§799.9780. TSCA immunotoxicity. — Inbound Citations

(1) This screening test provides limited information on systemic toxicity, neurotoxicity, and/or immunotoxicity following repeated exposure over a limited time period. In addition, it can be used to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition. It is not an alternative to, nor does it replace, the existing test guidelines in §§ 799.9370, 799.9380, 799.9620, and 799.9780 of this part.

(iii) If the test substance is administered dermally, use paragraphs (f)(5)(iii)(A) through (f)(5)(iii)(D) of this section.

(A) (1) In this test, it is desirable to determine a dose-response relationship as well as a NOEL. Therefore, at least three dose levels plus a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest dose level) group should be used. Doses should be spaced appropriately to produce test groups with a range of toxic effects. The data should be sufficient to produce a dose-response curve.

(2) The highest dose level should elicit signs of toxicity but not produce severe skin irritation or an incidence of fatality which would prevent a meaningful evaluation. If application of the test substance produces severe skin irritation, the concentration may be reduced, although this may result in a reduction in, or absence of, other toxic effects at the high dose level. If the skin has been badly damaged early in the study, it may be necessary to terminate the study and undertake a new one at lower concentrations.

(3) The intermediate dose levels should be spaced to produce a gradation of toxic effects.

(4) The lowest dose level should not produce any evidence of toxic effects.

(B) Shortly before testing, fur should be clipped from not less than 10% of the body surface area for application of the test substance. In order to dose approximately 10% of the body surface, the area starting at the scapulae (shoulders) to the wing of the ileum (hipbone) and half-way down the flank on each side of the animal should be shaved. Shaving should be carried out approximately 24 hrs before dosing. Repeated clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care should be taken to avoid abrading the skin which could alter its permeability.

(C) (1) Liquid test substances are generally used undiluted, except as indicated in paragraph (f)(5)(iii)(A)(2) of this section.

(2) Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account.

(3) The volume of application should be kept constant, e.g. less than 300 <greek-m≤L for the rat; different concentrations of test solution should be prepared for different dose levels.

(D) (1) The duration of exposure should be at least for 90 days.

(2) The animals should be treated with test substance for at least 6 hrs/day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day.

(3) The test substance should be applied uniformly over the treatment site.

(4) The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible.

(5) During the exposure period, the test substance should be held in contact with the skin with a porous gauze dressing. The test site should be further covered with nonirritating tape to retain the gauze dressing and the test substance and to ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance, but complete immobilization is not recommended.

(iii) If the test substance is administered dermally, use paragraphs (f)(5)(iii)(A) through (f)(5)(iii)(D) of this section.

(A) (1) In this test, it is desirable to determine a dose-response relationship as well as a NOEL. Therefore, at least three dose levels plus a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest dose level) group should be used. Doses should be spaced appropriately to produce test groups with a range of toxic effects. The data should be sufficient to produce a dose-response curve.

(2) The highest dose level should elicit signs of toxicity but not produce severe skin irritation or an incidence of fatality which would prevent a meaningful evaluation. If application of the test substance produces severe skin irritation, the concentration may be reduced, although this may result in a reduction in, or absence of, other toxic effects at the high dose level. If the skin has been badly damaged early in the study, it may be necessary to terminate the study and undertake a new one at lower concentrations.

(3) The intermediate dose levels should be spaced to produce a gradation of toxic effects.

(4) The lowest dose level should not produce any evidence of toxic effects.

(B) Shortly before testing, fur should be clipped from not less than 10% of the body surface area for application of the test substance. In order to dose approximately 10% of the body surface, the area starting at the scapulae (shoulders) to the wing of the ileum (hipbone) and half-way down the flank on each side of the animal should be shaved. Shaving should be carried out approximately 24 hrs before dosing. Repeated clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care should be taken to avoid abrading the skin which could alter its permeability.

(C) (1) Liquid test substances are generally used undiluted, except as indicated in paragraph (f)(5)(iii)(A)(2) of this section.

(2) Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account.

(3) The volume of application should be kept constant, e.g. less than 300 <greek-m≤L for the rat; different concentrations of test solution should be prepared for different dose levels.

(D) (1) The duration of exposure should be at least for 90 days.

(2) The animals should be treated with test substance for at least 6 hrs/day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day.

(3) The test substance should be applied uniformly over the treatment site.

(4) The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible.

