Assessment of immunotoxicity in female Fischer 344/N and Sprague Dawley rats and female B₆C₃F₁ mice exposed to hexavalent chromium via the drinking water

Figures & data

Table 1. Exposure endpoints.

Parameters measured
Assay cohort	A	B	C	D
Fischer 344/N and Sprague Dawley rats
1	IgM/IgG antibody forming cell assay	Serum IgM to SRBC		Body weights
2	Hematology			Body weights
3	Natural killer cell activity	Anti-CD3 mediated T Cell proliferation	Organ weights	Body weights
4	Cell quantification			Body weights
B6C3F1 mice
1	Organ weights, Hematology			Body weights
2	IgM/IgG antibody forming cell assay	Serum IgM to SRBC	Gross pathology	Body weights
3	Natural killer cell activity	Anti-CD3 mediated T Cell proliferation		Body weights
4	Cell quantification			Body weights
5	Natural killer cell activity	Anti-CD3 mediated T Cell proliferation	Mixed leukocyte response	Body weights
6	Mixed leukocyte response	Organ weights, Hematology		Body weights
7	IgM/IgG antibody forming cell assay	Serum IgM to SRBC		Body weights

Figure 1. Mean body weight gain in female Fischer 344/N and Sprague Dawley rats and B₆C₃F₁ mice following exposure to SDD in drinking water for 28 days. Mean body weight gain (g) reported for Days 1–8, 1–15, 1–22, and 1–29 (study termination). Mean body weight gain of (A) Fischer 344/N rats, (B) Sprague Dawley rats, and (C) B₆C₃F₁ mice. Data are expressed as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01 for exposed groups compared to vehicle contol. N = 32 for F344/N rats. N = 40 for SD rats and B₆C₃F₁ mice. VH: vehicle control.

Table 2. MCV and MCH levels in the blood of Fischer 344/N and Sprague Dawley rats.

		SDD (ppm)				CPS
	Vehicle	14.3	57.3	172	516	50 mg/kg
Fischer 344/N rats^a
MCV (fl)	54.8 ± 0.7	*56.5 ± 0.2	55.2 ± 0.1	55.9 ± 0.2	**56.9 ± 0.1	53.6 ± 0.1
MCH (pg)	19.1 ± 0.5	*20.4 ± 0.1	*20.5 ± 0.1	21.2 ± 1.3	19.9 ± 0.1	20.1 ± 0.1
Sprague Dawley rats^b
MCV (fl)	58.5 ± 0.5	58.9 ± 0.7	57.3 ± 0.6	57.0 ± 0.7	56.4 ± 0.5	57.1 ± 0.4
MCH (pg)	21.4 ± 0.2	21.3 ± 0.2	*20.8 ± 0.2	23.7 ± 2.4	**20.6 ± 0.2	20.8 ± 0.7

Data expressed as means ± SE.

^a N = 8.

^b N = 10.

* p ≤ 0.05, ** p ≤ 0.01 for exposed groups compared to vehicle control.

Table 3. MCV and MCH levels in the blood of B₆C₃F₁ mice.

		SDD (ppm)					CPS
	Vehicle	15.6	31.3	62.5	125	250	50 mg/kg
Study 1^a
MCV (fl)	50.4 ± 0.1	50.3 ± 0.2	50.0 ± 0.2	*49.6 ± 0.2	**49.2 ± 0.1	**47.4 ± 0.2	50.7 ± 0.2
MCH (pg)	16.9 ± 0.1	16.9 ± 0.1	16.7 ± 0.1	16.8 ± 0.1	**16.4 ± 0.1	**15.8 ± 0.1	**17.2 ± 0.1
Study 2^a
MCV (fl)	50.0 ± 0.2	49.8 ± 0.2	*49.4 ± 0.2	**49.2 ± 0.1	**49.0 ± 0.1	**47.2 ± 0.2	50.3 ± 0.2
MCH (pg)	16.8 ± 0.1	16.8 ± 0.1	16.8 ± 0.1	16.6 ± 0.1	16.6 ± 0.1	**15.9 ± 0.1	**17.1 ± 0.1

Data expressed as means ± SE.

