male mice was 7000 ppm (1186 mg/kg/day), the highest dietary concentration tested, while the NOEL for female mice was 2000 ppm (417 mg/kg/day) based on an increase in spleen weight for female mice fed 7000 ppm test substance.

The NOEL for immunotoxicology parameters in this study for male mice was 2000 ppm (327 mg/kg/day) test substance based on a lower primary humoral immune response to SRBC for males fed a diet containing 7000 ppm test substance. The NOEL for immunotoxicology parameters for female mice was 7000 ppm (1664 mg/kg/day) test substance, the highest dietary concentration tested.

Reference 2

Endpoint:

immunotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Substance name: Famoxadone Technical
Lot #: DPX-JE874-221
Purity: 97.28% by analysis

Species:

rat

Strain:

other: Crl:CD®(SD)IGS BR

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

800 ppm

Remarks:

Male: 55; Female: 57 mg/kg/day

Dose / conc.:

200 ppm

Remarks:

Male: 14; Female: 16 mg/kg/day

Dose / conc.:

100 ppm

Remarks:

Male: 7; Female: 8 mg/kg/day

Dose / conc.:

50 ppm

Remarks:

Male/Female: 4 mg/kg/day

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Positive control:

The known immunosuppressive agent, cyclophosphamide monohydrate, was used to validate the SRBC-specific IgM ELISA by evaluating the potential of cyclophosphamide monohydrate to suppress the primary humoral immune response to SRBC in male and female rats.

Statistics:

Body weights, body weight gains, food consumption, food efficiency data, and SRBC-specific serum IgM antibody log2 titer data were initially checked for normality and homogeneity of variances using the Shapiro-Wilk and Levene’s tests, respectively. For parametric data, the Dunnett’s test was used to compare the treatment groups to the control. For non-parametric data, the Dunn’s test was used to compare the treatment groups to the control. Additionally, for monotone data, a 2-sided Jonckheere’s trend test was used in a step-down manner.
Clinical sign incidence data were evaluated by the Cochran Armitage test for trend.

Organ weights (mean absolute and relative [percent of final body weight]) were analyzed by a one-way analysis of variance (ANOVA). Pairwise comparisons among test and control groups were made with the Dunnett's test.

Separate analyses were performed on the data collected for each gender. The significance level was judged at p < 0.05.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related or statistically significant differences in the incidence of clinical signs of toxicity in either males or females at any dietary concentration.

Mortality:

no mortality observed

Description (incidence):

All animals survived the duration of the treatment period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant low mean body weight when compared to controls occurred in the male and female groups fed 800 ppm famoxadone in the diet. For males, statistically significant decreases compared to control of 6, 9, 11, and 10% were observed for test days 7, 14, 21, and 28, respectively. The 800 ppm female group had statistically significant decreases compared to control of 7, 10, 11, and 13% in mean body weight for test days 7, 14, 21, and 28, respectively.

Statistically significant low mean body weight gain when compared to controls occurred in the male and female groups fed 800 ppm test substance in the diet. Males fed an 800 ppm diet had statistically significant low mean body weight gain compared to control for the 0-7, 7-14, and 14-21 day intervals, respectively. A statistically significant lower body weight gain of 22% compared to control was observed for the 100 ppm male group for the 14-21 day interval. The lower mean body weight gain that occurred for the 100 ppm male group was not observed in the presence of a dose-response and therefore, was not considered test substance-related. A statistically significant lower cumulative body weight gain of 24% compared to control was observed over the complete study interval of 0-28 days for the 800 ppm male group. Females fed an 800 ppm diet had statistically significant low mean body weight gains compared to control of 61 and 50%, respectively, for the 0-7 and 7-14 day intervals, respectively. During the first 2 weeks of the study, female weight gains were approximately half of the gains in control rats. A statistically significant lower cumulative body weight gain of 49% compared to control was observed over the complete study interval of 0-28 days for the 800 ppm female group. The dietary administration of 800 ppm famoxadone produced lower mean body weight and body weight gain in both male and female rats that are considered test substance-related and toxicologically adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

With the exception of the 800 ppm test substance male and female dose groups, the mean daily food consumption observed in test substance-treated groups was comparable to controls. For the 800 ppm male group, statistically significant low food consumption was observed for the 0-7, 7-14, and 14-21 day intervals. Over the course of the study, a statistically significant lower mean daily food consumption of 13% compared to control occurred in the 800 ppm male group. For the 800 ppm female group, statistically significant lower mean daily food consumption was observed for all weekly intervals resulting in a statistically significant lower mean daily food consumption of 18% compared to control over the course on the study.

