DINITROTOLUENE
Permanent Collection

Ambient Water Quality  Criteria
              Criteria and Standards Division
              Office of Water Planning and Standards
              U.S.  Environmental Protection Agency
              Washington, D.C.

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                        CRITERION  DOCUMENT



                         DINITROTOLUENES



CRITERIA



                           Aquatic Life



     2,3-d initrotoluene



          For 2,3-dinitrotoluene the criterion  to  protect  fresh-



water aquatic life as derived using the Guidelines  is  12 ug/1  as a



24-hour average and the concentration  should  not exceed  27  ug/1 at



any time.



          For 2,3-dinitrotoluene the criterion  to  protect  salt-



water aquatic life as derived using procedures  other  than  the



Guidelines is 4.4 ug/1 as a 24-hour average and the concentration



should not exceed 10 ug/1 at any time.



     2,4-d initrotoluene



          For 2,4-dinitrotoluene the criterion  to  protect  fresh-



water aquatic life as derived using procedures  other  than  the



Guidelines is 620 ug/1 as a 24-hour average and the concentration



should not exceed 1,400 ug/1 at any time.



          For saltwater aquatic life,  no criterion  for 2,4-di-



nitrotoluene can be derived using  the  Guidelines,  and  there  are



insufficient data to estimate a criterion using other  procedures.



                           Human Health



     For the maximum protection of human health from  the potential



carcinogenic effects of exposure to 2,4-dinitrotoluene through



ingestion of water and contaminated aquatic organisms, the  ambient



water concentration is zero.  Concentrations  of 2,4-dinitrotoluene



estimated to result in additional  lifetime cancer  risks  ranging



Erom no additional risk to an additional risk of 1  in  100,000  are

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presented in the Criterion  Formulation  section  of  this  document.
The Agency is considering setting  criteria  at an  interim  target
risk level in the  range  of  10~5, 10~6f  or 10~7  with  corresponding
criteria of 740 ng/1,  74.0  ng/lf and  7.4 ng/1,  respectively.

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                       DINITROTOLUENES



Introduction



     Dinitrotoluene  (DNT) is an  ingredient of explosives



for commercial and military use  because of its waterproofing



action and explosive potential.  Use is also made of DNT



as a chemical stabilizer in the  manufacture of smokeless



powder.



     DNT has been shown to enter the body through inhalation



of vapors or dust particles, ingestion of contaminated food,



and absorption through the skin.  As a result of exposure



to DNT, workers have experienced muscular weakness, headaches,



and dizziness; this exposure also has been suspected of



causing pallor, cyanosis,.and anemia.



     DNT is produced by nitration of toluene to nitrotoluene



to dinitrotoluene in a nitric and sulfuric acid solution



(Lopez, 1977).  In 1975, the production of 2,4- and 2,6-



DNT in the United States was 264,030 metric tons (U.S. Int.



Trade Comm., 1977).  The production of DNT is expected to



increase yearly at a rate of 20  to 25 percent (Sittig, 1974).



There are six isomers of dinitrotoluene, with the 2,4-isomer



being the most important (Snell and Ettre, 1971).  Often



this isomer alone is referred to as DNT (Manufacturing Chem-



ists Acsoc., 1966) or dinitrotoluol (Sax, 1963).



     Nitration of o-nitrotoluene yields mostly 2,4- and



2 ,6-dini trotoluene, CH-^CgE^ (NO2) 2'  i° tne ratio of about



65.35 (Wiseman, 1972).
                              A-l

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     2,4-DNT has a molecular weight of 182.14, a melting



point of 71°C, a boiling point of 300°C with decomposition,



and a density of 1.3208 at 71°C  (Weast, 1975).  Its solubility



in water is 270 mg/1 water at 22°C? 94 g/1 ether at 22°C



and 21.9 g/1 carbon disulfide at 17°C  (Kirk and Othmer,



1967).  It is also readily soluble in  ethanol at 15°C  (30.5



g/1)  (Kirk and Othmer,  1967).



      2,6-DNT has a melting point of 66°C, a density of 1.2833



at 111°C, and is soluble  in  alcohol  (Weast, 1975).



      Except  for  their  tendency  to decompose at elevated



temperatures, dinitrotoluenes are relatively stable.  At



250°C,  commercial grades  of  dinitrotoluene decompose at



non-sustaining  rates.   However,  at approximately 280 C rapid



self-sustaining  decomposition occurs.  Dinitrotoluenes may



burn  safely  if  unconfined, but  if confined may result  in



an explosion.   Decomposition may occur at lower temperatures



in the  presence  of  impurities  (Manufacturing Chemists Assoc.,



1966) .  Mixtures of  dinitrotoluene isomers are intermediates



in the  manufacture  of  toluene diisocyanates  (Wiseman,  1972}.



Because of  the  deactivating  effect of  the two nitro groups



in dinitrotoluenes,  the synthesis of  trinitrotoluene  (TNT)



does  not occur  as readily (Wiseman,  1972).
                               A-2

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                          REFERENCES







Kirk, R.E., and D.F. Othmer. 1967.  Kirk-Othmer Encyclopedia



of Chemical Technology. 2nd ed. John Wiley and Sons, Inc.,



New York.








Lopez, A.W. 1977. Toluene diisocynate.  A paper presented



at the Am. Ind. Chem. Eng. Conference, Houston, Tex., March 23







Manufacturing Chemists Association. 1966. Chemical safety



data sheet Sd-93, Dinitrotoluenes. Washington, D.C.







Sax, N.I. 1963. Dangerous properties of industrial materials.



Reinhold Publishing Corp., New York.







Sittig, M. 1974. Pollution control  in the organic chemical



industry- Noyes Data Corp., Park Ridge, N.J.







Snell, F.D., and L.S. Ettre, eds. 1971. Encyclopedia of



Industrial Chemical Analysis. Interscience Publishers, John



Wiley and Sons, Inc., New York.







U.S. International Trade Commission. 1977. Synthetic organic



chemicals, United States production and sales, 1975. U.S



Government Printing Office, Washington, D.C.
                              A-3

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Weast, R.C., ed. 1975. Handbook of chemistry and physics.
CRC Press, Cleveland, Ohio.

Wiseman, P- 1972. An  introduction to industrial organic
chemistry.  Interscience Publishers, John Wiley and Sons/
Inc., New York.
                              A-4

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AQUATIC LIFE TOXICOLOGY*



                        FRESHWATER  ORGANISMS



Introduction




     The data base  for  dinitrotoluenes  is  limited  but 2,3-dinitro-



toluene appears  to  be up  to  two orders  of  magnitude  more  adutely



toxic to freshwater fish  and  invertebrate  species  than



2,4-dinitrotoluene.  The  tested fish  and  invertebrate species  are



similarly sensitive to  these  two dinitrotoluenes.



Acute Toxicity




     The unadjusted 96-hour  LC50 for  the  fathead minnow and



2,4-dinitrotoluene  is 31,000  ug/1  (Table  1).   After  adjustment for



test methods and species  sensitivity, the  Final Fish Acute Value



for this compound is 4,300 ug/1.   The unadjusted 96-hour  LC50  for



the more toxic 2,3-dinitrotoluene  and the  bluegill  is 330 ug/1,



and this datum results  in a  Final  Fish  Acute Value of 46  ug/1



(Table 1).
*The reader is referred to the Guidelines  for Deriving Water



Quality Criteria for the Protection of Aquatic  Life  [43  FR  21506



(May 18, 1978) and 43 FR 29028 (July 5, 1978)]  in  order  to  better



understand the following discussion and recommendation.   The



following tables contain the appropriate data that were  found  in



the literature, and at the bottom of each  table are  the  calcula-



tions for deriving various measures of toxicity as described  in



the Guidelines.
                             B-l

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     Forty-eight-hour EC50 values  are  available  for Daphnia magna



for both 2,3- and 2,4-dinitrotoluene and  are  660  and 35,000 ug/1,



respectively  (Table 2).  The  Final Invertebrate  Acute Values are



27 and 1,400  ug/1 for  2,3- and  2,4-dinitrotoluene, respectively,



and since these  concentrations  are lower  than the comparable



concentrations for fish, they also become the Final Acute Values.



Chronic Toxicity



     The chronic value  for  2,3-dinitrotoluene,  derived from a



embryo-larval test with the  fathead minnow,  is  116 ug/1 (Table 3)



and is based  on  survival of  these  life stages (U.S. EPA,  1978).



The Final Fish Chronic  Value  is 17 ug/1/  and  this concentration



also becomes  the Final  Chronic  Value for  2,3-dinitrotoluene in the



absence of  data  on  any  invertebrate species.



Plant  Effects



     Cell numbers of  the alga,  Selenastrum capricornutum, were



reduced by  50 percent at a  concentration  of 2,3-dinitrotoluene of



1,370  ug/1  (Table 4).  A comparable inhibition in chlorophyll a_



occurred  at a concentration  of  1,620 ug/1.  No other data on



dinitrotoluenes  and  freshwater  plants  are available.



Residues



     No measured steady-state bioconcentration factor (BCF) is



available  for 2,4-dinitrotoluene.   A BCF  can  be estimated using



the octanol-water partition  coefficient of 100.   This coefficient



is used! to  derive an  estimated  BCF of  19  for  aquatic organisms



that cohtain  about  8  percent  lipids.   If  it is  known that the diet



ot~ the consuming species of  concern contains  a  significantly dif-



terent lipid  content, an appropriate adjustment in the estimated



BCF should  be made.





                              B-2                                /  .

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CRITERION FORMULATION

                     Freshwater-Aquatic Life

Summary of Available Data

     The concentrations below have been rounded  to  two  significant

figures.

2,3-d initrotoluene

     Final Fish Acute Value = 46 ug/1

     Final Invertebrate Acute Value =  27 ug/1

          Final Acute Value = 27 ug/1

     Final Fish Chronic Value = 17 ug/1

     Final Invertebrate Chronic Value  = not available

     Final Plant Value = 1,400 ug/1

     Residue Limited Toxicant Concentration = not available

          Final Chronic Value = 17 ug/1

          0.44 x Final Acute Value = 12 ug/1

2,4-dinitrotoluene

     Final Fish Acute Value = 4,300 ug/1

     Final Invertebrate Acute Value =  1,400 ug/1

          Final Acute Value = 1,400 ug/1

     Final Fish Chronic Value = not available

     Final Invertebrate Chronic Value  = not available

     Final Plant Value = not available

     Residue Limited Toxicant Concentration = not available

          Final Chronic Value = not available

          0.44 x Final Acute Value = 620 ug/1

2,3-dinitrotoluene

     The maximum concentration of 2,3-dinitrotoluene is  the  Final

Acute Value of 27 ug/1 and the 24-hour average concentration  is
                             B-3

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0.44 times the Final Acute Value.   No important adverse effects on



freshwater aquatic organisms  have  been reported to be caused by



concentrations lower than  the 24-hour average concentration.




     CRITERION:   For 2,3-dinitrotoluene the criterion to protect



freshwater aquatic life  as derived using the Guidelines is 12 ug/1



as a 24-hour average and  the  concentration should not exceed 27




u.g/1 at any  time.



2,4-dinitrotoluene



     No freshwater criterion  can be derived for 2,4-dinitrotoluene



using  the Guidelines because  no Final Chronic Value for either



fish or invertebrate species  or a good substitute for either value



is available.



     Results obtained  with 2,3-dinitrotoluene and freshwater or-



ganisms indicate  how a  criterion may be estimated for 2,4-dinitro-



toluene and  freshwater  organisms.



     For  2,3-dinitrotoluene  and freshwater organisms 0.44 times



the Final Acute Value  is  less than the Final Chronic Value based



on an  embryo-larval  test  with the fathead minnow.  Therefore/ a



reasonable  estimate of  a  criterion for 2,4-dinitrotoluene and



freshwater  organisms would be 0.44 times the Final Acute Value.



     The maximum  concentration of 2,4-dinitrotoluene is the Final



Acute  Value  of 1,400 ug/1  and the estimated 24-hour average con-



centration  is 0.44 times  the  Final Acute Value.  No important ad-



verso  effects on  freshwater  aquatic organisms have been reported



to be  caused by concentrations lower than the 24-hour average con-



centration.
                              B-4

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     CRITERION:  For 2,4-dinitrotoluene  the  criterion  to  protect



freshwater aquatic life as derived using procedures  other than  the



Guidelines is 620 ug/1 as a 24-hour average  and  the  concentration



should not exceed 1,400 ug/1 at any time.
                             B-5

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                               Table  i.  Freshwater fiah acute valueu  for  dinilrotoluenes
                                    Bioacaay  Ttbt      Chemical       Time
                                    ftJ._fr*iSN/4"    ^V-v. . .* W *   F\A*3ffrmm.**mtr\    < Fl •-• O\
                                                                                           Adjusted
                                                                                                      Ketc-t c-iiCfc
Kaihcad minnow,
Pituephales promelas
Bluegill.
lepomis macrochirus

S U 2,4-dinltro- 96
toluene
S U 2,3-dinitro- 96
toluene
31,000 17,000 U.S. Army. 1976

330 180 U.S. EPA, 1978

*  S - static
** U - unmeasured
   Geometric mean of adjusted  values:   2,3-dinitrotoluene - 180
                                                                                     160
03
I
                                         2,4-dinitrotoluene - 17,000 ng/1

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                               Table 2.   Freshwater invertebrate acute values for  diniLroLoluenea
                                     Bioaesay  Test      Chemical        Time

                                     ftetf)cxl*   cone ,**   pescrijitfon
                                        Adjusted

                              LC'jU       l.C t>l<

                              (u l/i)     (u.|/ 11  	 Heterence
Cladoceran,
Daphnia magna
Cladoceran,
Daphnia roagna
* S - static
** U - unmeasured
Geometric mean of adj
S U 2,3-dinltro- 48
toluene
S U 2,4-dinitro- 48
toluene


lusted values i 2 , 3-dinitrotoluene <* 560 \>
660 560 U.S. EPA, 1978
35,000 30,000 U.S. Army, 1976


g/1 J£ - 27 Mg/l
to
J
-J
2,4-dlnitrotoluene  » 30,000
                                                                                                   1.400 Mg/1

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B)
I
03
                                 3.    Freshwater fish chronic  values  for dinitrololuenea (U.S. EPA,  1978)
                                                              Chronic

                                                    Limits    Value

            Organism                      TfeBt*     lu
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DO
I
IO
                        Table 4.   Freshwater plant effects for dlnitrocoluonos  (U.S. EPA,  1978)
                                                 Concentration
          Organism                Effect          (ug/l>	


                                   2,3-dinlcrotoluene

          Alga.                    ECSO  96-hr          1,370
          Sfclenaatrum             cell  numbers
          caprlcornutum

          Alga,                    ECSO  96-hr          1,620
          Selenastrum             chlorophyll a
          caprlcornutum                      ~
          l-oweac  plant  value;   2,3-dlnlcrotoluene « 1,370 pg/1

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                       SALTWATER ORGANISMS



Introduction



     Acute toxicity tests  using static  conditions  have  been con-



ducted with 2,3-dinitrotoluene and  the  sheepshead  minnow,  the



mysid shrimp Mysidopsis bahia, and  an alga,  Skeletonema costatum.



The LC50 and EC50 values  range from 370 ug/1 for algal  cell num-



bers to 2,280 ug/1  for the sheepshead minnow.   No  other data on



any dinitrotoluene  are available.



Acute Toxicity



     The Final  Fish Acute  Value 'for 2,3-dinitroto4uene  is  340  ug/1



(Table 5) and is based on  a single  96-hour  static  test  with the



sheepshead minnow  (U.S. EPA,  1978).



