18.7
                                      15
                                     38.69
                                     6.79
                                     17.6
                                      15
                                     36.29
                                     6.00
                                     16.5
                                      15
                                     37.99
                                     7.86
                                     20.7

                                       A
                                      15
                                     38.18
                                     3.64
                                      9.5
                                      15
                                     38.70
                                     3.64
                                      9.4
                                      15
                                     36.29
                                     3.64
                                     10.0
                                      15
                                     37.99
                                     3.64
                                      9.6

                                       R
                                      15
                                     0.03
                                     0.01
                                     21.2
                                      15
                                     0.03
                                     0.01
                                     16.6
                                      15
                                     0.03
                                     0.00
                                     13.8
                                      15
                                     0.03
                                     0.01
                                     21.4
Ovaries
(mg)
                                       U
                                      15
                                     1.64
                                     0.10
                                      6.3
                                      15
                                     1.63
                                     0.13
                                      7.7
                                      15
                                1.40** (↓15)
                                     0.10
                                      6.9
                                      15
                                     1.58
                                     0.13
                                      8.0

                                       A
                                      15
                                     44.67
                                     5.10
                                     11.4
                                      15
                                     44.27
                                     5.10
                                     11.5
                                      15
                                25.36** (↓43)
                                     5.10
                                     20.1
                                      15
                                     39.78
                                     5.10
                                     12.8
Uterus, wet
(mg)
                                       U
                                      15
                                     262.4
                                     88.2
                                     33.6
                                      15
                                     261.3
                                     118.4
                                     45.3
                                      15
                                 198.1 (↓24)
                                     168.1
                                     84.8
                                      15
                                     226.3
                                     81.8
                                     36.2

                                       A
                                      15
                                     262.4
                                     62.1
                                     23.7
                                      15
                                     261.3
                                     62.06
                                     23.8
                                      15
                                 198.1 (↓24)
                                     62.06
                                     31.3
                                      15
                                     226.3
                                     62.1
                                     27.4
Uterus, blotted
(mg)
                                       U
                                      15
                                     231.1
                                     64.9
                                     28.1
                                      15
                                     222.7
                                     66.5
                                     29.9
                                      15
                                140.2** (↓39)
                                     94.28
                                     67.3
                                      15
                                     198.5
                                     67.3
                                     33.9

                                       A
                                      15
                                     231.1
                                     38.7
                                     16.7
                                      15
                                     222.7
                                     38.68
                                     17.4
                                      15
                                140.1** (↓39)
                                     38.68
                                     27.6
                                      15
                                     198.5
                                     38.7
                                     19.5
Thyroid, fixed
(mg)
                                       U
                                      15
                                     11.70
                                     1.35
                                     11.5
                                      15
                                     11.69
                                     1.84
                                     15.7
                                      15
                                10.11** (↓14)
                                     1.07
                                     10.6
                                      15
                                     11.57
                                     1.71
                                     14.8

                                       A
                                      15
                                     11.69
                                     0.79
                                      6.7
                                      15
                                     11.68
                                     0.79
                                      6.7
                                      15
                                10.11** (↓14)
                                     0.79
                                      7.8
                                      15
                                     11.57
                                     0.79
                                      6.8
a Data were obtained from the study report.  Percent difference from control is included in parentheses.
b 100 mg 1-Chloro-2-Nitrobenzene
U=Unadjusted for body weight on PND 21;  A =Adjusted for body weight on PND 21; R =Organ-to-body weight ratio (relative to body weight)
N=Number of animals examined; SD=Standard Deviation; CV=Coefficient of Variation
*Significantly different from controls at p<0.05.**Significantly different from controls at p<0.01.

Table 30:  Estrous Cyclicity Data of Female Rats Treated with Chemical S in the Female Pubertal Assay a
                               Treatment Groups
                                       N
                    Mean Age at First Vaginal Estrus (PND)
                           Mean Cycle Length (days)
                                  Cycling (%)
                             Regularly Cycling (%)
                           Cycle Status at Necropsy
                                  (# Females)

                                   Diestrus
                                   Proestrus
                                    Estrus
                                  Not Cycling
Vehicle
                                      15
                                      38
                                      5.6
                                     66.67
                                     80.00
                                      11
                                       1
                                       3
                                       5
Chemical S (10 mg/kg/day)
                                      15
                                      40
                                      3.5
                                     80.00
                                    66.67*
                                       7
                                       2
                                       4
                                       1
Chemical S (75 mg/kg/day)
                                      15
                                      ---
                                      ---
                                     20.00
                                     0.00*
                                       2
                                       1
                                       0
                                      ---
Positive Control b
                                      15
                                      40
                                      4.0
                                     80.00
                                    33.33*
                                       7
                                       0
                                       5
                                       0
a Data were obtained from the study report; b 100 mg 1-Chloro-2-Nitrobenzene
--- The considerable delay in onset of vaginal opening in the 75 mg/kg/day group (PND 38; sacrifice at PND 42) precluded the proper evaluation of the estrous cycle and mean age at first vaginal estrous.
N=Number of animals
*Significantly different from controls at p<0.05.
Table 31:  Hormone Levels and Clinical Chemistry Data of Female Rats Treated with Chemical S in the Female Pubertal Assay a
                                   Parameter
                                Vehicle Control
                                  Chemical S
                                10 mg/kg bw/day
                                  Chemical S
                                75 mg/kg bw/day
                              Positive Control b

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                               Thyroid Hormones
Total T4 (ug/dL)
                                      15
                                     3.404
                                     1.128
                                     33.2
                                      15
                                     3.600
                                     0.792
                                     22.0
                                      11
                                     5.391
                                     3.742
                                     69.4
                                      15
                                     3.004
                                     0.735
                                     24.5
TSH (ng/mL)
                                       9
                                     4.098
                                     4.507
                                     110.0
                                       9
                                     4.842
                                     3.559
                                     73.5
                                      15
                                     3.215
                                     0.849
                                     26.4
                                      12
                                     2.197
                                     1.973
                                     89.8
                              Clinical Chemistry
Glucose (mg/dL)
                                      15
                                     141.4
                                     17.76
                                     12.6
                                      15
                                     139.8
                                     12.77
                                      9.1
                                      15
                                122.1* (↓14)
                                     26.36
                                     21.6
                                      15
                                121.4* (↓14)
                                     19.87
                                     16.4
ALT (IU/L)
                                      15
                                     83.08
                                     21.28
                                     25.6
                                      15
                                     72.67
                                     11.61
                                     16.0
                                      15
                                     86.48
                                     20.02
                                     23.2
                                      15
                                     92.62
                                     25.42
                                     27.4
AST (IU/L)
                                      15
                                     272.0
                                     75.95
                                     27.9
                                      15
                                     260.8
                                     58.08
                                     22.3
                                      15
                                     248.8
                                     63.17
                                     25.4
                                      15
                                     232.0
                                     26.52
                                     11.4
Total Bilirubin (mg/dL)
                                      15
                                     0.07
                                     0.10
                                     142.6
                                      15
                                     0.06
                                     0.08
                                     151.6
                                      15
                                     0.12
                                     0.12
                                     102.6
                                      15
                                0.40** (↑471)
                                     0.17
                                     43.4
Creatinine (mg/dL)
                                      15
                                     0.31
                                     0.11
                                     36.4
                                      15
                                     0.32
                                     0.11
                                     36.0
                                      15
                                     0.27
                                     0.09
                                     33.1
                                      15
                                     0.34
                                     0.12
                                     34.4
BUN (mg/dL)
                                      15
                                     14.87
                                     1.72
                                     11.6

                                     15.11
                                     2.02
                                     13.4

                                     13.65
                                     3.34
                                     24.5