(5) During the exposure period, the test substance should be held in contact with the skin with a porous gauze dressing. The test site should be further covered with nonirritating tape to retain the gauze dressing and the test substance and to ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance, but complete immobilization is not recommended.

(iii) If the test substance is administered dermally, use paragraphs (f)(5)(iii)(A) through (f)(5)(iii)(D) of this section.

(A) (1) In this test, it is desirable to determine a dose-response relationship as well as a NOEL. Therefore, at least three dose levels plus a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest dose level) group should be used. Doses should be spaced appropriately to produce test groups with a range of toxic effects. The data should be sufficient to produce a dose-response curve.

(2) The highest dose level should elicit signs of toxicity but not produce severe skin irritation or an incidence of fatality which would prevent a meaningful evaluation. If application of the test substance produces severe skin irritation, the concentration may be reduced, although this may result in a reduction in, or absence of, other toxic effects at the high dose level. If the skin has been badly damaged early in the study, it may be necessary to terminate the study and undertake a new one at lower concentrations.

(3) The intermediate dose levels should be spaced to produce a gradation of toxic effects.

(4) The lowest dose level should not produce any evidence of toxic effects.

(B) Shortly before testing, fur should be clipped from not less than 10% of the body surface area for application of the test substance. In order to dose approximately 10% of the body surface, the area starting at the scapulae (shoulders) to the wing of the ileum (hipbone) and half-way down the flank on each side of the animal should be shaved. Shaving should be carried out approximately 24 hrs before dosing. Repeated clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care should be taken to avoid abrading the skin which could alter its permeability.

(C) (1) Liquid test substances are generally used undiluted, except as indicated in paragraph (f)(5)(iii)(A)(2) of this section.

(2) Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account.

(3) The volume of application should be kept constant, e.g. less than 300 <greek-m≤L for the rat; different concentrations of test solution should be prepared for different dose levels.

(D) (1) The duration of exposure should be at least for 90 days.

(2) The animals should be treated with test substance for at least 6 hrs/day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day.

(3) The test substance should be applied uniformly over the treatment site.

(4) The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible.

(5) During the exposure period, the test substance should be held in contact with the skin with a porous gauze dressing. The test site should be further covered with nonirritating tape to retain the gauze dressing and the test substance and to ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance, but complete immobilization is not recommended.

(iii) If the test substance is administered dermally, use paragraphs (f)(5)(iii)(A) through (f)(5)(iii)(D) of this section.

(A) (1) In this test, it is desirable to determine a dose-response relationship as well as a NOEL. Therefore, at least three dose levels plus a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest dose level) group should be used. Doses should be spaced appropriately to produce test groups with a range of toxic effects. The data should be sufficient to produce a dose-response curve.

(2) The highest dose level should elicit signs of toxicity but not produce severe skin irritation or an incidence of fatality which would prevent a meaningful evaluation. If application of the test substance produces severe skin irritation, the concentration may be reduced, although this may result in a reduction in, or absence of, other toxic effects at the high dose level. If the skin has been badly damaged early in the study, it may be necessary to terminate the study and undertake a new one at lower concentrations.

(3) The intermediate dose levels should be spaced to produce a gradation of toxic effects.

(4) The lowest dose level should not produce any evidence of toxic effects.

(B) Shortly before testing, fur should be clipped from not less than 10% of the body surface area for application of the test substance. In order to dose approximately 10% of the body surface, the area starting at the scapulae (shoulders) to the wing of the ileum (hipbone) and half-way down the flank on each side of the animal should be shaved. Shaving should be carried out approximately 24 hrs before dosing. Repeated clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care should be taken to avoid abrading the skin which could alter its permeability.

(C) (1) Liquid test substances are generally used undiluted, except as indicated in paragraph (f)(5)(iii)(A)(2) of this section.

(2) Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account.

(3) The volume of application should be kept constant, e.g. less than 300 <greek-m≤L for the rat; different concentrations of test solution should be prepared for different dose levels.

(D) (1) The duration of exposure should be at least for 90 days.

(2) The animals should be treated with test substance for at least 6 hrs/day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day.

(3) The test substance should be applied uniformly over the treatment site.

(4) The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible.

(5) During the exposure period, the test substance should be held in contact with the skin with a porous gauze dressing. The test site should be further covered with nonirritating tape to retain the gauze dressing and the test substance and to ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance, but complete immobilization is not recommended.