     The unadjusted 96-hour LC50  for 2,3-dinitrotoluene and



Mysidopsis bahia,  is  590  ug/1  (Table 6) and  after  adjustment for



test methods and species  sensitivity, a Final Invertebrate Acute



Value of 10 ug/1 is obtained.  This also becomes the  Final Acute



Value for 2,3-dinitrotoluene  and  saltwater  organisms  since the



comparable acute value for fish  is  higher.



Chronic Toxicity



     No chronic toxicity  data  are  available  for any dinitrotoluene



and saltwater organisms.



Plant Effects



     A 50 percent  reduction in cell numbers  of the alga,



Skeletonema costatum, occurred at a concentration  of  370 ug
                              B-10

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2,3-dinitrotoluene/l  (Table 7).  There was  a  50  percent  inhibition



of chlorophyll a_ production at 400 ug/l«



Residues



     No measured steady-state bioconcentration factor  (BCF)  is



available for 2,4-dinitrotoluene.  A BCF can  be  estimated  using



the octanol-water partition coefficient of  100..  This  coefficient



is used to derive an estimated BCF of 19 for  aquatic organisms



that contain about 8 percent lipids.  If it is known that  the diet



of the consuming species of concern contains  a significantly dif-



ferent lipid content, an appropriate adjustment  in the estimated



BCF should be made.
                             B-ll

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CRITERION FORMULATION
                       Saltwater-Aquatic Life
Summary of Available Data
     The concentrations  below have been rounded to two significant
figures.
2,3-dinitrotoluene
     Final Fish Acute  Value = 340 ug/1
     Final Invertebrate  Acute Value = 10 ug/1
           Final Acute  Value = 10 ug/1
     Final Fish Chronic  Value = not available
     Final Invertebrate  Chronic Value = not available
     Final Plant Value = 370 ug/1
     Residue Limited Toxicant Concentration = not available
           Final Chronic  Value = 370 ug/1 d for any dinitrotoluene
           0.44 x Final Acute Value = 4.4 ug/1 Value for either
     No saltwater criterion can be derived for any dinitrotoluene
 using  the  Guidelines because no Final Chronic Value for either
 fish or invertebrate species or a good substitute for either value
 is  available.
     Results obtained with 2,3-dinitrotoluene and freshwater
 organisms  indicate how a criterion may be estimated for 2,4-di-
 nitrotoluene and saltwater organisms.
      For 2,3-dinitrotoluene and freshwater organisms 0.44 times
 the Final  Acute Value is less than the Final Chronic Value based
 on  an  embryo-larval test with the fathead minnow.  Therefore, a
 reasonable estimate of a criterion for 2,3-dinitrotoluene and
 saltwater  organisms would be 0.44 times the Final Acute Value.
                              B-12

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     The maximum concentration of 2,3-dinitrotoluene  is  the Final



Acute Value of 10 ug/1 and the estimated  24-hour  average concen-



tration is 0.44 times the Final Acute Value.   No  important adverse



effects on saltwater aquatic organisms have been  reported to be



caused by concentrations lower than  the 24-hour average  concentra-



tion.



     CRITERION:  For 2,3-dinitrotoluene the criterion  to protect



saltwater aquatic life as derived using procedures  other than the



Guidelines is 4.4 ug/1 as a 24-hour  average and the concentration



should not exceed 10 ug/1 at any time.
                             B-13

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D3
                              Tdble  5.  Marine  fish acute values for dinitrotoluenes (U.S. EPA,  1978)


                                                                                           Adjusted
                                   BiOd&eay   Test       Chemical       Time      LCt>o      LLt>u
                                         i*_   £2fl£i.**   Description    ({Vra)      lltliiL
           Sheepshead minnow.         S        U           2,3-          96        2,280     1.246
           Cyprlnodon varifegatua                     dinitrotoluene
           *  S - static

           ** U • unmeasured
                                                                                        1 *? A A
              Geometric mean of adjusted values  for  2,3-dinltrotoluene - 1,246

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CO
I
»—
tn
                               Table  6.   Marine invertebrate acute values fur dinitrotoluencs (U.S.  EPA, 1978)



                                                                                            Adjusted

                                    BiOctssay   Test      Chemical       Time      LCbu      l.CLiu

                                    tt£lii2d*_   Cone,**    Description    (jug)     (u'l/i^
            Mysld shrimp,              S        U           2,3-         96         590        500
            Mysidopsia bahla                           dinitrotoluene
            *  S - static


            ** U = unmeasured


               Geometric mean of adjusted values  for  2,3-dlnltrotoluene - 500 |ig/l   —rs "  10  MB/I

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03

M
cn
                              Table 7.   Marine plant effects for dlnitrotoluenes (U.S. EPA. 1978)
                                                   Concentration
           organism               Effect           (ue/1)	
                                                       2.3-Dinttrotoluene
           Alga,                  ECSO 96-hr            400
           Skeletonema costatum   chlorophyll a

           Alga.                  ECSO 96-hr            370
           Skeleconema coatatum   cell numbers
           Lowest plant value:  2,3-dlnttrotoluene • 370 yg/l

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w
I
          *  S = static



          ** U = unmeasured

                                                                                      ^ O / £

             Geometric mean of adjusted values for 2,3-dinitrotoluene = 1,246 pg/1    —4—^— = 340 pg

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                        DINITROTOLUENE



                          REFERENCES







U.S. Army Research and Development Command.  1976.  Toxicity



of TNT wastewater  (pink water) to  ^aquatic organisms.  Final



Report, Contract DAMD17-75-C-5056.  Washington, D.C.







U.S. EPA. 1978.  In-depth studies on  health and environmental



impacts of selected water pollutants.  Contract No. 68-01-



4646.
                               B-18

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                      2,4-DINITROTOLUENE



Mammalian Toxicology and Human Health  Effects

                           EXPOSURE

Introduction

     2,4-Dinitrotoluene  (2,4-DNT)  is a pale  yellow  crystal-

line solid that is widely used as  a raw material  for  dyestuffs

and for urethane polymers through  a conversion  to the corres-

ponding diamine and then to diisocyanate  (Kirk  and  Othmer,

1967).  Some of its physical properties are  presented in

Table 1.  It is commercially prepared  in  the United States

by the direct dinitration of toluene.  This process  produces

a 80/20 ratio of 2,4-/2,6-isomers, which  on  fractionatioit
                                                         i
gives pure 2,4-DNT (Kirk and Othmer, 1967).  Precise  produc-

tion figures for 2,4-DNT are not available;  however,  the

U.S.  International Trade Commission (1977)  reported  a combin-

ed production of 272,610,000 pounds for the  2,4-and 2,6

DNT isomers in 1975.

     The name given by the Chemical Abstracts Service (1977)

for this compound is 1-methyl 2, 4-dinitrobenzene (CAS regis-

try number 121-14-2).  Other synonyms  for 2,4-DNT include

2,4-dinitrotoluol and toluene-2,4-dinitro.   2,4-DNT has

a moderate fire and explosion risk and it can be  detonated

only by a very strong initiator.

     Aside from its use by the dye and polyurethane manufactur-

ing industries, 2,4-DNT is used by the munition industry

as a modifier for smokeless powders and,  to  a limited  extent,
                              C-l

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                   TABLE 1
Some Physical Constants of 2,4-Dinitrotoluene
 (Data collected from Kirk and Othmer, 1967;
     St. John, et al. 1975; Weast, 1978)
  PROPERTY
                                        VALUE
Molecular weight
Melting Point,
Boiling point
Density
     15
    d4
     71
    d4

Vapor density  (air=l)
Vapor pressure at 25+2°C
Refractive index  (nQ)
Solubility, grams/liter
  Water, at 22°C
  Ethanol, at 15°C
  Diethyl ether, at 22°C
  Carbon disulfide, at 17°C
Heat of fusion (Hf)
                                 182.14
                                 69.5-70.5°C
                                 300°C  (dec.)
                                 1.521
                                 1.321
                                 6.27
                                 1.4 x 10~4torr
                                 1.442
                                 0.27
                                 30.46
                                 94
                                 21.9
                                 26.4 caI/gram
                 C-2

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as a gelatinizing and waterproofing agent  in military  and



commercial explosive compositions  (Hamilton and  Hardy,  1974).



2,4-DNT is also used as a chemical intermediate  in  the  pro-



duction of toluene diisocyanate  (TDI) which, in  turn,  is



consumed in the production of flexible and rigid polyurethane



foams and elastomers.  Most TDI producers, however,  use



toluene as the starting material, generating 2,4-DNT as



a captive intermediate  (Kirk and Othmer, 1967).



     The potential risk of exposure to 2,4-DNT is greatest



for workers in the dye and explosives industries and at



chemical plants producing TDI.  2,4-DNT is encountered  chief-



ly as a major component in the wastewater from munitions



industries.  The general population may experience  exposure



as a result of this discharge of 2,4-DNT into rivers and



streams from munition plants (NCI, 1978).  Aromatic  nitro



compounds are one of several classes of chemicals thought



to contribute to the increased cancer risk in dye and explo-



sive manufacturing industries (Wynder, et al. 1963).  The



structural relationship of 2,4-DNT to the known  carcinogen



2,4-toluenediamine (2,4-TDA) is also a factor in its selec-



tion for testing as a possible carcinogen  (NCI,  1978).



     The usual methods of identification and quantitative



determination of 2,4-DNT include spot tests (Ames and Yallop,



1966),  colorimetry (Goldman and Jacobs, 1953) , chromatographic



methods such as thin layer chromatography  (Yoshida,  et  al.



1967),  gas chromatography (Krzymien and Elias, 1975; Pella,



1976;  Fukuda, et al.  1977),  and HPLC (Walsh, et  al.  1973;



Doali  and Juhasz,  1974, Stanford, 1977; Natl.  Inst.  Occup.



Safety  Health Manual of Analytical Methods, 1978),  and  spec-




                              C-3

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troscopic methods such as  infrared  (Priestera, et al.  1960)
or ultraviolet  (Conduit, 1959) spectrophotometry, nuclear
magnetic resonance spectrometry  (Gehring and Reddy, 1968),
mass spectrometry  (Murrmann,  et  al.  1971; Plimmer and  Klingebiel,
1974; Zitrin and Yinon,  1976)  and  isotope dilution analysis
(St. John, et al. 1975,  1976).   In  many other  instances
where the residues of  explosives needed to  be  identified
after an explosion,  special  wet  chemical separation techniques
were used  (Hoffman and Byall,  1974;  Jenkins and  Yallop,
1970; Fukuda, et al. 1977).
                               C-4

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Ingestion from Water



     2,4-DNT has limited solubility  (270 ing/liter at 22°C)



in water as noted in Table 1.  Possible sources of 2,4-DNT



in the aqueous environment-either surface water, ground



water or drinking water-are from the dumping of chemical



wastes and from accidental loss during transfer and transport.



     Dinitrotoluene waste products are dumped into surface



water or sewage by manufacturing industries that make dyes,



isocyanates, polyurethans, and munitions.  The occurrence



of organic micropollutants due to the dumping of aromatic



nitro and amino compounds in the river water has been reported



by Meijers and Van der Leer (1976).  The pollution of the



rivers Rhine and Maas in the Netherlands by these aromatics



and oils was examined by extracting water samples in hexane



followed by analyzing the extracts by gas chromatograph/mass



spectrometry (GC/MS).  The results showed that the river



Rhine is heavily polluted by oil/ a number of aromatic hydro-



carbons, aromatic amines and aromatic nitro compounds includ-



ing 2,4-DNT.  The river Maas, however, is much less polluted



by these substances with the exception of oil.



     The second source of water contamination by 2,4-DNT



develops when the chemical is accidently spilled during



the process of transfer and/or transportation.  No specific



incidences of this type have been reported in the literature,



however.



     The ability of microorganisms to degrade 2,4-DNT and



related compounds has been studied by a number of investi-
                              C-5

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gators  (Schott, et al.  1943;  Ruchhoft,  et  al.  1945;  Ruchhoft
and Norris, 1946; Rogovskaya,  1951;  Nason,  1956;  Anon,  1970,
1971; Osmon and Klausmier,  1972;  Walsh,  et  al.  1973;  Nay,
1974; Traxler, et al.  1974; Won,  et  al.  1974;  McCormick,
et al.  1976; Parrish,  1977.)  Biotransformation of 2,4-DNT
does occur but its frequency  is much lower  than the  equivalent
activity  on 2,4,6-TNT.   The influence of aromatic nitrated
hydrocarbons including 2,4-DNT, on the activated sludge
process has been  extensively  studied (Bogatyrev,  1973;  Matsui,
et al.  1975; Roth and Murphy, 1978).  At concentrations
of 50 mg/liter of nitro aromatics, there was no effect  on
the  activated  sludge process.
Ingestion from Food
     The  likelihood  of 2,4-DNT existing in food is minimal,
since  it  is not  used as a pesticide or herbicide. There
is no  report  in  the  literature, however, on the toxic effect
of 2,4-DNT in  humans due to ingestion from food.
     A  bioconcentration factor (BCF) relates the concentration
of a chemical  in  water to the concentration in aquatic  organ-
isms,  but BCF's  are  not available for the edible portions
of all  four major groups of aquatic organisms consumed  in
the  United States.   Since data indicate that the BCF for
lipid-soluble  compounds is proportional to percent lipids,
BCF's  can be adjusted to edible portions using data  on  percent
lipids  and the amounts of various species consumed by Ameri-
cans.   A  recent  survey on fish and shellfish consumption
in the  United  States (Cordle, et  al. 1978)  found that the
per  capita consumption is 18.7 g/day.  From the data on
the  nineteen major species identified in the survey  and
                               C-6

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data on the fat content of the edible portion of  these  species

(Sidwell, et al. 1974), the relative consumption  of  the  four

major groups and the weighted average percent lipids  for each

group can be calculated:

                          Consumption       Weighted  Average
     Group                 (Percent)         Percent  Lipids

Freshwater fishes             12                  4.8

Saltwater fishes              61                  2.3

Saltwater molluscs             9                  1.2

Saltwater decapods            18                  1.2

Using the percentages  for consumption and lipids  for  each

of these groups, the weighted average percent lipids  is

2.3 for consumed fish  and shellfish.

     No measured steady-state bioconcentration factor  (BCF)

is available for 2,4-dinitrotoluene, but the equation "Log

BCF = 0.76 Log P - 0.23" can be used (Veith, et al. Manuscript)

to estimate the BCF for aquatic organisms that contain about.

eight percent lipids from the octanol-water partition coeffi-

cient (P).  Based on an octanol-water partition coefficient

of 100, the steady-state bioconcentration factor  for  2,4-

dinitrotoluene is estimated to be 19.  An adjustment  factor

of 2.3/8.0 = 0.2875 can be used to adjust the estimated

BCF from the 8.0 percent lipids on which the equation is

based to the 2.3 percent lipids that is the weighted  average

for consumed fish and  shellfish.  Thus, the weighted  average

bioconcentration factor for 2,4-dinitrotoluene and .the edible

portion of all aquatic organisms consumed by Americans is

calculated to be 19 x  0.2875 = 5.5.
                              C-7

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Inhalation
     The current estimate  in  the  United  States  for  the  number
of individuals involved  in the manufacture  of 2,4-DNT is
not available at present.   But the  U.S.  International Trade
Commission  (1977) reports  a combined  production of  272,610,000
pounds for  the 2,4- and  2,6-DNT  isomers  in  1975.  Since
DNT is produced  in such  large quantities, a considerable
population  may be at  risk.
     Inhalation  has been reported to  be  one of  the  major
routes of exposure of 2,4-DNT either  in  its particulate
or vapor state.  The  effects  from inhalation exposure to
2,4-DNT are caused by its  capacity  to produce anoxia due
to the formation of methemoglobin (See Effects  Section).
     There  are no data in  the literature on the ambient
atmospheric concentration  of  2,4-DNT. Thus, it is  not  possi-
ble to estimate  the extent of possible human exposure.
Dermal
     Since  2,4-DNT  is readily soluble in organic solvents
such as alcohol, ether,  etc., as  noted in Table 1,  it pene-
trates the  intact skin readily  (Patty, 1958; Hamblin, 1963).
From a survey  of the  literature  (Toxic and  Hazardous Indus-
trial Chemicals  Safety Manual, 1976;  Key, et al.  1977;  Proctor
and Hughes, 1978),  it is obvious  that skin  contact  is another
important route  for 2,4-DNT absorption in plant workers.
The quantitative data on the  threshold doses for  dermal
absorption  of  2,4-DNT are  unavailable in the literature.
However, the Occupational  Safety  and  Health Administration
(OSHA) recommends a threshold limit value  (TLV)  of  1.5  mg/m
of air including dermal  exposure  (Threshold Limit Values,
                              C-3

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1978).  This TLV was  set  by  analogy with  chemically  similar



nitro aromatic compounds  (American Conference  of Governmental



Industrial Hygienists, 1974).