                                     16.52
                                     3.22
                                     19.5
Total Protein (g/dL)
                                      15
                                     5.13
                                     0.53
                                     10.3
                                      15
                                     5.25
                                     0.37
                                      7.0
                                      15
                                 4.62* (↓10)
                                     0.50
                                     10.9
                                      15
                                     5.36
                                     0.46
                                      8.7
Albumin (g/dL)
                                      15
                                     4.02
                                     0.35
                                      8.6
                                      15
                                     4.15
                                     0.34
                                      8.2
                                      15
                                 3.68* (↓8)
                                     0.41
                                     11.1
                                      15
                                     4.18
                                     0.38
                                      9.1
Calcium (mg/dL)
                                      15
                                     9.11
                                     0.55
                                      6.0
                                      15
                                     9.24
                                     0.49
                                      5.3
                                      15
                                     9.36
                                     0.95
                                     10.1
                                      15
                                     9.70
                                     0.74
                                      7.7
Phosphorus (mg/dL)
                                      15
                                     7.96
                                     1.16
                                     14.5
                                      15
                                     8.05
                                     0.50
                                      6.2
                                      15
                                     8.08
                                     1.27
                                     15.7
                                      15
                                     8.16
                                     0.89
                                     11.0
Cholesterol (mg/dL)
                                      15
                                     71.68
                                     9.51
                                     13.3
                                      15
                                     75.30
                                     8.94
                                     11.9
                                      15
                                     77.36
                                     14.44
                                     18.7
                                      15
                                86.67** (↑21)
                                     10.89
                                     12.6
Urea (mg/dL)
                                      15
                                     31.85
                                     3.69
                                     11.6
                                      15
                                     32.36
                                     4.33
                                     13.4
                                      15
                                     29.22
                                     7.16
                                     24.5
                                      15
                                     35.38
                                     6.90
                                     19.5
ALP (lU/dL)
                                      15
                                     246.2
                                     52.46
                                     21.3
                                      15
                                     252.3
                                     58.17
                                     23.1
                                      15
                                     290.6
                                     79.36
                                     27.3
                                      15
                                     282.4
                                     52.75
                                     18.7
Globulin (g/dL)
                                      15
                                     1.11
                                     0.24
                                     21.5
                                      15
                                     1.10
                                     0.15
                                     13.6
                                      15
                                     0.94
                                     0.27
                                     29.0
                                      15
                                     1.18
                                     0.17
                                     14.5
Albumin/Globulin
                                      15
                                     3.72
                                     0.60
                                     16.1
                                      15
                                     3.84
                                     0.55
                                     14.4
                                      15
                                     4.15
                                     0.97
                                     23.3
                                      15
                                     3.60
                                     0.55
                                     15.2
   [a] Data were obtained from the study report.  Percent differences from controls are included in parentheses.

              viii. Pubertal Male Assay (Rat)
In a Male Pubertal Assay, 15 Wistar rats/dose group were treated daily via oral gavage with Chemical S (97.85% a.i.) in corn oil at doses of 0, 10, or 75 mg/kg bw/day from post-natal day (PND) 23 to 53.  An additional positive control group of 15 rats was similarly treated with 1-chloro-2-nitrobenzene in corn oil daily via oral gavage at a dose of 100 mg/kg bw/day.  Animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and body weight at day of attainment were recorded.  Following sacrifice on PND 53, blood was collected for clinical chemistry analyses, including selected hormones.  Total serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were determined using the enzyme-linked immunosorbent assay (ELISA).  Liver, adrenals, pituitary, thyroid, and urogenital organs were collected and weighed.  Microscopic examinations were performed on the right testes, right epididymis, thyroid, one kidney, liver, pituitary, adrenals, spleen, and gross lesions.
Summary data are presented in Tables 32 through 36. No treatment-related effects were observed on mortality or gross or microscopic pathology.  No effects were noted on the thyroid of the treated groups compared to vehicle controls:  thyroid colloid area and follicular cell height scores were similar; organ weights were similar; and no gross or microscopic lesion was reported.
At 10 mg/kg bw/day, no adverse, treatment-related effects were noted.  An increase (p<0.01) of 22% in adjusted dorsolateral prostate weight was noted compared to the vehicle controls, but the effect was considered incidental because the effect was a slight increase in weight without effects noted on other androgen-sensitive organs.  Increased incidences (number affected / number exposed) of slight to moderate salivation were observed in the 10 mg/kg bw/day group (2/15) compared to the vehicle controls (0/15).  This effect was considered minor and not adverse due to the lack of dose-response (i.e, absence of effect at the high dose).  Unadjusted and adjusted age at partial PPS was increased (p<0.01) by only 2.2 and 2.4 days, respectively; this  effect was not corroborated with other evidence of delayed sexual development such as changes in androgen-dependent tissue weights or serum testosterone concentrations.
In the high dose group (75 mg/kg bw/day), there was an increased incidence of slight to moderate salivation observed in all animals compared to none in the vehicle control group.  Body weights were decreased (p<0.05) by 10-22% from PND 27 to 53 compared to vehicle controls.  Adjusted and unadjusted final body weights were decreased (p<0.01) by 20% each, and adjusted and unadjusted overall body weight gains (PND 23-53) were decreased (p<0.01) by 26% each.  The adjusted body weight at PPS was decreased (p<0.05) by 11%.  Corresponding to the body weight decreases, food consumption was decreased (p<0.05) by 10-28% during the measured intervals from PND 23-30 through 51-53.
At 75 mg/kg bw/day, the unadjusted and adjusted ages at partial PPS was delayed (p<0.01) by 3.5 days compared to the control, and unadjusted and adjusted ages at PPS were delayed (p<0.01) by 5.9 days.  In addition, all of the controls completed PPS by necropsy day, as compared to only 12 of 15 of the 75 mg/kg bw/day males.  Testosterone was decreased (p<0.05) by 56% in the high dose group.  The following decreases (p<0.05) in unadjusted and adjusted androgen-sensitive tissues organ weights were observed compared to vehicle controls (% decrease):  (i) seminal vesicle and coagulating glands (with and without fluid; 48-55%); (ii) ventral prostate (42% each); (iii) dorsolateral prostate (26% each); (iv) LABC (43-44%); (v) left and right epididymides (24 - 25%); and (iv) left and right testes (15-17%).
At 75 mg/kg bw/day, the following differences (p<0.05) were noted in organ weights compared to controls:  (i) unadjusted and adjusted liver weights decreased by 13% each and relative (to body) liver weight increased by 8%; (ii) unadjusted and adjusted kidney weight decreased by 24% each; and (iii) unadjusted and adjusted adrenal weights decreased by 4 - 12%.  These effects were not considered adverse due to slight or minor magnitude without additional evidence from clinical chemistry or gross or microscopic pathology.  In addition, alanine aminotransferase (ALT) was increased (p<0.05) by 21%, and alkaline phosphatase (ALP) was decreased (p<0.01) by 34%.  These slight effects on ALT and ALP were not considered adverse.
In the positive control group, slight to moderate salivation was also observed (12/15) compared to 0/15 vehicle controls.  An incidental decrease (p<0.05) was noted in mean body weight on PND 29, and a minor increase (p<0.05) was observed in adjusted body weight at PPS.  An incidental decrease (p<0.01) in food consumption was noted on PND 23-30.  Unadjusted and adjusted ages at partial PPS were increased (p<0.05) by 2.8-3.0 days, and unadjusted and adjusted ages at PPS were increased (p<0.01) by 3.0-3.1 days.  The following differences (p<0.05) were observed in organ weights:  (i) unadjusted, adjusted, and relative liver weights were increased by 56-60%; (ii) relative kidney weight were increased by 7%; (iii) unadjusted and adjusted ventral prostate weights were decreased by 18% each; (iv) adjusted left and right epididymides weights were decreased by 10% each; and (v) adjusted left and right testes weights were decreased by 6-7%.  Total T4 was decreased (p<0.01) by 17%.  The following increases (p<0.05) were noted in clinical chemistry parameters:  ALT (58%), aspartate aminotransferase (AST; 18%), albumin (5%), and cholesterol (21%).  Spleen size was increased in all animals of the positive control group compared to no animals in the other groups.  All animals were observed with liver degeneration (with single cell necrosis) and spleen extramedullary hematopoiesis (with sinusoidal congestion) compared to none in the vehicle control group.
Chemical S was negative for A and T pathways in the pubertal male assay.
Table 32:  Selected Body Weight of Male Rats Treated with Chemical S in the Male Pubertal Assay a
                                      PND
                                Vehicle Control
                                  Chemical S
                                 10 mg/kg/day
                                  Chemical S
                                 75 mg/kg/day
                              Positive Control b

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                      23
                                      15
                                     43.52
                                     4.59
                                     10.5
                                      15
                                     44.71
                                     3.77
                                      8.4
                                      15
                                     43.61
                                     3.59
                                      8.2
                                      15
                                     44.35
                                     3.86
                                      8.7
                                      27
                                      15
                                     58.53
                                     6.45
                                     11.0
                                      15
                                     60.17
                                     4.93
                                      8.2
                                      15
                                52.93* (↓10)
                                     5.59
                                     10.6
                                      15
                                     54.07
                                     5.24
                                      9.7
                                      41
                                      15
                                    128.91
                                     12.75
                                      9.9
                                      15
                                    130.91
                                     11.48
                                      8.8
                                      15
                               100.15** (↓22)
                                     13.66
                                     13.6
                                      15
                                    122.09
                                     14.73
                                     12.1
                                      53
                                      15
                                    190.02
                                     14.29
                                      7.5
                                      15
                                    192.85
                                     13.44
                                      7.0
                                      15
                               151.49** (↓20)
                                     22.80
                                     15.1
                                      15
                                    186.53
                                     18.27
                                      9.8
   a	Data were obtained from the study report.  Percent difference from control is included in parentheses.
   b	100 mg/kg/day 1-Chloro-2-Nitrobenzene
   N	Number of animals examined
   SD	Standard Deviation
   CV	Coefficient of Variation
   *	Significantly different from controls at p<0.05.
   **	Significantly different from controls at p<0.01.

