### Page 1 of 12



# Addendum to the ToxSAC Report on Fluopyram (Dec. 23, 2009 **)**

**(from RAB 3 Risk Assessment Team, 1/13/2010)** On July 16, 2009, ToxSAC met to evaluate the proposed toxicity endpoints and FQPA safety factors for fluopy

ram . The members of ToxSAC evaluated the available data

. The toxicity data derived from repeated dosing demonstrated that fluopy ram predominantly produced liver, thy roid, and kidney

effects and reduced body weight

. Most of the toxicity endpoints and points of departure for risk assessment selected for risk assessment were based on the liver and thy roid effects. Thyroid effects including thyroid hormonal changes were seen in several studies in rats and mice with repeated dosing. However, the available data on thy roid hormonal changes were inconsistent and sparse. The ToxSAC raised a concern about the potential influence of thyroid hormone on the development of the young animals since the thy roid was not examined in the pups. The ToxSAC recommended a comparative thy roid hormone assay

on fluopy

ram and a 10x safety factor to account for the uncertainty associated with the lack of this study

. All the data on fluop yram registration are undergoing a global review ; the major global partners are Germany and Canada. Both Canada and Germany evaluated the toxi cology data and concluded that the available data demonstrated that the thy roid effect to be secondary to the liver effect. The data showed that there were increased liver weight, liver hypertrophy, and increased liver enzy me activities . The increase d liver enzy me activities enhanced thyroid hormone metabolism leading to a decrease in circulating thyroid hormone and perturbation on the pituitary

-thy

roid axis. An increase in TSH level was seen in 14

- day mechanistic studies in rats and mice. Both Germany and Canada determined that the thy roid effect was secondary to the liver toxicity

. HED Division director was briefed on this difference and the relevant data were presented at the meeting. The attendees at this meeting were Paula Deschamp , Sheila Healy, Elizab eth Mendez, Whang Phang, and Tina Levine (Division Director)

. In this meeting the information and data summaries contained in Attachments 1, 2, &3 were presented and discussed

. **After careful consideration of all data, the RAB3 Risk Assessment Team determi ne**  **s that**  **the available data do not support the requirement of a comparative thyroid assay and associated 10x FQPA safety factor for the following reasons:** 
**1. The existing data demonstrated slight changes in thyroid hormonal level**  **; the effect**  **of hormonal changes was not considered to be a major effect.** 
**2. There was no increase in sensitivity in fetuses which were exposed**  **during p re** **- natal**  **duration and pups which were exposed during pre** 

**- natal and post** 

**- natal periods.** 
**3. The data suggest that the**  **non -** **neopla stic**  **thyroid effect was associated with the**  **liver affect.** 

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# Attachment 1 **Fluopyram Discussion**  **16**

**- December** 

**- 2009**  **Objective** To present the current status on endpoints/uncertainty factors proposed for EPA risk assessment and the implications for US registrations and global harmonization. **Background** Fluopy ram is a new broad

- spectrum s ystemic fungicide of the carboxamide group. The proposed use is to control fungal diseases in practically all crops. It acts by inhibiting succinate dehydrogenase in the cell respiration of the fungus (mitochondrial respiration Complex II), thus blocking electron transport. Fluop yram is a global review chemical in partnership with Canada and Germany. Germany has the work

- share lead for review of toxicology data. **Summary of Toxicological**  **Findings** Decreased body weight found in practically all studies; target organ effects are as follows:

- Liver in rats, mice & dogs

- increased liver weight, hepatocellular hypertrophy, vacuolation, increased liver microsomal enzymes (P

-450, B ROD,& EROD activities in rats), hepatocellular adenomas and carcinomas.

- Kidneys

- increase organ weight associated with hyaline droplet nephropathy in rats

- Thy roid in rats and mice: increased thyroid weight, slight changes in thyroid hormone in the studies where thyroid hormones were measure, hypertrophy of the follicular cell, hyperplasia, and thyroid follicular cell adenomas

. (There are data to suggest that the thyroid effects are due to liver microsomal enzyme induction leading to enhanced catabolism o f T3 & T4 in the liver and an increase in TSH level via feed back mechanism).

- The data of a recovery group in a 90

- day oral toxicity study in rats showed that, after the treated rats were maintained on the control diet for 1 month, liver effects seen in the treated rats returned to normal; do did the thy

roid gland

. 

