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

Amendment 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 REREGISTRATION 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., Jeffries, T. K., Yano, B. L. (1994). 2,4-Dichlorophenoxyacetic acid: Subchronic 

Neurotoxicity Study in Fischer 344 Rats. The Toxicology Research Laboratory, Dow Chemical Co.; Study No. 

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 Unpublished.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 No. K-002372-064, June 23, 1993. MRID 43293901. Document No. 011614. 

Unpublished. 

EXECUTIVE SUMMARY: In this 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], respectively. 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 weight was observed 

throughout the first year 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 weight was very slight 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 

year 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-year 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 findings 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 females 

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 decreased 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 urinalysis finding was a decrease 

in specific gravity , which was observed in both sexes and time points at the mid- and high-dose levels.

Thyroid 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 weights [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 histiocytosis in females at high dose], 

adipose tissue [atrophy in mid- and high-dose females], testes [atrophy at high dose], and thyroid [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 body 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), decreased 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 hypertrophy 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), and lungs (males).

NEUROTOXICITY STUDY

No treatment-related, hand-held, FOB observations were noted at any of 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. In agreement with the 

chronic toxicity portion of the study, an increased incidence of bilateral retinal degeneration was observed in the 

high-dose females. 

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 study 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 Tables 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 Tables 27-28 [pages 136-139] of the report