[2,4 -D] Chronic Neurotoxicity Screening Battery - Rat [§82 - 7/OPPTS 870.6200]

Am endment to DER for MRID 43293901 - Chronic Neurotoxicity Screening Battery [Rat] [2,4 -DICHLOROPHENOXYACETIC ACID] This amendment provides an EXECUTIVE SUMMARY and data tables to upgrade the original DER [Document No. 011614]. EPA Reviewer: Linda L. Taylor, Ph.D. Reregistration Branch I, Health Effects Division (7509C) EPA Secondary

Reviewer: Whang Phang, Ph.D. Reregistration Branch I, Health Effects Division (7509C) TXR# 0051360 AMENDED DATA EVALUATION RECORD STUDY TYPE : chronic neurotoxicity
- rat § 82
- 7/OPPTS 870.6200 P.C. CODE : 030001 REREGI STRATION CASE NO. : 818706 CASWELL NO. : 315 CAS NO. : 94
- 75
- 7 TEST MATERIAL(PURITY) : 2,4
- Dichlorophenoxyacetic acid [96.7%] SYNONYMS : 2,4
- D SPONSOR : Industry

Task Force II on 2,4
- D Research Data CITATION : Mattsson, J. L. , Je ffries, T. K., Yano, B. L. (1994). 2 ,4
- Dichlorophenoxyacetic acid: Subchronic Neurotoxicity

S tudy in Fischer 344 Rats. The Toxicology Research Laboratory, Dow Chemical Co.; Study N o. K
- 002372
- 064N, June 28, 1994. MRID 43293901. Document No. 011614. Unpublished. Jeffries, T. K., Yano, B. L., and Ormand, J. R. (1995). 2,4
- Dichlorophenoxyacetic acid: Chronic Toxicity /Oncogenicity

Study in Fischer 344 Rats. 1
- Year Interim Report. The Toxicology Research Laboratory, Dow Chemical Co.; Study N o. K
- 002372
- 064, June 23, 1993. MRID 4329390 1. Document No. 011614. Unpublished. EXECUTIVE SUMMARY : Inthis chronic toxicity /carcinogenicity study

with a chronic neurotoxicity screening battery substudy [MRID 43293901], 50 Fischer 344 rats/sex/group [main study]; 15/sex/group [substudy] were administered 2,4
- dichlorophenoxyacetic acid [96.4%] via the diet for up to 24 months at concentrations of 0 [basal diet], 5 mg/kg/day, 75 mg/kg/day, and 150 mg/kg/day [achieved doses were 4.6, 71.2, and 141.1 mg/kg/day

[ males] and 4.6, 68.0, and 138.9 mg/kg/day [females], respectivel y. Additionally, 10 rats/sex/group were sacrificed at 12 months [interim sacrifice]. NOTE: This DER presents the results of the interim [one
-

year] sacrifice and neurotoxicity substudy. There were no treatment- related deaths or clinical signs of toxicity
. Decreased body w eight was observed throughout the first ye ar of the study at the high
- dose level in both sexes [males 91%/females 88% of control at the interim sacrifice] and at the mid
- dose level in females [94% of control at the interim sacrifice]. At 90 days, the decrease in body

w eight was very

s light in the high
- dose males [96% of control] and somewhat greater in the mid
- [95% of control] and high
- dose [90% of control] females. Body-weight gains were decreased throughout the first ye ar at the mid
- and high
- dose levels in both sexes, although statistical significance was not always attained in males at the mid
- dose level [2
- week interval (males: mid
- dose 90%/high
- dose 84%; females: mid
- dose 81%/high
- dose 63% of control); 3
- month interval (males: mid
- dose 98%/high
- dose 88%; females: mid
- dose 87%/high
- dose 75% of control); and 1
- ye ar interval (males: mid
- dose 95%/high
- dose 82%; females: mid
- dose 89%/high
- dose 73% of control). Consistent with the decreased body
- weight gains was a slight decrease in food consumption, which was observed in both sexes at the high
- dose level.[2,4 -D] Chronic Neurotoxicity Screening Battery - Rat [§82 - 7/OPPTS 870.6200]