^a N = 8.

* p ≤ 0.05, **p ≤ 0.01 for exposed groups compared to vehicle control.

Table 4. Spleen lymphocyte sub-populations in Fischer 344/N and Sprague Dawley rats.

		SDD (ppm)				CPS	AAGM1
	Vehicle	14.3	57.3	172	516	50 mg/kg	1:10
Fischer 344/N rats^a
Spleen cells ×10^7	41.57 ± 1.92	46.23 ± 2.52	44.69 ± 2.04	46.77 ± 2.51	40.13 ± 2.66	**11.89 ± 0.80	39.36 ± 1.95
CD45^+b	190.3 ± 9.6	218.2 ± 11.6	218.4 ± 10.2	215.5 ± 13.1	197.6 ± 15.2	**12.2 ± 1.3	–
% Value	45.7 ± 0.8	47.2 ± 0.6	48.9 ± 0.7	46.2 ± 1.6	49.2 ± 1.5	**10.2 ± 0.6
CD5^+c	175.9 ± 9.0	193.5 ± 10.0	180.2 ± 9.0	187.9 ± 13.1	151.7 ± 13.2	**84.7 ± 5.4	–
% Value	42.3 ± 0.5	42.0 ± 0.8	40.3 ± 0.6	40.1 ± 1.7	38.1 ± 2.3	**71.5 ± 1.0
CD4^+CD5^+d	93.0 ± 4.8	100.1 ± 6.3	94.3 ± 4.6	99.1 ± 8.3	76.5 ± 7.2	**26.1 ± 1.7	–
% Value	22.4 ± 0.4	21.6 ± 0.5	21.1 ± 0.4	21.1 ± 1.2	19.3 ± 1.4	22.0 ± 0.6
CD8^+CD5^+e	73.3 ± 4.2	80.8 ± 4.9	77.9 ± 4.5	83.1 ± 5.9	65.2 ± 6.2	*58.2 ± 3.5	–
% Value	17.6 ± 0.4	17.4 ± 0.2	17.4 ± 0.5	17.7 ± 0.7	16.3 ± 1.0	**49.2 ± 1.1
NKR-P1A^+CD8^+f	12.6 ± 0.5	15.4 ± 1.3	15.2 ± 1.0	**20.6 ± 1.4	16.7 ± 2.8	–	**0.6 ± 0.1
% Value	3.1 ± 0.2	3.3 ± 0.2	3.4 ± 0.2	**4.4 ± 0.2	*4.1 ± 0.5		**0.2 ± 0.1
HIS36^+g	27.1 ± 1.7	*37.4 ± 3.5	30.0 ± 2.6	34.1 ± 3.2	25.1 ± 1.7	**8.6 ± 1.0	–
% Value	6.6 ± 0.3	8.0 ± 0.5	6.7 ± 0.5	7.3 ± 0.4	6.4 ± 0.4	7.1 ± 0.5
Sprague Dawley rats^h
Spleen cells ×10^7	70.72 ± 5.96	57.76 ± 4.68	59.29 ± 4.02	60.20 ± 4.18	66.99 ± 6.10	**8.66 ± 0.87	–
CD45^+b	343.8 ± 46.6	273.1 ± 31.6	297.9 ± 27.9	285.4 ± 25.2	327.5 ± 44.5	**11.7 ± 1.7	–
% Value	47.2 ± 2.2	46.3 ± 2.1	48.2 ± 2.0	46.9 ± 1.4	47.7 ± 2.4	**14.4 ± 1.9
CD5^+c	263.0 ± 26.2	201.0 ± 14.7i	203.5 ± 14.4	215.3 ± 16.3	239.8 ± 21.5i	**52.0 ± 6.6	–
% Value	37.6 ± 1.5	35.7 ± 1.3	33.3 ± 1.4	35.9 ± 1.3	35.8 ± 2.5	**58.6 ± 3.1
CD4^+CD5^+d	156.1 ± 20.2	123.9 ± 10.0	119.5 ± 12.4	126.1 ± 11.5	146.9 ± 14.0	**22.9 ± 3.2	–
% Value	21.9 ± 1.3	21.7 ± 1.0	19.2 ± 0.9	20.9 ± 1.1	22.3 ± 1.4	25.9 ± 2.5
CD8^+CD5^+e	86.5 ± 7.3	61.7 ± 2.4	72.5 ± 7.3	76.5 ± 5.5	69.8 ± 7.1	**28.1 ± 3.8	–
% Value	12.6 ± 0.9	11.1 ± 0.7	12.0 ± 1.5	12.9 ± 0.7	10.7 ± 0.7	**31.3 ± 2.0
NKR-P1A^+CD8^+f	15.7 ± 2.2	16.2 ± 1.4	15.3 ± 1.7	14.8 ± 1.0	14.6 ± 1.7	–	–
% Value	2.3 ± 0.3	2.9 ± 0.3	2.5 ± 0.2	2.5 ± 0.1	2.2 ± 0.2
HIS36^+g	20.2 ± 2.1	24.8 ± 1.7	24.6 ± 4.2	21.4 ± 3.1	26.2 ± 2.1	**10.6 ± 1.1	–
% Value	2.9 ± 0.2	*4.4 ± 0.3	3.9 ± 0.5	3.5 ± 0.3	*4.1 ± 0.4	**13.2 ± 1.8