     Because of the availability of only  limited data  on



the human exposure to 2,4-DNT/  it is difficult  to assess



quantitatively the contribution of each route  of exposure



to the total dose; it is  likely that the  greatest contri-



bution comes via inhalation, particularly in an occupational



setting.  The next most likely  route is dermal  and the least



likely is ingestion.



                       PHARMACOKINETICS



Absorption, Distribution, and Excretion



     2,4-DNT is absorbed  mainly by inhalation  of its vapor



or by percutaneous absorption of its solution  in organic



solvents.  Hodgson, et al.  (1977) recently reported  a  study



on the comparative absorption, distribution, and excretion



of 2,4,6-TNT and isomers  of  DNT in rats.   It was noticed


         14
that the   C-ring labeled nitrotoluenes were well absorbed



after oral administration in the rat.  The absorption  was



essentially complete in 24 hours with  60  to 90 percent of



the dose being absorbed.  The extent of absorption occurred



in the following order:



2,4-DNT = 3,4-DNT >3,5-DNT = 2,4,6-TNT =  2,5-DNT >2,3-DNT =



2,6-DNT.  The liver, kidneys and blood contained small amounts



of radioactivity.   The ratio of radioactivity  in tissue/plasma


                          14
indicated a retention of   C in both the  liver and kidneys,


                             14
while negligible amounts of   C were found in  the other


             14
tissues.  No   C was recovered in the  expired air; most



of the absorbed radioactivity was eliminated in the  urine.





                              C-9

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     14
When   C-labeled nitrotoluenes  were  administered to bile



duct-cannulated rats,  10.3  to 27.3 percent of the 14C was



recovered  in  the bile,  suggesting that biliary excretion



is also an  important  elimination pathway.   Thin layer chroma-



tographic  analysis  of the  urine from rats  treated with 2,4,6-



TNT or dinitrotoluene indicated extensive  metabolism of



the parent  compounds.   However, this study does not report



the characterization  of the metabolic products from dinitro-



toluenes  and  2,4,6-TNT.



     Another  study  examining the excretion and distribution



of  tritium-labeled  2,4-dinitrotoluene ( H-2,4-DNT)  in the



rat has  been  reported recently (Mori, et al.  1977).  Approxi-



mately  21.3 percent of the radioactivity was  excreted in



the feces on  the  first day after a single  oral administration



of  H-2,4-DNT.  The amount of radioactivity excreted in



the feces on  the  second and third days were 4.1 and 1.1



percent  of the  administered dose, respectively.  About 13.5



percent  of the  radioactivity administered  was excreted in



the urine on  the  first day.- but after the  second day the



urinary  excretion  of  radioactivity was found  in only trace



quantities.  In all,  about 46 percent of the  radioactivity



administered  was  excreted  in the feces and urine during



the 7 days (see Table 2).



     In  the same  experiment, relatively high  amounts of



radioactivity were  found in adipose  tissue, skin, and liver
       ?


of  the  rats seven  days after administration;  the relative



amounts  of radioactivity remaining  in other organs were



not significant (Table 3).   This investigation by the single



oral administration of  H-2,4-DNT suggests that 2,4-DNT



remains  in the  liver,  skin, and adipose tissue.


                               C-10

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                         TABLE 2

      Urinary and Fecal Excretion of Radioactivity,
         Expressed as Percentages of Administered
                      Radioactivity
                 (From Mori,  et al.  1977)
Day
1st
2nd
3rd
4th
5th
6th
7th
Urine (%)
13.52+1.44
0.61+0.12
0.66+0.12
0.48+0.18
0.28+0.08
0.19+0.09
0.15+0.03
Feces (%)
21.34+3.10
4.11+0.53
1.25+0.41
0.78+0.12
0.77+0.14
0.84+0.21
1.23+0.02
Values are indicated as means and deviations of three  rats
                       C-ll

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                                  TABLE 3

               Remaining Radioactivity in the Tissues of Rat
              Seven Days  after  Administration of 3H-2,  4-DNT
                          (From  Mori,  et  al.  1977)
Tissue
Brain
Heart
Lung
Liver
Spleen
Pancreas
Kidney
Adrenal
S tomach
Small intestine
Large intestine
Testis
Mesenteriolum
Adipose tissue
Skin

dpm per 100.,mg
Tissue x 10
0.93
0.99
1.14
1,98
0.81
1.30
0.98
2.11
0.80
0.99
1.02
0.85
0.82
13.99
0.79
Radioactivity
Total dpm x 10
1.19
0.49
1.12
17.23
0.36
0.71
1.77
0.03
0.60
4.56
0.84
1.98
1.54
68.30
25.53

% of Dose
0.03
0.01
0.03
0.40
0.01
0.02
0.04
trace
0.01
0.10
0.02
0.04
0.04
1.60
0.60
Mean of three rats given 50 mg of  H-2,4-DNT/kg p.o.
Weights of skin and adipose tissue were roughly calculated as:
skin = body weight x 1/25; adipose tissue = body weight x 1/40
                                C-12

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Metabolism



     No report has yet been published on  the metabolic  fate



of 2,4-DNT  in humans.  Even the  two  studies  (Hodgson, et



al. 1977; Mori, et al. 1977) which describe the  absorption,



distribution and excretion of 2,4-DNT in  rats do not give



details on  the characterization  of metabolites and metabolic



pathways.



     The isolation, identification and synthesis of biotrans-



formation products from 2,4-DNT  have been reported by McCormick,



et al.  (1978) from a detailed study  on the microbial trans-



formation of 2,4-DNT by Mucrosporium Sp.  (Strain QM 9651).



The biotransformation products were  identified by thin  layer



chromatography (by using silica  gel plates with  fluorescent



indicator to visualize the metabolites and developing in



benzene-hexane 50:50 percent v/v solvent mixtures) and  then



were followed by GC/MS.  The metabolites  identified were



2-amino-4-nitrotoluene, 4-amino-2-nitrotoluene,  2,2'-dinitro-



4,4'-azoxytoluene, 4,4'-dinitro-2,2'-azoxytoluene, and  4-



acetamido-2-nitro-toluene; a third azoxy compound, believed



to be a "mixed" type (i.e. 2,4'-azoxy or 4,2'-azoxy), was



also isolated, but not identified.  These authors present



a scheme for the biotransformation of 2,4-DNT (Figure 1).



Although no 2,4-toluenediamine (2,4-TDA) was detected in



the present system, complete reduction of both nitro groups



to amino groups has been reported in the biotransformation



of 2,4-DNT by anaerobic bacterial systems (McCormick, et



al. 1976);  hence,  2,4-TDA is also included in Figure 1.
                              C-13

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                     CH3
                        NHOH
                        NO2
                                                            NH2
                                    (I)
                          FIGURE  1.

Proposed Pathways for  the formation  of  Biotransforraation
Products from 2,4-Dinitrotoluene  (A)
(Taken from McCormick, et al.  1978)
The hypothetical nitroso and  hydroxylamino intermediates are enclosed
brackets.  The potential formation of 2,4-toluenediamine (L)  is
indicated by dashed arrows.

(B)  2-Nitroso-4-nitrotoluene;  (C) 2-Hydroxylamino-4-nitrotoluene;
(D)  4,4'-Dinitro-2,2'-azoxytoluene;  (E)  2-Amino-4-nitrotoluene;
(P)  4-Nitroso-2-nitrotoluene;  (G) 4-Hydroxylamino-2-nitrotoluene;
(H)  4-Amino-2-nitrotoluene;  (I)  2,2'-Dinitro-4,4'azoxytoluene;
(J)  4,2'-Dinitro-2,4'-azoxytoluene;  (K)  4-Acetamido-2-nitrotoluene
                               C-14

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     In a study of the microbial transformation of  2,4-DNT,
2,4,6-TNT and other nitroaromatic compounds by anaerobic
bacterial systems  (McCormick, et al. 1976), these compounds
were reduced by hydrogen in the presence of enzyme  prepara-
tions from Veillonella alkalescens.  Consistent with the
proposed reduction pathways, R-NO2 H2^R-NQ H2;R^-NHOH H2	^
R-NH2, 3 moles of H~ were utilized per mole of nitro group.
From the rates of reduction of 40 mono-, di-, and trinitroaro-
matic compounds by Veillonella alkalescens, it was  noticed
that reactivity of the nitro group depended on other substi-
tuents and on the position of the nitro groups relative
to these substituents.  The order of reduction rate of nitro
compounds is consistent with the "electronegativity rule"
(Shikata and Tachi, 1938):
          -NO2 > -COOH >~CH3 > -H > -OH >NH2
In the case of nitrotoluenes, the para nitro group was the
most readily reduced, the 4-nitro position of 2,4-DNT being
reduced first.  The "nitro-reductase" activity of Veillonella
alkalescens extracts was associated with protein fractions,
one having some ferredoxin-like properties and the other
possessing hydrogenase activity.  The question of whether
ferredoxin acts as a nonspecific reductase for nitroaromatic
compounds remains unresolved.
     Since the microbial transformation pathway of  2,4-DNT
(McCormick, et al. 1978)  is similar to that of 2,4,6-TNT
(McCormick, et al. 1976), it can be assumed that these two
compounds may behave-similarly during biochemical trans-
formation in animals and humans.  Hence, it is reasonable
to discuss a few studies on the metabolism of 2,4,6-TNT
in animals and humans in this context.
                              C-15

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     The explosive 2,4,6-TNT has  been  extensively  investi-
gated because of the toxic symptoms which  it  produces  in
people engaged in  its manufacture (Palmer,  et al.  1943;
Schwartz, 1944; Dobbin Crawford,  1954;  Goodwin,  1972;  Djerassi
and Vitany, 1975;  Morton, et al.  1976).   It is generally
agreed that its toxicity  is due  to its metabolic products
(Won, et al.  1974, 1976;  Carpenter, et al.  1978).   Earlier
studies  (White and Hay, 1901;  Moore,  1918;  Schereschewsky,
1918; Voegtlin, et al. 1920) have shown that  the urine of
2,4,6-TNT workers  and of  experimental  animals receiving
2,4,6-TNT orally or  by injection contained  2,2,6,6-tetranitro-
4,4'-azoxytoluene  and 2-  or 4-aminodinitrotoluene.   The
investigations of  Channon, et  al. (1944)  showed that rabbits,
when given  small oral doses of 2,4,6-TNT,  excreted 2-  and
4-aminodinitrotoluenes and 4-hydroxylamino-2,6-dinitrotoluene.
Of  the  two  amino compounds excreted,  the 4-amino-2,6-dinitro-
toluene  was found  in larger quantities and  the 4-hydroxy-
lamino-2,6-dinitrotoluene was  obviously an  intermediate
in  the  reduction of  2,4,6-TNT  to the  corresponding amino
compound.   The 4-amino-2,6-dinitrotoluene was also formed
when 2,4,6-TNT was incubated with an  acetone  extract of
pig liver  (Bueding and Jolliffe, 1946).   When administered
to  pigs,  some 24 to  30 percent of the 2,4,6-TNT appears
in  the  urine  as compounds containing  a diazotizable amino
group.   In  man, 2,4,6-TNT appears to  be converted  to the
same metabolites as  in the rabbit (Williams,  1959).  Dale
(1921)  showed that 2,2',6,6'-tetranitro-4,4'-azoxytoluene
could be isolated  from the urine of 2,4,6-TNT workers, a
                               C-16

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fact which  indicates  that  2,4,6-TNT  is  reduced  in  man  to



4-hydroxylaminc~2,6-dinitrotoluene.   Lemberg  and Callaghan



(1944) also detected  the 4-amino-2,6-dinitrotoluene  and



2-amino-4,6-dinitrotoluene  in human  urine. These authors



stated that the qualitative and quantitative  distribution



of 2,4,6-TNT metabolites in human  urine is similar to  that



found in rabbit urine.  A  scheme for  the biotransformation



of 2,4,6-TNT is presented  in Figure  2.   It is interesting



to note that no study in the literature reports the  formation



of 2,4,6-triaminotoluene as a metabolic product of 2,4,6-



TNT, though such a possibility cannot be ruled out.



     Thus, from an analogy of metabolism of 2,4,6-TNT  with



that of 2,4-DNT (compare Figures 1 and  2), one might expect



most of the products presented in Figure 1 to be present



in the urine of humans and animals exposed to 2,4-DNT.



Most of these metabolites are either  toxic (Fairchild, et
                                                         i


al. 1977)  or suspected carcinogens (Christensen, et  al.



1976) .



                           EFFECTS



Acute, Sub-acute, and Chronic Toxicity



     Acute toxic effects of 2,4-DNT  include methemoglobinemia



followed by cyanosis.  The inhalation of the  fumes or  dust,



the ingestion of the compound, or the absorption by  the



skin through contact of 2,4-DNT bring about a chemical change



of the blood oxyhemoglobin into methemoglobin (basically,



oxidation of Fe(II) to Fe(III)).  The onset of symptoms



of methemoglobinemia due to the absorption of 2,4-DNT  is



often insidious and may be delayed up to four hours; headache

-------
                                             NO2
                                                            NH2
                                    N02     N02
                                       (H)
                           FIGURE 2

Proposed Pathways  for  the  formation  of  Biotransformation
Products from 2,4, 6-Trinitrotoluene  (A)  (Taken from Williams,
1959; Won, et al.  1974).   The  hypothetical  nitroso inter-
mediat^es are enclosed  in brackets.   The potential formation
of 2/4-Diamino-6-nitrotoluene  (J)  is indicated by dashed arrows.
    4-Nitroso-2,6-dinitrotoluene;  (C)  4-Hydroxylamino-2,6-
dinitrotoluene;  (D) 2,2',  6,6 '-Tetranitro-4,4 '-azoxy toluene;  (E)
4-Amino-2,6-dinitrotoluene;  (F)  2-Nitroso-4,6-dinitrotoluene;
(G) 2-Hydroxylamino-4,6-dinitrotoluene;  (H)  4r4',  6 ,6 '-Tetranitro-
2, 2 '-azoxy toluene;  (I) 2-aminQ-4,6-dinitrotoluene
                              C-18

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is commonly the first symptom and may become quite  intense



as the severity of methemoglobinemia progresses.  The  follow-



ing symptoms have been reported as a result of varying doses



of 2,4-DNT:  vertigo, fatigue, dizziness, weakness, nausea,



vomiting, dyspnea, drowsiness, arthralgia, insomnia, tremor,



paralysis, unconsciousness, chest pain, shortness of breath,



palpitation (rapid throbbing of heart), anorexia  (lack of



appetite), and loss of weight (Koelsch, 1917; Von Oettingen,



1941; Mangelsdorff, 1952, 1956; Hamblin, 1963; Toxic and



Hazardous Industrial Chemicals Safety Manual, 1976; Key,



et al. 1977; Proctor and Hughes, 1978).  2,4-DNT also produces



Heinz bodies (granules in red blood cells due to damage



of the hemoglobin molecules) in the cat (Bredow and Jung,



1942).  Human subjects are similarly susceptible, and workers



handling such compounds as nitrobenzenes, nitrotoluenes



and phenylhydrazines occasionally exhibit Heinz bodies in



their blood (Hughes and Treon, 1954; De Bruin, 1976).



