Immunotoxicity studies of sulfolane following developmental exposure in Hsd:Sprague Dawley SD rats and adult exposure in B6C3F1/N mice

Figures & data

Table 1. Study cohorts and endpoints assessed in sulfolane-treated adult female B6C3F1/N mice (n = 8/treatment group) and F1 Harlan Sprague Dawley rats (n = 12/sex/treatment group).

Cohort	Endpoints examined
1	Gross pathology Enhanced immunopathology Hematology (Complete blood count with differential and reticulocyte counts)
2	Antibody response to SRBC
3	Antibody response to KLH
4	T-cell proliferation NK cell activity Immunophenotyping of the spleen
5	CTL response to influenza infection

CTL: cytotoxic T-lymphocyte; KLH: keyhole limpet hemocyanin; NK: natural killer; SRBC: sheep red blood cell.

Table 2. Mean water consumption and sulfolane intake during the perinatal and post-weaning period in HSD rats (Cohort 1) exposed to sulfolane in drinking water.

	Generation/ sex	Sulfolane in drinking water (mg/L)
		0	30	100	300	1000
Gestation (GD 6–21)
Water consumption^a	F0/F	0.0 ± 0.0	121.4 ± 2.7	121.9 ± 2.0	120.9 ± 2.0	127.1 ± 2.9
Sulfolane intake^b		0.0 ± 0.0	3.6 ± 0.1	12.2 ± 0.2	36.3 ± 0.6	127.1 ± 2.9
Lactation (PND 1–28^c)
Water consumption		0.0 ± 0.0	254.6 ± 5.9	266.7 ± 5.9	267.7 ± 6.5	270.0 ± 6.8
Sulfolane intake		0.0 ± 0.0	8.0 ± 0.2	26.8 ± 0.6	80.8 ± 1.1	270.0 ± 6.8
Postweaning (PND 35–91)
Water consumption	F1/M	86.3 ± 1.8	85.8 ± 3.8	95.3 ± 2.4	84.0 ± 2.2	78.4 ± 2.2
	F1/F	104.3 ± 3.5	109.6 ± 2.4	101.7 ± 1.9	110.3 ± 3.8	127.7 ± 2.3
Sulfolane intake	F1/M	0.0 ± 0.0	2.6 ± 0.1	9.5 ± 0.2	25.2 ± 0.7	78.4 ± 2.3
	F1/F	0.0 ± 0.0	3.3 ± 0.1	10.2 ± 0.2	33.1 ± 1.1	127.7 ± 2.3

Mean ± SE. N = 30–38/treatment group (F₀ dams); N = 12/treatment group (F₁ offspring).

^aUnits are in g water/kg BW/day.

^bUnits are in mg sulfolane/kg BW/day.

^cReported for F0 females but includes consumption by F1 offspring beginning on or around PND 13 until weaning on PND 28.

Table 3. Organ weights in male and female F1 rats exposed developmentally to sulfolane.

