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Directorate-General for Health and Food Safety
1- Introduction
The Interagency Co-ordinating Committee for validation of Alternative Methods (ICCVAM) assembled a peer review panel to evaluate a submission on the utility of the mouse local lymph node assay (LLNA) for hazard assessment of potential human contact sensitisers. The review dealt with the following aspects of the test method:
- method description
- data quality
- test performance and reliability (repeatability/reproducibility),
and other scientific considerations.
The evaluations were summarized in a conclusion addressing the acceptability of the LLNA as a stand alone alternative to the currently accepted guinea pig methods and an improvement for animal welfare. A public meeting of the peer review panel was held September 17, 1998 in Gaithersburg, Maryland, USA, to make a final recommendation. The final report from the meeting was published in February 1999: The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of chemicals/Compounds. NIH Publication No: 99-4494.
The peer review panel unanimously recommended the LLNA as a stand-alone alternative for contact sensitisation hazard assessment, provided that the following protocol modifications were made:
1. The protocol should specify the use of female mice only, until a systematic comparison of data between male and female mice has been conducted,
2. It was decided that animals should be individually identified.
3. Body weight data should be collected at the start and end of the assay.
4. DPM should be determined on lymph nodes from individual animals and it was not allowed to pool lymph nodes within the different dose groups.
5. Statistical analysis should be performed.
6. A single dose of a moderate sensitiser should be included as a positive control.
7. The decision process should include a stimulation index >3, statistical significance and dose response evaluation.
8. 3H-methyl thymidine or 125I-iododeoxyuridine may be used in the LLNA.
9. An illustration should be added to the protocol, indicating the nodes draining the exposure site that are to be harvested.
The peer review panel recommended that retrospective data audits should be conducted on the intra- and inter-laboratory validation studies presented by the sponsors.
The LLNA is regarded as a definite improvement with respect to animal welfare (refinement) over currently accepted methods.
The validity of the LLNA has been endorsed by ECVAM (European Centre for the Validation of Alternative Methods) on 21 March 2000 according to the statement adopted by ESAC (ECVAM Scientific Advisory Committee)( SCCNFP/0333/00).
2- Test method description
A LLNA test usually includes 4-5 control mice and 3-5 groups of test mice treated with different concentrations of test chemical. The submission contains a thorough protocol. The scientific basis for the test is measurement of the incorporation of 3H-methyl thymidine into lymphocytes in draining lymph nodes of animals topically exposed to the test article as a measurement of sensitisation. The endpoint of interest is a stimulation index giving the ratio of thymidine incorporation in lymph nodes from dosed animals compared to the incorporation in lymph nodes from control animals. The test is positive when the stimulation index exceeds 3 (SI > 3). Information is provided on the appropriate choice of vehicles and the selection of doses, including the need for dose response. The great majority of the published data represents the testing of simple chemicals. The use of the method to assess skin sensitising potential of mixtures and extracts is not addressed in this validation.
An aspect of the protocol that could cause inter-laboratory differences in procedure is a description of the lymph nodes to be examined. It should be the auricular lymph nodes, however, this is not standard anatomical nomenclature, so it is possible that different laboratories could be removing different nodes for radio assay. The locating of the proper nodes might be difficult, when there is no induction by the test material.
The majority of the data presented in submission resulted from exposure to test articles applied in a mixture of acetone: olive oil. There is limited experience with other suitable vehicles. This is a matter of concern.
The dose selection process as defined by the protocol is based on previous experience in guinea pig tests, structure analysis, and solubility factors. If the LLNA is to be used as a stand-alone assay on new substances, reference to guinea pig tests might appear inappropriate. Concentrations to be tested should be based on toxicity and solubility. The standard protocol describes that 3-5 concentrations are selected among 10 possibilities ranging from 0,1% to 100%. It is important to test high concentrations in order to avoid false negatives. An example of this potential problem is with ethylene diamine, eugenol, hexylcinnamic aldehyde and penicillin G.
The protocol specifies that a vehicle group and 3-5 test groups should be assayed. Assuming that the appropriate concentrations are chosen, this study design is appropriate for a toxicology study. However, in the absence of any data on toxicity or solubility, details regarding how test concentrations should be chosen are necessary.
The LLNA protocol describes the use of CBA mouse in the LLNA. However, the choice of strain has been made without a systematic comparison of alternatives. A better description of the responder properties of various mouse strains would be useful for evaluation of the robustness of the LLNA. The protocol permits the use of both male and female mice, but only one sex in one experiment. Generally, male mice tend to have stronger inflammatory responses. Often, female mice are used in experiments because they are considered to show less inter-mouse variation.