     Inactivation of the hemoglobin under the effect of



2,4-DNT and related compounds has been noted by Vasilenko,



et al. (1972).   These authors observed the transformation



of hemoglobin into methemoglobin, nitrosylhemoglobin, and



sulfhemoglobin when rats received 0.1 to 0.2 LD50 of 2,4-



DNT orally for  a period of 30 days.  An increase in the



levels of methemoglobin and sulfhemoglobin was accompanied



by a decrease in oxyhemoglobin, but the total level of hemo-



globin remained unchanged.



     Methemoglobin formation of nitrotoluenes in relation



to the number  and positioning of nitro groups was studied



by Kovalenko (1973).  when administered orally at doses




                              C-19

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corresponding to 0.1 to 0.2 LD50 values  to  rats  for  one



to three months, the hemotoxicity of  the nitrotoluenes de-



creased in the order:  trinitrotoluene >dinitrotoluene >
                                                       Vy


m-nitrotoluener p-nitrotoluene> 0-nitrotoluene.



     Cyanosis due  to the  absorption of 2,4-DNT occurs when



the methemoglobin  concentration is 15 percent or more. The



symptoms observed  include blueness in the lips,  the  nose,



and the ear lobes.   The individual usually feels  well, has



no complaints, and insists that nothing  is  wrong until the



methemoglobin concentration approaches approximately 40



percent, when there usually is  weakness  and dizziness; at



levels of  about  70 percent methemoglobin there may be ataxia,



dyspnea on mild  exertion, tachycardia, nausea, vomiting,



and drowsiness  (Hamblin,  1963).  Because of the  increased



vapor pressure with higher ambient temperatures, there is,



in general,  an  increased  susceptibility  to  cyanosis  from



exposure to  2,4-DNT (Linen, 1974).



     Some  earlier  studies provide useful information on



the toxicity of  2,4-DNT.   Animal experiments reported by



White, et  al.  (1902)  indicate that 2,4-DNT  is comparatively



less toxic than  1,3-dinitrobenzene.   They found  that cats



may tolerate the  repeated oraj.  administration of 2 or 4 ml



of a 1 percent  solution  in cod  liver  oil, until  a  total



of 24 ml has been  given,  without any  toxic  effect.   Similarly



Zieger  (1913) observed no toxic effects  from the inhalation



of vapors, whereas in  the experience  of  Kuhls  (1908), the



subcutaneous injection  in cats  of 0.05 to 0.5 g  of 2,4-DNT



dissolved  in mineral oil  resulted in  death  after periods



of 2 to 23 days.   Regarding the possibility of absorption





                              C-20

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through the skin, Dambleff  (1908) found  in rabbits no  indica-
tion of a toxic action by this route, and similarly Kuhls  (1908)
observed in cats no toxic effects from the cutaneous adminis-
tration of 0.3 g/kg body weight, while Zieger  (1913) found
two doses of 5 g each were fatal to cats in eight hours.
     A list of the toxic doses for a number of animal  species
is presented in Table 4.  The rat oral LD50 values listed
in Table 4 are comparable to those of nitrobenzene and 2,6-
DNT.  The mouse oral toxicity follows the order:  aniline >
1,3,5-trinitrobenzene >2,6-DNT >3-nitrotoluene = 4-nitrotol-
uene = 2,5-DNT >2,4-DNT ?2-nitrotoluene.

                           TABLE 4
        Acute  Toxic Levels  of  2,4-Dinitrotoluene  for
                      Different Species
        (Data collected  from  Spector, 1956; Fairchild,
              et al.  1977;  Vernot, et al. 1977)
Species
Rat
Mouse
Cat

Route
Oral
Oral
Oral
S.C.
Toxicity
LD50
LD50
MLD
LDLo
Dose
(mg/kg)
268
1625
27
50-500
     S.C.  - subcutaneous;  LDLo - lowest published lethal dose;
     LD50  - lethal dose 50 percent kill; MLD - minimum lethal dose
                              C-21

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     With-- regard-: to  the  human  toxicity/of :2, 4-DNT, toxic
effects may'only  occasionally  be observed:from.the handling
of the- pure-material.   In addition to the complaints'discussed
above  due to methemoglobinemia,  more severe cases involving
dyspnea, dizziness,  sleepiness,  and painrin"the joints (es-
pecially in the  knee)  have-been  reported'(Perkins,,1919).
Perkins  (1919) also  pointed-out  that :during the purification
of• the crude 2,4-DNT cakes,  toxic vapors may be inhaled
and-the material may be sufficiently absorbed through the
skin' to cause  toxic  effects.   Floret"(1929) reported a'severe.
case of 2',4-DNT  poisoning, in  which:-the .patient - (a plant.,
worker) suffered from severe cyanosis and^complained"later
of headache, palpitation of-heart, oppression in'the-chest,,
insomnia and'lack of appetite.  Upon examination, medical
findings  indicated tremors of-varying intensity :in the hands,
arms,, head, extended"fingers  and- coat ing
which use-2,4-DNT'.,  The chief  symptomsnof a group of
workers  so  exposed were an unpleasant metallic -taste,  weakness,
headache, loss^of appetite,  and  dizziness.  Two^-thirds of
the  men  in  the group-selected  for- study/had these complaints
                               C-22-

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at one time or another during the 12-month exposure period.



One-half of the group developed clinical signs of  intoxi-



cation, chiefly pallor, cyanosis and low-grade anemia.



Jaundice was observed in two patients.  No instances of



permanent physical impairment were found.  The symptoms



described by these workers are presented in Table  5; Table



6 presents the chief findings from clinical examinations



of these workers.



     There is no report in the literature that discusses



the mechanism of toxic action of 2,4-DNT per se.   Usually



its toxic action is presented along with other structurally



related aromatic nitro and amino compounds.  Most  of the



aromatic nitro and aminp compounds are not in themselves



cyanogenic, but oxidation-reduction enzyme systems promote



biotransformation to known active derivatives that arise



from either reduction of the nitro group or oxidation of



the amine.  Most of the aromatic nitro and amino compounds



that have been investigated, regardless of species, including



man, come to a point of equilibrium,



          Hemoblobin^    ^Methemoglobin,



beyond which,  in spite of further dosage, no appreciable



increase in methemoglobin concentration can be obtained



(Hamblin, 1963).   Bodansky (1951) also points out  that there



normally exists an equilibrium in blood between hemoglobin



and methemoglobin,  which is usually shifted far to the right.



He believes that this shift is regulated by various oxidizing



and reducing substances produced during in vivo metabolism;
                              C-23

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                       TABLE 5

Symptoms Presented by 154 2,4-Dinitrotoluene Workers
              (From McGee,  et al. 1942)
Screening
House
Symptom Number of
Wor kmen
Unpleasant taste
in mouth
Weakness
Headache
Inappetence
Dizziness
Nausea
Insomnia
Pain in extremities
Vomi t i ng
Numbness and tingling
Loss of weight
(5 pounds or more)
Diarrhea
62
51
48
42
43
39
37
26
22
18
7
3
Coating
House and
Air dry
Number of
Workmen
34
27
28
30
25
18
20
14
13
11
3
5
Total
Number Percent
96
78
76
72
68
57
57
40
35
29
10
8
62
51
49
47
44
37
37
26
23
19
6.5
5.2
                       C-24

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                      TABLE 6

Clinical Findings in 154 2,4-Dinitrotoluene Workers
             (From McGee, et al. 1942)
Screening
House
Finding (Number of
Workmen)
Pallor
Cyanosis
Anemia
Leucocytosis
Hypotension
Skin rash
Leukopenia
Hepatitis and
Jaundice
40
38
28
12
8
2
2
1
Coating
House
(Number of
Workmen)
15
14
8
7
1
4
3
1
Total
55
52
36
19
9
6
5
2
Percent
36
34
23
12
5.
3.
3.
1.




8
9
2
4
                      C-25

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he believes such a concept  helps  to  explain  the  difference
in degree of methemoglobin  formation in  various  species,
as well as the differing  rates  of reduction  of methemoglobin
to hemoglobin.  Methemoglobin-forming capacity  in the cat
of some aromatic nitro  and  amino  compounds including  2,4-
DNT are presented  in  Table  7.
     From a ten year  study  on  the biological monitoring
for industrial exposure to  cyanogenic aromatic nitro  and
amino  compounds, Linch  (1974)  establishes a  reasonably good
relationship between  causative  agent structure  and biochemical
hazard in order to rank the relative hazard  of  these  chemicals.
In this study, dinitrotoluenes  are ranked No.  12 (1 most
potent, 13 least potent)  indicating  that 2,4-DNT does not
produce cyanosis as  rapidly as  other cyanogenic  aromatic
nitro  and amino compounds.   From  the similarities of  its
toxic  effects with other  structurally related aromatic nitro
compounds, and also  from the available information of its
metabolic pathway  (as presented in Figure 1),  a  possible
cyanosis mechanism for  2,4-DNT  is presented  in Figure 3.
     Subacute toxicity  of 2,4-DNT in dogs, rats, and  mice
was studied by Ellis, et al. (1976).  2,4-DNT was given
orally to dogs  in  daily doses  of  1,  5, or 25 mg/kg and to
rats and mice  in  feed as 0.07,  0.2,  or 0.7 percent of their
diet for 13 weeks.   Toxic effects in the dogs and rats includ-
ed inhibition of muscular coordination in the hind legs,
rigidity in extension of  the hind legs,  decreased appetite,
and weight loss.   Only  the  appetite  and  weight effects were
observed in mice.   The  highest  doses were lethal to some
animals in all  three  species, while  the  lowest doses  produced
                               C-26

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                        TABLE 7
    Methemoglobin-forming Capacity of Some Aromatic
            Nitro and Amino Compounds in Cat
   (Data collected from Hamblin, 1963; De Bruin,  1976)
        Compound
Molecular ratio*
Nitrobenzene
1,3-Dinitrobenzene
1,3,5-Trinitrobenzene
2-Nitrotoluene
3-Nitrotoluene
4-Nitrotoluene
2,4-Dinitrotoluene
2,6-Dinitrotoluene
2,4,6-Trinitrotoluene
Aniline
Phenylhydroxylamine
3-Aminonitrobenzene
1,3-Diaminobenzene
Nitrosobenzene
   0.86
   7.1
   4.8
   0.05
   0.04
   Very slight
   1.4
   0.55
   1.7
   2.5 (2.7)
  34.0
   3.0
   1.4
   8.6 '
 Molar ratio of methemoglobin formed to dose of test compound
                           C-27

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                                Methemoglobin
                                  reductase
                          Hemoglobin       Methemoglobin
                 CH3
                      NO2
                            NAD*
                 NHOH
   4-HYDROXYUAMINO— 2— NITROTOLUENE
                           \
            RAPID
                       NADH + H+
                                                        CH3
                                                            NO2
                                                        NO
                          4—NITROSO—2—NITROTOLUENE
                              /
                             /
(0)
 cyanopathic
intermediates
(H)
SLOW
                 CHS
                                     CH3
               (Q
                     .NO2
                 NH2
       4—AMINO—2—NITROTOLUENE
                                         NO2
                                     NO2


                             2.4—DINITROTOI UENE
                                FIGURE 3


Suggested Metabolic Pathway  for Cyanosis by  2,4-Dinitrotoluene
based upon data from related compounds.
                                      C-28

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no toxic effects.  All  species  showed  methemoglobinemia



and anemia with reticulocytosis.  Characteristic  tissue



lesions were extramedullary hematopoeisis  in  the  spleen



and liver, gliosis and  demy-elination  in the  brain,  and



atrophy with aspermatogenesis in  the testes.   2,6-DNT  tested



similarly in dogs  (Ellis, et al.  1976) at  4,  20,  or  100



mg/kg/day and  in rats and mice  at 0.01, 0.05,  and 0.25 percent



in their diet, produced similar effects.   It  was  concluded



that the primary subacute toxic effects of  2,4- and  2,6-



DNT are seen in the red cells,  nervous system, and testes.



     Chronic exposure of 2,4-DNT may produce  liver damage,



jaundice and reversible anemia  due to  blood damage (Linch,



1974; Key, et  al. 1977; Proctor and Hughes, 1978).   Liver



injury may be more common than  cyanosis, especially  if the



diet is deficient in protein (von Oettingen,  1941; Gleason,



et al. 1969).  Kovalenko (1973)  reports that  the  chronic



exposure of 2,4-DNT in  rats caused anemia accompanied by



reticulocytosis, a decrease in  the level of sulfhydryl groups,



and an increase in that of fibrinogen  in the  blood.



     Influence of diet on the chronic  toxicity of 2,4-DNT



in mice was studied by Clayton  and Baumann  (1944).   Mice



fed with 2,4-DNT grew better on diets  high  in  fat than those



fed on other diets.  Those animals maintained  on  diets low



in fat and fed 2,4-DNT showed a retardation in the rate



of growth, and many died within five weeks.  Mice raised



to maturity on the low fat diet or on  a procarcinogenic



diet were less resistant to toxicity from parenteral 2,4-



DNT than mice  raised on the other diets.





                              C-29

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     From another study on  the  effect  of  fat  and  calories
on the resistance of mice to chronic toxicity of  2,4-DNT,
Clayton and Baumann  (1948)  observed that  mice ingesting
2,4-DNT grew less and died  faster  when fed  a  diet moderately
low in fat  (0.46 percent) than  when fed the same  amount
of 2,4-DNT per calorie  in diets containing  5  or 30 percent
of added  fat.  Pat likewise appeared to minimize  the toxic
effects of  2,4-DNT in rats.  When  the  effects of  a low calorie
intake are  corrected for, 2,4-DNT  per  se retarded growth
only slightly-  Clayton and Baumann  (1948)  noted  that many
different fats and oils appeared equally active  in minimizing
the toxic effects of 2,4-DNT.
     The  effect of diet on  the  susceptibility of  the rat
to chronic  poisoning by 2,4-DNT was also studied  in detail
by Shils  and Goldwater  (1953).   A  high intake of  fat, in
the form  of corn oil, was found to be  definitely  beneficial
with respect to  the  survival of rats subsisting on a low-
protein  intake and receiving 2,4-DNT parenterally.  Increased
amounts of  protein with a low  fat  diet prevented  death,
regardless  of  the mode  of 2,4-DNT  administration.
Synergism and/or Antagonism
     Ingestion of alcohol has  a synergistic effect on the
toxicity  of 2,4-DNT-  Friedlander  (1900)  discussed a patient
who exhibited  acute  confusion  and  retrograde  amnesia after
exposure  to 2,4-DNT  and drinking a small amount of beer.
This synergistic effect of  alcohol on  the toxicity of 2,4-
DNT was also noted by McGee, et al.  (1942).  Of  the group
of 154 male workers  exposed to  2,4-DNT in military screening
and coating  houses,  23  showed  a reduced tolerance for alcohol
                              C-30

-------
and 31 stated that their toxic symptoms had  been  aggravated



by ingesting alcohol.  Some workers  reported  that they  had



found it  impossible to drink any alcoholic beverage within



two to three hours after finishing a shift without experienc-



ing reactions such as substernal pressure, precardial "palpi-



tation",  fullness in the head, and severe acute illness.



