**ATTACHMENT II - APERC COMMENTS ON TSCA SECTION 21 PETITION**

**Docket ID Number: EPA-HQ-OPPT-2007-0490**

**SUPPORTING DATA ON VITELLOGENIN AND RELATED STUDIES FROM:** Staples,
C., Mihaich, E., Carbone, J., Woodburn, K., and Klecka, G. (2004). A
Weight of Evidence Analysis of the Chronic Ecotoxicity of Nonylphenol
Ethoxylates, Nonylphenol Ether Carboxylates, and Nonylphenol. [Human and
Ecological Risk Assessment]{.underline}, [10]{.underline}, 999-1017.

**Note**: Full citations for study references are available in the
Staples et al. (2004) manuscript.

+----+------------+-----------------+------------------------+-------+
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| ed |            |                 |                        |       |
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+====+============+=================+========================+=======+
| Ma | Test       | Endpoint        | Comments               | Refe  |
| te | System     | Description     |                        | rence |
| ri |            |                 |                        |       |
| al |            |                 |                        |       |
+----+------------+-----------------+------------------------+-------+
|    | [In Vitro  |                 |                        |       |
|    | Tests]{.   |                 |                        |       |
|    | underline} |                 |                        |       |
+----+------------+-----------------+------------------------+-------+
| N  | VTG        | relative        | NPE40: no activity     | Jo    |
| PE | induction  | potency as      |                        | bling |
| 40 | in male    | compared to     | NPE9: 2E-7             | and   |
|    | rainbow    | 17-*B*          |                        | Su    |
| NP | trout      | estradiol       | NPE2: 6 E-6            | mpter |
| E9 | h          | (which equals   |                        | (     |
|    | epatocytes | 1)              | NP: 9E-6               | 1993) |
| NP |            |                 |                        |       |
| E2 |            |                 |                        |       |
|    |            |                 |                        |       |
| NP |            |                 |                        |       |
+----+------------+-----------------+------------------------+-------+
| NP | VTG        | dose response   | No statistics used,    | White |
| E2 | induction  | assessment of   | however, substantial   | et    |
|    | in         | VTG production  | increase in VTG        | al.   |
| NP | cultured   |                 | induction at 1E-5 M    | (     |
|    | male       |                 | concentrations of NPE2 | 1994) |
|    | rainbow    |                 | and NP compared to     |       |
|    | trout      |                 | 1E-6 and 1E-7 M        |       |
|    | h          |                 |                        |       |
|    | epatocytes |                 |                        |       |
+----+------------+-----------------+------------------------+-------+
|    | [In Vivo   |                 |                        |       |
|    | Tests]{.   |                 |                        |       |
|    | underline} |                 |                        |       |
+----+------------+-----------------+------------------------+-------+
| NP | Exposed    | VTG induction   | 100- to 1000-fold      | Jo    |
| E2 | male adult | in blood plasma | increase in serum VTG  | bling |
|    | rainbow    | from exposure   | concentration for both | et    |
| NP | trout (*O. | to nominal      | NPE2 and NP, as        | al.   |
|    | mykiss*)   | 30μg/L          | compared to controls   | (     |
|    | for 3      |                 |                        | 1996) |
|    | weeks      |                 |                        |       |
+----+------------+-----------------+------------------------+-------+
| NP | Exposed    | Dose response   | VTG induced at 20.3,   | Jo    |
|    | male adult | assessment of   | but not 5.02 μg/L.     | bling |
|    | rainbow    | VTG induction,  |                        | et    |
|    | trout (*O. | GSI measured in | GSI reduced at 54.3,   | al.   |
|    | mykiss*)   | same fish       | but not 20.3 μg/L      | (     |
|    | for 3      |                 |                        | 1996) |
|    | weeks      |                 |                        |       |
+----+------------+-----------------+------------------------+-------+