2 Ophthalmology

f indings were comparable among the groups at the interim sacrifice. Decreased RBC [mid
- and high
- dose females (6 & 12 months) and high
- dose males (12 months)], HCT [mid
- and high
- dose females and high
- dose males (6 & 12 months)], HGB [ high
- dose females (6 months)], WBC 9high
- dose females (6 months)], and platelet counts [mid
- and high
- dose females and high
- dose males (6 & 12 months) were observed. A dose
-related increase in aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase was observed in the mid
- and high
- dose males at 6 months but not at 12 months. The mid
- and high
- dose female s displayed a dose
-related increase in alkaline phosphatase values at both the 6 and 12
- month intervals. Cholesterol levels were decreased in the mid
- (males at 12 months only ) and high
- dose rats of both sexes at both time intervals. T4 values were decreas ed at both time points in both sexes at the mid
- and high
- dose levels, although the mid
- dose males at 6 months did not attain statistical significance. The only urinal ys is finding was a decrease in specific gravity , which was observed in both sexes and time points at the mid
- and high
- dose levels. Thy roid weights [absolute and relative] were statistically
- significantly increased in the mid
- dose females [ 20% greater than control] and in both sexes at the high
- dose level [ 20% greater than control] at the 12
- month interim sacrifice. Decreased testes weights [absolute and relative to brain] were observed at the high
- dose level at 12 months [  15% lower than control]. Kidney w eights [absolute and relative] were increased at the 12
- months sacrifice in males at the mid
- [7%
- 12% greater than control] and high
- dose [8%
- 16% greater than control] levels [dose
-related]. Gross pathology findings included decreased fat in high
- dose females [4/10] and multifocal pale foci in the lungs [1/10 mid
- dose, 10/10 high
- dose females]. Microscopically, there were increased incidences of lesions in the bone marrow [decreased hematopoiesis in high
- dose females], eyes [bilateral retina degeneration in 1 male and 10 females at high dose], kidney [proximal tubule degeneration in mid
- and high
- dose males and females], liver [altered tinctorial properties in mid
- dose females and both sexes at high dose], lungs [multifocal , subacute to chronic inflammation (mid
- dose females, both sexes at high dose), alveolar histiocy tosis in females at high dose], adipose tissue [atrophy

i n mid
- and high
- dose females], testes [atrophy at high dose], and thy

roid [decreased secretory material, epithelial cells
-high
- dose females]. The NOAEL is 5 mg/kg/day. The LOAEL of 75 mg/kg/day is based on decreased bo dy weight (females)/body-weight gain (both sexes), alterations in hematology [decreased RBC, HCT, and platelets (females)], clinical chemistry parameters [increased alanine and aspartate aminotransferases (males), alkaline phosphatase (both sexes), decreas ed cholesterol (both sexes), and decreased T4 (both sexes)], and urinalysis [decreased specific gravity (both sexes)], increased kidney weights (males), increased incidence of degeneration of the descending proximal tubules (both sexes), hepatocellular hyp ertrophy with altered tinctorial properties (females), lung inflammation (females), adipose tissue atrophy (females). At the high - dose level, there also were microscopic lesions in the eyes (both sexes), liver (males), testes (males), thyroid (females), an d lungs (males). NEUROTOXICITY STUDY No treatment-related, hand
- held, FOB observations were noted at any o f the evaluation periods. Relative forelimb grip strength was significantly increased in both sexes at the high
- dose level, but there was no treatment-related change in absolute grip strength. There was no treatment- related effect on motor activity
. Inagreement with the chronic toxicity

portion of the study, an increased incidence of bilateral retinal degeneration was observed in the high
- dose female s. The NOAEL for neurotoxicity is 75 mg/kg/day, based on increased relative forelimb grip strength and increased incidence of bilateral retinal degeneration at the LOAEL of 150 mg/kg/day.[2,4 -D] Chronic Neurotoxicity Screening Battery - Rat [§82 - 7/OPPTS 870.6200]