Table 33:  Selected Food Consumption Data of Male Rats Treated with Chemical S in the Male Pubertal Assay [a]
                                      PND
                                Vehicle Control
                                  Chemical S
                                10 mg/kg bw/day
                                  Chemical S
                                75 mg/kg bw/day
                              Positive Control b

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                     23-30
                                       8
                                     9.37
                                     0.79
                                      8.5
                                       8
                                     9.60
                                     0.66
                                      6.8
                                       8
                                6.86** (↓27)
                                     1.11
                                     16.2
                                       8
                                7.78** (↓17)
                                     0.89
                                     11.4
                                     30-37
                                       8
                                     15.00
                                     0.90
                                      6.0
                                       8
                                     15.07
                                     0.98
                                      6.5
                                       8
                                10.82** (↓28)
                                     1.65
                                     15.2
                                       8
                                     13.79
                                     1.40
                                     10.1
                                     37-44
                                       8
                                     18.28
                                     0.77
                                      4.2
                                       8
                                     18.51
                                     1.12
                                      6.0
                                       8
                                13.49** (↓26)
                                     2.33
                                     17.3
                                       8
                                     17.78
                                     1.17
                                      6.6
                                     44-51
                                       8
                                     18.69
                                     1.12
                                      6.0
                                       8
                                     18.40
                                     0.75
                                      4.1
                                       8
                                15.93** (↓15)
                                     2.15
                                     13.5
                                       8
                                     19.69
                                     0.96
                                      4.9
                                     51-53
                                       8
                                     19.65
                                     1.12
                                      5.7
                                       8
                                     19.74
                                     1.15
                                      5.8
                                       8
                                17.61* (↓10)
                                     1.95
                                     11.1
                                       8
                                     20.50
                                     1.43
                                      7.0
      a Data were obtained from the study report. Percent difference from control is included in parentheses.
      b100 mg/kg/day 1-Chloro-2-Nitrobenzene
      N=Number of animals examined
      SD=Standard Deviation
      CV=Coefficient of Variation
      *Significantly different from controls at p<0.05
      **Significantly different from controls at p<0.01
Table 34:  General Growth and Preputial Separation of Rats Treated with Chemical S in the Male Pubertal Assay a
                              Parameter Evaluated
                                Vehicle Control
                                  Chemical S
                                10 mg/kg bw/day
                                  Chemical S
                                75 mg/kg bw/day
                              Positive Control b

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                        Initial Body Weight (PND 23; g)
                                       U
                                      15
                                     43.5
                                     4.59
                                     10.5
                                      15
                                     44.7
                                     3.77
                                      8.4
                                      15
                                     43.6
                                     3.59
                                      8.2
                                      15
                                     44.4
                                     3.86
                                      8.7

                                       A
                                      15
                                     43.9
                                     0.65
                                      1.5
                                      15
                                     44.2
                                     0.65
                                      1.5
                                      15
                                     43.9
                                     0.65
                                      1.5
                                      15
                                     44.2
                                     0.65
                                      1.5
                            Body Weight at PPS (g)
                                       U
                                      15
                                      162
                                     14.3
                                      8.8
                                      15
                                      169
                                     13.1
                                      7.8
                                       3
                                      149
                                     16.2
                                     10.9
                                      14
                                      174
                                     11.2
                                      6.4

                                       A
                                      15
                                      163
                                     12.8
                                      7.9
                                      15
                                      169
                                     12.8
                                      7.6
                                       3
                                 145* (↓11)
                                     13.2
                                      9.1
                                      14
                                  174* (↑7)
                                     12.8
                                      7.3
                         Final Body Weight (PND 53; g)
                                       U
                                      15
                                      190
                                     14.3
                                      7.5
                                      15
                                      193
                                     13.4
                                      7.0
                                      15
                                 152** (↓20)
                                     22.8
                                     15.1
                                      15
                                      187
                                     18.3
                                      9.8

                                       A
                                      15
                                      191
                                     7.89
                                      4.1
                                      15
                                      192
                                     7.90
                                      4.1
                                      15
                                 152** (↓20)
                                     7.89
                                      5.2
                                      15
                                      186
                                     7.89
                                      4.2
                       Final Body Weight (% of control)
                                       U
                                      NA
                                     98.5
                                     79.7
                                     98.2

                                       A
                                      NA
                                     99.5
                                     79.7
                                     97.6
                        Body Weight Gain (PND 23-53; g)
                                       U
                                      15
                                      147
                                     11.3
                                      7.7
                                      15
                                      148
                                     11.4
                                      7.7
                                      15
                                 108** (↓26)
                                     21.5
                                     20.0
                                      15
                                      142
                                     15.9
                                     11.2

                                       A
                                      15
                                      147
                                      7.7
                                      5.2
                                      15
                                      148
                                     7.67
                                      5.2
                                      15
                                 108** (↓26)
                                     7.66
                                      7.1
                                      15
                                      142
                                     7.66
                                      5.4
                           Age at Partial PPS (PND)
                                       U
                                      15
                                     34.9
                                     2.13
                                      6.1
                                      15
                                 37.1** (↑6)
                                     1.92
                                      5.2
                                      15
                                38.3** (↑10)
                                     1.05
                                      2.7
                                      15
                                 37.7* (↑8)
                                     1.33
                                      3.5

                                       A
                                      15
                                     34.8
                                     0.79
                                      2.3
                                      15
                                 37.2** (↑7)
                                     0.79
                                      2.1
                                      15
                                38.3** (↑10)
                                     0.79
                                      2.1
                                      15
                                 37.8** (↑8)
                                     0.79
                                      2.1
                               Age at PPS (PND)
                                       U
                                      15
                                     47.5
                                     2.50
                                      5.3
                                      15
                                     48.3
                                     2.61
                                      5.4
                                      15
                                53.4** (↑12)
                                     1.24
                                      2.3
                                      15
                                 50.5** (↑6)
                                     2.83
                                      5.6

                                       A
                                      15
                                     47.5
                                     1.13
                                      2.4
                                      15
                                     48.5
                                     1.13
                                      2.3
                                      15
                                53.3** (↑12)
                                     1.13
                                      2.1
                                      15
                                 50.6** (↑7)
                                     1.12
                                      2.2
                         Proportion unseparated (#/N)
                                     0/15
                                     0/15
                                     12/15
                                     1/15
      a Data were obtained from the study report.  Percent difference from control  is included in parentheses.
      b 100 mg/kg/day 1-Chloro-2-Nitrobenzene
      U =Unadjusted for body weight on PND 21; A=Adjusted for body weight on PND 21;
      N=Number of animals examined
      SD=Standard Deviation; CV=Coefficient of Variation; NA=Not applicable
      * Significantly different from controls at p<0.05;** Significantly different from controls at p<0.01.
Table 35:  Organ Weight Data of Male Rats Treated with Chemical S in the Male Pubertal Assay a
                                     Organ
                                Vehicle Control
                                  Chemical S
                                10 mg/kg bw/day
                                  Chemical S
                                75 mg/kg bw/day
                              Positive Control b

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
Liver (g)
                                       U
                                      15
                                     8.46
                                     0.82
                                      9.6
                                      15
                                     8.62
                                     0.789
                                      9.1
                                      15
                                 7.33* (↓13)
                                     1.37
                                     18.7
                                      15
                                13.3** (↑57)
                                     1.77
                                     13.3

                                       A
                                      15
                                     8.53
                                     0.55
                                      6.4
                                      15
                                     8.53
                                     0.548
                                      6.4
                                      15
                                7.38** (↓13)
                                     0.55
                                      7.4
                                      15
                                13.3** (↑56)
                                     0.55
                                      4.1

                                       R
                                      15
                                     4.45
                                     0.22
                                      5.0
                                      15
                                     4.47
                                     0.227
                                      5.1
                                      15
                                 4.82** (↑8)
                                     0.38
                                      7.9
                                      15
                                7.11** (↑60)
                                     0.40
                                      5.6
Kidneys (g)
                                       U
                                      15
                                     1.25
                                     0.12
                                      9.8
                                      15
                                     1.21
                                     0.104
                                      8.6
                                      15
                                0.94** (↓24)
                                     0.14
                                     14.7
                                      15
                                     1.30
                                     0.16
                                     11.9

                                       A
                                      15
                                     1.25
                                     0.06
                                      4.9
                                      15
                                     1.20
                                     0.061
                                      5.1
                                      15
                                0.95** (↓24)
                                     0.06
                                      6.5
                                      15
                                     1.30
                                     0.06
                                      4.7