     The  ingest ion of alcohol normally causes  increased



susceptibility to cyanosis; thus, alcohol in  any  form should



never be  administered to a victim of 2,4-DNT  poisoning.



Furthermore, since the body eliminates 2,4-DNT rather slowly,



abstention from alcoholic beverages  should be practiced



for several days after 2,4-DNT exposure (Von  Oettingen,



1941; Key, et al. 1977; Proctor and Hughes 1978).



Teratogenicity



     No studies were found in the literature  which addressed



the teratogenicity of 2,4-DNT or the other isomers of dinitro-



toluene.



Mutagenicity



     The  data available in the literature on  the  mutagenicity



of 2,4-DNT are limited and rather confusing.  Studies by



Hodgson,  et al.  (1976)  show some positive results.  The



mutagenic effect of 2,4-DNT on germinal cells was  studied



by these  authors using the dominant lethal assay  on rats



fed a diet containing 2,4-DNT for 13 weeks.  Females mated



to males  treated with 0.2 percent 2,4-DNT showed  a significant



increase  in the number of dead implants/total implants over



control animals.



     Hodgson,  et al.  (1976)  also studied somatic  cell mutation



effects by cytogenetic analysis of lymphocyte and  kidney



                              031

-------
cultures derived from  rats  fed  0.2  percent  of  2,4-DNT for
19 weeks.  No  increase in the  frequency  of  translocations
or chromatid breaks was  observed  in either  the lymphocyte
or kidney cultures.  However,  significant increases in the
frequency of chromatid gaps were  observed in kidney cultures
after five weeks and in lymphocytes at 19 weeks.   This would
suggest  that 2,4-DNT has a  potential for inducing damage
in somatic cells.   In  vitro studies using the  CHO-K1 test
system were negative.   On the  other hand, microbial tests
using Salmonella typhimurium TA 1535 indicated that 2,4-
DNT  is capable of  producing base-pair mutations.
     There are two other reports  in the  literature (Simmon,
et al. 1977; Cotruvo,  et al. 1977)  which discuss  the mutagenic
effects  of products from ozonation  or chlorination reactions
of 2,4-DNT and other related di-  and trinitrotoluenes.
In the study by Simmon, et  al.  (1977), a number of compounds
present  in waste water from munitions plants were examined
before and after ozonation  or  chlorination to  determine
whether  such activity  was affected  by the treatment.  Test
materials  included 1,3-dinitrobenzene; 2,4-DNT; 3,5-DNT,
2,4,6-TNT; 2,4,6-TNT production waste water; hexahydro-1,3,5-
trinitro-s-triazine (RDX);  octahydro-1,3,5,7-tetranitro-
s-tetrazine  (HMX); components  of  photolyzed 2,4,6-TNT; pent-
aerythritol  tetranitrate, and  trinitroresorcinol.  The ir\
vitro mutagenic assays used were  the Salmonella/microsome
assay  (Ames, et al.  1973) with strains TA 1535, TA 1537,
TA 1538, TA  98, and TA 100  and mitotic recombination in
the  yeast, Saccharomyces cerevisiae D3.   A metabolic acti-
vation system  using the postmitochondrial supernatant fraction
                               C-32

-------
of liver from rats pretreated with Aroclor  1254  was  included



in each assay procedure.  Under  these conditions,  neither



ozonation nor chlorination significantly altered the mutagenic



activity of these nitro aromatic materials  tested  including



2,4-DNT.



     In the investigation of mutagenicity of products of



ozonation in water by Cotruvo, et al. (1977), compounds



such as 2,4-DNT, phenol, hydroquinone and nitrilotriacetic



acid were found to give anomalous results in Saccharomyces



after ozonation.  Although elevated activity was indicated



in some of the experiments, it was not dose-related.  At



the concentrations tested  (0.08 jug/plate, highest  dose),



2,4-DNT was not mutagenic in the Salmonella assay  before



or after ozonation.  The highest concentration tested in



the Saccharomyces assay, 0.004 percent was not mutagenic



or toxic.   There was generally a higher number of  mitotic



recombinants after ozonation, but the response was not dose-



related.  The products of ozonation of TNT condensate water



(a mixture of complex nitroaromatics containing  primarily



2,4-and 2,6-DNT's) were also tested for mutagenicity.  Two



new products (m/e 166 and 270) were found in the GC/MC profile.



The fragmentation pattern of the m/e 166 compound  was found



to be consistent with a nitrosonitrotoluene but  was not



confirmed.   Prior to ozonation, the TNT condensate water



mixture was mutagenic in Salmonella assays but not in Saccha-



romyces.  After ozonation, the mixture was weakly mutagenic



in only one experiment with TA 1535 and TA 100 in  the absence



of metabolic activation; thus, activity was considerably



reduced afer ozonation.  A duplicate experiment  showed no




                              C-33

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activity.  These mutagenicity  results  are  presented  in Table


8.


Carcinogencity


     There are  two  reports  in  the  literature  (NCI/ 1978;


Lee, et al. 1978) which  address  the  carcinogenicity  of 2,4-


DNT.  A bioassay of practical-grade  2,4-DNT for  possible


carcinogencity  (NCI,  1978)  was conducted  using Fisher  344


rats and B6C3F1 mice.  2,4-DNT was administered  in the feed


to male and female  rats;  the  low and high  time-weighted


average doses were  17.6  and 44.0 mg/kg/day for male  rats


and 25.3 and 63.4 mg/kg/day for  female rats,  respectively.


For male and female mice,  the  low  and  high time-weighted


average doses were  16.3  and 81.5 mg/kg/day, respectively.


Both rats  and mice  were  treated  with 2,4-DNT  for 78  weeks.


In  the male rats, a significantly  higher  incidence of  fibroma


of  the skin and subcutaneous  tissue  occurred  in  the  high


and low dose groups when compared  to their respective  controls


 (Table 9).  A statistically significant incidence of fibroade-


noma of the mammary gland occurred in  the  treated female


rats of  the high dose group (Table 10).  It should be  noted


that the above-mentioned tumors  were benign.


     There were certain  unusual  neoplasms  (i.e., hemangio-


sarcoma  in the  subcutis,  hemangiosarcoma  of the  urinary


bladder, and prostrate gland  adenocarcinoma)  that occurred


at  low incidences  in  high dose male  rats  but  did not occur


 in  either  low dose  or control  male rats.   The authors  (NCI,


1973) considered that these tumors were not related  to chemical
       i

administration.
                               C-34

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                                                    TABLE  8
O
I
ui
                                            Mutagenic  Assay  Results
                                            of Munitions Compounds
                                          (From  Cotruvo,  et  al.  1977)
Munitions
Compounds
Initial
Concentration
(ppm)
Reaction
Time
(min)
Reacted pH
Salmonella
Activity
Saccha-
romyces
Activity
Comments
   2,4-Dinitrotoluene        28.3
   TNT condensate water      35.4
20
100
96
8.4/3.8
9.3    7.2/3.6
-/+     elevated activity
        in high dose, not
        dose related

-/-     activity found in
        one test, reduced
        by ozonation

-------
o
                                                   TABLE 9

                                Summary of the Significant Primary Tumors  at
                        Specific Sites in Male Rats Treated with 2,4-Dinitrotoluene
                                               (From NCI,  1978)
TOPOGRAPHY: MORPHOLOGY
Subcutaneous Tissue or Skin: Fibroma
P Values0
Relative Risk (Control)
Lower Limit
Upper Limit
Weeks to First Observed Tumor
LOW DOSE
CONTROL
0/46(0.00)
	
	
	
HIGH DOSE
CONTROL LOW DOSE
0/25(0.00) 7/49(0.14)
	 P = 0.008
	 Infinite
1.827
	 Infinite
96
HIGH DOSE
13/49(0.27)
P = 0.003
Infinite
2.106
Infinite
85
     Treated  groups  received  time-weighted  average concentrations of 17.6 and 44.0 mg/kg/day in feed.

     Number of  tumor-bearing  animals/number of  animals  examined at site (proportion).
    Q
     The  probability level  for  the  Fisher exact test  for  the comparison of a treated group with the
    control group  is given  beneath  the  incidence of tumors  in the treated group when P<0.05;  other-
    wise,  not significant  (N.S.)  is indicated.   A negative  designation (N)  indicates a lower incidence
    in  the treated group than in  the  control group.

     The  95%  confidence  interval  of the  relative risk of  the treated group to the  control group.

-------
     For the mice, there were no tumors  in either sex having

a statistically significant positive association between

administration of 2,4-DNT and incidence  of tumor.  As such

there is no convincing evidence of tumorigenicity in B6C3F1

mice at the dose levels of 2,4-DNT used  in these experiments.

The possibility of a negative association between administra-

tion and incidence was observed for pituitary adenomas in

female mice and for alveolar/bronchiolar neoplasms in male

mice.

     At this point, it is relevant to present some of the

comments made regarding this carcinogenesis study by the

Data Evaluation/Risk Assessment Subgroup of the Clearinghouse

on Envirnomental Carcinogens: (NCI, 1978)

     1.    The tumors in the treated rats must be viewed
          with concern, especially since the maximum tolerat-
          ed dose may not have been attained.
     2.    Since 2,4-DNT is an intermediate in the production
          of dyes, there may be considerable human exposure
          from its residues in dye products.  Hence, there
          may be a potential for human risk because of the
          increased tumor incidence seen in the treated rats.
     3.    The biological activity of 2,4-DNT may be due
          to its possible conversion to  the diamine compound,
          2,4-toluenediamine.  The rate of its enzymatic
          conversion may limit its activity.
     4.    These data do not allow an assessment of human
          risk.
     5.    In view of the significant number of benign tumors in
          the treated rats and widespread human exposure, 2,4-DNT
          should be considered for retest using another species and
          route of exposure, especially dermal.

-------
                                             TABLE  10

                           Summary of  the Significant  Primary Tumors  at
                  Specific Sites  in Female Rats  Treated  with  2,4-Dinitrotoluene*
                                          (From NCI,  1978)
LOW DOSE HIGH DOSE
TOPOGRAPHY: MORPHOLOGY CONTROL CONTROL
Mammary Gland:
P Values0
Relative Risk
Weeks to First
Fibroadenomab 9/48(0.19) 4/23(0.17)
	 	
(Control) d 	 	
Lower Limit 	 	
Upper Limit 	 	
Observed Tumor 92 109
LOW DOSE
12/49(0.24)
N.S.
1.306
0.559
3.183
83
HIGH DOSE
23/50(0.46)
P = 0.016
2.645
1.062
9.435
69
 Treated groups received time-weighted average concentrations of 25.3 and 63.4 mg/kg/day in feed.

 Number of tumor-bearing animals/number of animals examined at site  (proportion).

GThe probability level for the Fisher exact test for the comparison of a treated group with the
control group is given beneath the incidence of tumors in the treated group when P^O.05; other-
wise, not significant (N.S.)  is indicated.  A negative designation  (N) indicates lower incidence
in the treated group than in the control group.

 The 95% confidence interval of the relative risk of the treated group to the-control group.

-------
     Another bioass'ay of practical  grade  2,4-DNT for  possible
carcinogenicity was conducted  by  Lee,  et  al.  (1978)  using
CD^ rats  (Charles River Breeding  Laboratory,  Wilmington,
Mass.)  The high dose, with  2,4-DNT intake of 34.0 mg/kg/day
in male rats and 45.0 mg/kg/day in  female rats,  was quite
toxic, caus.ing decreased weight gain and  shortened life
span.  Target organs  included  the blood (toxic anemia),
the liver  (hep'atocellular  carcinoma),  the testis (aspermato-
genesis) ,  and connective tissue in  male rats  (fibromas),
and the mammary  tissue  in  female rats (fibroadenomas).
No specific effects were seen  on the reproductive process,
on chromosomes,  or on the  metabolism of 2,4-DNT.  The middle
dose, with 2,4-DNT intake  of 3.90 mg/kg/day in male rats
and 5.10  mg/kg/day in female rats,  was somewhat  toxic.
It caused similar effects  in some, 'more susceptible,  individual
rats.  The low dose,  with  2,4-DNT intake  of 0.57 and  0.71
mg/kg/day in male and female rats respectively,  had no apparent
toxic effects.   The carcinogenicity results for  male  and
female rats are  summarized in  Tables 11 and 12.
     Since 2,4-toluenediamine  (2,4-TDA)  is a  possible metabolic
product of 2,4-DNT  (as  seen  in Figure 1)  and  is  mentioned
in the critique  of the  above study,  it is reasonable  to
discuss briefly  the carcinogenicity and mutagenicity  of
2,4-TDA.
     2,4-TDA  is  widely  used  in the  production of human hair
dyes.  Umeda  (1955) reported that the repeated subcutaneous
injections of 2,4-TDA induced  rhabdomyosarcomas  in 100  percent
of rats treated.  Rats  fed diets  containing 2,4-TDA developed
hepatocellular carcinomas  (Ito,  et  al.  1969).  Similarly
Swiss-Webster mice fed  2,4-TDA showed  a high  incidence  of
                               C-38

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                    TABLE  11

  Summary of the Male Rats with Apparent Tumors
After being Fed  2,4-Dinitrotoluene  for  18 months
             (From Lee,  et al. 1978)
Dose
(mg/kg/day)
0
0.57
3.90
34.0
Tumor/Total
1/37
0/37
0/29
17/23
Percent
3
0
0
74
                    TABLE 12

 Summary  of  the  Female Rats  with Apparent Tumors.
After being Fed 2,4-Dinitrotoluene for 18 months
             (From Lee,  et al.  1978)
Dose
(mg/kg/day
0
0.71
5.10
45.0
Tumor/Total
8/29
11/40
10/27
28/32
Percent
28
28
37
88
                      C-39

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lung neoplasms  (Stoats, 1972).  In contrast,  the  recent
study by Giles, et al.  (1976)  indicates  that  the  2,4-TDA
and other hair dye ingredients did not augment  the development
of primary lung neoplasms  in mice.  Skin neoplasms were
seen in most groups of  Swiss-Webster mice, but  the incidence
of these tumors in treated animals when  compared  to  control
mice, was not significant.  The 2,4-TDA  under these  experimen-
tal conditions was found to be nontoxic  and noncarcinogenic
to the skin of mice.
     On the other hand, it has been shown  that  2,4-TDA  is
a mutagen in several  systems.  A good correlation between
mutagenicity of 2,4-TDA in the Salmonella/ microsome test
and morphological transformation in hamster embryo cell
system was observed by  Shah, et al.  (1977).   2,4-TDA usually
requires metabolic activation  by rat liver microsomal enzymes
(S9) for mutagenesis  in tester strains TA  1538  and TA 98
(McCann, et al. 1975; Shah, et al. 1977; Dybing,  et  al.
1977; Pienta, et al.  1977).  In contrast,  transformation
of hamster cells was  induced without the addition of external
enzymes  (Shah,  et al. 1977), presumably  because the  cells
can metabolize  2,4-TDA  to  its  active derivatives.  There
was no mutagenic activity  in the strain  TA 100, indicating
that 2,4-TDA is not a base pair mutagen. The dose-response
curves obtained with  tester strains TA 1538 and TA 98 demon-
strated that 2,4-TDA  is metabolized by the S9 to  a frameshift
mutagen  (Shah,  et al. 1977).   2,4-TDA was  also  found to
be mutagenic in the sex-linked recessive lethal test in
Drosophilia melanogaster male  germ cells (Blijleven,  1977;
_I^H^MMB»«.^A^M«^^H^MBW ~-^^^—^—~—^^^*-^——^~            ^
Fahmy and Fahmy, 1977;  Venitt, 1978).
                              C-40

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                    CRITERION FORMULATION



Existing Guidelines and Standards



     At present, no standard for exposure to 2,4-DNT  in



drinking or ambient water has been set in the United  States.