The protocol allows for pooling of the draining lymph nodes from each test group or the analysis of pooled nodes from individual animals. The submitters state that the 3-fold increase is an arbitrary number chosen based on the performance of the assay with a group of known sensitisers. Extensive experience with the assay supported the 3-fold increase as a good indicator of the sensitising ability of chemicals. The peer review panel had significant concerns about the lack of emphasis on statistical analysis in the submission. The requirement of statistical analysis was regarded as an important improvement to the protocol. It would confirm whether or not an apparently high SI (> 3) is due to chance variation, thereby reducing possible false positives. It may detect that apparently low SI (< address the use of controls. The inclusions of a single concentration of a moderate sensitiser as a positive control together with the vehicle control would provide validity to the assay indication that all procedures involved in the assay were conducted properly.
The strengths of the LLNA are the quantitative nature of the assay, the inclusion of a dose response in the assay, the ability of the assay to test coloured substances, improved animal welfare and reduced time required to conduct the study.
3- Test method data quality
The experiments in the submission appear to have been conducted in the spirit of Good Laboratory Practice (GLP). Formal audited reports were not prepared as the data was primarily intended for publication. Data record forms supplied for the peer review panel indicated that record keeping and data collection was adequate.
4- Test method performance
The peer review panel with the assistance of ICCVAM compared the LLNA to the guinea pig assays in terms of specificity, sensitivity, positive predictivity and negative predictivity. The purpose of this evaluation was to determine if the LLNA as a test for hazard identification is equivalent to or superior to the guinea pig assays. In order to make that comparison, the guinea pig assay would have to undergo the same rigorous evaluation as the LLNA. However, such an evaluation has not been performed. Therefore it was decided to compare the performance of LLNA to available guinea pig data, and also including available sources of human data that were viewed as the "gold standard". Of the 200 chemicals tested in the LLNA, 97 were also tested in guinea pig maximization or Buehler assay, an additional 29 were tested using non-standard guinea pig tests, and 39 were tested using the human maximization test (HMT).
The comparative evaluation of the LLNA database included 57 comparisons between GPMT/BA versus human maximization test giving a positive predictivity on 100% and a negative predictivity of 16%, and an accuracy of 72%. The comparison between LLNA versus human maximization test included 74 comparisons giving a positive predictivity of 96%, negative predictivity on 17% and accuracy on 72%. The comparison between LLNA versus GPMT/BA included 97 comparisons, a positive predictivity on 93%, and negative predictivity on 80% and accuracy on 89%. Thus it appears that the LLNA assay is equal to the current guinea pig assays in determining sensitisation potential of chemicals.
The LLNA seems to be predictive for strong and moderate human contact allergens as with the guinea pig test. There is risk of both false positive and false negative results. The local lymph node assay is deficient in detecting sensitisation by low grade to moderate contact sensitisers, some metals, and organometal compounds. The LLNA was less sensitive compared to the GPMT with types of agents as metals, benzocaine, 4-chloroaniline, neomycin sulfate, streptomycin sulfate, and sulfanilic acid. All were positive in the GPMT. The peer review panel was concerned that some strong irritants like SLS gave false positive results however, the animal assays for contact sensitisation cannot stand alone. The results have to be evaluated in comparison with other available dermato-toxicological and chemical data.
5- Determination of test method reliability
The LLNA submission presents qualitative data, which demonstrate adequate intra- and inter-laboratory repeatability and reproducibility.
Reviews of the other scientific literatures support the use of the local lymph node assay as an alternative assay to identify contact allergens. In general, the test method can be readily transferred among properly equipped and staffed laboratories. The method is tolerant of minor protocol changes. A concern is what to do with waste material containing radioactive isotopes. The training and expertise in biology required to perform the LLNA is substantial. The technical skills required are significant, but not prohibitive.
ositive or negative result.
In the future the LLNA may be modified and improved by developing cell culture, systems for ex-vivo incorporation of radioactive treasures. The sensitivity may be improved by giving the animals vitamin acetate enriched diet, the administration route of radioactive thymidine may be changed to peritoneal administration, and DMSO may be considered as a vehicle to increase the sensitivity. Further, it should be considered if the LLNA could be used for detection of chemicals causing systemic allergy and immediate type hypersensitivity.