                                       R
                                      15
                                     0.656
                                     0.04
                                      6.8
                                      15
                                     0.628
                                     0.036
                                      5.8
                                      15
                                     0.626
                                     0.05
                                      8.2
                                      15
                                0.699** (↑7)
                                     0.65
                                     22.7
Pituitary (mg)
                                       U
                                      15
                                      5.6
                                      1.6
                                     29.4
                                      15
                                      5.6
                                      0.9
                                     16.4
                                      15
                                      4.9
                                      1.2
                                     25.3
                                      15
                                      5.3
                                      1.1
                                     21.3

                                       A
                                      15
                                     5.60
                                     0.66
                                     11.8
                                      15
                                     5.65
                                     0.659
                                     11.7
                                      15
                                     4.86
                                     0.66
                                     13.5
                                      15
                                     5.35
                                     0.66
                                     12.3

                                       R
                                      15
                                     0.003
                                     0.00
                                     27.7
                                      15
                                     0.003
                                     0.001
                                     18.7
                                      15
                                     0.003
                                     0.00
                                     27.5
                                      15
                                     0.003
                                     0.00
                                     22.9
Adrenals (mg)
                                       U
                                      15
                                     49.1
                                      3.8
                                      7.7
                                      15
                                     48.7
                                      6.6
                                     13.6
                                      15
                                 43.1* (↓4)
                                      5.8
                                     13.4
                                      15
                                     48.8
                                      8.2
                                     16.9

                                       A
                                      15
                                     49.2
                                     3.11
                                      6.3
                                      15
                                     48.4
                                     3.11
                                      6.4
                                      15
                                43.3** (↓12)
                                     3.11
                                      7.2
                                      15
                                     48.7
                                     3.11
                                      6.4

                                       R
                                      15
                                     0.026
                                     0.00
                                      8.8
                                      15
                                     0.025
                                     0.004
                                     15.5
                                      15
                                     0.029
                                     0.00
                                     16.6
                                      15
                                     0.026
                                     0.00
                                     16.7
Seminal vesicle + CG, with fluid (mg)
                                       U
                                      15
                                      246
                                     72.9
                                     29.7
                                      15
                                      252
                                     62.8
                                     25.0
                                      15
                                 112** (↓55)
                                     29.7
                                     26.6
                                      15
                                      217
                                     70.5
                                     32.5

                                       A
                                      15
                                      248
                                     29.0
                                     11.7
                                      15
                                      248
                                     29.0
                                     11.7
                                      15
                                 113** (↓54)
                                     28.9
                                     25.5
                                      15
                                      216
                                     28.9
                                     13.4
Seminal vesicle + CG, without fluid (mg)
                                       U
                                      15
                                      198
                                     45.3
                                     22.9
                                      15
                                      209
                                     45.0
                                     21.5
                                      15
                                 103** (↓48)
                                     25.0
                                     24.3
                                      15
                                      185
                                     55.6
                                     30.1

                                       A
                                      15
                                      199
                                     20.3
                                     10.2
                                      15
                                      206
                                     20.3
                                      9.9
                                      15
                                 104** (↓48)
                                     20.3
                                     19.5
                                      15
                                      184
                                     20.3
                                     11.0
Ventral prostate (mg)
                                       U
                                      15
                                      113
                                     16.4
                                     14.5
                                      15
                                      119
                                     17.7
                                     14.9
                                      15
                                65.3** (↓42)
                                     15.3
                                     23.5
                                      15
                                 92.8* (↓18)
                                     24.5
                                     26.4

                                       A
                                      15
                                      113
                                     9.15
                                      8.1
                                      15
                                      118
                                     9.16
                                      7.7
                                      15
                                65.8** (↓42)
                                     9.14
                                     13.9
                                      15
                                92.6** (↓18)
                                     9.14
                                      9.9
Dorsolateral prostate (mg)
                                       U
                                      15
                                     37.3
                                      9.7
                                     26.1
                                      15
                                     45.6
                                     12.4
                                     27.2
                                      15
                                 27.5* (↓26)
                                      7.7
                                     28.1
                                      15
                                     41.6
                                      8.2
                                     19.8

                                       A
                                      15
                                     37.3
                                     5.05
                                     13.5
                                      15
                                45.6** (↑22)
                                     5.06
                                     11.1
                                      15
                                27.6** (↓26)
                                     5.05
                                     18.3
                                      15
                                     41.6
                                     5.05
                                     12.1
LABC (mg)
                                       U
                                      15
                                      296
                                     48.1
                                     16.2
                                      15
                                      285
                                     56.1
                                     19.7
                                      15
                                 167** (↓44)
                                     49.3
                                     29.5
                                      15
                                      277
                                     53.4
                                     19.3

                                       A
                                      15
                                      298
                                     24.1
                                      8.1
                                      15
                                      282
                                     24.1
                                      8.6
                                      15
                                 168** (↓43)
                                     24.1
                                     14.3
                                      15
                                      277
                                     24.1
                                      8.7
Epididymis, left (mg)
                                       U
                                      15
                                      216
                                     39.3
                                     18.2
                                      15
                                      221
                                     26.8
                                     12.1
                                      15
                                 161** (↓25)
                                     31.1
                                     19.3
                                      15
                                      196
                                     24.4
                                     12.5

                                       A
                                      15
                                      217
                                     13.9
                                      6.4
                                      15
                                      219
                                     13.9
                                      6.4
                                      15
                                 162** (↓25)
                                     13.9
                                      8.6
                                      15
                                 195* (↓10)
                                     13.9
                                      7.1
Epididymis, right (mg)
                                       U
                                      15
                                      216
                                     38.5
                                     17.8
                                      15
                                      221
                                     25.2
                                     11.4
                                      15
                                 164** (↓24)
                                     29.8
                                     18.2
                                      15
                                      195
                                     22.2
                                     11.4

                                       A
                                      15
                                      217
                                     14.4
                                      6.6
                                      15
                                      219
                                     14.4
                                      6.6
                                      15
                                 164** (↓24)
                                     14.4
                                      8.8
                                      15
                                 195* (↓10)
                                     14.4
                                      7.4
Testis, left (mg)
                                       U
                                      15
                                     1227
                                      108
                                      8.8
                                      15
                                     1236
                                     90.9
                                      7.4
                                      15
                                1039** (↓15)
                                      160
                                     15.4
                                      15
                                     1160
                                     92.8
                                      8.0

                                       A
                                      15
                                     1232
                                     52.4
                                      4.3
                                      15
                                     1228
                                     52.5
                                      4.3
                                      15
                                1043** (↓15)
                                     52.4
                                      5.0
                                      15
                                 1158* (↓6)
                                     52.4
                                      4.5
Testis, right (mg)
                                       U
                                      15
                                     1224
                                      113
                                      9.2
                                      15
                                     1224
                                     95.1
                                      7.8
                                      15
                                1019** (↓17)
                                      170
                                     16.7
                                      15
                                     1147
                                     89.4
                                      7.8

                                       A
                                      15
                                     1230
                                     54.4
                                      4.4
                                      15
                                     1216
                                     54.5
                                      4.5
                                      15
                                1023** (↓17)
                                     54.4
                                      5.3
                                      15
                                 1145* (↓7)
                                     54.4
                                      4.7
Thyroid, fixed
(mg)
                                       U
                                      15
                                     13.1
                                      1.7
                                     13.0
                                      15
                                     13.8
                                      2.0
                                     14.3
                                      15
                                     12.1
                                      2.4
                                     20.2
                                      15
                                     13.5
                                      2.5
                                     18.6

                                       A
                                      15
                                     13.1
                                     1.14
                                      8.7
                                      15
                                     13.8
                                     1.14
                                      8.2
                                      15
                                     12.1
                                     1.14
                                      9.4
                                      15
                                     13.5
                                     1.14
                                      8.4

a Data were obtained from the study report.  Percent difference from control is included in parentheses.
b 100 mg 1-Chloro-2-Nitrobenzene
U=Unadjusted for body weight on PND 21
A=Adjusted for body weight on PND 21
R=Organ-to-body weight ratio (relative to body weight)
N=Number of animals examined
SD=Standard Deviation
CV=Coefficient of Variation
*Significantly different from controls at p<0.05
**Significantly different from controls at p<0.01

Table 36:  Hormone Levels and Clinical Chemistry Data of Male Rats Treated with Chemical S in the Male Pubertal Assay a
                                   Parameter
                                Vehicle Control
                                  Chemical S
                                10 mg/kg bw/day
                                  Chemical S
                                75 mg/kg bw/day
                              Positive Control b