However, a Russian study  (Korolev, et al. 1977) recommends



that a maximum permissible concentration in the surface



waters should be set at a level of 0.5 mg/1 for each  DNT



isomer.



     The American Conference of Governmental Industrial



Hygienists (Am. Conf. Gov. Ind. Hyg.) recommends a threshold



limit value-time weighted average  (TLV-TWA) concentration



of 1.5 mg of 2,4-DNT per cubic meter of air (1.5 mg/m )



including dermal exposure for a normal eight-hour workday



of 40-hour workweek (Am. Conf. Gov. Ind. Hyg. 1978).   This



value represents the highest level to which nearly all workers



may be repeatedly exposed, day afer day, without adverse



effect.  This TLV-TWA was set by analogy with chemically



similar nitro aromatic compounds.  A threshold limit  value-



short term exposure level (TLV-STEL) of 5 mg of 2,4-DNT/m



of air was also set by the ACGIH  (Am. Conf.  Gov- Ind. Hyg.



1978).  The TLV-STEL is defined as the maximal allowable



concentration to which workers can be exposed for a period



of up to 15 minutes continuously without suffering from



1) irritation, 2)  chronic or irreversible tissue change,



or 3) narcosis of sufficient degree to increase accident



pront-ness, impair self-rescue, or materially reduce work



efficiency.  No more than four exposures to the TLV-STEL



per day are permitted, with at least 60 minutes between



exposure periods,  and the daily TLV-TWA must also not  be



exceeded.                        . .
                              C-41

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Current Levels of Exposure


     No data on the extent  of  human  exposure  to  2,4-DNT


are available in the  literature.   However,  a  study of  the


concentration of explosives in air by  isotope dilution analy-


sis  (St. John, et al.  1975)  reported a concentration of


184 ppb V/V  (=1.384 mg/m3)  of  2,4-DNT  in air  at  25°C,  which


is very close to the  TLV-TWA value noted above.


Special Groups at Risk


     The main group expected to be at  high  risk  for exposure


to 2,4-DNT  is industrial workers involved in  the manufacturing


or handling  of 2,4-DNT in places such  as ammunition, dye,


and  polyurethane plants.


Basis  and Derivation  of Criterion


     The data from  the bioassay of 2,4-DNT  for possible


carcinogenicity obtained by the National Cancer  Institute


 (1978)  and  Lee, et  al.  (1978)  were used for the  determina-


tion of  a water quality criterion for  the protection of


human  health.  It should be noted at this point, however,


that the Data Evaluation/Risk  Assessment Subgroup of the


Clearinghouse on Environmental Carcinogens  (NCI, 1978) expres-


sed  reservations about the adequacy  of this bioassay for


use  in assessing human risk.  Nevertheless, the  criterion


was  developed  from  the animal  carcinogenicity data from


these  two  studies by  utilizing the linear non-threshold


model  (see  Appendix I).  The rat carcinogencity  studies
      t

with dietary administration of 2,4-DNT showed increased


 incidences  of  fibroadenomas of the subcutaneous  tissue and


 inanition  in male -rats and fibroadenomas of the  mammary


gland  and  inanition in female  rats.


                               C-42

-------
          Under  the Consent Decree  in NRDC  vs Train/  criteria

     are  to state  "recommended maximum permissible  concentrations

     (including  where appropriate,  zero) consistent with  the

     protection  of aquatic organisms, human health, and recreation-

     al activities." 2,4-DNT  is suspected of being  a  human  carcino-

     gen.  Because there  is no recognized safe concentration

     for  a.human carcinogen,  the.recommended concentration  of

     2,4-DNT in  water for maximum protection of human health

     is zero.

          Because attaining a zero  concentration  level may  be

     infeasible  in some cases and in order  to assist  the  Agency

     and  States  in the possible future development  of water quality

     regulations, the concentrations of 2,4-DNT corresponding

     to several  incremental lifetime cancer risk  levels have

     been estimated.  A cancer risk level provides  an estimate

     of the additional incidence of cancer  that may be expected

     in an exposed population.  A risk of 10~  for  example, indi-

     cates a probability of one additional  case of  cancer for

     every 100,000 people exposed,  a risk of 10"  indicates one

     additional  case of cancer for  every million  people exposed,

     and  so forth.

          In the Federal Register notice of availability  of draft

     ambient water quality criteria, EPA stated that  it is  consid-

     ering setting criteria at an interim target  risk level of

     10~\ 10~  or 10~  as shown in the table below.

Exposure Assumptions               Risk Levels and  Corresponding Criteria  (1)

     (per day)                      £      1£~7         10/~6          1CTD

2 liters of drinking water             7.4  ng/1     74.0  ng/1        740 ng/1
and consumption of 18.7
grams fish and shellfish. (2)

Consumption of fish and                 .156 jug/1     1.56 jug/1   15.6jug/l
shellfish only.
                                   C-43

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(1)   Calculated by applying a modified "one-hit" extrapolation
     model described in the FR 15926, 1979 to the animal
     bioassay data presented in Appendix I and in Table
     9.  Since the extrapolation model is linear at low
     doses, the additional lifetime risk is directly propor-
     tional to the water concentration.  Therefore, water
     concentrations corresponding to other risk levels can
     be derived by multiplying or dividing one of the risk
     levels and corresponding water concentrations shown
     in the table by factors such as 10- 100, 1000 and so
     forth.
(2)  Approximately five percent of the DNT exposure results
     from the consumption of aquatic organisms which exhibit
     an average bioconcentration potential of 5.5 fold.
     The remaining 95 percent of DNT exposure results from
     drinking water.
     Concentration levels were derived assuming a lifetime
exposure to various amounts of DNT,  (1) occurring from the
consumption of both drinking water and aquatic life grown
in waters containing the corresponding DNT concentrations
and, (2) occurring solely from consumption of aquatic life
grown  in the waters containing the corresponding DNT concen-
trations.  Although total exposure information for chloroform
is discussed and an estimate of the contributions from other
sources of exposure can be made, this data will not be factor-
ed into ambient water quality criteria.  The criteria present-
ed, therefore, assume an incremental risk from ambient water
exposure only.
                              C-44

-------
     Results obtained  from  the  linear  non-threshold  model
give a value of 740 ng/liter  as  the  lowest  value  obtained,
the dose  level which establishes  a carcinogenicity risk
level in  water for humans of  1  in 100,000.  This  was obtained
from the  study of fibroadenomas of the mammary  gland and
inanition of the female rats  (Lee, et  al.,  1978).  It should
be noted  that this level is 1/500 the  level of  0.5 mg/liter
for surface water recommended in  the U.S.S.R.   (Korolev,
et al. 1977).
     Using the TLV-TWA value of 1.5 mg/m  of air  for 2,4-
DNT recommended by the Am. Conf.  Gov-  Ind. Hyg.  (1978),
the daily occupational exposure gives  a value of  5.4 mg
of 2,4-DNT per day (see Appendix  II  for calculation).  At
an ambient water level of 740 ng/liter, assuming  a daily
intake of 2 liters and a daily aquatic organism intake of
18.7  g with a bioaccumulation factor  of 5.5, it  can be
shown (see Appendix II for calculation) that the  daily intake
of 2,4-DNT is 0.0016 mg/day which is substantially below
the occupational exposure level and hence, will not  pose
a significant additional burden of exposure by  those at
risk occupationally.   This proposed level in ambient water
leads to an intake (0.0016 mg/day) which would  cause an
insignificant effect in terms of  contribution to  methemoglobi-
nemia (25 mg of 2,4-DNT/liter)  (Cartwright, 1977; Proctor
and Hughes, 1978).  It would thus appear that the linear
non-threshold model,  using female rat data on fibroadenomas
of the mammary gland and inanition (Lee, et al. 1978) provides
a level of ambient water exposure which achieves  a high
margin of safety.
                              C-45

-------
     It should be noted that data are urgently  needed  in



the following areas to evaluate properly any  hazard  from



2,4-DNT:



1.   Monitoring of workers exposed  to 2,4-DNT in  industries



     manufacturing or using the chemical.



2.   Monitoring of public water supplies and  industrial



     and municipal effluents to determine  an  expected  range



     of concentrations under differing  environmental conditions



3.   More detailed studies on  the pharmacokinetics of  2,4-
                                  *


     DNT using several animal  species and  if  possible, occu-



     pationally exposed humans.



4.   Evaluation of chronic toxicity and teratogenicity using



     currently acceptable techniques.



5.   Detailed and definitive mutagenicity  studies of 2,4-



     DNT and  its metabolites using  several assay  systems



     such as:



     a)   Salmonella/microsomal, b)  dominant  lethal, c)



     Drosophila, arid  d) host mediated assay.



6.   More definitive  studies on the carcinogenicity  of 2,4-



     DNT and  its metabolites using  several animal species
      *


      (and if  possible, occupationally exposed humans)  using



     oral and dermal  routes.
                              C-46

-------
                          REFERENCES




American Conference of Governmental Industrial Hygienists.


1974.  Dinitrotoluene.  Documentation of the threshold limit


values for substances in workroom air. 3rd ed., 2nd printing,


Cincinnati.




American Conference of Governmental Industrial Hygienists.

           P
1978. TLV's :  Threshold limit values for chemical substances


and physical agents in the workroom environment with intended


changes for 1978.




Ames, B.N., et al.  1973a.  An improved bacterial test system


for the detection and classification of mutagens and carcino-


gens.  Proc. Natl. Acad. Sci.  70: 782.




Ames, B.N., et al.  1973b.  Carcinogens are mutagens: a


simple test system combining liver homogenates for activation


and bacteria for detection.  Proc. Natl. Acad. Sci.  70:


2281.




Ames, S.A.H.,  and H.J. Yallop.  1966.  The identification


of industrial blasting explosives of the gelignite type.


Jour. Forensic Sci. Soc.  6: 185.




Blijleven, W.G.H.  1977.  Mutagenicity of four hair dyes


in Drosophila melanogaster.  Mutat. Res.  48: 181.
                             C-47

-------
Bodansky, O.  1951.  Methemoglobinemia  and methemoglobin-


producing compounds.  Pharm. Rev.   3: 144.



                                i
Bogatyrev, 0.  1973.  Influence of  aromatic  nitrated  hydro-


carbons on the activated  sludge process.  Acta  Hydrochim.


Hydrobiology. 1:  455.
              •t



Brandt, A.D.  1943.  Engineering  control  of  air contamination


of the working environment.  Pages 198-266 _in W.M.  Gafafer


ed. Manual of industrial  hygiene  and medical service  in


war industries.   W.B. Saunders Co., Philadelphia.




Bredow, M.V., and F. Jung.   1942.   Methemoglobin formation.


XXV.  Comparative toxicity  of some  aromatic  nitro derivatives,


Arch. Exp. Pathol.  Pharmakol.  200: 335.




Bueding,  E.,  and  N.  Jolliffe.  1946.  Metabolism of  trinitro-


toluene  (TNT) _in  vitro.   Jour. Pharmacol. Exp.  Ther.   88:

300.




Burrows,  D.,  and  J.C. Dacre.  1975.  Toxicity to aquatic

organisms and chemistry of  nine selected  waterborn pollutants


from munitions manufacture.  Literature evaluation.   Gov.

Rep. Announce Index 75: 45.  A review. AD-A Rep. No.  010660.

Natl. Tech.  Inf.  Serv-, Springfield, Va.
                               C-48

-------
Carpenter, D.F., et al.  1978.  Microbial  transformation


   14
of   C-labeled 2,4,6-trinitrotoluene in an activated sludge


system.  Appl. Environ. Microbiol.  35: 949.
Cartwright, G.E.  1977.  Methemoglobinemia and sulfhemoglo-


binemia. Pages 1710-1713  _in G.W. Thorn, et al., eds. Harrison's



principles of internal medicine, 8th ed. McGraw-Hill, New


York.






Channon, H.J., et al.  1944.  The metabolism of 2,4,6-trini-



trotoluene ( -TNT).  Biochem. Jour.  38: 70.






Chemical Abstracts Service.  1977.  The chemical abstracts



service  (CAS) ninth collective index. Vol. 76-85. Am. Chem.


Soc., Washington, D.C.






Christensen, H.E., et al., eds. 1976.  Suspected carcinogens.


2nd ed. A subfile of the NIOSH registry of toxic effects



of chemical substances.   (USDHEW Publ. No. 77- 149). U.S.



Government Printing Office, Washington, D.C.






Clayton, C.C., and C.A. Baumann.  1944.  Some effects of


diet on the resistance of mice toward 2,4-dinitrotoluene.


Arch. Biochem.  5: 115.






Clayton, C.C., and C.A. Baumann.  1948.  Effect of fat and


calories on the resistance of mice to 2,4-dinitrotoluene.


Arch. Biochem.  16: 415.
                              C-49

-------
Conduit, C.P.  19.59-  Ultraviolet-and infrared spectra of
some aromatic nitro compounds..   Jour. Chem.  Soc.  London
(P. 4): 3273.

Cordle, P.,  et al., 1978.  Human exposure to  polychlorinated
biphenyls  and polybrominated biphenyls.  Environ.  Health
Perspect.   24: 157.

Cotruvo, J.A., et  al.   1977.  Investigation  of mutagenic
effects of products of. ozonation reactions in water.   Ann.
N.Y. Acad.  Sci.   298:  124.

Dale,  H.H.   1921.  Characterization of a metabolite from
2,4,6-TNT.  Spec.  Rep.  Sex. Med. Res. Council No.  58.  London.

Dambleff,  J. 1908.   On the knowledge of the toxic action
of  nitrated benzenes  especially from the skin.  Inaug. Diss.
Wurzburg.

DeBruin, A.  1976. Anomalies in hemoglobinT-methemoglobinemia.
Pages  1259-1282  Ln Biochemical, toxicology of environmental
agents. Elsevier/North Holland Biomedical Press,  The Netherlands,

Djerassi,  L.S.,  and L.  Vitany.   1975..  Hemolytic  episode
in  G6  PD deficient workers exposed to TNT.  Br.  Jour. Ind.
Med.   32:  54,
                               C-50

-------
Doali, J.O, and A.A. Juhasz.  1974.  Application of high



speed liquid chromatography to the qualitative analysis



of compounds of propellant and explosives interest.  Jour.



Chromatogr. Sci.  12: 51.







Dobbin Crawford, M.A.  1954.  Aplastic anemia due to trini-



trotoluene intoxication.  Br. Med. Jour.  2: 430.







Dybing, E., et al.  1977.  Use of the Salmonella mutagenicity



test in drug metabolism studies.  Acta Pharmacol. Toxicol.



41(S4): 31.(Abstract.)







Ellis, H.V. Ill, et al.  1976.  Subacute toxicity of 2,4-



dinitrotoluene and 2,6-dinitrotoluene.  Toxicol. Appl. Pharma-



col.  37: 116.  (Abstract from 15th Annu. Meet. Soc.Toxicol.



March 14-18.).