### Harmonization Issues 1) Carcinogenicity : EPA, PMRA, and Germany agree that the MOA argument for carcinogenicity

has not been made because dose levels tested for MOA studies were too high (greater than the dose which produced tumor in some studies).

- EPA: Carcinogenicity : increased incidence of liver tumors in rats and thy roid tumors in mice. The registrant submitted data to demonstrate a mode of action for both liver and thy roid tumors. However, these data were insufficient. The CARC classified fluopy ram as “Likely to be Carcinogenic to Human” and a unit risk, Q 1 

### *, of 1.55x10 - 2 

### was calculated based upon female rat liver tumors (adenomas and carcinomas combined).

- PMRA has calculated unit risks based on both thyroid adenomas (1.72x10 - 2

### ) and liver tumors (1.21x10 - 2 

### ), whereas Germany is going to classify fluop yram according to its own system and conducts the risk using RfD approach.

### Page 3 of 12

2) Non

- cancer thyroid effects :

o ToxSAC had evaluated the proposed toxicity endpoints and recommended a comparative thyroid assay in rats to be conducted. Due to lack of reliable thyroid hormonal measurements in repeated dose studies and uncertainty of the thyroid hormonal effects in the pups. This leads to databas e uncertainty, and a 10x FQPA safety factor is recommended to be applied for assessing the risk.

o Currently, PMRA and Germany do not feel that thy

roid effect is a concern because they

believe it is a secondary

effect due to increased metabolism of the thy roid hormone in the liver.

### Page 4 of 12



## Attachment 2 **Summary Data of Liver and Thyroid Effects Found in Toxicity Studies with**  **Fluopyram**
**Dose levels (mg/kg/day)**  **Liver Effects Thyroid Effects** 
**28**

**- Day Oral Tox. rats**  4.0/4.6 (M/F) 

### None None 31.0/36.1 Liver enzy me increase None 256/263 Liver weight increases, hypertrophy Thy roid weight increases with follicular cell hy pertrophy **90**

**- Day oral tox.** 

**- rats**  3.1/3.6 None None 12.5/14.6 None None 60.5/70.1 ↑ liver weight (+25%), hy pertrophy, & macrovacuolation ↑ thyroid weight (+4%), thyroid follicular cell hy pertrophy . TSH, T4 and T3 were increased. 204/230 ↑ liver weight (+53%), hy pertrophy, & macrovacuolation ↑ thyroid weight (+10%), thyroid follicular cell hy pertrophy . TSH, T4 and T3 were increased. 1 month recovery group showed (60.5/70.1 mg/kg) livers were comparable to those of the controls. 1 month recovery group showed (60.5/70.1 mg/kg) thyroid glands were comparable to those of the controls. TS H, T3, & T4 returned to the control levels. 

### C **arc inogenicity**  **study**

**- mice**  4.2/5.3 None None 20.9/26.8 ↑ Liver weight (11

- 20%),

hy pertrophy, single cell degeneration Thy roid follicular cell hyperplasia

105/129 ↑ Liver weight (11

- 20%),

hy pertrophy, single cell degeneration, vacuolation Thy roid follicular cell hyperplasia, adenomas **Chronic/carcinogenicity s tudy** 

**- rats**  1.20/1.68 None None

### Page 5 of 12

**Dose levels (mg/kg/day)**  **Liver Effects Thyroid Effects** 
6.0/8.6 Hy

pertocellular hy

pertrophy, marcovacuolation Thy roid follicular cell hypertrophy
28/89 ↑ Liver weight (17% M, 39% F),

hy pertrophy, single cell degeneration, liver tumor in females ↑ thrroid weight (8% M, 23% F), f ollicular cell hypertrophy
**2**

**- Generation Reproduction Study** 

**- rats**  2.7/3.2 (M/F) 

### None (parent & offspring) None (parent & offspring) 15.1/17.6 

### None (parent & offspring) None (parent & offspring) 83.1/96.3 

### Increase liver weight and hepto

centrilobular hy

pertrophy parent & F1 generation. Thy roid gland was weighed and examined no effect was reported for P & F1 generations.. **The numerical details of the above summarized data are presented in the tables following this page**

.