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                               Thyroid Hormones
Total T4 (ug/dL)
                                      15
                                     4.178
                                     0.543
                                     13.0
                                      14
                                     4.493
                                     0.438
                                      9.8
                                      15
                                     3.971
                                     0.486
                                     12.3
                                      15
                                3.487** (↓17)
                                     0.466
                                     13.4
TSH (ng/mL)
                                       9
                                     2.363
                                     1.877
                                     79.4
                                       9
                                     2.568
                                     1.796
                                     69.9
                                      14
                                     1.932
                                     1.739
                                     90.0
                                      10
                                     1.173
                                     1.105
                                     94.1
Testosterone (ng/mL)
                                      14
                                     1.836
                                     1.059
                                     57.7
                                      15
                                     2.215
                                     1.274
                                     57.5
                                      14
                                0.814* (↓56)
                                     0.593
                                     72.8
                                      14
                                     1.621
                                     1.068
                                     65.9
                              Clinical Chemistry
Glucose (mg/dL)
                                      15
                                    173.17
                                     30.45
                                     17.6
                                      15
                                    165.27
                                     18.19
                                     11.0
                                      15
                                    162.02
                                     35.63
                                     22.0
                                      15
                                    150.30
                                     23.07
                                     15.4
ALT (IU/L)
                                      15
                                     78.74
                                     16.37
                                     20.8
                                      15
                                     77.43
                                     9.47
                                     12.2
                                      15
                                94.92* (↑21)
                                     20.06
                                     21.1
                                      15
                               124.21**
(↑58)
                                     37.28
                                     30.0
AST (IU/L)
                                      15
                                    162.74
                                     29.93
                                     18.4
                                      15
                                    153.18
                                     15.51
                                     10.1
                                      15
                                    187.61
                                     46.06
                                     24.6
                                      15
                                191.63* (↑18)
                                     35.19
                                     18.4
Total Bilirubin (mg/dL)
                                      15
                                     0.00
                                     0.00
                                      0.0
                                      15
                                     0.00
                                     0.00
                                      0.0
                                      15
                                     0.03
                                     0.07
                                     235.4
                                      15
                                     0.24
                                     0.17
                                     72.6
Creatinine (mg/dL)
                                      15
                                     0.39
                                     0.10
                                     26.2
                                      15
                                     0.36
                                     0.14
                                     37.8
                                      15
                                     0.34
                                     0.09
                                     28.2
                                      15
                                     0.37
                                     0.13
                                     34.8
Total Protein (g/dL)
                                      15
                                     7.09
                                     0.71
                                     10.1
                                      15
                                     7.25
                                     0.69
                                      9.6
                                      15
                                     6.52
                                     0.56
                                      8.6
                                      15
                                     7.40
                                     0.60
                                      8.1
Albumin (g/dL)
                                      15
                                     4.50
                                     0.18
                                      4.1
                                      15
                                     4.51
                                     0.09
                                      2.0
                                      15
                                     4.40
                                     0.24
                                      5.6
                                      15
                                 4.74** (↑5)
                                     0.14
                                      2.9
Calcium (mg/dL)
                                      15
                                     12.43
                                     0.45
                                      3.7
                                      15
                                     12.52
                                     0.72
                                      5.7
                                      15
                                     12.16
                                     0.60
                                      4.9
                                      15
                                     12.49
                                     0.53
                                      4.2
Phosphorus (mg/dL)
                                      15
                                     10.75
                                     1.13
                                     10.5
                                      15
                                     11.22
                                     0.93
                                      8.3
                                      15
                                     9.97
                                     1.01
                                     10.1
                                      15
                                     11.38
                                     0.96
                                      8.4
Cholesterol (mg/dL)
                                      15
                                     81.93
                                     14.76
                                     18.0
                                      15
                                     78.05
                                     9.78
                                     12.5
                                      15
                                     84.35
                                     11.31
                                     13.4
                                      15
                                99.28** (↑21)
                                     10.36
                                     10.4
Urea (mg/dL)
                                      15
                                     34.88
                                     5.89
                                     16.9
                                      15
                                     30.36
                                     4.08
                                     13.4
                                      15
                                     31.99
                                     8.19
                                     25.6
                                      15
                                     39.70
                                     5.23
                                     13.2
ALP (lU/dL)
                                      15
                                    438.57
                                    117.14
                                     26.7
                                      15
                                    401.32
                                     74.31
                                     18.5
                                      15
                               288.12**
(↓34)
                                     54.19
                                     18.8
                                      15
                                    369.47
                                    105.55
                                     28.6
BUN (mg/dL)
                                      15
                                     16.29
                                     2.75
                                     16.9
                                      15
                                     14.18
                                     1.90
                                     13.4
                                      15
                                     14.94
                                     3.83
                                     25.6
                                      15
                                     18.54
                                     2.44
                                     13.2
Globulin (g/dL)
                                      15
                                     2.58
                                     0.64
                                     24.6
                                      15
                                     2.73
                                     0.64
                                     23.4
                                      15
                                     2.12
                                     0.49
                                     23.0
                                      15
                                     2.66
                                     0.55
                                     20.7
Albumin/
Globulin
                                      15
                                     1.84
                                     0.44
                                     23.7
                                      15
                                     1.73
                                     0.36
                                     20.9
                                      15
                                     2.17
                                     0.49
                                     22.4
                                      15
                                     1.86
                                     0.40
                                     21.7
a Data were obtained from the study report.  Percent difference from control is included in parentheses.
SD=Standard Deviation;  CV=Coefficient of Variation
*Significantly different from controls at p<0.05
**Significantly different from controls at p<0.01

              ix. Fish Short-term Reproduction Assay (FSTRA)
The 21-day FSTRA of Chemical S with fathead minnows (Pimephales promelas) was conducted under flow-through conditions.  Adult fish, 20 spawning groups (2 males and 4 females in each group: 6 months) were exposed to Chemical S at nominal concentrations of 0 (negative control; NC), 0 (solvent control; SC; dimethylformamide (DMF) at 0.02mL/L), 0.16, 8.0 and 4.0 ug ai/L while mean-measured concentrations were <LOQ (<0.010), <LOQ, 0.0834, 0.527, and 1.66 ug ai/L, respectively.  The test system was maintained at 24.8 to 25.4[o]C and a pH of 7.91 to 8.35.
Spawning frequency occurred at least every 3 to 4 days and fecundity in negative and solvent control was 26 and 34 eggs/female/day/replicate, respectively (range: NC=21-36, SC=24-40); fertilization success in the NC and SC groups averaged 93 and 90%, respectively (range: NC=89-98, SC=87-92).  Fertility was significantly reduced (p<0.05) by 13% (relative to negative control) for the 1.66 ug a.i./L treatment group (Table 37).  Fecundity was not significantly reduced from negative control (p>0.05) at any treatment concentration; however, there was a 31% increase in fecundity in the solvent control and a 41 and 50% increase in the low and middle test concentrations with a 38% decrease at the high concentration compared to the negative control (Table 38).
Male plasma vitellogenin (VTG) was significantly increased (p=0.045) by 90% relative to the negative control in the 1.66 ug a.i./L treatment group.  There was no significant difference in VTG for females (p>0.05).  The increase in male plasma VTG in this treatment group was coincident with a 38% mortality rate for males and an overall (i.e., male and female fish combined) mortality rate of 50%.  Plasma vitellogenin was initially analyzed using a trend test (Jonckeere-Terpstra) and all reported values (analyzed using replicate means).  The individual fish values for male VTG are depicted in Figure 13. This figure illustrates that across the negative control and all Chemical S treatment groups, there is one individual fish value in each group that appears to be substantially greater than the other replicate means; additionally, all the individual values in the highest treatment group are within the range of the negative control values.  A discussion of the observed variability and potential outliers associated with male VTG measurements was presented in the FSTRA section of the May 2013 SAP white paper (USEPA 2013), and the results for Chemical S reflect that discussion. Furthermore, if induction of vitellogenesis in males was occurring due to Chemical S interacting with the estrogen signaling pathway, the overall change of response in the exposed fish is expected to be greater than the approximate 2-fold increase (based on treatment means) observed in this study for males in the highest treatment.  Therefore, based on a more rigorous analysis of the male plasma VTG data, the initial statistical significance is considered an artifact of the observed variability.  This conclusion is further supported by the relatively limited extent of up-regulation of vitellogenesis in the fish at the highest treatment group compared to the control.
Figure 13.    Male plasma VTG (ng/mL) from Chemical S where the high treatment group was statistically different (p<=0.05;Jonckeere-Terpstra using treatment replicate means and all available data)  compared to the negative control.  The median of the individual  is represented by the solid line, with the box representing the 25[th] and 75[th] percentile and the whiskers are the range of individual values, with potential outliers outside the whiskers (defined as greater than the median plus three times the interquartile range).