Fahmy, M.J.,  and O.G. Fahmy.  1977.  Mutagenicity of hair



dye components relative to the carcinogen benzidine in Droso-



phila melanogaster.  Mutat. Res.  56: 31.







Fairchild, E.J., et al. eds. 1977. Registry of toxic effects



of chemical substances. USDHEW Publ.No.  (NIOSH) 78-104-B.



U.S. Government Printing Office, Washington, D.C.
                              C-51

-------
Floret, F.  1929.  Medical opinions on industrial poisonings.
Centr. Gewerbehyg.  Unfallverhut.  16: 280.

Friedlander, A.  1900.  On the clinical picture of poisoning
with benzene and toluene derivatives with  special reference
to the so-called anilinism.  Neurol. Centrlbl.  19: 155.

Fukuda, H., et al.  1977.  Detection of nitroglycerine and
nitro compounds  in fragments after dynamite explosion.
Kagaku Keisatsu  Kenkyusho Hokoku  30: 263.

Gehring,  D.G., and G.S. Reddy.  1968.  Nuclear magnetic
resonance examination and determination of the di- and trini-
trotoluene isomers in 2,4,6-trinitrotoluene.  Anal. Chem.
40: 792.

Giles, A.L., et  al.  1976.  Dermal carcinogenicity study
by mouse-skin painting with 2,4-toluenediamine alone or
in representative hair dye formulations.   Jour. Toxicol.
Environ.  Health  1: 433.

Gleason,  M.N., et al. 1969. In Clinical toxicology of commer-
cial products, 3rd ed. The Williams and Wilkins Co., Baltimore.

Goldman,  F.H., and M.B. Jacobs.   1953.  Chemical methods
in industrial hygiene. Interscience Publishers Inc., New
York.
                               C-52

-------
Goodwin, J.W.  1972.  Twenty years handling TNT in a shell



loading plant.  Am. Ind. Hyg. Assoc. Jour.  33: 41.







Hamblin, D.O.  1963.  Aromatic nitro and amino compounds.



Pages 2105-2169 _in F.A. Patty, et al.,  eds. Industrial hygiene



and toxicology. Vol. II: Toxicology. John Wiley and Sons.,



New York.







Hamilton, A., and H.L. Hardy- 1974. In Industrial toxico-



logy. Publishing Sciences Group, Inc.,  Massachusetts.







Hodgson, J.R., et al. 1976.  Mutation studies on 2,4-dinitro-



toluene. Mutat. Res. 38: 387-   (Abstract from the 7th Annu.



Meet. Am. Environ. Mutagen Soc., Atlanta, March 12-15.).







Hodgson, J.R., et al.  1977.  Comparative absorption, distri-



bution, excretion, and metabolism of 2,4,6-trinitroluene



(TNT) and isomers of dinitrotoluene  (DNT) in rats.  Fed.



Proc.  36:  996.







Hoffman, C.M., and E.B. Byall.  1974.  Identification of



explosive residues in bomb scene investigations.  Jour.



Forensic Sci.  19: 54.







Hughes, J.P., and J.F. Treon.  1954.  Erythrocytic inclusion



bodies in the blood of chemical workers.  Arch. Ind. Hyg.



Occup.  Med.  10: 192.
                              C-53

-------
Ito, N., et al.  1969.  The development of carcinoma  in
liver of rats treated with toluenediamine and the antagonistic
effects with other chemicals.  Cancer Res.  29: 1137.

Jenkins, R., and H.J. Yallop.  1970.  The identification
of explosives in trace quantities on objects near an  explosion,
Explosivstoffe  18: 139.

Key, M.M., et al. eds. 1977. Pages  278-279 in Occupational
diseases:  a guide to their recognition.U.S.Dept.Health.Edu.
Welfare.  U.S. Government Printing  Office, Washington, D.C.

Kirk, R.E., and D.F. Othmer.   1967.  Encyclopedia of  chemical
technology- Vol. 13. John Wiley  and Sons., New York.

Koelsch, F-  1917.  Contributions to the toxicology of aro-
matic nitro compounds.  Zentr, Gewerbehyg, Unfallverhut.
5:  60.

Korolev, A.A., et al.  1977.   Experimental data for hygienic
standardization of dinitrotoluene and trinitrobenzene in
reservoir waters.  Gig. Sanit. Iss.  10: 17-

Kovalenko, I.I.  1973.  Hemotoxicity of nitrotoluenes in
relation to number and positioning  of nitro groups.   Farmakol.
Toxicol.  (Kiev.) 8: 137.
                               C-54

-------
Krzymien, M., and L. Elias.  1975.  A continuous  flow  trace



vapor source.  Lab. Tech. Rep. LTR-UA-32. Natl.   Res.  Counc.



Can., Unsteady Aerodyn. Lab.








Kuhls, F.  1908.  Quantitative experiments on  the absorption



of poisons through the skin.  Inaug. Dissert.  Wurzburg.







Lee, C.C., et al. 1978.  Mammalian toxicity of munition



compounds. Phase III: Effects of life-time exposure.   Part



I: 2,4-Dinitrotoluene. U.S. Army Med.  Res. Dev.  Command.



Contract No. DAMD-17-74-C-4073. Rep. No.  7, September.







Lemberg, R., and J.P. Callaghan.  1944.  Metabolism of symmet-



rical trinitrotoluene.  Nature (London) 154: 768.







Lewin, L. 1921. The poisoning from trinitrotoluene.  Arch.



Exp". Pathol. Pharmakol.  89: 340.







Linch, A.L. 1974.  Biological monitoring for industrial



exposure to cyanogenic aromatic nitro and amino compounds.



Am. Ind. Hyg. Assoc. Jour.  35: 426.







Mangelsdorff, A.F.  1952.  Methemoglobinemia - recognition,



treatment, and pevention.  Ind. Med. Surg.  21: 395.







Mangelsdorff, A.F.  1956.  Treatment of methemoglobinemia.



Am. Med. Assoc. Arch. Ind. Health  14: 148.
                              C-55

-------
Matsui, S., et al.  1975.  Activated  sludge  degradability
of organic substances  in  the waste water of  the  Kashiraa
Pertroleum and Petrochemical Industrial Complex  in  Japan.
Prog. Water Technol.   7:  645.

McCann, J., et al.  1975.  Detection  of carcinogens as muta-
gens in the Salmonella/microsome  test: assay of  300 chemicals,
Proc. Natl. Acad.  Sci.  72: 5135.

McCormick, N.G.,  et al.   1976.  Microbial  transformation
of 2,4,6-trinitrotoluene  and other nitroaromatic compounds.
Appl. Environ. Microbiol.  31:  949.

McCormick, N.G.,  et al.   1978.  Identification of biotrans-
formation products from 2,4-dinitrotoluene.   Appl.  Environ.
Microbiol.  35:  945.

flcGee, L.C.,  et  al.   1942.  Metabolic disturbances  in workers
exposed to dinitrotoluene.  Am. Jour. Dig. Dis.   9: 329.

Meijers, A.P.,  and R.C. Van der Leer. 1976. The occurrence
of organic micropollutants in  the river Rhine and the river
Maas  in 1974.  Water  Res. 10:  597.

Moore, B.  1918.   Causation and prevention of trinitrotoluene
poisoning.  Jour.  Am.  Med. Assoc.  70: 412.
                               C-56

-------
Mori, M., et al.  1977.  Studies on the metabolism and toxi-
city of dinitrotoluenes — on excretion and distribution
of tritium-labelled 2,4-dinitrotoluene (3H-2,4-DNT) in the
rat.  Radioisotopes 26: 780.
Morton, A.R., et al.  1976.  Biological effects of trinitro-
toluene from exposure below the threshold limit value.
Am. Ind. Hyg. Assoc. Jour.  37: 56.

Nason, A.  1956.  Enzymatic steps in the assimilation of
nitrate and nitrite in fungi and green plants. Pages 109-
136 in Inorganic nitrogen metabolism. W.D. McElroy, and
B. Glass, eds.  John Hopkins Press., Baltimore.

National Cancer Institute. 1978. Bioassay of 2,4-dinitrotoluene
for possible carcinogenicity. Carcinogenesis Tech. Rep.
Ser. No. 54. USDHEW (NIH) Publ.  No. 78-1360. U.S. Government
Printing Office, Washington, B.C.

National Institute for Occupational Safety and Health. 1977.
Information profiles on potential occupational hazards.
Nitrotoluenes. Contract No. 210-77-0120.

National Institute for Occupational Safety and Health. 1978.
DinLtrotoluene.  Ln NIOSH manual of analytical methods.
Vol. 4. U.S. Government Printing Office, Washington, D.C.

Nay, M.W.,  Jr., 1974.  Biological treatability of trinitro-
toluene manufacturing waste water.  Jour. Water Pollut.
Control Fed.  46:  485.
                               C-57

-------
Osmon, J.L., and R.E. Klausmier.   1972.  The microbial  degra-
dation of explosives.  Dev.  Ind. Microbiol.  14:  247.

Palmer, W.L., et al.  1943.  Toxic necrosis of  the  liver
from  trinitrotoluene.  Jour. Am. Med.  Assoc.  123:  1025.

Parrish, P.w.   1977.  Fungal transformation of  2,4-dinitro-
toluene and  2,4,6-trinitrotoluene.  Appl.  Environ.  Microbiol,
34:   232.

Patty, F.A.  1958.   The  mode of  entry  and  action  of toxic
materials.   Page 162 in  F.A. Patty, ed.  Industrial hygiene
and  toxicology. Vol.  I.  General principles. Interscience
Publishers,  Inc.,  New York.

Pella, P.A.  1976.   Generator  for  producing trace vapor
concentrations  of  2,4,6-trinitrotoluene, 2,4-dinitrotoluene,
and  ethylene glycol dinitrate  for  calibrating explosives
vapor detectors.   Anal.  Chem.   48: 1632.

Perkins, R.G.   1919.  A  study  of the munitions  intoxications
in France.  U.S. Pub.  Health  Rep.  34: 2335.

Pienta, R.J., et al.  1977.  Correlation of bacterial muta-
genicity and hamster cell transformation with tumorigenicity
induced by  2,4-toluenediamine.   Cancer Lett.  3:  45.
                               C-58

-------
Plimmer, J.R., and U.I. Klingebiel.  1974.  The application



of mass spectrometry to the study of nitroaniline-derived



herbicides. Pages 99-112 ir\ Mass spectrometry and NMR spectro-



scopy in pesticide chemistry- Plenum Press., New York.







Priestera, F., et al.  1960.  Analysis of explosives using



infrared spectroscopy.  Anal. Chem.  32: 495.







Proctor, N.H., and J.P. Hughes.  1978.  Chemical hazards



of the workplace. J.B. Lippincott Co., Philadelphia/Toronto.







Rogovskaya, T.I.  1951.  The effect of trinitrotoluene on



the microorganisms and biochemical processes of self purifi-



cation in waters.  Mikrobiologiya  20: 480.







Roth, M., and J.M. Murphy, Jr.  1978.  Correlation of oxygen



demand and total organic carbon tests on waste waters from



ammunitions plants.  Proc. Ind. Waste Conf.  32: 674.







Ruchhoft, C.C,,  et al.  1945.  TNT wastes from shell-loading



plants.   Ind. Eng. Chem.  37: 937.







Ruchhoft, C.C.,  and F.I. Norris.  1946.  Estimation of ammoni-



um picrate in wastes from bomb and shell-loading plants.



Ind.  Eng. Chem.  38: 480.







Schott,  S., et al.  1943.   TNT wastes.  Ind. Eng. Chem.



35: 1122.





                              C-59

-------
Schereschewsky, J.W.  1918.  Trinitrotoluene:  practical
points in its safe handling. U..S.  Pub.  Health Serv.   Rep.
434.

Schwartz, L.  1944.  Dermatitis  from  explosives.   Jour.
Am. Med. Assoc.   125: 186.

Shah, M.J., et al.   1977.  Comparative  studies of  bacterial
mutation and hamster cell  transformation induced by  2,4-
toluenediamine.   Proc. Am. Assoc.  Cancer Res.   18: 23.
(Abstract.)

Shikata, M., and  I.  Tachi.   1938.   Polarographic studies
with  the dropping mercury  cathode.  LXXIX.   The electronega-
tivity rules of the  reduction  of organic compounds.   Collect.
Czech. Chem. Conunun.  10:  368.

Shils, M.E., and  L.J. Goldwater.  1953.   Effect of diet
on  the susceptibility of  the rat to poisoning  by 2,4-dini-
trotoluene.  Am.  Med. Assoc. Arch.  Ind.  Hyg. Occup.  Med.
8:   262.

Sidwell, V.D., et al. 1974.  Composition of  the edible portion
of  raw  (fresh or  frozen)  crustaceans,  finfish, and mollusks.
I.  Protein, fat,  moisture, ash,  carbohydrate,  energy value,
and cholesterol.  Mar. Fish.  Rev. 36:  21.
                               C-60

-------
Simmon, V.F., et al.  1977.  Munitions wastewater treatments:
does chlorination or ozonation of individual components
produce microbial mutagens?  Toxicol. Appl. Pharmacol.
41: 197.  (Abstract from the 16th Annu. Meet. Soc.  Toxicol.,
Toronto, Can., March 27-30.)

Spector, W.S. ed. 1956. Handbook of toxicology.  Vol. I.
Acute toxicities of solids, liquids, and gases to labora-
tory animals. W.B. Saunders Co., Philadelphia/London.

St. John, G.A.,  et al.  1975.  Determination of the concen-
tration of explosives in air by isotope dilution analysis.
Forensic Sci.  6: 53.

St. John, G.A.,  et al.  1976.  Determination of the concen-
tration of explosives in air by isotope dilution.  Edgewood
Arsenal Spec. Publ. EO-SO-76001. U.S. Dep. Army (Proc.
6th Annu. Symp.  Trace Anal. Detect. Environ., 1975.

Stanford, T.B.,  Jr.  1977.  The determination of tetryl
and 2,3-, 2,4-,  2,5-, 2,6-, 3,4-, and 3,5-dinitrotoluenes
using high performance liquid chromatography. AD Rep. AO42598.Natl<
Tech. Inf. Serv-

Stoats, J.  1972.  Standardized nomenclature for inbred
strains of mice: fifth listing.  Cancer Res.  32: 1609.
                              C-61

-------
Toxic and Hazardous Industrial Chemicals Safety Manual.
1976.  Toxic and hazardous  industrial chemicals safety manual
for handling and disposal with toxicity and  hazard  data.
The Inter. Tech. Inst., Japan.

Traxler, R.W., et al.   1974.  Bacterial degradation of
TNT.  Dev. Ind. Microbiol.   16:  71.

Umeda, M.  1955.  Production of  rat  sarcoma  by  injections
of propylene glycol solution of  m-toluenediamine.   Gann.
46:  597.

U.S. Army. 1970. Special  study of  the effect of alpha TNT
on microbial systems  and  the determination of the biodegrad-
ability  of alpha TNT.  U.S.  Army  Environ. Hyg. Agency San.
Eng.  Spec. Study No.  24-017-70/71.  Edgewood Arsenal, Md.

U.S. Army. 1971. Evaluation of toxicity of selected TNT
wastes of  fish. Phase I.  Acute toxicity of alpha TNT to
bluegills. U.S. Army  Environ. Hyg. Agency San.  Eng.  Spec.
Study No.  24-007-70/71. Edgewood Arsenal. Md.

U.S.  International  Trade  Commission.  1975.   Synthetic organic
chemicals: United States  production  and sales.  USITC Publ.
804  Washington, D.C.
                               C-62

-------
Vasilenko, N.M., et al.  1972.  Inactivation of the blood
respiratory pigment under the effect of aromatic nitro and
amino compounds from the benzene series.  Sovrem. Probl.
Biokhim. Dykhaniya Klin., 2nd Mater. Vses. Konf., 1971.
1: 411.