| NP | Exposed    | Dose response   | VTG induction at 8.3   | H     |
|    | female     | assessment of   | μg/L and higher,       | arris |
|    | adult      | VTG induction,  | plasma E2 reduced at   | et    |
|    | rainbow    | plasma levels   | 85.6 μg/L, plasma FSH  | al.   |
|    | trout (*O. | of follicle     | and pituitary gene     | (     |
|    | mykiss*)   | stimulating     | expression reduced at  | 2001) |
|    | for 18     | hormone (LSH)   | all concentrations,    |       |
|    | weeks      | and gene        | pituitary LH reduced   |       |
|    |            | expression of   | at 85.6 μg/L, LH gene  |       |
|    |            | LSH in the      | expression reduced at  |       |
|    |            | pituitary,      | 8.3 μg/L, transitory   |       |
|    |            | plasma and      | slight increase in     |       |
|    |            | pituitary       | plasma LH, GSI reduced |       |
|    |            | levels of       | at 85.6, but not 8.3   |       |
|    |            | luteinizing     | μg/L                   |       |
|    |            | hormone (LH)    |                        |       |
|    |            | and gene        |                        |       |
|    |            | expression in   |                        |       |
|    |            | pituitary,      |                        |       |
|    |            | plasma E2       |                        |       |
|    |            | levels, GSI     |                        |       |
|    |            | measured in     |                        |       |
|    |            | same fish       |                        |       |
+----+------------+-----------------+------------------------+-------+
| NP | Exposed    | Solvent         | No effects on plasma   | Moore |
|    | Atlantic   | control, 5 to   | VTG, plasma Na^+^ or   | et    |
|    | salmon     | 20 μg/L         | Cl^-^ ions, gill Na^+^ | al.   |
|    | (*S.       |                 | K^+^ ATPase activity   | (     |
|    | salar*)    |                 | at any concentration   | 2003) |
|    | smolts for |                 |                        |       |
|    | 30 days    |                 |                        |       |
+----+------------+-----------------+------------------------+-------+
| NP | Exposed    | VTG induction,  | No induction of VTG at | Allen |
|    | male adult | gonad           | highest mean measured  | et    |
|    | flounder   | histopathology, | concentration of 24.5  | al.   |
|    | (*P.       | GSI and HSI     | μg/L, no effects on    | (     |
|    | flesus*)   | measured in     | gonad histology        | 1999) |
|    | for 3      | same fish       |                        |       |
|    | weeks      |                 |                        |       |
+----+------------+-----------------+------------------------+-------+
| N  | 42-d       | Plasma VTG,     | No induction of VTG at | Ni    |
| PE | fl         | 17β-estradiol   | any concentration      | chols |
| -9 | ow-through | (E2),           | tested (highest 7.9    | et    |
|    | with       | testosterone    | μg/l), No differences  | al.   |
|    | fathead    | (T)             | between treatments and | (     |
|    | minnows    |                 | control for plasma E2  | 2001) |
|    | (*P.       |                 | or T                   |       |
|    | promelas*) |                 |                        |       |
+----+------------+-----------------+------------------------+-------+
| NP | 21-d       | Plasma VTG      | Plasma VTG induction   | Dus   |
|    | fl         |                 | in fish exposed to NP  | sault |
| NP | ow-through |                 | started to appear at   | et al |
| E1 | rainbow    |                 | water concentrations   | (     |
|    | trout      |                 | of 13 µg/L but more    | 2005) |
| NP | (*On       |                 | typically occurred at  |       |
| EC | corhynchus |                 | water concentrations   |       |
|    | mykiss*)   |                 | of 25 µg/L of NP.      |       |
|    |            |                 | Measurements of the    |       |
|    |            |                 | relative potency of    |       |
|    |            |                 | NP1EO and NP1EC        |       |
|    |            |                 | compared to NP,        |       |
|    |            |                 | yielded ratios of 0.22 |       |
|    |            |                 | and 0.03, respectively |       |