In conclusion the LLNA assay performed well and gave equivalent results to guinea pig methods for the hazard identification of strong to moderate chemical sensitisers. More refinement seems to be required to correctly classify irritants in the LLNA or it must be accepted that some irritants will give false positive results.
A number of cosmetic ingredients have been tested in this assay: e.g. para-aminobenzoic acid,
abietic acid, ammonium thioglycolate, benzalkonium chloride, chloromethyl-isothiazolinone, cinnamic aldehyde, citral, cocoamidopropylbetaine, dibromo-dicyanobutane, dihydroeugenol, eugenol, formaldehyde, geraniol, hexylcinnamic aldehyde, hydroquinone, hydroxycitronellal, imidazolidinyl urea, isoeugenol, isopropanol, isopropylisoeugenol, lanolin, 6-methylcoumarin, methylisoeugenol, methyl salicylate, musk ambrette, octyl gallate, para-phenylenediamine, propylene glycol, propyl gallate, propylparaben, resorcinol, sodium lauryl sulfate, toluenediamine bismaleimide, toluenesulfonamide formaldehyde resin, polyoxyethylene sorbitan ester (Tween 80).
6- Opinion of the SCCNFP
Based on the ICCVAM peer review document, the statement endorsed by ESAC, and on the opinion of the SCCNFP on the "Predictive testing of potentially cutaneous sensitising cosmetic ingredients or mixtures of ingredients" (SCCNFP/0120/99) the SCCNFP is of the opinion that the LLNA is a recommended method for assessing the sensitisation potential of cosmetic ingredients. However, according to the considerations presented above, it is accepted that the LLNA cannot replace the current guinea pig assays in all cases.
7- References
- National Institute of Environmental Health Sciences - USA (1999) The Murine Local Lymph Node Assay: a test method for assessing the allergic contact dermatitis potential of chemicals/compounds. NIH Publication N° 99-4494.
- European Centre for the Validation of Alternative Methods (ECVAM) (2000) Statement on the validity of the Local Lymph Node Assay for skin sensitisation testing endorsed on 21 March 2000 (SCCNFP/0333/00).
- Scientific Committee on Cosmetic Products and Non-Food Products intended for Consumers (2000) The predictive testing of potentially cutaneous sensitising cosmetic ingredients or mixtures of ingredients. Opinion adopted on 17 February 2000 (SCCNFP/0120/99).
The Interagency Co-ordinating Committee for validation of Alternative Methods (ICCVAM) assembled a peer review panel to evaluate a submission on the utility of the mouse local lymph node assay (LLNA) for hazard assessment of potential human contact sensitisers. The review dealt with the following aspects of the test method:
- method description
- data quality
- test performance and reliability (repeatability/reproducibility),
and other scientific considerations.
The evaluations were summarized in a conclusion addressing the acceptability of the LLNA as a stand alone alternative to the currently accepted guinea pig methods and an improvement for animal welfare. A public meeting of the peer review panel was held September 17, 1998 in Gaithersburg, Maryland, USA, to make a final recommendation. The final report from the meeting was published in February 1999: The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of chemicals/Compounds. NIH Publication No: 99-4494.
The peer review panel unanimously recommended the LLNA as a stand-alone alternative for contact sensitisation hazard assessment, provided that the following protocol modifications were made:
1. The protocol should specify the use of female mice only, until a systematic comparison of data between male and female mice has been conducted,
2. It was decided that animals should be individually identified.
3. Body weight data should be collected at the start and end of the assay.
4. DPM should be determined on lymph nodes from individual animals and it was not allowed to pool lymph nodes within the different dose groups.
5. Statistical analysis should be performed.
6. A single dose of a moderate sensitiser should be included as a positive control.
7. The decision process should include a stimulation index >3, statistical significance and dose response evaluation.
8. 3H-methyl thymidine or 125I-iododeoxyuridine may be used in the LLNA.
9. An illustration should be added to the protocol, indicating the nodes draining the exposure site that are to be harvested.
The peer review panel recommended that retrospective data audits should be conducted on the intra- and inter-laboratory validation studies presented by the sponsors.
The LLNA is regarded as a definite improvement with respect to animal welfare (refinement) over currently accepted methods.
The validity of the LLNA has been endorsed by ECVAM (European Centre for the Validation of Alternative Methods) on 21 March 2000 according to the statement adopted by ESAC (ECVAM Scientific Advisory Committee)( SCCNFP/0333/00).