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# Attachment 3 **Organ weight changes in the 28** 

**- day rat study with**  **fluopyram**
**Dosage level (ppm )**  **0 50**  **400 3200**  Males Liver weight Absolute (g) 11.13 ± 0.93 11.18 ± 0.93 (+0.4 % ) 12.45 ± 1.15

(+12 % ) 17.11 ± 1.76 **

(+54 % ) Bodyweight

-relative 2.864 ± 0.210 2.811 ± 0.137 (

-2 % ) 3.055 ± 0.176

(+7 % ) 4.401 ± 0.239 **

(+54 % ) Brain

-relative 567.042 ± 57.354 565.078 ± 48.426

(0 % ) 607.382 ± 50.971

(+7 % ) 878.091 ± 86.733

(+55 % ) Kidney weight Absolute (g) 2.63 ± 0.09 2.49 ± 0.18 (

- 5 % ) 3.16 ± 0.24 **

(+20 % ) 3.13 ± 0. 20 **

(+19 % ) Bodyweight

-relative 0.676 ± 0.016 0.627 ± 0.053 (

- 7 % ) 0.777 ± 0.052

(+15 % ) 0.801 ± 0.094

(+18 % ) Thyroid weight Absolute (g) 0.0176 ± 0.0040 0.0197 ± 0.0023 (+12 % ) 0.0222 ± 0.0026

(+26 % ) 0.0252 ± 0.0033 **

(+43 % ) Bodyweight

-relative 0.00456 ± 0.00118 0.00499 ±

0.00085 (+9 % ) 0.00548 ± 0.00086

(+20 % ) 0.00653 ±

0.00109 ** (+41 % )
Females Liver weight Absolute (g) 5.84 ± 0.36 6.24 ± 0.32 (+7 % ) 6.76 ± 0.46

(+16 % ) 9.64 ± 1.00 **

(+65 % ) Bodyweight

-relative 2.665 ± 0.200 2.773 ± 0.064 (+4 % ) 3.072 ± 0.151

(+15 % ) 4.599 ± 0.349 **

(+73 % ) *: p≤0.05; **: p≤0.01; Figures in parentheses are % differences from control

### Page 7 of 12

**Liver weight changes after 90 days of treatment (% change when compared to controls) in rats** 
**Dietary level (ppm )**  **0 50**  **200 1000**  **3200** Males Mean absolute liver Weight (g) 10.94 ± 0.86 10.82 ± 0.63 (

- 1 % ) 12.02 ± 1.19

(+10 % ) 13.63 ± 1.13 **

(+25 % ) 16.77 ± 1.70 **

(+53 % ) Mean liver to body weight ratio 2.282 ± 0.096 2.277 ± 0.101 (0 % ) 2.400 ± 0.151

(+5 % ) 2.736 ± 0.188**

(+20 % ) 3.669 ± 0.223**

(+61 % ) Females Mean absolute liver Weight (g) 5.74 ± 0.58 5.94 ± 0.32 (+3 % ) 6.40 ± 0.43

(+11 % ) 7.09 ± 0.58**

(+24 % ) 9.09 ± 1.55**

(+58 % ) Mean liver to body weight ratio 2.123 ±0.096 2.235 ± 0.105 (+5 % ) 2.366 ± 0.0 71

(+11 % ) 2.697 ± 0.172**

(+27 % ) 3.699 ± 0.503**

(+74 % ) * = p < 0.05. **= p < 0.01


### Thyroid weight changes after 90 days of treatment(% change when compared to controls) **in rats**
**Dietary level (ppm )**  **0 50**  **200 1000**  **3200** Males Mean absolute thyroid weight (g) 0.0272 ± 0.0052 0.0224 ± 0.0058 (

- 18 % ) 0.0269 ± 0.0048

(

- 1 % ) 0.0282 ± 0.0060

(+4 % ) 0.0299±0.0051

(+10 % ) Mean thyroid to bodyweight ratio 0.00568 ±

0.00107 0.00471 ±

0.00123 (

- 17 % ) 0.00496 ±

0.00081 (

- 13 % ) 0.00565 ±

0.00106 (

- 1 % ) 0.00657 ±

0.00121 (+16 % ) Females Mean absolute thyroid weight (g) 0.0185 ± 0.0021 0.0203 ± 0.0040 (+10 % ) 0.0164 ± 0.0042