+----+------------+-----------------+------------------------+-------+
| N  | Japanese   | 100-d, R,       | NPE-9 and NPE-4: all   | Balch |
| PE | medaka     | endpoints:      | endpoints NOEC 1000    | and   |
| -9 |            | secondary sex   | μg/L, highest          | Met   |
|    | *Oryzias   | c               | concentration tested   | calfe |
| N  | latipes*   | haracteristics, |                        | (     |
| PE |            | male papillary  | NPE-1: secondary       | 2005) |
| -4 |            | processes,      | characteristics NOEC   |       |
|    |            | testis-ova      | (LOEC) 100 (300) μg/L, |       |
| N  |            |                 | all other endpoints    |       |
| PE |            |                 | NOEC 300 μg/L          |       |
| -1 |            |                 |                        |       |
|    |            |                 | NPEC1: all endpoints   |       |
| N  |            |                 | NOEC 3000 μg/L,        |       |
| PE |            |                 | highest concentration  |       |
| C1 |            |                 | tested                 |       |
|    |            |                 |                        |       |
| NP |            |                 | NP: secondary          |       |
|    |            |                 | characteristics NOEC   |       |
|    |            |                 | (LOEC) 10 (30) μg/L,   |       |
|    |            |                 | male processes NOEC    |       |
|    |            |                 | 100 μg/L, testis-ova   |       |
|    |            |                 | 30 (100)               |       |
+----+------------+-----------------+------------------------+-------+
| NP | 28 to 31-d | Measured serum  | As compared to         | Ville |
|    | exposure   | sex steroids,   | controls, no effects   | neuve |
|    | to carp    | VTG induction,  | on somatic growth or   | et    |
|    | (*Cyprinus | gonadal         | liver or gonad         | al.   |
|    | carpio*)   | histology, mean | weights, no            | (     |
|    |            | measured        | differences in plasma  | 2002) |
|    |            | concentrations  | E2 or testosterone, no |       |
|    |            | up to 5.36 μg/L | differences in plasma  |       |
|    |            |                 | VTG, no                |       |
|    |            |                 | histopathological      |       |
|    |            |                 | changes observed in    |       |
|    |            |                 | testes,                |       |
|    |            |                 | hepatopancreas, brain  |       |
|    |            |                 | or gills               |       |
+----+------------+-----------------+------------------------+-------+
| NP | Paire      | Secondary sex   | Reduction in number of | Ha    |
|    | d-breeding | c               | tubercles but not the  | rries |
|    | assay with | haracteristics, | size of the fat pad    | et    |
|    | fathead    | serum VTG       | noted at 8.1 to 57.7   | al.   |
|    | minnows    |                 | μg/L. Serum VTG        | (     |
|    | (*P.       |                 | induced at 8.1 to 57.7 | 2000) |
|    | p          |                 | μg/L.                  |       |
|    | romelas*), |                 |                        |       |
|    | fl         |                 |                        |       |
|    | ow-through |                 |                        |       |
+----+------------+-----------------+------------------------+-------+
| NP | 42-d       | Plasma VTG,     | Dose related decrease  | Giesy |
|    | fl         | 17β-estradiol   | in plasma VTG in       | et    |
|    | ow-through | (E2)            | females. No dose       | al.   |
|    | with       |                 | related change in      | (     |
|    | fathead    |                 | plasma VTG in males.   | 2000) |
|    | minnows    |                 |                        |       |
|    | (*P.       |                 | \~Equal elevation of   |       |
|    | promelas*) |                 | plasma E2 at all doses |       |
|    |            |                 | except the highest     |       |
|    |            |                 | (3.4 μg/L) in females  |       |
|    |            |                 | and males.             |       |
+----+------------+-----------------+------------------------+-------+
| N  | 42-d       | Observations of | NPE-9 - No effects on  | M     |