A significant 39% increase (p<0.05) relative to control in male gonado-somatic index (GSI) was observed at the 1.66 ug ai/L group. Female GSI was not significantly (p>0.05) different.  Nuptial tubercle scores were also significantly reduced by 33% (p<0.05) in male fish at the 1.66 ug ai/L level; no tubercles were observed for females.   The study author reported no substantial differences in gonadal staging or histopathological effects in the male testes or female ovaries for any treatment group compared to the control.  Measurements of sex steroids were not reported neither was information provided on dorsal nape pads.  Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control.
There was a significant (p<0.05) reduction in survival (50% reduction compared to negative control) at the 1.66 ug ai/L treatment level.  There appeared to be inadequate reporting of secondary sex characteristics and clinical signs. The study author reported pectoral fin hemorrhage in one male from the control group.  There were no incidences reported in the 1.66 ug ai/L treatment group despite a significant reduction in survival.  There were no significant differences in male or female body weight between the control and treatment groups at test termination; body length at test termination was not reported (Table 39).

The percent fertility in the negative control (93%) and solvent control (90%) was slightly less than the guideline criterion minimum of 95%.  Additionally, the dissolved oxygen dropped below 60% in several solvent control treatment group replicates, with a reported minimum of 47%. Only the range was reported, so the reviewer could not determine how many or for which days dissolved oxygen dropped below the minimum criteria. However, once a decrease was observed, aeration was added to each test chamber on Day 10.  Finally, the analytical confirmation samples varied by 26-130%.  As such the CV for the all treatments groups was greater than the 20% variance criteria.  However, given the low solubility and propensity to sorb to glass for Chemical S, deviations from the 20% may have made maintaining exposure in the water column more difficult.
While these deviations to the performance criteria were observed, they did not impact the ability to interpret the results of the assay.
Overall, while significant changes were reported in male fish including decreased fertility and male nuptial tubercles and increased male GSI and VTG, these effects were only observed at the same test concentration in which there was a significant increase in mortality. Additionally, for male VTG, while a significant increase was initially reported, upon further evaluation, it was not determined to be statistically different from the controls based on the range of reported values and the magnitude of change.   At concentrations where no apparent overt toxicity occurred, there were no endocrine-related responses. The pesticidal mode of action of Chemical S involves the uncoupling of mitochondrial oxidative phosphorylation resulting in the depletion of ATP, which may have affected the overall energy reserves of the fish and may have in turn impacted energy inputs into reproduction.  Given the mode of action for Chemical S along with the observed decreases in fertility and tubercles and increases in GSI which lack endocrine-specificity and were coincident with effects on survival, there is uncertainty whether these effects were due to a possible endocrine interaction with the A (antagonism) or E pathway (agonism) or due to the fish's response to the toxic action of the chemical.
Although there is uncertainty, based on the overt toxicity observed at the highest treatment level, the lack of specificity in endpoints that were significantly affected, Chemical S is classified as "Possible" in the FSTRA for impacts to E or T signaling modalities.
Table 37:  Chemical S - Reproductive and HPG Endpoints[1] for Male Fathead Minnow in the FSTRA
                                   Treatment
                                 (ug a.i./L)
                                [mean measured]
                                Tubercle Score
                                      GSI
                     Gonadal Staging and Histopathology[2]
                                  Plasma VTG

                                    Median
                                 % Difference
                               Effect? (Yes/No)
% Difference
Control (dilution water only),
 if used
                                     36.5
                                       0
                                      No
                                       0
Solvent control
                                     35.4
                                     -15.8
                                      No
                                     -53.3
0.0834
                                     31.0
                                     4.44
                                      No
                                     -14.4
0.527
                                     39.0
                                     -4.44
                                      No
                                     61.9
1.66
                                    24.0[3]
                                    39.3[3]
                                      No
                                    89.8[3]
	Abbreviations: Conc. Concentration.  GSI Gonado-Somatic Index.    [VTG] Vitellogenin.
	[1]  Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean   water) control.
		[2]  Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.
		[3]  Statistically significant at p<0.05.

Table 38: Chemical S -Reproductive and HPG Endpoints1 for Female Fathead Minnow in the FSTRA

                                   Treatment
                                 (ug a.i./L)
                                [mean measured]
                                   Fecundity
                               Fertility Success
                                Tubercle Score
                                      GSI
                     Gonadal Staging and Histopathology[2]
                                  Plasma VTG

                                 % Difference
                                 % Difference
                                    Median
                                 % Difference
                               Effect? (Yes/No)
                                 % Difference
Control (dilution water only), if used
                                       0
                                       0
                                       0
                                       0
                                      No
                                       0
Solvent control
                                     30.8
                                      3.2
                                       0
                                      7.1
                                      No
                                     32.8
0.0834
                                     40.6
                                     -4.81
                                       0
                                     -4.98
                                      No
                                     39.6
0.527
                                     50.0
                                     -4.01
                                       0
                                     1.60
                                      No
                                     5.20
1.66
                                     -38.4
                                   -13.0[3]
                                       0
                                     23.8
                                      No
                                     35.0
Abbreviations: Conc. Concentration.  Diff. Difference.   [Fert.] Fertilization.  [GSI] Gonado-Somatic Index.
    [VTG] Vitellogenin.
[1]  Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control.
2  Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.
[3]  Statistically significant at p<0.05.
Table 39:  Chemical S - Growth Endpoints[1] in the Fish Short-Term Reproduction Assay (FSTRA)
                                   Treatment
                                  (mg a.i./L)
                                [mean measured]
                                  Body Weight

                                     Males
                                    Females

                                    % Diff.
                                    % Diff.
Control (dilution water only), if used
                                       0
                                       0
Solvent control
                                     -7.9
                                     -4.1
0.0000834
                                     -1.26
                                     2.00
0.000527
                                     -3.43
                                     -4.23
0.00166
                                     -3.51
                                     2.76
Abbreviations: [Diff.] Difference.
   1 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water) control.  Termination body lengths not reported.

 xi.   Amphibian Metamorphosis Assay
The 21-day assay of Chemical S on amphibian metamorphosis of African clawed frog (Xenopus laevis) was studied under flow-through conditions.  Amphibian larvae (80 Nieuwkoop-Faber [NF] Stage 51 tadpoles, 20 in each of 4 replicates) were exposed to nominal concentrations of 0 (negative and solvent controls), 0.09, 0.300, and 1.00 ug a.i./L.  Time-weighted average (TWA) concentrations were <0.010 (<LOQ; controls), 0.0156, 0.0711, and 0.307 ug a.i./L.
The test system was maintained at 21.5 to 22.6°C and a pH of 7.9 to 8.4. Chemical S did not significantly increase or decrease normalized hind-limb length (HLL) at either Day 7 or 21. Similarly, there was no significant acceleration or delay of median Day 7 and 21 NF developmental stage at any treatment level.  Several individuals were excluded from the analysis of the Day 21 continuous endpoints (SVL, body weight, and normalized HLL) because they developed to NF Stage 61; one tadpole from the negative control and between 4-5 tadpoles in each of the solvent control and treated groups were excluded from the Day 21 continuous endpoint analysis for this reason.  Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control.
No asynchronous development was observed in any treatment group.  Effects on thyroid gland histopathology were observed in both controls and the TWA 0.0156, 0.0711, and 0.307 ug a.i./L.  Histopathological effects included mild follicular cell hyperplasia and hypertrophy, though there was no apparent treatment-related effect because the incidence of these diagnoses was similar in controls and treatments.  The study author did not report clinical observations.
There was poor recovery (as low as 8.8% of nominal at test termination in the highest treatment level) and high variability of measured exposure concentrations, with overall %CVs ranging from 33-69% in all treatment levels; this exceeded the performance criterion maximum CV of 20%. The very low recoveries and reported instability (likely due to low solubility and high Koc) of Chemical S in the test system make the results of this amphibian metamorphosis study difficult to interpret.  An additional performance criterion which was not met was the decline of DO to 3.24 mg/L (i.e., 39% saturation), below the recommended minimum of 3.5 mg/L (40% saturation); this occurred in one replicate vessel from the vehicle control group on Day 13 and all test vessels were aerated upon this discovery to correct the saturation issue. Iodide in the laboratory water (13 ug/L) was higher than the guideline recommended level (<=10 ug/L); however, the analysis was last performed one year prior to the conduct of the study and it is unclear if the water analyzed was representative of the test water, which was reported to be a combination of laboratory well water and laboratory well water that had been filtered through reverse osmosis.  All other performance and validity criteria were satisfied in this study.
Overall, exposure levels in the AMA were determined to be inadequate, although there were no HPT-related effects within the range of concentrations tested.