3 This chronic toxicity

/neurotoxicity

s tudy in the rat is classified ACCEPTABLE/Guideline, and it satisfies the guideline requirement [OPPTS 870. 6200; §82
- 7] for a subchronic neurotoxicity study in the rat.[2,4 -D] Chronic Neurotoxicity Screening Battery - Rat [§82 - 7/OPPTS 870.6200]

4 Table 1. Body
- Weight/Body
- Weight Gain Parameter/Day/Sex/Dose 0 mg/kg/day 5 mg/kg/day 75 mg/kg/day 150 mg/kg/day BODY WEIGHT [g] MALES

- 2 6 27 90 365 FEMALES

- 2 6 27 90 365 197.6±11.5

217.5±13.1 246.0±15.9 308.3±22.1 409.8±28.5 129.2±4.9 139.2±5.6 160.5±7.4 187.0±9.1 217.5±10.9 197.6±9.9

216.8±12.0 247.7±13.9 311.8±20.0 415.5±23.5 127.9±5.6 138.4±5.6 160.3±7.6 186.3±8.6 217.6±12.4 197.1±9.2

214.4±11.1 243.3±14.6 305.3±20.4 400.0±30.2 126.6±5.0* [98]  134.1±5.7* [96] 151.1±6.3* [94] 176.8±8.0* [95] 205.6±11.8* [95] 197.8±10.6 213.5±11.9 239.5±14.6* [97] 295.2±19.4* [96] 372.5±26.6* [91] 126.2±5.2* [98] 130.7±5.1* [94] 145.8±6.1* [91] 169.3±7.3* [91] 190.7±9.1* [88] BODY
- WEIGHT GAIN [g] MALES

- 2
- 6

- 2
- 13

- 2
- 90

- 2
- 365 FEMALES

- 2
- 6

- 2
- 13

- 2
- 90

- 2
- 365 19.9±4.1 33.0±5.7 110.7±15.0 212.2±20.4 10.0±2.7 17.5±3.4 57.8±7.6 88.3±10.4 19.2±5.8

33.3±5.0 114.3±14.7 217.9±19.2 10.5±2.8 19.1±3.1* 58.4±6.5 89.7±10.7 17.3±3.5* [87]

29.8±6.4* [90] 108.3±14.6 202.4±25.8* [95] 7.5±3.5* [75] 14.2±3.3* [81] 50.2±6.6* [87] 79.0±10.6* [89] 15.7±3.6* [79]

28.0±5.6* [85] 97.4±12.9* [88] 175.0±20.5* [82] 4.5±3.7* [45] 11.0±3.5* [63] 43.1±5.9* [75] 64.5±8.0* [73]

 [% of control]; data from Tables 9
- 12 [pages 96
- 107] of the report[2,4 -D] Chronic Neurotoxicity Screening Battery - Rat [§82 - 7/OPPTS 870.6200]

5 Table 2. Clinical Pathology Data
- MALES Dose/Month/Parameter 0 mg/kg/day 5 mg/kg/day 75 mg/kg/day 150 mg/kg/day platelets [x10E3/cumm]

6 12 615±41 586±65 613±32

598±25 604±36

572±33 529±25* [86]

474±45* [81] RBC[ x10E6/cumm] 6 12 9.82±0.35

9.31±0.28 9.85±0.30

9.20±0.22 10.11±0.26

9.36±0.19 9.60±0.34

8.55±0.35* [92] HGB [g/dL] 6 12 17.2±0.5

15.0±0.5 17.2±0.6

15.1±0.3 17.5±0.5

15.1±0.3 17.1±0.4

14.9±0.7 HCT {%} 6 12 61.9±2.2

58.5±1.8 61.9±2.1

58.1±1.5 62.6±1.7

58.2±1.2 59.3±1.7* [86]

