ASSESSMENT OF PROTOCOL VARIABLES IN THE NTP EVALUATION OF CYTOTOTXICITY
ASSAYS

Balb/C 3T3 Cells

I.  What is the acceptable solvent concentration?

> Two solvents, DMSO and EtOH, were assayed in the 3T3 assay to
> determine acceptable concentrations. Multiple exposure times were
> assessed since the final assay exposure time was not yet established.
> Various cell seeding concentrations were tested since these
> experiments were run concurrently with others which used to determine
> optimal seeding density.

**Table 1.**

  ------ --------- ----- ------ ------- ------ ----------- ------ -------- ----------- --------
  EtOH                                                                                 

         Date      2%    1%     0.50%          Seeding                                 
                                               Density                                 

  48h    2/26/02   58%   72%    100%           9X10^3^                                 
                                               cells/ml                                

         2/26/02   49%   73%    102%           4.5X10^3^                               
                                               cells/ml                                

                                                                                       

                                                                                       

  72h    2/26/02   67%   75%    105%           9X10^3^                                 
                                               cells/ml                                

         2/26/02   68%   82%    108%           4.5X10^3^                               
                                               cells/ml                                

                                                                                       

                                                                                       

  DMSO                                                                                 

         Date      2%    1%     0.5%    0.4%   0.3%        0.2%   0.1%     Seeding     
                                                                           Density     

  24h    3/19/02         76%    91%     92%    99%         100%   101.6%   2X10^4^     
                                                                           cells/ml    

                                                                                       

  48h    2/26/02   25%   54%    83%                                        9X10^3^     
                                                                           cells/ml    

         2/26/02   27%   56%    78%                                        4.5X10^3^   
                                                                           cells/ml    

         3/19/02         116%   123%    122%   120%        117%   108.8%   1X10^4^     
                                                                           cells/ml    

                                                                                       

  72h    2/26/02   20%   52%    86%                                        9X10^3^     
                                                                           cells/ml    

         2/26/02   19%   56%    93%                                        4.5X10^3^   
                                                                           cells/ml    

         3/19/02         58%    89%     102%   102%        112%   110.1%   5X10^3^     
                                                                           cells/ml    

                                                                                       
  ------ --------- ----- ------ ------- ------ ----------- ------ -------- ----------- --------

> We concluded from these experiments that 0.5% EtOH was the optimal
> EtOH concentration (little to no toxicity), and that 0.5% was probably
> acceptable for DMSO as a trade-off between slight toxicity and ability
> to test chemicals to higher does levels.
>
> From about the middle of March on, we used 0.5% in all of our
> experiments where DMSO was called for as a solvent. This gave us a
> number of opportunities to further determine the toxicity of DMSO by
> comparing the solvent control wells with the media control wells in
> the same experiment.

**Table 2.**

+------------------+-----------------+---------------+-----------------+
| # DMSO           |                 |               |                 |
+------------------+-----------------+---------------+-----------------+
| Date & Exposure  | OD Assay Medium | OD Solvent    | \% Survival in  |
| Time             | Wells           | Wells         | Solvent         |
+------------------+-----------------+---------------+-----------------+
| 24h 3/19/02      | 0.502           | 0.474         | 94.5%           |
+------------------+-----------------+---------------+-----------------+
|                  | 0.441           | 0.394         | 89.4%           |
+------------------+-----------------+---------------+-----------------+
|                  |                 |               |                 |
+------------------+-----------------+---------------+-----------------+
| 48h 3/19/02      | 0.587           | 0.536         | 91.4%           |
+------------------+-----------------+---------------+-----------------+
|                  | 0.582           | 0.545         | 93.6%           |
+------------------+-----------------+---------------+-----------------+
|                  |                 |               |                 |
+------------------+-----------------+---------------+-----------------+
| 72h 3/19/02      | 0.687           | 0.601         | 87.6%           |
+------------------+-----------------+---------------+-----------------+
|                  | 0.666           | 0.588         | 88.3%           |
+------------------+-----------------+---------------+-----------------+

> The average survival in 0.5% DMSO from Table 2 was 90.8%.

II. Doubling Time Experiments

We ran a series of experiments designed primarily to determine the
appropriate original seeding density for 24, 48, and 72 h exposure
times. We judged our results on visual observations of the cells at the
conclusion of the experiment (control cells should be just confluent at
24, 48, or 72h), and on the shape of the growth curve.

![](media/image1.pct)**Figure 1.**

![](media/image2.pct)**Figure 2.**

We have concluded from these growth curves that our 3T3 cells have a
doubling time of about 19 h and that cell concentration of: 1X104
cells/ml (24h); 5X103 cells/ml (48h); and 2.5X103 (72h) are acceptable.

III. Exposure Duration

The exposure question was first raised by Richard Clothier who indicated
that a paper by Riddell, et al. showed a number of chemicals whose
toxicity changed greatly between a 24 h and a 72 h exposure (for 25/50
materials there was little change and for 25/50 materials there was a
change). We examined the paper and chose to investigate six chemicals
that showed some of the largest differences between 24h and 72h.

Our initial studies gave similar results to those of Riddell et al.
However we felt that the cell number for the longer exposures was not
optimal, and we conducted additional studies to determine a standard
seeding density for each exposure period. Using this methodology we
looked at the 6 materials in a standardized fashion at 24, 48 and 72h.

Our results are shown in Fig. 3.

![](media/image3.pct)**Figure 3.**

In this figure the historic Halle, et al. data are shown as small blue
dots and the regression line as a dark black line. To add perspective we
have included the Riddell, et al. data as a light blue diamond (24h) or
a dark blue diamond (72h). Arrows emerging from certain points indicate
that the value is less than or greater than that point. Our values are
graphed in increasing shades of green from light (24h) to dark (72h).
All green values are averages of at least two separate experiments. It
appears that our data are somewhat different than Riddell, et al., i.e
most differences are not as great as originally seen. Nonetheless the
values, as expected, do become more toxic with increased exposure time.
We feel that 48 hrs is probably the optimal time for these data if the
Halle regression is considered some type of a standard.

Next we asked whether a 48 h exposure time would affect our earlier
results with the 11 chemicals presented in the Guidance Document. If
these numbers were changed significantly, this might cause us to make
significant modification to our guidance.

To assess the effect of increasing exposure time on the 11 chemicals, we
tested them with exposure times of 24h, 48h and 72h as shown in Fig. 4.

![](media/image4.pct)**Figure 4.**

The data shown on the graph are averages of duplicate experiments. It
can be seen that although each of the chemicals becomes more toxic with
increased exposure, all points are still within the 0.5 log range of the
regression line. It again appears that 48 h exposure fits the regression
more closely, however we regraphed the data in Fig. 5 to show the
regression line and statistics for each of the new sets of data.

Figure 5.

![](media/image5.png){width="6.245833333333334in"
height="4.788194444444445in"}

In this figure it can be seen that all the regression lines for the 3
new time points plus the Guidance Document data (red triangles) fall
with in the regression boundaries. It again appears that the 48 hour
values best fit the original regression line.

We now feel that for the 3T3 cells an extended exposure period (\>24h)
should be used, and that 48h seems to help identify the more toxic
compounds while not over estimating the less toxic ones.