A. Other Scientifically Relevant Information (OSRI) for Chemical S
The registrant elected to conduct the entire Tier 1 assay.  Therefore, no OSRI were submitted to the agency.  However, Chemical S has Part 158 mammalian and ecotoxicity studies previously submitted to the Agency. These studies are discussed below.
      1. Mammalian OSRI Data
In a subchronic toxicity study, Chemical S was administered in the feed to 6 Carworth Farm E (CFE) rats/sex/group at 0, 50, 100, 300 or 600 ppm (equivalent to 2.5, 5, 15 or 30 mg/kg bw/day) for 3 months.  Testes weights and histopathology were evaluated. Body weight was decreased at 300 (6% in males and 7% in females and 600 ppm (13% in males and 16% in females).  A slight decrease in food efficiency occurred at 600 ppm in males (27% from wk 0-4 and 12% from wk 0-13) and females (26% for wk 0-4 and 17% for Week 0-13).  The decreases in body weights may have been the result of the unpalatability of the diet.  No histological findings were seen in the testes. The NOAEL is 5 mg/kg bw/day and the LOAEL is 15 mg/kg bw/day based on elevated alkaline phosphatase in male rats.
In a 2-year rat toxicity study, Chemical S (97% a.i.) was administered in the diet to CFE  rats (72/sex/group) for up to two years at 50, 100, 300 or 600 ppm (equivalent to 2.5, 5, 15 or 30 mg/kg bw/day. For controls 144 rats/sex were used.  Organ weights were obtained for testes. Histopathology was conducted on the testes, prostate, ovaries, uterus, mammary glands, pituitary and thyroid glands. The diet was unpalatable to both males and females and resulted in decreases in body weights, body weight gains, and food consumption, especially during the early stages of the study.  There was a significant body weight depression in males and females at 15 and 30 mg/kg bw/day, correlated with decreased food consumption.  Only the 30 mg/kg bw/day dose groups showed a nominal trend to decreased food efficiency for weeks 0-52.  It should be noted that the subchronic and 2-generation studies on reproduction generally show a similar nominal decrease in food efficiency at 15 and 30 mg/kg bw/day, respectively. Mortality of females at the 30 mg/kg bw/day (33% versus 56% in controls) was less than corresponding controls.  Male mortality was unchanged.  Increased absolute and relative testes weights were noted at 15 and 30 mg/kg/day, but no treatment histological correlates were noted. No treatment-related histopathological lesions were observed in the prostate, ovaries, uterus, mammary glands, pituitary and thyroid glands. The only effects on the intestines was a suggestion of an increased trend for soft stools, especially at the two highest dietary treatment levels (incidence in males was 6%, 12%, 21%, 42% and 71% from control to 30 mg/kg bw/day, respectively).  Mild dilation of the glands in the fundic mucosa of the stomach may have shown a slight increased trend in males, but it was not considered toxicologically significant. For chronic toxicity, the NOAEL was 5 mg/kg bw/day and LOAEL was 15 mg/kg bw/day based on treatment-related alkaline phosphatase increase in males and females.
In a carcinogenicity study in mice, Chemical S (97% pure) was administered in the diet to 120 control and 60 CFE mice/sex/group at 0, 50, 100, 300, or 600 ppm (0, 7.5, 15, 45 or 90 mg/kg bw/day) for 18-months. Testes weights were obtained. Histopathological examinations were conducted for the gonads, prostate, uterus, pituitary and thyroid glands.  Body weights were significantly (p <0.001) reduced compared with control values from week 4 (first week determined) to termination at 45 and 90 mg/kg bw/day doses for both males (3% and 17%, respectively), and females (4% and 13%,)).  At weeks 36 and 72, male body weights at 300 and 600 ppm were reduced by 5% and 16%-18%, respectively, and female body weights were reduced 6%-3% and 16%, respectively.  At termination, male body weights were reduced at the same dose levels. The NOAEL was 15 mg/kg/day and the LOAEL was 45 mg/kg/day based on body weight reductions.
In a developmental toxicity study, 27 mated Specific Pathogen Free Wistar rats per group were administered Chemical S (98.9-99.1% a.i. ) by gavage in an aqueous solution at doses of 0, 15, 30, and 60 mg/kg bw/day on gestation days (GDs) 6-15, inclusive.  On GD 20, all surviving dams were euthanized and then necropsied, and all fetuses were examined externally, weighed and sacrificed.  Several high-dose animals exhibited diarrhea at varying times between GDs 6 and 20 (7/27 vs. 2/27 for controls). For maternal toxicity, the NOAEL was 15 mg/kg bw/day and the LOAEL was 30 mg/kg bw/day, based on decreased body weight gain. There were no treatment-related differences in the intrauterine parameters between the treated and control groups and there were no significant increases in litter or fetal incidences of any individual structural abnormalities for any treated group. For developmental toxicity, the NOAEL was 60 mg/kg bw/day, the highest dose tested; a LOAEL was not established.
In another developmental toxicity study, 18-23 pregnant New Zealand white rabbits per group were administered Chemical S (98.7% a.i) by gelatin capsule at doses of 0, 1, 5, or 10 mg/kg bw/day on gestation days (GD) 6-18, inclusive.  On GD 29, all surviving does were sacrificed and the uteri were weighed and examined.  Treatment-related deaths occurred on or after GD 19 in two mid-dose and four high-dose animals.  The number of animals that aborted was 3, 1, 2, and 12 in the control, low-, mid-, and high-dose groups, respectively.  Anorexia (subjective observations of not eating and passing few feces) was observed in 2/18, 2/18, 11/18, and 23/23 animals, respectively, and was often associated with abortion and/or whole litter loss.  Lesions of the gastric mucosa were found in the mid- and high-dose animals that died intercurrently, were sacrificed following abortion, or were examined at terminal sacrifice.  The overall incidence rates for lesions in the stomach were 2, 2, 6, and 8 animals in the control, low-, mid-, and high-dose groups, respectively.  For maternal toxicity, the NOAEL was 1 mg/kg bw/day and the LOAEL was 5 mg/kg bw/day based on clinical signs of toxicity and gross lesions of the stomach. No treatment-related effects on any cesarean section parameter were observed in the low- or mid-dose groups. No treatment-related external, visceral, or skeletal malformations/variations were observed in this study. For developmental toxicity, the NOAEL was 5 mg/kg bw/day and the LOAEL was 10 mg/kg bw/day based on increased numbers of resorptions/litter and post-implantation loss/litter and decreased fetal body weights.
In a two-generation reproduction study, Chemical S (99.4% a.i.) was fed to groups of 30 male and 30 female F0 and F1 rats at dietary concentrations of 0, 40, 75, 250, or 500 ppm.  The dietary concentrations corresponded to doses of 2.39, 4.47, 14.8, and 30.0 mg/kg bw/day for F0 males; 3.18, 5.92, 20.0, and 45.7 mg/kg bw/day for F1 males; 2.76, 5.13, 16.6, and 33.6 mg/kg bw/day for F0 females; and 3.68, 6.83, 23.9, and 54.1 mg/kg bw/day for F1 females averaged over the premating period only.  The F0 male and female rats received treated or control diets for 72 days prior to mating and during mating, gestation, and lactation of one litter.  The F1 pups were weaned onto the same diets as their parents and were fed their respective diets for 105 days prior to mating and during mating, gestation, and lactation of one litter.  The F0 males were sacrificed after 124-126 days of feeding, F0 females after 116-137 days, F1 males after 159-177 days, and F1 females after 160-178 days. Testes were weighed. Histopathological examinations were conducted for epididymides, testes, seminal vesicles, prostate, coagulating glands, ovaries, uterus, vagina, pituitary and thyroid glands. Sexual maturation, estrous cycle length and periodicity, and sperm parameters were not evaluated in this study.
No treatment-related effect were seen on survival, clinical signs, gross  or microscopic findings in adult F0 or F1 male or female rats at any dose, or on body weights, body weight gain, food consumption, or food efficiency at 40, 75, or 250 ppm.  Body weights and overall body weight gain, food consumption, and food efficiency were significantly (p<0.05) decreased in both sexes and generations administered 500 ppm of the test material compared with the controls.  The F0 males weighed 8-19% less than controls throughout the premating/postmating period and gained 33% less weight; F1 males weighed 23-25% less and gained 24% less weight.  The F0 females weighed 7-12% less (except for week 1) and gained 41% less weight during the premating period, and F1 females weighed 18-30% less and gained 20% less weight.  The F0 and F1 males consumed 12% less food averaged over the entire premating period, F0 females consumed 13% less, and F1 females consumed 7% less.
No treatment-related effects were observed on the following parameters for F1 or F2 offspring: live birth, viability, lactation, or litter survival indices, the number of litter or pups born (live or stillborn), the sex ratio at birth, or the pup weight at birth.  Pup growth was significantly (p<0.05) inhibited by 250 and 500 ppm of the test material in the diet.  At 500 ppm, F1 pups weighed 6% (N.S.), 16%, 20%, and 24% less than controls on lactation days 4 (precull), 7, 14, and 21, respectively, and gained 28% less weight between day 0 and 21.  The F2 pups at 500 ppm weighed 13%, 19%, 22%, and 29% less, respectively, than controls and gained 33% less weight between day 0 and 21.  At 250 ppm, F1 pups had body weights similar to those of the controls throughout the lactation period; whereas, F2 pups weighed 9%, 12%, 9% (p<0.05), and 10% (p<0.05) less than controls on lactation days 4 (precull), 7, 14, and 21, respectively, and gained 11% less weight.  The F2 pup weights were statistically significantly decreased from control values on lactational day 14 and 21 at 250 ppm. Between lactational day 0- 4, F2 pups showed a weight gain decrement of 19% at 250 ppm.
For parental/systemic toxicity, the NOAEL was14.8 mg/kg bw/day for males and 16.6 mg/kg bw/day for females and LOAEL were 30.0 mg/kg bw/day for males and 33.6 mg/kg bw/day for females based on decreased body weights, body weight gain, food consumption, and food efficiency in F0 and F1 males and females.  For reproductive toxicity, the NOAEL was 30 mg/kg bw/day in males and 33.6 mg/kg bw/day in females; a LOAEL was not established. There were no effects on fertility at the highest dose tested. For offspring toxicity, the NOAEL was 5.13 mg/kg/day and the LOAEL was 16.6 mg/kg/day based on decreased pup weight and weight gain in F2 litters.
Chemical S was administered in capsules to 8 control and 4 Beagle dogs/sex/group at 0, 2.5, 5, 15, 30 or 60 mg/kg/day for 2 years.  Organ weights were obtained for testes and thyroid. Most dogs dosed with Chemical S were reported to have exhibited vomiting and diarrhea.   It was also reported that after one-year animals dosed at 2.5-15 mg/kg/day, vomiting and diarrhea had subsided.  In the 30 and 60 mg/kg bw/day groups vomiting and diarrhea continued after one year, but to a lesser extent.  Convulsions were seen in one female from each of the 0, 2.5, 15 and 30 mg/kg bw/day dose levels.  One male at 2.5 mg/kg bw/day was affected.  Male (21%) and female (23%) body weights were significantly reduced at 60 mg/kg bw/day.  Organ weights showed not effects. The vomiting and diarrhea could be expected from a test material that irritated the gut. Possibly this vomiting and diarrhea resulted in the decreased body weight at 60 mg/kg bw/day.  Vomiting and diarrhea from gut irritation is an adverse effect, but it may also reduce the dose administered, which occurred over the first year of the study.  The NOAEL is 5 mg/kg bw/day and LOAEL is 15 mg/kg bw/day based on excess vomiting and diarrhea with a body weight decrement at 60 mg/kg bw/day.
2.   Ecotoxicity OSRI Data
Rainbow trout (Onchorynchus mykiss) were exposed to the technical grade active ingredient (TGAI) (98.6% a.i.) under flow-through conditions in a 60-day early life stage test.  DMF was used as a solvent.  The study author reported that larval survival, length, and weight, were significantly less than in the solvent control at measured concentrations of 0.61 ug/L and above.  The NOAEL values were 0.31 ug/L for larval survival and 0.61 ug/L for embryo hatching.  The study was considered scientifically sound; however, raw data were not submitted and the reviewer was therefore unable to confirm the study author's conclusions.
The 96-hour acute LC50 value for Chemical S using rainbow trout was 1.7 ug a.i./L (based on nominal concentrations) tested under static conditions.
In a one generation avian reproduction test with bobwhite quail, nominal dietary concentrations of the TGAI at 50 and 150 ppm had no effect on bobwhite reproductive parameters during the 21-week exposure period.  At 500 ppm there appeared to be a reduction in the number of eggs laid per hen, and an increase in the number of hens that exhibited lesions of old egg yolk peritonitis at terminal sacrifice. The NOAEC was 150 ppm.
Nominal dietary concentrations of the TGAI at 50 and 150 ppm had no effect on mallard duck  reproductive  parameters during a 19-week exposure period.  At 500 ppm there was a reduction in the number of eggs laid per hen. There was an increase in the number of females in all treatment groups that exhibited lesions of old egg yolk peritonitis at terminal sacrifice.
The freshwater invertebrate waterflea, Daphnia magna, was exposed to the TGAI in a 21-day chronic toxicity test.  The NOAEL was 16 ug/L (measured) or 25 ug/L (nominal).  There was a significant difference between the solvent control and the test chemical at the two highest treatment concentrations (50 and 100 ug/L, nominal) with respect to growth, percent adult survival, and total number of young.  These NOAEL values are above the reported solubility of Chemical S which is about 13 ug/L.
No additional data with frog or other amphibian species have been submitted.

