# Presentation: 147638

## NICEATM			ICCVAM
National Toxicology Program  Interagency 	Interagency Coordinating Committee Center for the Evaluation Of Alternative 	on the Validation of Alternative 	 Toxicological Methods 			Methods

**NICEATM****	****	****	****ICCVAM**
**National Toxicology Program  Interagency ****	****Interagency Coordinating Committee Center for the Evaluation Of Alternative ****	****on the Validation of Alternative ****	**** Toxicological Methods ****	****	****	****Methods**
**	****	****	****	**

**Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM)**
**Test Method**
**BRD Summary**

**Expert Panel Meeting**
**January 11-12, 2005**
**Bethesda, Maryland **

## Current U.S. Regulatory Status of HET-CAM

**ICCVAM agencies were surveyed and, to the best of their knowledge, HET-CAM test method data have not been submitted to U.S. Regulatory Agencies**

## Primary HET-CAM Data Sources

| Study | Accuracy 
(Severes/Total) |  |  | Intralab (Severes/Total) |  | Interlab 
(Severes/Total) |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | GHS | EPA | EU | CVs | GHS classific. | CVs | GHS classific. |
| Gettings et al. (1991) | 3/9 | 3/9 | 2/9 | - | - | - | - |
| CEC (1991) | - | - | 11/32 | - | - | 14 | - |
| Gettings et al. (1994) | 1/18 | 1/18 | 1/18 | - | - | - | - |
| Bagley et al. (1992) | 0/3 | 0/3 | 0/3 | - | - | - | - |
| Vinardell and Macian (1994) | 0/2 | 0/2 | 0/2 | - | - | - | - |
| Balls et al. (1995) (Q)
Balls et al. (1995) (S) | 15/45
11/17 | 10/45
6/12 | 14/48
11/19 | -
- | -
- | 40
8 | 15/29
11/5 |
| Kojima et al. (1995) | 3/5 | 3/5 | 3/5 | - | - | - | - |
| Gettings et al. (1996) | 8/23 | 10/25 | 6/25 | - | - | - | - |
| Spielmann et al. (1996) | - | - | 45/118 | - | - | - | - |
| Hagino et al. (1999) | 8/16 | 6/14 | 7/17 | - | - | 8 | 8/8 |

**CV = coefficient of variation; classific. = classification**** **

## Other HET-CAM Reports Considered

**39 other reports were identified that could not be used for an evaluation of accuracy or reliability due to the lack of:**
**comparative *****in vivo *****rabbit test***** *****data **
**quantitative *****in vitro *****data**
**These reports discussed in Section 9**
**To the extent possible, data requested from authors of studies considered most useful**

## HET-CAM Analysis Methods (1)

**Irritation Score (A) (IS(A))**
**Irritation responses are evaluated at 0.5, 2, 5 minutes**
**Time-dependent score are assigned to each endpoint**
**IS(A) is calculated by adding assigned scores**
**IS(B)**
**Time of first appearance of endpoint is noted after application of test substance**
**IS(B) is calculated using empirically derived formula**
**Q-Score**
**Calculated as ratio of test substance irritation score to investigator determined reference standard irritation score**

## HET-CAM Analysis Method (2)

**S-Score**
**Calculated as the highest total HET-CAM score for any endpoint evaluated**
**IS and ITC**
**Two analysis methods used **
**Irritation score calculated as IS(A) or IS(B)**
**Threshold concentration defined as the lowest concentration required to produce a slight response after substance application**

## HET-CAM Database

**246 different substances evaluated in 253 tests**
**Distribution of substances among analysis methods **
**IS(A) = 64 substances (43 formulations, 21 chemicals)**
**IS(B) = 86 substances (52 formulations, 34 chemicals) **
**S-Score = 20 substances (all chemicals)**
**Q-Score = 49 substances (all chemicals)**
**IS and ITC = 118 substances (all chemicals or pharmaceutical intermediates)**
**20 Chemical classes tested***
**Most frequent classes: alcohols, carboxylic acids, amines, and formulations**
**15 Product classes tested***
**Most frequent classes: cosmetics, solvents, hair shampoos, soaps/surfactants**