55.3±2.7* [95] alanine aminotransferase [U/L]

6 12 65±8 66±18 63±14

61±15 120±61* [185]

91±21* [138] 122±26* [188]

68±15 aspartate aminotransferase [U/L]

6 12 116±16 106±21 111±21

95±20 162±64* [140]

132±39 [125] 172±27* [148]

103±15 blood urea nitrogen [mg/dL]

6 12 17±1 15±2 17±1

13±1 [87] 18±1

14±2 18±3

13±1 [87] creatinine [mg/dL] 6 12 0.7±0.0 0.6±0.0 0.8±0.1

0.6±0.0 0.8±0.1

0.7±0.1* [117] 0.9±0.1* [129]

0.7±0.1* [117] alkaline phosphatase [U/L]

6 12 73±7 57±4 75±7

57±5 85±11* [116]

58±6 92±9* [126]

55±7 glucose [mg/dL] 6 12 112±16 117±9 116±17

124±4 108±15

111±7 102±15 [91]

100±10* [85] cholesterol [mg/dL] 6 12 86±10 87±17 80±3

83±11 84±10

69±11* [79] 71±8* [83]

53±6* [61] triglycerides [mg/dL]

6 12 140±27 101±29 139±36

105±29 171±38

89±21 [88] 129±12

70±9* [69]  [% of control]; n=10; *p<0.05; data from Tables 15
- 16 [pages 112
- 115] and Tables23
- 26 [pages 128
- 144] of the report

Table 3. Clinical Pathology Data
- FEMALES Dose/Month/Parameter 0 mg/kg/day 5 mg/kg/day 75 mg/kg/day 150 mg/kg/day platelets [x10E3/cumm]]

6 12 18 24 599±39 583±23 591±40 562±92 609±44

548±120 [94] 613±47 549±139 461±30* [77]

471±48* [81] 496±33* [84] 551±130 403±30* [67]

486±44* [84] 491±33* [83] 523±135 [93][2,4 -D] Chronic Neurotoxicity Screening Battery - Rat [§82 - 7/OPPTS 870.6200]

6 Table 3. Clinical Pathology Data
- FEMALES Dose/Month/Parameter 0 mg/kg/day 5 mg/kg/day 75 mg/kg/day 150 mg/kg/day RBC[ x10E6/cumm] 6 12 18 24 9.11±0.21

8.37±0.29 8.61±0.18 8.28±1.60 9.05±0.22

8.46±0.50 8.41±0.21 7.91±1.53 [96] 8.71±0.16* [96]

7.78±0.48* [93] 7.83±0.93* [91] 7.44±1.49* [90] 8.22±0.28

7.59±0.25 7.61±0.25* [88] 7.01±1.77* [85] HGB [g/dL] 6 12 18 24 17.2±0.3 14.8±0.4 16.1±0.3 14.7±2.6 17.1±0.3

15.0±0.9 15.8±0.3* [98] 14.6±1.9 16.9±0.4

14.4±0.9 14.8±2.1* [92] 13.9±2.3[95] 16.6±0.4* [97]

14.5±0.6 14.9±0.3* [93] 13.3±3.0 [90] HCT [&] 6 12 18 24 60.6±1.2 57.3±1.9 46.8±1.2 43.6±7.6 60.3±2.0

57.7±3.4 xx.5±0.9

 43.6±4.3 58.4±1.2* [96]

52.6±3.1* [92] 44.0±4.4 41.6±6.4 [95] 55.9±2.3* [92]

51.8±2.2* [90] 43.5±1.4* [93] 40.9±6.5 [94] alanine aminotransferase [U/L]