Veith, G.D., et al. An evaluation of using partition coeffi-
cients and water solubility to estimate bioconcentration
factors for oganic chemicals in fish.  (Manuscript).

Venitt, S.  1978.  Mutagenicity of hair dyes: some more
evidence and the problems of its interpretation.  Mutat.
Res. 53: 214.  (Abstract from 2nd Int. Conf. Envrion. Mutagens,
July 11-15, 1977, Edinburgh, Great Br,)

Vernot, E.H., et al.  1977.  Acute toxicity and skin corro-
sion data for some organic and inorganic compounds and aqueous
solutions.  Toxicol. Appl. Pharmacol.  42: 417.

Voegtlin, C., et al.  1920.  Trinitrotoluene poisoning -
its nature, poisoning, and prevention. U.S. Pub. Health
Serv. Hyg. Bull.  126: 7.

Von Oettingen, W.F.  1941.  The aromatic amino and nitro
compounds, their toxicity and potential dangers.  U.S. Pub.
Health Serv. Bull. No. 271. U.S. Government Printing Office,
Washington, D.C.
                               C-63

-------
Walsh, J..T., et al.  1973.  Application  of  liquid  chromato-
graphy to pollution abatement  studies  of  munition wastes.
Anal. Chem.  45: 1215.

Weast. R.C., ed, 1978. CRC  handbook  of chemistry and  physics.
A ready-reference  book of chemical and physical data.  CRC
Press, Inc., Cleveland,  Ohio.

White, R.P., and J. Hay.  1901.   Some  recent inquires and
researches  into the poisonous  properties  of  naphthalene
and  the  aromatic compounds.  Lancet  2: 582.

White, R.P., et al.   1902.   Some  notes from  an inquiry into
the  action  of dinitrobenzene upon the  urine  of man. Lancet
160:  1393.

Williams, R.T.  1959.  Metabolism of aromatic nitro compounds.
Pages 410-427 in Detoxication  mechanisms.  John Wiley  and
Sons, Inc.,  New York.

Won,  W.D.,  et al.   1974.  Metabolic  disposition of  2,4,6-
trinitrotoluene.   Appl.  Microblol.   27: 513.

Won,  W.D.,  et al.   1976.  Toxicity and mutagenicity of 2,4,6-
trinitrotoluene and  its  microbial metabolites. Appl.  Environ,
Microbiol.   31: 576.
                               C-64

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Wynder, E.L., et al.  1963.  An epidemiological investiga-
tion of cancer of the bladder.  Cancer  16: 1388.

Zieger, J.  1913.  Studies on the effect of nitrobenzene,
dinitrobenzene, nitrotoleuene and dinitrotoluene with absorp-
tion through the lungs and the skin.  Inaug. Dissert. Wurzburg

Zitrin, S., and J.  Yinon.  1976.  Chemical ionization mass
spectrometry of explosives. Pages 369-381 in Advances in
mass spectrometry in biochemistry and medicine. Vol. 1.
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                          APPENDIX I

      Carcinogenicity Risk Assessment by Extrapolation
            from Laboratory Animal  Toxicity Tests

     An assessment of health risks  associated with exposures
of a general environmental nature requires prediction  of
effects from low level exposures of lifetime duration.
Carcinogenic risks effects from environmental exposures
must normally be estimated from animal data obtained at
much higher levels because of the difficulty in detecting
a small increase in tumor  induction resulting from long-
term low level exposure.  Because the carcinogenic process
is generally believed  to  be irreversible, self-replicating,
and often originating  from a single somatic cell mutation,
assumptions of threshold  levels of  effect are believed to be
invalid for many, if  not  all, cancer-causative compounds.
Although many models  have been proposed  for extrapolation
from animal data to human risk assessment, the one utilized
here was chosen to facilitate uniform treatment of the variety
of chemical compounds  that are discussed  in the development
of those water criterion  documents  that  deal with animal
carcinogens.
     It is recognized  that the process of evaluating existing
studies and resultant  data in preparation for application
of mathematical methods involves  a  high  level of professional
judgment.  Many questions will necessarily arise due to
the unique characteristics of the specific compounds under
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discussion and the tremendous variability in completeness

and comparability among the available studies.

     A general explanation of the evaluation and extrapolation

procedures to be used are as follows:




     1.   Since the compounds discussed are known, or suspect

          carcinogens, emphasis was placed on those studies

          with carcinogenic or mutagenic endpoints.  In

          particular, those studies dealing with" mammalian

          species.

     2.   The extrapolation method employed is a mathematical

          procedure that uses a single dose and observed

          response of a toxicologic experiment to estimate

          a dose level for humans that will not increase

          the risk of tumors by more than a specified level

          (1 in 100,000)  (Personal communication, Dr. Todd

          Thorsland,  CAG, U.S.EPA, Washington, D.C.). Clearly

          this method is predicated on sound toxicologic

          test procedures.  Hence, each included study was

          evaluated for adherence to sound toxicological

          and statistical principles.

     3.   Judgment was exercised in prioritizing the signifi-

          cance of toxicologic studies that use different

          routes of administration.  In general, the preferred

          route of exposure is oral (food, water, or gavage)

          followed by intraperitoneal, intravenous, inhalation,

          or dermal routes of administration for the same

          species.  However, in some instances, consideration

          of absorption rates required that other routes

          be evaluated.
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     The NCI's Ad Hoc committee on  the  Evaluation  of  Low  Levels
of Environmental Chemical Carcinogens outlined  two conditions
that would render the extrapolations of animal  carcinogenesis
to man inappropriate.  This committee reported  to  the Surgeon
General as follows:
     Any substance which  is shown conclusively  to  cause
     tumors  in animals should  be considered  carcinogenic
     and therefore a potential hazard for  man.   Exceptions
     should  be considered only where the carcinogenic effect
     is clearly  shown the results from  physical rather than
     chemical induction or where the route of administration
     is shown to be grossly inappropriate  in terms of conceiv-
     able human  exposure.
4.   After selection of the sound toxicologic studies that
     form the basis for development of  a recommended  criteria,
     a, single dose and observed response were selected for
     the most "sensitive" sex  (if both  males and females
     were tested) according to the  following method:   Select
     the lowest  dose that yields a  tumor response  rate
     that is greater than the  control rate.   If the standard
     controls and media control response rates  are not signif-
     icantly different  (d<0.05), a  combined  rate was  calcul-
     ated from controls.
5.   The extrapolation methods were applied  independently
     to each selected dose and response pair.   The lowest
     projected dose was selected as the "safe level"  based
     on the  available toxicologic studies, if judgment indica-
     ted equal confidence in  the various dose-response pairs.
6.   The calculated safe  dose  was evaluated  along  with the
     results from human studies to  develop a recommended
     criteria.
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Calculation of Estimated Safe Levels for Humans;

     The specific data analyses performed along with required
input data are described in Mathematical Description of
Extrapolation Method.  This model provides the additional
risk associated with ingestion of 2 liters of water per
day and contaminated aquatic foods.  Any other risks associated
with air, food, or other exposure are not addressed by this
model.  A copy of the working data sheet is also included.

      Mathematical  Description  of  Extrapolation  Method

A.   Necessary information:
     nt = No.  of animals (males or females) exposed to selected
          dose that developed tumors (all sites combined
          unless tumors appear to be related to route of
          administration, e.g., peritoneal tumors would
          not be included if intraperitoneal injection method
          is used).

     Nt = Total number of animals  (male or females) exposed
          to selected dose level.

     nc = Number of control animals (males or females)  with
          tumors.

     NC = Total number of control animals (males or females).
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     Le = Actual maximum lifespan for test  animals.

     le = Length of exposure  (no. of hours, days, weeks,
          etc.).

     d =  Average dose per unit of  time  (mg/kg).

     w =  Average weight of test animals  (kg).

B.   Necessary information from general literature:

     70 kg = Average weight of man.

     L =  Theoretical average length of life  for  test  species,
          unless specified in articles.   (See attached table
          for appropriate values.)

     p =  Average weight of fish consumed per day,  assumed
          18.7 grams.

C.   Necessary ecological information.

     R =  Bioaccumulation factor for edible portions of
          fish  (supplied by Environmental Research  Laboratory,
          Duluth)

           (Note: If a bioaccumulation factor  is provided
          for the total  fish  or for some part other than
          the total edible portion  (such as the fat) an
          attempt should be made to estimate  factor for
          edible portion.)

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D. Mathematical Model:



          Pt = PC +  (1-Pc)     1  -  e"

     Where:

     Pt = nt - NT =  Proportion of  test  animals with tumors.

     PC = nc - NC =  Proportion of  control animals with tumors
     D = 	 = Lifespan weighted  average  dose level
           Le         (mg/kg)/(unit of Time)
    I     1 - Pt
B = V-ln
         -\ FD x t3 "I where t = li£esPan  £or  test  animals = Le
1 - PC  I  I *—       -1           length of  life  for species  L
        3 | —     (Note: It  is assumed  that  average weight of man = 70 kg
B1 = B
If and only if B1  0.1, then


     «• • 1°;' *7°      = Safe level  (mg/1)  for
          ~"O  \t* T r\xr ^
If B'20.1, then


     SL = ^B'^ti +°RxF) x  70 = Safe  level (mg/1) for  man

     (Note:  It is assumed average daily consumption  of water  is

     2 liters/day)
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                              APPENDIX  II

     1.   Calculation of Daily Occupational  Exposure  level  of
          2,4-Dinitrotoluene based on its Threshold Limit Value-
          Time Weighted Average  (TLV-TWA) concentration  (Am. Conf.  Gov,
          Ind. Hyg. 1978):
          TLV-TWA for 2,4-DNT =1.5 mg/m  of air  for  a normal
                                 8-hour  workday  or  40-hour workweek

                              =  1.5 x 10~3rng	
                                          liter  of  air

                               =  1-5
                                     liter  of  air
Therefore, the daily occupational  level  for
          2,4-DNT = 1.5 jag     x  7.5  liter  of  air  x  60  minute  x  8  hour
                        liter        minute            hour      day
                   = 5,400
                   = 5. 4 mg
     where 7.5 liter of air  is  the  ventilation  rate  for  an  average
     70 kg man doing moderately hard  work  (Kamon,  1979) .
     2.   Calculation of Daily  Intake Level  of  2,4-DNT:
          The assumptions used  for  this  calculation  are:
          a)   Bioaccumulation  factor of 5.5 as determined  for  the
               blue-gill sunfish (U.S. EPA  report,  Duluth, Minnesota),
          b)   Average  weight of aquatic organisms consumed per  day
                 is 18.7 g, and
          c)   Consumption of water per  person  per day
               is 2 liters over a period of  70  years.
          d)   A concentration  of 2,4-DNT  in water of 740 ng/1
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Bioaccumulation factor of 2,4-DNT = 5.5

The concentration of 2,4-DNT in fish =

          740 x 5.5 x 0.0187 = 76 ng from aquatic organisms
Daily intake of

2,4-DNT from 2 liters

of drinking water = 740 ng/1 x 2 = 1480 ng
Total intake/day  = 1480 + 76 mg
                    or 1556 ng
               (1.55 /ug or .00155 mg)
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                         APPENDIX  III
    Summary and Conclusions"Regarding the Carcinogenicity
                    of  2,4-Dinitrotoluene*
     2,4-Dinitrotoluene  (2,4-DNT) is a pale yellow crystalline
solid with a melting point of 70°C and has a moderate fire
explosion risk.  A combined U.S. production of approximately
272 billion pounds of 2,4- and 2,6-dinitrotoluene isomers
was reported in 1975.  2,4-DNT is widely used as a raw ma-
terial for dyestuffs and for urethane polymers, as a modifier
for smokeless powders, and as a  gelatinizing and waterproofing
agent in military and commercial explosives.
     The reports concerning the  mutagenicity of 2,4-DNT
are limited and their results conflicting.  However, this
compound was found to be mutagenic in the dominant lethal
assay in rats and in microbial tests using Salmonella typhimu-
rium TA1535 indicating base-pair substitution.
     Two reports concerning the  carcinogenicity of 2,4-DNT
are in the literature.  The first is a National Cancer Insti-
tute  (NCI) two-year bioassay in  male and female Fisher 344
rats and B6C3F1 mice fed 2,4-DNT (1978).  The major pathologic
findings were present in the rats.  These included fibromas
of the skin and subcutaneous tissues in males and fibroadenomas
of the mammary gland in  the females.  These tumors are benign
and were dose-related.  The mice had no statistically signifi-
cant carcinogenic response to the administration of 2,4-
dinitrotoluene.
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     The second study relating oral administration of  2,4-


DNT to carcinogenicity was a bioassay in male and female

Charles River CD rats and CD-I mice fed 2,4-DNT for  two

years (Lee, et al. 1978) .  The major pathologic findings

in the rats included a significant increase of hepatocellular

carcinomas (p = 7.1 x 10~ )  and neoplastic nodules  (p  =  .01)

in the liver of females, mammary gland tumors of the female
             _c
(p = 8.3 x 10  )  and the suspicious increase of hepatocellular

carcinomas of the liver in males.  All of these rat  tumors

were in high dose animals.  The pathologic finding in  the

mice was the highly significant  (p = 1.5 x 10" ) increase

of kidney tumors in the males of the middle dose group.


     The induction of hepatocellular carcinomas, hepatocellu-


lar neoplastic nodules and mammary tumors in female  rats

and kidney tumors in male mice from the administration of

2,4-dinitrotoluene indicates that it is likely to be a human
                                   V
carcinogen.

     The water quality criterion for 2,4-dinitrotoluene

is based on the induction of mammary tumors, hepatocellular

carcinomas, and hepatocellular neoplastic nodules in female

Charles River CD rats fed 200 ppm 2,4-DNT for 24 months

(Lee, et al. 1978).  It is concluded that the water concentra-

tion of 2,4-dinitrotoluene should be less than 740 ng/1

in order to keep the lifetime cancer risk below 10
*This summary has been prepared and approved by the Carcino-
 gens Assessment Group of EPA on June 19, 1979.
                              C-75

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                  Summary of Pertinent Data
     The water quality criterion  for  2,4-dinitrotoluene
is derived from the oncogenic effects observed  in  the mammary
gland and liver of female Charles River CD rats  fed  200
ppm in the diet.  The time-weighted average  dose of  45 mg/kg/day
was given in the feed for 24 months,  with the surviving
animals sacrificed one month later.   The mammary tumor inci-
dence was 11/23 and 33/35 in the  control and treated groups,
respectively.  The incidence of hepatocellular  carcinomas
and neoplastic nodules was  0/23 and 24/34 in the control
and treated groups, respectively.  Assuming  a fish bioconcen-
tration factor of 5.5, the  criterion  is calculated from
the following parameters:
     nt mammary =33          d - 45  mg/kg/day
      t mammary =35          R = 5.5
     nc mammary =11          L = 25  months
     Nc mammary =23          W = 0.464 .kg
     nt liver   =24          F = 0.0187 kg/day
     Nt liver   = 34
     nc liver   =  0
     Nc liver   = 23
     le         = 24 months
     Le         = 25 months
                                                   g
     Based on these parameters, the one-hit  slope,  H, is
2.95 x 10   for mammary  tumors and 1.53 x 10"   for hepatocell-
ular carcinomas and hepatocellular neoplastic nodules.
The resulting water concentration of  2,4-dinitrotoluene
calculated to keep the individual lifetime cancer  risk below
 10~5  is  740  ng/1.
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