2- Test method description
A LLNA test usually includes 4-5 control mice and 3-5 groups of test mice treated with different concentrations of test chemical. The submission contains a thorough protocol. The scientific basis for the test is measurement of the incorporation of 3H-methyl thymidine into lymphocytes in draining lymph nodes of animals topically exposed to the test article as a measurement of sensitisation. The endpoint of interest is a stimulation index giving the ratio of thymidine incorporation in lymph nodes from dosed animals compared to the incorporation in lymph nodes from control animals. The test is positive when the stimulation index exceeds 3 (SI > 3). Information is provided on the appropriate choice of vehicles and the selection of doses, including the need for dose response. The great majority of the published data represents the testing of simple chemicals. The use of the method to assess skin sensitising potential of mixtures and extracts is not addressed in this validation.
An aspect of the protocol that could cause inter-laboratory differences in procedure is a description of the lymph nodes to be examined. It should be the auricular lymph nodes, however, this is not standard anatomical nomenclature, so it is possible that different laboratories could be removing different nodes for radio assay. The locating of the proper nodes might be difficult, when there is no induction by the test material.
The majority of the data presented in submission resulted from exposure to test articles applied in a mixture of acetone: olive oil. There is limited experience with other suitable vehicles. This is a matter of concern.
The dose selection process as defined by the protocol is based on previous experience in guinea pig tests, structure analysis, and solubility factors. If the LLNA is to be used as a stand-alone assay on new substances, reference to guinea pig tests might appear inappropriate. Concentrations to be tested should be based on toxicity and solubility. The standard protocol describes that 3-5 concentrations are selected among 10 possibilities ranging from 0,1% to 100%. It is important to test high concentrations in order to avoid false negatives. An example of this potential problem is with ethylene diamine, eugenol, hexylcinnamic aldehyde and penicillin G.
The protocol specifies that a vehicle group and 3-5 test groups should be assayed. Assuming that the appropriate concentrations are chosen, this study design is appropriate for a toxicology study. However, in the absence of any data on toxicity or solubility, details regarding how test concentrations should be chosen are necessary.
The LLNA protocol describes the use of CBA mouse in the LLNA. However, the choice of strain has been made without a systematic comparison of alternatives. A better description of the responder properties of various mouse strains would be useful for evaluation of the robustness of the LLNA. The protocol permits the use of both male and female mice, but only one sex in one experiment. Generally, male mice tend to have stronger inflammatory responses. Often, female mice are used in experiments because they are considered to show less inter-mouse variation.
The protocol allows for pooling of the draining lymph nodes from each test group or the analysis of pooled nodes from individual animals. The submitters state that the 3-fold increase is an arbitrary number chosen based on the performance of the assay with a group of known sensitisers. Extensive experience with the assay supported the 3-fold increase as a good indicator of the sensitising ability of chemicals. The peer review panel had significant concerns about the lack of emphasis on statistical analysis in the submission. The requirement of statistical analysis was regarded as an important improvement to the protocol. It would confirm whether or not an apparently high SI (> 3) is due to chance variation, thereby reducing possible false positives. It may detect that apparently low SI (< address the use of controls. The inclusions of a single concentration of a moderate sensitiser as a positive control together with the vehicle control would provide validity to the assay indication that all procedures involved in the assay were conducted properly.
The strengths of the LLNA are the quantitative nature of the assay, the inclusion of a dose response in the assay, the ability of the assay to test coloured substances, improved animal welfare and reduced time required to conduct the study.
3- Test method data quality
The experiments in the submission appear to have been conducted in the spirit of Good Laboratory Practice (GLP). Formal audited reports were not prepared as the data was primarily intended for publication. Data record forms supplied for the peer review panel indicated that record keeping and data collection was adequate.
4- Test method performance
The peer review panel with the assistance of ICCVAM compared the LLNA to the guinea pig assays in terms of specificity, sensitivity, positive predictivity and negative predictivity. The purpose of this evaluation was to determine if the LLNA as a test for hazard identification is equivalent to or superior to the guinea pig assays. In order to make that comparison, the guinea pig assay would have to undergo the same rigorous evaluation as the LLNA. However, such an evaluation has not been performed. Therefore it was decided to compare the performance of LLNA to available guinea pig data, and also including available sources of human data that were viewed as the "gold standard". Of the 200 chemicals tested in the LLNA, 97 were also tested in guinea pig maximization or Buehler assay, an additional 29 were tested using non-standard guinea pig tests, and 39 were tested using the human maximization test (HMT).