| PE | flo        | secondary sex   | tubercle or fat pad    | iles- |
| -9 | w-through, | characteristics | size, on survival, or  | Richa |
|    | fathead    | and gonadal     | on testicular or       | rdson |
| NP | minnows    | histology.      | ovarian histology at   | et    |
|    | (*P.       |                 | highest level (5.5     | al.   |
|    | p          |                 | μg/L).                 | (     |
|    | romelas*). |                 |                        | 1999) |
|    |            |                 | Ex I: NP - No effects  |       |
|    |            |                 | on tubercle or fat pad |       |
|    |            |                 | size, or survival at   |       |
|    |            |                 | highest level (3.4     |       |
|    |            |                 | μg/L). Increase in     |       |
|    |            |                 | severity score of male |       |
|    |            |                 | gonads at 1.6 but not  |       |
|    |            |                 | 0.4 μg/L.              |       |
|    |            |                 |                        |       |
|    |            |                 | Ex II: NP - data not   |       |
|    |            |                 | usable as solvent      |       |
|    |            |                 | control completely     |       |
|    |            |                 | inhibited egg          |       |
|    |            |                 | production (see        |       |
|    |            |                 | companion paper, Giesy |       |
|    |            |                 | et al. 2001)           |       |
+----+------------+-----------------+------------------------+-------+
| NP | 9-d        | Induction of    | Significant induction  | Ped   |
|    | flo        | plasma VTG      | of VTG at 76 μg/L      | erson |
|    | w-through, |                 |                        | et    |
|    | rainbow    |                 |                        | al.   |
|    | trout (*O. |                 |                        | (     |
|    | mykiss*)   |                 |                        | 1999) |
+----+------------+-----------------+------------------------+-------+
| NP | 1 year     | Induction of    | VTG induction: (1.05)  | Acke  |
|    | flo        | VTG, ZRP        | μg/L                   | rmann |
|    | w-through, | expression in   |                        | et    |
|    | rainbow    | liver           | ZRP expression: 1.05   | al.   |
|    | trout (*O. |                 | (10.17) μg/L           | (     |
|    | mykiss*)   |                 |                        | 2002) |
+----+------------+-----------------+------------------------+-------+
| NP | 3-d        | Induction of    | VTG induced at 4 to    | Kwak  |
|    | renewal,   | VTG nRNA in     | 100 μg/L. Increases in | et    |
|    | swordtail  | liver, scoring  | numbers of             | al.   |
|    | fish (*X.  | of male gonadal | necrotic/apoptotic     | (     |
|    | helleri*)  | cells for       | male gonad cells at 4  | 2001) |
|    |            | nec             | to 100 μg/L.           |       |
|    |            | rosis/apoptosis |                        |       |
+----+------------+-----------------+------------------------+-------+
| NP | Sheepshead | Following a     | hepatic VTG rapidly    | H     |
|    | minnow     | 16-d renewal    | diminished following   | emmer |
|    |            | exposure,       | cessation of exposure, | et    |
|    | *          | hepatic VTG     | plasma VTG clearance   | al.   |
|    | Cyprinodon | nRNA regulation | concentration and time | (     |
|    | v          | and plasma VTG  | dependent, plasma      | 2002) |
|    | ariegatus* | clearance rates | clearance slower than  |       |
|    |            | were measured   | hepatic clearance      |       |
+----+------------+-----------------+------------------------+-------+

E2 is 17*B*-estradiol, EE2 is ethinylestradiol, a synthetic estrogen,
VTG is vitellogenin, FSP means female-specific proteins, T means
testosterone, NOEC means no observed effect concentration, LOEC means
lowest observed effect concentration, ECx is an effect concentration
caused at x percent level, F0 is the parental generation and F1 is the
offspring generation of the F0, NPE are nonylphenol ethoxylates, NP is
nonylphenol, NPEC are nonylphenol ether carboxylates, GSI is
gonadosomatic index, LSI is the liver-somatic index, GLP refers to the
use of Good Laboratory Practices.