(i) If the antibody PFC assay is performed, the criteria listed under paragraphs (g)(1)(i)(A) through (g)(1)(i)(F) of this section shall be adhered to. Assays described in the references under paragraphs (j)(2) and (j)(4) of this section may be used.

(A) The T cell-dependent antigen, SRBC, shall be injected intravenously or intraperitoneally, usually at 24 days after the first dosing with the test substance. 5 Although the optimum response time is usually 4 days after immunization, some strains of test animal may deviate from this time point. The strain to be used shall be evaluated for the optimum day for PFC formation after immunization.

(B) The activity of each new batch of complement shall be determined. For any given study, the SRBCs shall be from a single sheep, or pool of sheep, for which the shelf life and dose for optimum response has been determined.

(C) Modifications of the PFC assay described in paragraph (g)(1)(i) of this section exist and may prove useful; however, the complete citation shall be made for the method used, any modifications to the method shall be reported, and the source and, where appropriate, the activity or purity of important reagents shall be given. Justification or rationale shall be provided for each protocol modification. Discussions of modifications of the PFC assay are available in the references under paragraphs (j)(5),(j)(6), and (j)(10) of this section

(D) Samples shall be randomized and shall be coded for PFC analysis, so that the analyst is unaware of the treatment group of each sample examined.

(E) Spleen cell viability shall be determined.

(F) The numbers of IgM PFC per spleen, and the number of IgM PFC per 10 6 spleen cells shall be reported.

(i) If the antibody PFC assay is performed, the criteria listed under paragraphs (g)(1)(i)(A) through (g)(1)(i)(F) of this section shall be adhered to. Assays described in the references under paragraphs (j)(2) and (j)(4) of this section may be used.

(A) The T cell-dependent antigen, SRBC, shall be injected intravenously or intraperitoneally, usually at 24 days after the first dosing with the test substance. 5 Although the optimum response time is usually 4 days after immunization, some strains of test animal may deviate from this time point. The strain to be used shall be evaluated for the optimum day for PFC formation after immunization.

(B) The activity of each new batch of complement shall be determined. For any given study, the SRBCs shall be from a single sheep, or pool of sheep, for which the shelf life and dose for optimum response has been determined.

(C) Modifications of the PFC assay described in paragraph (g)(1)(i) of this section exist and may prove useful; however, the complete citation shall be made for the method used, any modifications to the method shall be reported, and the source and, where appropriate, the activity or purity of important reagents shall be given. Justification or rationale shall be provided for each protocol modification. Discussions of modifications of the PFC assay are available in the references under paragraphs (j)(5),(j)(6), and (j)(10) of this section

(D) Samples shall be randomized and shall be coded for PFC analysis, so that the analyst is unaware of the treatment group of each sample examined.

(E) Spleen cell viability shall be determined.

(F) The numbers of IgM PFC per spleen, and the number of IgM PFC per 10 6 spleen cells shall be reported.

(i) If the antibody PFC assay is performed, the criteria listed under paragraphs (g)(1)(i)(A) through (g)(1)(i)(F) of this section shall be adhered to. Assays described in the references under paragraphs (j)(2) and (j)(4) of this section may be used.

(A) The T cell-dependent antigen, SRBC, shall be injected intravenously or intraperitoneally, usually at 24 days after the first dosing with the test substance. 5 Although the optimum response time is usually 4 days after immunization, some strains of test animal may deviate from this time point. The strain to be used shall be evaluated for the optimum day for PFC formation after immunization.

(B) The activity of each new batch of complement shall be determined. For any given study, the SRBCs shall be from a single sheep, or pool of sheep, for which the shelf life and dose for optimum response has been determined.

(C) Modifications of the PFC assay described in paragraph (g)(1)(i) of this section exist and may prove useful; however, the complete citation shall be made for the method used, any modifications to the method shall be reported, and the source and, where appropriate, the activity or purity of important reagents shall be given. Justification or rationale shall be provided for each protocol modification. Discussions of modifications of the PFC assay are available in the references under paragraphs (j)(5),(j)(6), and (j)(10) of this section

(D) Samples shall be randomized and shall be coded for PFC analysis, so that the analyst is unaware of the treatment group of each sample examined.

(E) Spleen cell viability shall be determined.

(F) The numbers of IgM PFC per spleen, and the number of IgM PFC per 10 6 spleen cells shall be reported.