(

- 11 % ) 0.0184 ± 0.0038

(

- 1 % ) 0.0206 ± 0.0026

(+11 % ) Mean thyroid to bodyweight ratio 0.00689 ±

0.00093 0.00767 ±

0.00163 (+11 % ) 0.00610 ±

0.001 56 (

- 11 % ) 0.00696 ±

0.00116 (0 % ) 0.00841 ±

0.00097* (+22 % ) * = p < 0.05

### Page 8 of 12

**90**

**- Day Oral Toxicity Study** 

**- rats**  **Thyroid Hormons and TSH Levels in Fluopyram Treated Rats**

**- 3 Week**  **Dose level (Mg/kg/day)**  **M/F TSH**  **T3 T4** 

**Males Females**  **Males Females**  **Males Females** 
0/0 **6.32 ± 2.58 3.51 ± 0.59**  **1.33 ± 0.19 1.22 ± 0.24**  **61.9 ± 15.1 34.0 ± 6.5**  3.06/3.63 **6.58 ± 2.67 3.70 ± 1.31**  **1.37 ± 0.13 1.41 ± 0.28**  **57.8 ± 6.7 45.2 ± 10.9**  12.5/14.6 **5.71 ± 2.00 4.13 ± 0.96**  **1.31 ± 0.17 1.21 ± 0.27**  **59.1 ± 9.0 36.7 ± 6.9**  60.5/70.1 **7.35 ± 1.18**  **4.14 ± 0.88 1.50 ± 0.24**  **1.37 ± 0.21 65.8 ± 12.5**  **44.5 ± 9.8** 204/230 **10.30 ± 4.95  ** 5.99 ± 1.13**** **1.53 ± 0.23 1.51 ± 0.13** * **55.4 ± 10.5 52.3 ± 10.5** ** * p<0.05 **p<0.01
**90**

**- Day Oral Toxicity Study** 

**- rats**  **Thyroid Hormons and TSH L evels in Fluopyram Treated Rats** 

**- 13 Week**  **Dose level (Mg/kg/day)**  **M/F TSH**  **T3 T4** 

**Males Females**  **Males Females**  **Males Females** 
0/0 **4.44 ± 1.13 4.16 ± 1.45**  **1.14 ± 0.29 1.46 ± 0.46**  **48.4 ± 11.0 34.1 ± 11.0**  3.06/3.63 **6.09 ± 1.93 3.62 ± 0.49**  **1.27 ± 0.21 1.45 ± 0.**  **31 53.5 ± 14.3**  **38.3 ± 12.1** 12.5/14.6 **5.76 ± 1.69 3.73 ± 0.87**  **1.22 ± 0.34 1.44 ± 0.22**  **51.7 ± 4.8 44.8 ± 9.2**  60.5/70.1 **6.82 ± 1.97 3.94 ± 0.69**  **1.39 ± 0.35 1.60 ± 0.38**  **69.4 ± 11.4  ** 47.1 ± 11.6** 204/230 **8.35 ± 3.12  ** 5.42 ± 1.90** **1.60 ± 0.26  ** 1.73 ± 0.24** **5 8.2 ± 12.0**  **38.6 ± 12.2** * p<0.05 **p<0.01
**90**

**- Day Oral Toxicity Study** 

**- rats** 

**Thyroid Hormons and TSH Levels in Fluopyram Treated Rats**

**- 1** 

**- Month Recovery Groups**  **Dose level (Mg/kg/day)**  **M/F TSH**  **T3 T4** 

**Males Females**  **Males Females**  **Males Fe**  **males**
0/0 **7.34 ± 2.08 3.93 ± 1.45**  **1.26 ± 0.49 1.23 ± 0.17**  **47.2 ± 8.1 29.0 ± 4.3**  204/230 **6.34 ± 1.73 3.51 ± 1.03**  **1.37 ± 0.27 1.36 ± 0.20**  **43.6 ± 8.0 39.8 ± 11.7**  * p<0.05 **p<0.01

### Page 9 of 12

**L iver**  **weight changes**  **at scheduled**  **sacrifice, chro**  **nic phase**  **(% change**  **when compared**  **to**