6 12 18 24 51±8 37±10 42±8 48±28 50±19

42±20 40±5 50±28 56±16

37±8 34±12 61±78 40±6*

33±10 36±4 55±62 aspartate aminotransferase [U/L]

6 12 18 24 96±11 71±15 80±15 92±60 101±35

80±34 70±12 105±110 103±14

63±10 72±8 136±291 [148] 98±13 68±16 81±9 134±216 [146] blood urea nitrogen [mg/dL]

6 12 18 24 20±3 14±1 20±1 20±10 20±3

14±1 18±2 16±2 21±3

15±2 21±3 20±6 21±2

13±2 19±2 25±28 creatinine [mg/dL] 6 12 18 24 0.8±0.1 0.7±0.1 0.7±0.0 0.7±0.1 0.8±0.1

0.7±0.1 0.7±0.0* 0.7±0.0 0.9±0.1* [113]

0.8±0.1* [114] 0.9±0.0* [129] 0.8±0.1* [114] 0.9±0.1* [113]

0.7±0.0* 0.9±0.1* [129] 0.8±0.1* [114] alkaline phosphatase [U/L]

6 12 18 24 47±9 32±3 37±5 47±28 46±4

32±6 40±10 63±50 59±7* [126]

47±8* [147] 69±48* [186] 84±77* [179] 73±16* [155]

54±10* [169] 76±18* [205] 99±52* [211] glucose [mg/dL] 6 12 18 24 96±12 109±8 97±6 94±18 96±10

114±10 103±10 94±11 88±9 [92]

112±9 86±11* [89] 82±18* [87] 72±8[75]

113±11 79±6* [81] 77±11* [82] cholesterol [mg/dL] 6 12 18 24 116±11 105±13 155±9 169±38 123±5

110±16 148±14 166±29 81±6* [70]

54±7* [51] 76±11* [49] 104±57* [62] 76±8* [66]

57±7* [54] 81±4* [52] 109±36* [64] triglycerides [mg/dL]

6 77±14 88±22 88±16 83±6[2,4 -D] Chronic Neurotoxicity Screening Battery - Rat [§82 - 7/OPPTS 870.6200]

7 Table 3. Clinical Pathology Data
- FEMALES Dose/Month/Parameter 0 mg/kg/day 5 mg/kg/day 75 mg/kg/day 150 mg/kg/day

12 18 24 52±8 136±46 183±85 56±18

129±34 180±98 54±6

93±14* [68] 126±52* [69] 56±4

88±14* [65] 105±21* [57]  [% of control]; n=10 [6, 12, 18 months]/n=20 [24 months]; *p<0.05; data from Tables 15
- 16 [pages 112
- 115] and Tables23
- 26 [pages 128
- 144] of the report

Table 4. T4 Results [μg/dL] Dose/Sex/Month 0 mg/kg/day 5 mg/kg/day 75 mg/kg/day 150 mg/kg/day MALES 6 12 3.2±0.5 3.0±0.4 3.4±0.6 3.1±0.3 2.7±0.2 [84]

2.6±0.3* [87] 2.1±0.9* [66] 0.9±0.2* [30] FE MALES 6 12 2.6±0.5 2.3±0.4 2.7±0.4 2.2±0.4 1.1±0.3* [42]

0.8±0.2* [35] 0.9±0.2* [35] 0.7±0.2* [30]  [% of control]; n=10 [6, 12, 18 months]/n=20 [24 months]; *p<0.05; data from T ables 27
- 28 [pages 136
- 139] of the report[2,4 -D] Chronic Neurotoxicity Screening Battery - Rat [§82 - 7/OPPTS 870.6200]

8 Table 5. Organ Weight Data
- MALES [12 months] Dose/Organ 0 mg/kg/day 5 mg/kg/day 75 mg/kg/day 150 mg/kg/day brain absolute [g] relative
- to -body [g/100] 2.039±0.105 0.536±0.016 2.000±0.066