(i) If the antibody PFC assay is performed, the criteria listed under paragraphs (g)(1)(i)(A) through (g)(1)(i)(F) of this section shall be adhered to. Assays described in the references under paragraphs (j)(2) and (j)(4) of this section may be used.

(A) The T cell-dependent antigen, SRBC, shall be injected intravenously or intraperitoneally, usually at 24 days after the first dosing with the test substance. 5 Although the optimum response time is usually 4 days after immunization, some strains of test animal may deviate from this time point. The strain to be used shall be evaluated for the optimum day for PFC formation after immunization.

(B) The activity of each new batch of complement shall be determined. For any given study, the SRBCs shall be from a single sheep, or pool of sheep, for which the shelf life and dose for optimum response has been determined.

(C) Modifications of the PFC assay described in paragraph (g)(1)(i) of this section exist and may prove useful; however, the complete citation shall be made for the method used, any modifications to the method shall be reported, and the source and, where appropriate, the activity or purity of important reagents shall be given. Justification or rationale shall be provided for each protocol modification. Discussions of modifications of the PFC assay are available in the references under paragraphs (j)(5),(j)(6), and (j)(10) of this section

(D) Samples shall be randomized and shall be coded for PFC analysis, so that the analyst is unaware of the treatment group of each sample examined.

(E) Spleen cell viability shall be determined.

(F) The numbers of IgM PFC per spleen, and the number of IgM PFC per 10 6 spleen cells shall be reported.

(i) If the antibody PFC assay is performed, the criteria listed under paragraphs (g)(1)(i)(A) through (g)(1)(i)(F) of this section shall be adhered to. Assays described in the references under paragraphs (j)(2) and (j)(4) of this section may be used.

(A) The T cell-dependent antigen, SRBC, shall be injected intravenously or intraperitoneally, usually at 24 days after the first dosing with the test substance. 5 Although the optimum response time is usually 4 days after immunization, some strains of test animal may deviate from this time point. The strain to be used shall be evaluated for the optimum day for PFC formation after immunization.

(B) The activity of each new batch of complement shall be determined. For any given study, the SRBCs shall be from a single sheep, or pool of sheep, for which the shelf life and dose for optimum response has been determined.

(C) Modifications of the PFC assay described in paragraph (g)(1)(i) of this section exist and may prove useful; however, the complete citation shall be made for the method used, any modifications to the method shall be reported, and the source and, where appropriate, the activity or purity of important reagents shall be given. Justification or rationale shall be provided for each protocol modification. Discussions of modifications of the PFC assay are available in the references under paragraphs (j)(5),(j)(6), and (j)(10) of this section

(D) Samples shall be randomized and shall be coded for PFC analysis, so that the analyst is unaware of the treatment group of each sample examined.

(E) Spleen cell viability shall be determined.

(F) The numbers of IgM PFC per spleen, and the number of IgM PFC per 10 6 spleen cells shall be reported.

(i) If the antibody PFC assay is performed, the criteria listed under paragraphs (g)(1)(i)(A) through (g)(1)(i)(F) of this section shall be adhered to. Assays described in the references under paragraphs (j)(2) and (j)(4) of this section may be used.

(A) The T cell-dependent antigen, SRBC, shall be injected intravenously or intraperitoneally, usually at 24 days after the first dosing with the test substance. 5 Although the optimum response time is usually 4 days after immunization, some strains of test animal may deviate from this time point. The strain to be used shall be evaluated for the optimum day for PFC formation after immunization.

(B) The activity of each new batch of complement shall be determined. For any given study, the SRBCs shall be from a single sheep, or pool of sheep, for which the shelf life and dose for optimum response has been determined.

(C) Modifications of the PFC assay described in paragraph (g)(1)(i) of this section exist and may prove useful; however, the complete citation shall be made for the method used, any modifications to the method shall be reported, and the source and, where appropriate, the activity or purity of important reagents shall be given. Justification or rationale shall be provided for each protocol modification. Discussions of modifications of the PFC assay are available in the references under paragraphs (j)(5),(j)(6), and (j)(10) of this section

(D) Samples shall be randomized and shall be coded for PFC analysis, so that the analyst is unaware of the treatment group of each sample examined.

(E) Spleen cell viability shall be determined.

(F) The numbers of IgM PFC per spleen, and the number of IgM PFC per 10 6 spleen cells shall be reported.