The comparative evaluation of the LLNA database included 57 comparisons between GPMT/BA versus human maximization test giving a positive predictivity on 100% and a negative predictivity of 16%, and an accuracy of 72%. The comparison between LLNA versus human maximization test included 74 comparisons giving a positive predictivity of 96%, negative predictivity on 17% and accuracy on 72%. The comparison between LLNA versus GPMT/BA included 97 comparisons, a positive predictivity on 93%, and negative predictivity on 80% and accuracy on 89%. Thus it appears that the LLNA assay is equal to the current guinea pig assays in determining sensitisation potential of chemicals.
The LLNA seems to be predictive for strong and moderate human contact allergens as with the guinea pig test. There is risk of both false positive and false negative results. The local lymph node assay is deficient in detecting sensitisation by low grade to moderate contact sensitisers, some metals, and organometal compounds. The LLNA was less sensitive compared to the GPMT with types of agents as metals, benzocaine, 4-chloroaniline, neomycin sulfate, streptomycin sulfate, and sulfanilic acid. All were positive in the GPMT. The peer review panel was concerned that some strong irritants like SLS gave false positive results however, the animal assays for contact sensitisation cannot stand alone. The results have to be evaluated in comparison with other available dermato-toxicological and chemical data.
5- Determination of test method reliability
The LLNA submission presents qualitative data, which demonstrate adequate intra- and inter-laboratory repeatability and reproducibility.
Reviews of the other scientific literatures support the use of the local lymph node assay as an alternative assay to identify contact allergens. In general, the test method can be readily transferred among properly equipped and staffed laboratories. The method is tolerant of minor protocol changes. A concern is what to do with waste material containing radioactive isotopes. The training and expertise in biology required to perform the LLNA is substantial. The technical skills required are significant, but not prohibitive.
ositive or negative result.
In the future the LLNA may be modified and improved by developing cell culture, systems for ex-vivo incorporation of radioactive treasures. The sensitivity may be improved by giving the animals vitamin acetate enriched diet, the administration route of radioactive thymidine may be changed to peritoneal administration, and DMSO may be considered as a vehicle to increase the sensitivity. Further, it should be considered if the LLNA could be used for detection of chemicals causing systemic allergy and immediate type hypersensitivity.
In conclusion the LLNA assay performed well and gave equivalent results to guinea pig methods for the hazard identification of strong to moderate chemical sensitisers. More refinement seems to be required to correctly classify irritants in the LLNA or it must be accepted that some irritants will give false positive results.
A number of cosmetic ingredients have been tested in this assay: e.g. para-aminobenzoic acid,
abietic acid, ammonium thioglycolate, benzalkonium chloride, chloromethyl-isothiazolinone, cinnamic aldehyde, citral, cocoamidopropylbetaine, dibromo-dicyanobutane, dihydroeugenol, eugenol, formaldehyde, geraniol, hexylcinnamic aldehyde, hydroquinone, hydroxycitronellal, imidazolidinyl urea, isoeugenol, isopropanol, isopropylisoeugenol, lanolin, 6-methylcoumarin, methylisoeugenol, methyl salicylate, musk ambrette, octyl gallate, para-phenylenediamine, propylene glycol, propyl gallate, propylparaben, resorcinol, sodium lauryl sulfate, toluenediamine bismaleimide, toluenesulfonamide formaldehyde resin, polyoxyethylene sorbitan ester (Tween 80).
6- Opinion of the SCCNFP
Based on the ICCVAM peer review document, the statement endorsed by ESAC, and on the opinion of the SCCNFP on the "Predictive testing of potentially cutaneous sensitising cosmetic ingredients or mixtures of ingredients" (SCCNFP/0120/99) the SCCNFP is of the opinion that the LLNA is a recommended method for assessing the sensitisation potential of cosmetic ingredients. However, according to the considerations presented above, it is accepted that the LLNA cannot replace the current guinea pig assays in all cases.
7- References
- National Institute of Environmental Health Sciences - USA (1999) The Murine Local Lymph Node Assay: a test method for assessing the allergic contact dermatitis potential of chemicals/compounds. NIH Publication N° 99-4494.
- European Centre for the Validation of Alternative Methods (ECVAM) (2000) Statement on the validity of the Local Lymph Node Assay for skin sensitisation testing endorsed on 21 March 2000 (SCCNFP/0333/00).
- Scientific Committee on Cosmetic Products and Non-Food Products intended for Consumers (2000) The predictive testing of potentially cutaneous sensitising cosmetic ingredients or mixtures of ingredients. Opinion adopted on 17 February 2000 (SCCNFP/0120/99).