0.515±0.017 2.025±0.062

0.557±0.040 1.988±0.095 [97]  0.056±0.031 liver absolute [g] relative
- to -body [g/100] relative
- to -brain [g/100] 9.508±0.662 2.496±0.062 466.26±19.50 9.630±0.661

2.476±0.111 481.45±30.38 8.543±0.677* [90]

2.338±0.080* [94] 421.69±28.47* [90] 8.942±0.370 2.520±0.125 450.74±30.15 kidney absolute [g] relative
- to -body [g/100] relative
- to -brain [g/100] 2.231±0.105 0.586±0.019 109.47±3.49 2.255±0.111

0.580±0.017 112.75±4.24 2.391±0.150* [107]

0.655±0.026* [112] 118.09±6.45* [108] 2.409±0.125* [108] 0.678±0.013* [116] 121.32±6.73* [111] testes absolute [g] relative
- to -body [g/100] relative
- to -brain [g/100] 3.158±0.293 0.832±0.092 155.10±14.49 3.247±0.128

0.836±0.032 162.35±5.67 3.198±0.193

0.877±0.052 157.96±8.63 2.669±0.540* [85]

0.748±0.139 [90] 134.09±26.32* [86] thyroid absolute [g] relative
- to -body [g/100] relative
- to -brain [g/100] 0.027±0.002 0.0071±0.0006 1.3178±0.1091 0.025±0.002

0.0064±0.0006 1.2517±0.1068 0.026±0.003

0.0070±0.0006 1.2582±0.1198 0.032±0.003* [119]

0.0091±0.0009* [128] 1.6308±0.1805* [124] terminal body weight [g] 380.8±21.9 388.6±14.8 365.5±28.3 355.4±18.8* [93]

 [% of control]; n=10; *p<0.05; data from Tables 27
- 28 [pages 136
- 139] of the report

Table 6. Organ Weight Data
- FEMALES [12 months] Dose/Organ 0 mg/kg/day 5 mg/kg/day 75 mg/kg/day 150 mg/kg/day brain absolute [g] relative
- to -body [g/100] 1.833±0.058 0.926±0.037 1.852±0.051

0.887±0.045 1.837±0.049

0.942±0.060 1.796±0.056

1.013±0.056* [109]  liver absolute [g] relative
- to -body [g/100] relative
- to -brain [g/100] 5.294±0.350 2.673±0.154 288.80±15.35 5.560±0.418

2.655±0.125 300.34±23.66 5.354±0.445

2.735±0.118 291.59±24.39 5.499±0.422

3.094±0.171* [116] 305.97±19.45 [106] kidney absolute [g] relative
- to -body [g/100] relative
- to -brain [g/100] 1.440±0.069 0.727±0.029 78.55±2.53 1.481±0.112

0.708±0.041 80.05±6.75 1.558±0.092

0.797±0.036* [110] 84.89±5.60 [108] 1.544±0.148 [107] 0.868±0.061* [119] 85.94±7.63* [109] ovaries absolute [g] relative
- to -body [g/100] relative
- to -brain [g/100] 0.057±0.013 0.029±0.006 3.11±0.70 0.056±0.012

0.027±0.005 3.03±0.64 0.058±0.005

0.029±0.003 3.13±0.24 0.052±0.009 [91] 0.029±0.005 2.87±0.44 [92] thyroid absolute [g] relative
- to -body [g/100] relative
- to -brain [g/100] 0.018±0.001 0.0093±0.0008 1.0052±0.0872 0.019±0.002

0.0091±0.0010 1.0276±0.1280 0.022±0.001* [122]

0.0111±0.0006* [119] 1.1816±0.0659* [118] 0.022±0.003* [122] 0.0126±0.0012* [135] 1.2463±0.1265* [124] terminal body weight [g] 198.2±10.5 209.3±10.5 195.7±13.1 177.7±9.8* [90]

 [% of control];*p<0.05; n=10; data from T ables 27
- 28 [pages 136
- 139] of the report