*** Classes with at least 3 entries**

## Distribution of Tests Among Analysis Methods

| Method | Number of Testing Laboratories |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| IS(A) | 47 | - | - | - | 13 | 4 | - |
| IS(B) | 54 | - | 13 | 1 | 1 | 4 | 14 |
| S-Score | 2 | 7 | 6 | 5 | - | - | - |
| Q-Score | 2 | 6 | 1 | 40 | - | - | - |
| IS and ITC | - | 118 | - | - | - | - | - |

## Major HET-CAM Protocol Variations

| Study | # Eggs |  |  | Inc. Temp/
Humidity | Quantity Tested | Rinsing | Endpoints Evaluated** | Method of Analysis |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Neg  | Treat | Pos |  |  |  |  |  |
| Gettings et al. (1991) | - | - | - | - | - | - | H, VL, C | IS(B) |
| CEC (1991) | - | 6 | - | 37.5/62.5% | 0.3 mL or 0.1 g | 20 secs after | H, L, C | IS(B) |
| Gettings et al. (1994) | - | 3 | - | 38/60% | 0.3 mL | - | H, L, C | IS(A)
IS(B) |
| Gettings et al. (1996) | - | -
3 | - | -
38/60% | 0.1 mL 0.3 mL | - | D, H, C
H, L, C | IS(A)
IS(B) |
| Bagley et al. (1992) | 2 | 4 | - | 37.5/62.5% | 0.3 mL or 0.1 g | 20 secs after | HY, H, C | IS(A) |
| Vinardell and Macian (1994) | 2 | 6 | 2 | - | 0.3 mL | - | H, V, C | IS(B) |
| Balls et al. (1995) | - | - | - | - | - | 3 mins after* | H, L, C | S-Score, 
Q-Score |
| Kojima et al. (1995) | - | 4 | - | 37.6/~70% | 0.2 mL | 20 secs after | HY, H, C | IS(A) |
| Hagino et al. (1999) | - | 4 | - | 37.6/~70% | 0.2 mL or 0.2 g | 20 secs after | HY, H, C | IS(A) |
| Spielmann et al. (1996) | - | 3 | - | - | - | 5 mins after* | H, L, C | IS & ITC |

*** For non-transparent substances only**
**** H = hemorrhage, VL = vascular lysis, C = coagulation, HY = hyperemia, L = lysis, D = dilation, V = vasoconstriction**

## Accuracy Analysis

**Ability to correctly identify ocular corrosives and severe irritants determined for**
**GHS classification system (Category 1)**
**EPA classification system (Category I)**
**EU classification system (R41)**
**Accuracy statistics calculated:**
**for each HET-CAM test method protocol, by report and where appropriate **
**classifications were pooled into one classification per substance (i.e., majority call among studies used)**
**using individual studies, where a balanced design existed (multiple substances in multiple labs)**

## Analysis Method Accuracy - GHS

| Analysis Method | Accuracy | Sensitivity | Specificity | False Positive Rate | False Negative Rate |
| --- | --- | --- | --- | --- | --- |
| IS(A) | 75% (46/61) | 67% (12/18) | 79% (34/43) | 21% (9/43) | 33% (6/18) |
| IS(B) | 85% (44/52) | 100% (12/12) | 80% (32/40) | 20% (8/40) | 0% 
(0/12) |
| Q-Score | 62% (28/45) | 100% (15/15) | 43% (13/30) | 57% (17/30) | 0% 
(0/15) |
| S-Score | 47% (8/17) | 36% 
(4/11) | 67% 
(4/6) | 33% 
(2/6) | 64% (7/11) |

## Recommended HET-CAM Version Accuracy

| Statistic | GHS (n=52) | EPA (n=54) | EU (n=86)* |
| --- | --- | --- | --- |
| Accuracy | 85% (44/52) | 83% (45/54) | 73% (63/86) |
| Sensitivity | 100% (12/12) | 93% (13/14) | 95% (19/20) |
| Specificity | 80% (32/40) | 80% (32/40) | 67% (44/66) |
| False 
Positive Rate | 20% (8/40) | 20% (8/40) | 33% (22/66) |
| False Negative Rate | 0% (0/12) | 7% (1/14) | 5% (1/20) |

*** Additional 32 chemicals available for EU analysis only**
**(individual animal data not available for GHS or EPA classification)**