**controls) (Carcinogenicity Study in Mice)** 

**Sex Males**  **Fem ales**  Dose level of fluopyram (ppm) 0 30 150 750 0 30 150 750 Mean absolute liver weight (g) 1.26

± 0.10 1.24

± 0.09 (

-2 % ) 1.40*

± 0.15 (+11 % ) 1.47**

± 0.13 (+17 % ) 1.09

± 0.13 1.07

± 0.18 (

-2 % ) 1.31**

± 0.12 (+20 % ) 1.40**

± 0.14 (+28 % ) Mean liver to body weight ratio ( % ) 4.53

± 0.366 4.52

± 0.232 (0 % ) 5.20**

0.503 (+15 % ) 5.68**

0.235 (+25 % ) 4.70

± 0.404 4.61

± 0.688 (

-2 % ) 5.51**

± 0.365 (+17 % ) 6.12**

± 0 .325 (+30 % ) *: p≤0.05;**: p≤0.01



### Incidence **and severity**  **of microscopic**  **changes in**  **the thyroid**  **gland, all**  **animals, chronic** 

**phase (12** 

**- Months) (Carcinogenicity study in mice)** 

**Sex Males**  **Dose level of fluopyram (ppm)**  **0 30**  **150 750**  Number of animals 9 1 0 9 10 Follicular cell hyperplasia: focal/multifocal Minimal 0 0 2 1 Slight 0 0 0 1 Total 0 0 2 2



### Incidence and severity of microscopic changes in the thyroid gland, all animals, **carcinogenicity phase in mice** 

**Sex Males**  **Fem ales**  **Dose level of fluopyra**  **m (ppm** **) 0**  **30 150**  **750 0**  **30 150**  **750** Number of animals 50 50 50 50 48 50 50 50 Follicular cell hyperplasia: focal/multifocal Minimal 0 2 10 18 11 4 10 10 Slight 3 2 5 6 5 2 5 12 Moderate 1 2 2 5 1 2 3 6 Marked 0 0 3 3 0 0 1 4 Severe 0 0 1 0 0 0 0 1 Total 4 6 21** 32** 17 8* 19 33** **: p≤0.01

### Page 10 of 12

**Liver weight changes at scheduled sacrifice of the chronic phase (12 month)**  **(% change when compared to controls)**  **(Rat Carcinogenicity Study)**

**Sex Males**  **Fem ales**  **Dose level of fluopyram**  **(ppm )**  **0 30**  **150 750**  **0 30**  **15 0**  **1500** Mean absolute liver weight (g) 12.21

± 1.33 12.70

± 1.50 (+4 % ) 12.43

± 1.05 (+2 % ) 14.31 *

± 1.92 (+17 % ) 7.51

± 0.99 7.37

± 1.01 (

-2 % ) 7.67

± 1.43 (+2 % ) 10.44 **

± 1.61 (+39 % ) Mean liver to body weight ratio 1.86

± 0.12 1.93

± 0.18 (+4 % ) 1.88

± 0.15 (+1 % ) 2.19 **

± 0.18 (+17 % ) 2.17

± 0.19 2.19

± 0.22 (+1 % ) 2.26

± 0.20 (+4 % ) 3.35 **

± 0.33 (+54 % ) *: p≤0.05; **: p≤0.01



### Rat l**iver weight changes at scheduled sacrifice of the carcinogenicity phase (% change when compared to controls)** 

**Sex Males**  **Fem ales**  **Dose level of fluopyram**  **(ppm) 0**  **30 150**  **750/375 0**  **30 150**  **1500**
Mean absolute liver weight (g) 12.56

± 1.71 11.09 *

± 1.12 (

- 12 % ) 14.02 *

± 2.06 (+12 % ) 13.24

± 2.25 (+5 % ) 9.46

± 2.19 9.69

± 2.12 (+2 % ) 9.89

± 2.12 (+5 % ) 13.16 **

± 2.87 (+39 % ) Mean liver to body weight ratio 2.06

± 0.33 1.94

± 0.26 (

- 6 % ) 2.22

± 0.39 (+8 % ) 2.32

± 0.43 (+12 % ) 2.37

± 0.37 2.31

± 0.30 (

- 3 % ) 2.52

± 0.35 (+6 % ) 3.70 **

± 0.59 (+56 % ) *: p≤0.05; **: p≤0.01



### Incidence **and severity**  **of microscopic**  **changes in**  **the liver,**  **all animals**  **of the** 

**carcinogenicity phase**

**Sex Males**  **Fem ales**  **Dose level fluopyram (ppm** **) 0**  **30 150**  **750/375 0**  **30 150**  **1500** Number of animals examined 60 60 60 58 60 60 60 59 Centrilobular to panlobular hepatocellular hypertrophy: diffuse Minimal 1 1 14 15 0 0 0 6 Slight 0 0 0 15 0 0 0 21 Moderate 0 0 0 0 0 0 0 21 Total 1 1 14** 30** 0 0 0 48** Focus(i) of hepatocellular alteration: clear: focal/multifocal Minimal 8 6 6 11 0 3 3 8 Slight 2 2 1 5 1 1 1 3 Total 10 8 7 16 1 4 4 11** Focus(i) of hepatocellular alteration: eosinophilic: focal/multifocal Minimal 12 20 18 15 23 17 14 25 Slight 4 3 10 12 6 7 15 13 Moderate 0 1 2 1 0 2 1 7