Data expressed as means ± SE.

^aN = 8

^bB cell, OX-33⁺; ^cT cell, OX-19⁺; ^dT helper cell, OX-38⁺OX-19⁺; ^eCytotoxic T cell, OX-8⁺OX-19⁺; ^fNatural killer cell, NKR-P1A⁺OX-8⁺; ^gMacrophage, HIS36⁺.

^hN = 7 for 57.3 ppm, N = 8 for vehicle, and N = 10 for exposed groups unless otherwise noted.

ⁱN = 9.

*p ≤ 0.05, **p ≤ 0.01 for exposed groups compared to vehicle control.

Table 5. Spleen lymphocyte sub-populations in B₆C₃F₁ mice.

		SDD (ppm)					CPS	AAGM1
	Vehicle	15.6	31.3	62.5	125	250	50 mg/kg	1:10
B6C3F1 Mice^a
Spleen cells ×10^7	19.64 ± 0.92	21.15 ± 1.04	19.16 ± 1.08	19.94 ± 0.79	19.37 ± 0.72	19.04 ± 0.72	**7.31 ± 0.41	20.92 ± 0.70
Ig^+c	99.0 ± 5.2	111.8 ± 5.6	98.0 ± 5.4	102.2 ± 4.2^b	95.8 ± 5.6^b	101.9 ± 4.2	**26.0 ± 1.7	–
% Value	50.3 ± 0.8	52.9 ± 0.9	51.2 ± 0.7	52.0 ± 0.8	49.0 ± 0.9	*53.5 ± 0.7	**35.6 ± 1.0
CD3^+d	64.1 ± 1.8	69.2 ± 4.5	64.7 ± 3.1	64.4 ± 3.2	69.9 ± 3.6	64.7 ± 3.0	**41.4 ± 2.1	–
% Value	32.9 ± 0.8	32.6 ± 1.0	33.9 ± 0.8	32.3 ± 1.1	36.0 ± 1.0	34.0 ± 0.8	**56.9 ± 1.3
CD4^+CD8^-e	38.3 ± 1.9	43.1 ± 2.1	39.0 ± 2.3	42.3 ± 1.6	41.4 ± 2.0	43.6 ± 1.8	**17.0 ± 1.5	–
% Value	19.5 ± 0.2	*20.4 ± 0.2	20.4 ± 0.5	**21.3 ± 0.5	**21.3 ± 0.4	**22.9 ± 0.2	**23.1 ± 1.1
CD4^-CD8^+f	20.6 ± 0.7	21.7 ± 1.1	21.0 ± 1.4	22.0 ± 1.1	22.9 ± 1.0	23.2 ± 0.9	**10.1 ± 1.0	–
% Value	10.6 ± 0.3	10.3 ± 0.2	10.9 ± 0.4	11.0 ± 0.2	**11.8 ± 0.2	**12.2 ± 0.2	**13.6 ± 0.9
CD4^+CD8^+g	0.2 ± 0.1	0.2 ± 0.1	0.2 ± 0.1	0.2 ± 0.1	0.2 ± 0.1	0.2 ± 0.1	**0.1 ± 0.1	–
% Value	0.1 ± 0.1	0.1 ± 0.1	0.1 ± 0.1	0.1 ± 0.1	0.1 ± 0.1	0.1 ± 0.1	0.1 ± 0.1
NK1.1^+CD3^-h	6.8 ± 0.3	7.1 ± 0.4	6.6 ± 0.5	6.9 ± 0.5	7.5 ± 0.4	6.0 ± 0.3	–	**0.4 ± 0.1
% Value	3.5 ± 0.1	3.4 ± 0.1	3.4 ± 0.1	3.5 ± 0.2	3.9 ± 0.2	3.2 ± 0.1
Mac-3^+i	6.6 ± 0.2	6.3 ± 0.2	*5.3 ± 0.4	5.6 ± 0.3	**4.4 ± 0.3	6.1 ± 0.4	**3.0 ± 0.2	–
% Value	3.4 ± 0.2	3.0 ± 0.1	**2.7 ± 0.1	**2.8 ± 0.1	**2.3 ± 0.1	3.2 ± 0.1	*4.1 ± 0.2