The mean daily intake of test substance for male rats fed diets containing 0, 50, 100, 200, or 800 ppm was 0, 4, 7, 14, and 55 mg/kg/day. The mean daily intake of famoxadone for female rats fed diets containing 0, 50, 100, 200, or 800 ppm was 0, 4, 8, 16, and 57 mg/kg/day.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Male rats fed an 800 ppm diet had statistically significant decreases compared to control of 14 and 13%, respectively, in mean daily food efficiency for the 0-7 and 14-21 day intervals. For the overall study interval 0-28, a statistically significant lower food efficiency of 13% occurred for the 800 ppm male group. For the 14- to 21-day interval, statistically significant lower efficiency was observed for the 100 ppm male group. No dose response was present and the lower efficiency was therefore not considered test substance-related. Female rats fed an 800 ppm diet had statistically significant decreases compared control of 47 and 41%, respectively, in mean daily food efficiency for the 0-7 and 7-14 day intervals, respectively. For the overall interval 0-28, a statistically significant lower food efficiency of 12 and 38% occurred for the 200 and 800 ppm female groups, respectively. The 800 ppm food consumption and food efficiency data suggest that part of the body weight effects may reflect reduced food consumption possibly due to poor palatability. The lower food consumption and food efficiency of the 800 ppm male and female dose groups are consistent with their lower body weight and weight gain, and are therefore considered to be test substance-related and toxicologically adverse. The low food efficiency of the 200 ppm female dose group did not correspond with significant effects on body weight and body weight gain and therefore was not considered toxicologically adverse.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Male rats fed an 800 ppm diet had statistically significant increases in mean absolute and relative spleen weights, when compared to the male controls. Females in the 800 ppm group had a statistically significant increase in relative spleen weight. Similarly, in a 90-day feeding study with test substance, 800 ppm produced a significant increase in absolute and relative spleen weights as well as alterations in splenic histopathology of both males and females. In the 90-day study, both male and female rats in the 800 ppm group had mild hemolytic anemia with statistically significant decreases in all relevant indicators of circulating red blood cell mass (red blood cell count, hematocrit, hemoglobin). The increased spleen weights and altered splenic histopathology (i.e., increases in hemosiderin and extramedullary hematopoesis) were consistent with and considered secondary to the hemolytic anemia. Therefore, the increase in spleen weight observed in this study for the 800 ppm male and female test group was considered test substance related but secondary to the test substance-induced hemolytic anemia, and not considered an immunotoxic effect. Thymus weight parameters were similar to controls for all test groups. In previous studies, doses up to 1600 ppm and 200 ppm test substance, respectively, in a 90-day and two-year feeding study did not alter thymic histopathology in males or females.

Humoral immunity examinations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences in the primary humoral immune response to SRBC for male and female rats at any dietary concentration of test substance. Nonstatistically significant higher values in the primary humoral immune response occurred for both male and female groups at all dietary concentrations. The increases in the primary humoral immune response of the males and females did not exhibit a dose-response and therefore were not considered to be test substance-related. The dietary administration of test substance did not suppress the primary humoral immune response to SRBC for either male or female rats.

A statistically significant decrease compared to control of 62 and 60%, respectively, in the primary humoral immune response to SRBC was observed for male and female rats dosed with 20 mg/kg/day cyclophosphamide monohydrate for 6 days. Additionally, for the pooled positive control sera, a 59 and 56% decrease, respectively, in the primary humoral immune response to SRBC was observed for males and females. Therefore, the SRBC-specific ELISA test system was valid for this feeding study with test substance.

Dose descriptor:

NOEL

Remarks:

for systemic toxicity

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

food efficiency

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOEL

Remarks:

for immunotoxicity

Effect level:

800 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

immunology

Remarks on result:

other: highest dose tested

Conclusions:

NOEL for immunotoxicity (Rat): 800 ppm (55 mg/kg/day for males and 57 mg/kg/day for females), highest dose tested

Executive summary:

This study was conducted following US EPA guideline OPPTS 870.7800. The objective of this study was to evaluate the potential of test substance to suppress the primary humoral immune response to sheep red blood cells (SRBC) when incorporated into nutritionally adequate diet and fed to male and female Crl:CD^®(SD)IGS BR rats for at least 28 days. Five groups of 10 male and 5 groups of 10 female rats were fed either 0, 0, 50, 100, 200, or 800 ppm famoxadone. Body weights were measured once per week and clinical signs were recorded at least once weekly. Food consumption was determined weekly. To evaluate the primary humoral immune response, all animals were injected on test day 22 with SRBC and sacrificed on test day 28. Following sacrifice, serum was collected and analyzed for SRBC-specific IgM antibody, and the spleen and thymus were removed from each animal and weighed. Sera previously collected from rats injected with SRBC and dosed with the known immunosuppressive agent cyclophosphamide monohydrate were analyzed for SRBC-specific IgM antibody concurrently with the study samples as a positive control.

The mean daily intake of test substance for male rats fed diets containing 0, 50, 100, 200, or 800 ppm was 0, 4, 7, 14, and 55 mg/kg/day. The mean daily intake of test substance for female rats fed diets containing 0, 50, 100, 200, or 800 ppm was 0, 4, 8, 16, and 57 mg/kg/day.

The dietary administration of 800 ppm test substance produced lower body weight, body weight gain, food consumption and food efficiency in both male and female rats that are considered test substance-related and toxicologically adverse.

A test substance-related increase in spleen weight for both male and female rats fed 800 ppm in the diet was observed but was considered secondary to test substance-induced hemolytic anemia, based on findings from hematology evaluations in previous studies. The dietary administration of test substance did not suppress the primary humoral immune response to SRBC for either male or female rats fed any dietary concentration.

The no-observed-effect level (NOEL) for systemic toxicity, other than immunotoxicity, in this study for males and females was 200 ppm (14 mg/kg/day for males and 16 mg/kg/day for females) based on the decreases in body weight parameters, food consumption, food efficiency, and the increase in spleen weight observed in rats fed 800 ppm test substance diets.

The NOEL for immunotoxicology endpoints in this study for both male and female rats was 800 ppm (55 mg/kg/day for males and 57 mg/kg/day for females) test substance, the highest dietary concentration tested.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Quality of whole database:

Guideline immunotoxicity studies were available in rats and mice.

Justification for classification or non-classification

A small decrease in the primary humoural immune response to SRBC was observed in the 7000 ppm male mice, and although concluded as potentially indicative of minimal immunotoxicity, the biological significance was considered equivocal. No immunotoxic effects were observed in female mice or male and female rats.  Therefore, the test substance is not classified for immunotoxiciy according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.