## HET-CAM GHS Accuracy By Chemical/Physical Class

| Class | # of Substances |  |  | False Positive Rate |  | False Negative Rate |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Total | Cat 1 | Cat 2A, 2B, NI | % | n | % | n |
| Overall | 52 | 12 | 40 | 20 | 8/40 | 0 | 0/12 |
| Formulation | 50 | 12 | 38 | 18 | 7/38 | 0 | 0/12 |
| - Hydroalcoholic formulation | 9 | 3 | 6 | 33 | 2/6 | 0 | 0/3 |
| - Oil/Water emulsion | 18 | 1 | 17 | 24 | 4/17 | 0 | 0/1 |
| - Surfactant-based formulation | 23 | 8 | 15 | 7 | 1/15 | 0 | 0/8 |
| Surfactant | 2 | 0 | 2 | 50 | 1/2 | - | - |
| Liquids | 52 | 12 | 40 | 20 | 8/40 | 0 | 0/12 |

## Additional HET-CAM Accuracy Analyses (EU)

| Statistic | EU 
(n=86) | Spielmann et al. (1996) - IS10
(n=112) | Spielmann et al. (1996) - IS100
(n=108) |
| --- | --- | --- | --- |
| Accuracy | 73% (63/86) | 68% (76/112) | 57% (62/108) |
| Sensitivity | 95% (19/20) | 80% (32/40) | 88% (35/40) |
| Specificity | 67% (44/66) | 61% (44/72) | 40% (27/68) |
| False 
Positive Rate | 33% (22/66) | 39% (28/72) | 60% (41/68) |
| False Negative Rate | 5% (1/20) | 20% (8/40) | 12% (5/40) |

## Limitations of HET-CAM IS(B) Accuracy

**Impact of differences in test method protocols between studies is unknown; limits conclusions**
**Most substances evaluated using IS(B) analysis method were: **
**Nonsevere substances **
**Formulations**
**Tested as solutions or liquids**
**Limited information on analysis method ability to accurately identify a variety of chemical classes, product classes, and physicochemical properties (i.e., solids)**

##  HET-CAM IS(B) Reliability Analysis

**Intralaboratory Repeatability and Reproducibility **
**Not conducted due to the lack of published intralaboratory HET-CAM data**
**Interlaboratory Reproducibility**
**Qualitative analysis: Extent of agreement between testing laboratories when identifying corrosives and severe irritants**
**Quantitative analysis: Coefficient of variation (CV)**

## HET-CAM IS(B) Agreement Among Laboratories

| % Interlaboratory Agreement | EU (3-5 labs, 32 substances) |  |
| --- | --- | --- |
|  | % | n |
| 100% (all) | 47 | 15/32 |
| ≥60% (all) | 91 | 29/32 |
| 100% (severes) | 70 | 7/10 |
| ≥60%(severes) | 100 | 10/10 |

## HET-CAM IS(B) Interlaboratory %CV Values

| Coefficient of Variation Analysis | CEC (1991) |
| --- | --- |
| Mean 
(all substances) | 34.1
(n=14) |
| Median 
(all substances) | 33.1
(n=14) |
| Range
(all substances) | 6.6-74.9
(n=14) |

***n = number of substances**
**Interlaboratory %CV values based on results from five laboratories**
**CV = Standard deviation/mean**

## Limitations of IS(B) Reliability

**Intralaboratory reliability unknown due to lack of published data**
**Interlaboratory reproducibility based on a small number of substances (n=14)**

## Draft HET-CAM BRD Proposals

**A proposed version of HET-CAM, which evaluates development of hemorrhage, lysis, and coagulation of vessels on CAM**
**A proposed standardized protocol**
**Proposed test method protocol follows the method provided by ZEBET with IS(B) analysis method**
**Decision criteria previously described by Kalweit et al. (1987)**
**Proposed test method protocol requires the use of positive and negative controls**
**Proposed additional optimization studies, including:**
**Retrospective analysis of decision criteria used to identify corrosives and severe irritants**
**Evaluation of additional endpoints (e.g., trypan blue absorption) for potential inclusion in the calculation of irritancy potential**
**Once optimized, additional validation studies to further characterize accuracy and reliability of the optimized test method version **