A. Discussion

   i. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis
As previously noted, the EDSP Tier 1 screening data and OSRI were evaluated together to determine whether Chemical S has potential to interact with the HPG axis or its downstream pathways related to steroidogenesis or sex steroid signaling.  The Tier 1 in vitro assays relevant to this analysis include the AR, binding, ER binding, ERTA, aromatase, and steroidogenesis assay.  Relevant in vivo assays include the male and female pubertal rat, uterotrophic, Hershberger, and the FSTRA assays.
Effects on Estrogen Pathway: Table 40 below summarizes the results of the available EDSP Tier 1 screening assay results and OSRI results relevant for determining the potential for Chemical S to interact with the estrogen pathway.  The various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects.
Chemical S was classified as "not interactive" in the in vitro estrogen receptor binding assay and negative in the ERα transcriptional activation and Uterotrophic assays.  Based on these results, Chemical S does not appear to disrupt the estrogen signaling pathway through estrogen receptor agonism or antagonism.  Chemical S showed an equivocal response in the in vitro aromatase assay and was negative in the steroidogenesis assay.  However, there is some evidence that Chemical S may have the potential to interact with the estrogen pathway in the in vivo Female Pubertal assay.   At the low dose (10 mg/kg bw/day), significant (p<= 0.01) delays ( ~ 2 days) in vaginal opening (VO) and decreased body weight at VO were seen in the absence of overt toxicity.   At the high dose (75 mg/kg bw/day), estrogen-related effects were manifested as delayed VO (~ 7 days; p<= 0.01); decreased body weight at VO; decreased weights of the ovaries and uterus but in the presence of overt toxicity (salivation, and/or decreased body weights and food consumption).  In the FSTRA assay, effects that may indicate potential interaction with the estrogen signaling pathway included alterations in fertility (13% decrease), GSI (39% increase in males), and 33% decrease in male nuptial tubercles. Additionally, while an increase in male VTG was initially reported (90% increase), upon further review of the data for this endpoint, it was concluded that this response was not different from the control and was not consistent with the magnitude of response expected with an upregulation of VTG due to an estrogen agonism.  Furthermore, effects were only observed at a concentration in which survival was markedly affected (50% overall mortality observed).
The pesticidal mode of action of Chemical S involves the uncoupling of mitochondrial oxidative phosphorylation and resulting in the depletion of ATP.  Based on the available data, another plausible mode of toxic action is related to its irritation properties including irritation that compromises the integrity of the gastro-intestinal tract in mammals leading to restricted caloric intake due to reduced food consumption.  For the female pubertal assay at the high dose where overt toxicity was observed, the directionality of the reported responses is consistent with depletion of ATP and restricted caloric intake.  As the pesticidal mode of action for Chemical S eventually leads to a depletion of ATP, there is uncertainty in whether the responses observed in the FSTRA are potentially due to an interaction with the estrogen pathway or due to the a lack of energy resources available for reproduction output.    Additionally, the responses in the female rat pubertal, in the absence of overt toxicity do not suggest an agonistic interaction with the estrogen pathway.
The results of mammalian Part 158 studies did not reveal any potential of Chemical S to interact with the estrogen pathway. There were reported decreases in reproduction in birds and freshwater invertebrates, but the reported endpoints are non-endocrine specific.
Based on WoE evaluation of EDSP Tier 1 screening assay and OSRI results, in the absence of overt toxicity, Chemical S demonstrated a potential to interact with the estrogen pathway presumably through the perturbation of the HPG axis in the Female Pubertal assay. However, data from the remaining assays are not sufficiently compelling to support such a perturbation.

Table 40:  Estrogenic/Anti-Estrogenic Pathway for Chemical S
 Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Chemical S[a]
                        Study Type/Literature Citation

 ER Binding
 ER Activation
 Steroidogenesis
 Uterine Weight
 Ovarian Weight
 Ovarian/Gonad Staging and Histopathology
 Pituitary Weight
 Estrous Cyclicity[b]
 Age & Weight at VO
 Fertility/Fecundity
 Vitellogenin
 Fish Gonadal Weight (GSI)
 Overt Toxicity Observed[c]
                                  EDSP Tier 1
 ER Binding
                                Not interactive

 ERTA

                                    Negative

 Aromatase

                                   Equivocal

 Steroidogenesis

                                    Negative

 Uterotrophic