### Page 11 of 12

**Sex Males**  **Fem ales**  **Dose level fluopyram (ppm** **) 0**  **30 150**  **750/375 0**  **30 150**  **1500** Marked 0 0 1 0 0 0 0 3 Total 16 24 31* 28** 29 26 30 48** Hepatocellular vacuolation: focal/multifocal Minimal 10 4 13 7 5 10 7 13 Slight 0 2 3 0 1 4 2 9 Moderate 0 1 0 0 0 0 0 0 Total 10 7 16 7 6 14** 9 22** *: p≤0.05, **: p≤0.01



### Rat t**hyroid gland weight changes at schedul ed sacrifice of the chronic phase (% change** 

**when compared to controls) (Carcinogenicity Study)**  **Sex Males**  **Fem ales**  **Dose level of fluopyram**  **(ppm) 0**  **30 150**  **750 0**  **30 150**  **1500**
Mean absolute thyroid gland weight (g) 0.0272

± 0.0059 0.0280

± 0.0036 (+3 % ) 0.0244

± 0.0066 (

- 10 % ) 0.0295

± 0.0059 (+8 % ) 0.0201

± 0.0052 0.0196

± 0.0063 (

-2 % ) 0.0213

± 0.0048 (+6 % ) 0.0248

± 0.0058 (+23 % ) Mean thyroid gland to body weight ratio 0. 0042

± 0.0009 0.0043

± 0.0006 (+3 % ) 0.0038

± 0.0012 (

- 10 % ) 0.0045

± 0.0008 (+9 % ) 0.0058

± 0.0013 0.0057

± 0.0015 (

- 1 % ) 0.0063

± 0.0013 (+9 % ) 0.0080 **

± 0.0019 (+38 % ) **: p≤0.01



### Incidence **and severity**  **of microscopic**  **changes in**  **the rat**  **thyroid gland,**  **all animals**  **of the** 

**chronic phase (12 months)** 

**Sex Males**  **Fem ales**  Dose level of f

luopy ram (ppm) 0 30 150 750 0 30 150 1500 Number of animals examined 10 10 10 10 10 10 10 10 Follicular cell hypertrophy: diffuse Minimal 0 0 2 2 0 0 0 4 Slight 0 0 0 2 0 0 0 1 Total 0 0 2 4 0 0 0 5 Colloid alteration Minimal 4 4 3 2 1 0 0 3 Slight 0 1 1 2 0 0 0 1 Moderate 0 0 0 3 0 0 0 0 Total 4 5 4 7 1 0 0 4

### Page 12 of 12

**Incidence and**  **severity of**  **microscopic changes**  **in the**  **rat thyroid**  **gland, all**  **animals of**  **the**

**carcinogenicity phase**

**Sex Males**  **Fem ales**  **Dose level of fluopyram (ppm)**  **0 30**  **150 750/375**  **0 30**  **150 1500**  Number of animals examined 58 59 57 54 60 60 60 58 Follicular cell hyperplasia: focal/multifocal Minimal 0 0 0 2 1 1 0 2 Slight 0 0 0 1 1 1 1 2 Moderate 1 0 0 0 0 0 0 0 Marked 0 0 1 0 0 0 0 0 Total 1 0 1 3 2 2 1 4 Follicular cell hypertrophy: diffuse Minimal 1 0 3 5 0 0 1 7 Slight 0 0 1 2 0 0 0 4 Moderate 0 0 0 0 0 0 0 1 Total 1 0 4 7** 0 0 1 12** Colloid alteration Minimal 18 5 12 10 5 6 10 17 Slight 8 9 16 20 2 1 5 15 Moderate 2 1 4 9 0 0 1 3 Marked 0 0 0 0 0 0 0 1 Total 28 15 32 39** 7 7 16* 36** *: p≤0.05, **: p≤0.01