Data expressed as means ± SE.

^a N = 8.

^b N = 7.

^c B cell.

^d Mature T cell.

^e T helper cell.

^f Cytotoxic T cell.

^g Immature T cell.

^h Natural killer cell.

ⁱ Macrophage.

^j N = 7 for 57.3 ppm, N = 8 for vehicle, and N = 10 for exposed groups unless otherwise noted.

^k N = 9.

* p ≤ 0.05, **p ≤ 0.01 for exposed groups compared to vehicle control.

Figure 2. IgM antibody-forming cell (AFC) response to sheep red blood cells (SRBC) in female Fischer 344/N and Sprague Dawley rats and B₆C₃F₁ mice following exposure to SDD in drinking water for 28 days. The AFC response to SRBC was evaluated in splenocyte suspensions prepared from previously immunized animals and reported as either specific activity (AFC/10⁶ spleen cells) or total activity (AFC/spleen). Specific activity (AFC/10⁶ spleen cells) in (A) F344/N rats, (B) SD rats, and (C) B₆C₃F₁ mice. Total activity (AFC/spleen) in (D) F344/N rats, (E) SD rats, and (F) B₆C₃F₁ mice. Data are expressed as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01 for exposed groups compared to vehicle contol. N = 7 (VH, 14.3 and 172 ppm) or 8 (57.3 and 516 ppm) for F344/N rats. N = 10 for SD rats. N = 8 for B₆C₃F₁ mice. VH: vehicle control; CPS: cyclophosphamide positive control (50 mg/kg).

Table 6. Serum IgM antibody titers to SRBC in Sprague Dawley rats and B₆C₃F₁ mice.

		SDD (ppm)					CPS
	Vehicle	14.3	57.3	172	516	-	50 mg/kg
Sprague Dawley rats^a
Serum Titers (Log2)	8.343 ± 0.322	8.841 ± 0.460	8.174 ± 0.270	8.252 ± 0.366	8.270 ± 0.316	–	**3.396 ± 0.322
		SDD (ppm)					CPS
	Vehicle	15.6	31.3	62.5	125	250	50 mg/kg
B6C3F1 mice^b
Serum Titers (OD) 0.5	30 ± 3	50 ± 9	37 ± 4	34 ± 4	38 ± 4	33 ± 4	**8 ± 0
Serum Titers (Log2)	4.890 ± 0.120	5.475 ± 0.258	5.172 ± 0.148	4.996 ± 0.174	5.179 ± 0.151	4.984 ± 0.148	**3.000 ± 0.000

Data presented as means ± SE.

^a N = 10.

^b N = 8.

** p ≤ 0.01 for exposed groups compared to vehicle control.