	Sulfolane (mg/L)					CPS (n = 8) 15 mg/kg
	0	30	100	300	1000
F1 MALES
Terminal BW	449.0 ± 7.4	453.0 ± 7.8	425.7 ± 3.3	443.1 ± 10.0	438.7 ± 5.5	364.8 ± 5.9**
Spleen	0.75 ± 0.02^#	0.76 ± 0.03	0.73 ± 0.02	0.79 ± 0.03	0.82 ± 0.03	0.45 ± 0.01**
Spleen:BW ratio	1.67 ± 0.04^##	1.68 ± 0.05	1.70 ± 0.04	1.79 ± 0.04	1.86 ± 0.06**	1.24 ± 0.05**
Thymus	0.40 ± 0.02	0.41 ± 0.02	0.37 ± 0.02	0.41 ± 0.03	0.38 ± 0.02	0.18 ± 0.01**
Thymus: BW ratio	0.88 ± 0.03	0.90 ± 0.04	0.88 ± 0.05	0.92 ± 0.05	0.86 ± 0.04	0.49 ± 0.03**
Adrenal	0.024 ± 0.001^#	0.022 ± 0.001	0.022 ± 0.001	0.021 ± 0.001	0.021 ± 0.001	0.021 ± 0.001*
Adrenal:BW ratio	0.05 ± 0.00	0.05 ± 0.00	0.05 ± 0.00	0.05 ± 0.00	0.05 ± 0.00	0.06 ± 0.00
Liver	16.4 ± 0.5	16.6 ± 0.4	15.6 ± 0.3	15.9 ± 0.6	16.7 ± 0.4	12.5 ± 0.4**
Liver:BW ratio	36.4 ± 0.7	36.7 ± 0.4	36.4 ± 0.6	35.7 ± 0.6	37.9 ± 0.7	34.1 ± 0.9
Kidney	1.53 ± 0.03	1.53 ± 0.03	1.45 ± 0.03	1.48 ± 0.04	1.55 ± 0.03	1.19 ± 0.05**
Kidney:BW ratio	3.42 ± 0.08	3.37 ± 0.05	3.41 ± 0.08	3.35 ± 0.06	3.54 ± 0.06	3.25 ± 0.10
F1 FEMALES
Terminal BW	265.3 ± 4.5^#	264.8 ± 6.2	274.8 ± 5.4	263.3 ± 7.3	246.8 ± 2.5	216.9 ± 4.0**
Spleen	0.56 ± 0.02	0.58 ± 0.02	0.581 ± 0.02	0.58 ± 0.03	0.58 ± 0.02	0.37 ± 0.01**
Spleen:BW ratio	2.11 ± 0.06	2.20 ± 0.05	2.11 ± 0.05	2.20 ± 0.09	2.36 ± 0.08	1.68 ± 0.04**
Thymus	0.27 ± 0.01	0.28 ± 0.01	0.29 ± 0.01	0.31 ± 0.02*	0.25 ± 0.01	0.15 ± 0.02**
Thymus: BW ratio	1.01 ± 0.03	1.07 ± 0.03	1.06 ± 0.05	1.19 ± 0.06*	1.02 ± 0.07	0.68 ± 0.07**
Adrenal	0.032 ± 0.001	0.029 ± 0.001	0.033 ± 0.001	0.032 ± 0.001	0.030 ± 0.001	0.032 ± 0.002
Adrenal:BW ratio	0.12 ± 0.00	0.11 ± 0.00	0.12 ± 0.00	0.12 ± 0.01	0.12 ± 0.00	0.15 ± 0.01*
Liver	9.34 ± 0.30	9.32 ± 0.35	9.52 ± 0.22	9.03 ± 0.37	9.15 ± 0.26	7.09 ± 0.16**
Liver:BW ratio	35.3 ± 1.1	35.2 ± 1.0	34.3 ± 0.5	34.2 ± 0.7	37.1 ± 0.9	32.7 ± 0.53
Kidney	0.92 ± 0.02^#	0.90 ± 0.02	0.94 ± 0.03	0.87 ± 0.02	0.84 ± 0.02*	0.78 ± 0.02**
Kidney:BW ratio	3.46 ± 0.07	3.41 ± 0.06	3.43 ± 0.08	3.30 ± 0.07	3.40 ± 0.07	3.59 ± 0.06

Values represent mean ± SEM (n = 11–12/group, unless noted otherwise).

Absolute weights in g; organ:BW ratios in mg/g BW.

BW: bodyweight; CPS: cyclophosphamide.

^#Significant decreasing trend p < 0.05; ^##p < 0.01.

Bold values indicate significant pairwise differences when compared to the vehicle control group *p < 0.05; **p < 0.01.

Table 4. Select hematological parameters following daily gavage exposure to sulfolane in female mice.

	Sulfolane (mg/kg)						CPS
	0	1	10	30	100	300
Animals (N)	7	5	6	7	6	7	7
Leukocyte Count	5.86 ± 0.81	7.57 ± 0.84	6.45 ± 0.37	4.97 ± 0.56	5.50 ± 0.52	6.54 ± 1.23	1.60 ± 0.14**
Lymphocyte Count	4.84 ± 0.66	6.31 ± 0.66	5.11 ± 0.40	4.11 ± 0.50	4.49 ± 0.45	5.21 ± 1.01	1.38 ± 0.11**
Neutrophil Count	0.71 ± 0.11	0.72 ± 0.12^1	0.80 ± 0.16	0.53 ± 0.05	0.67 ± 0.05	0.87 ± 0.14	0.14 ± 0.03**
Monocyte Count	0.13 ± 0.02	0.15 ± 0.01	0.11 ± 0.02	0.11 ± 0.01^1	0.13 ± 0.02	0.13 ± 0.02	0.02 ± 0.01**
Eosinophil Count	0.10 ± 0.02	0.26 ± 0.04*	0.34 ± 0.11	0.13 ± 0.03	0.15 ± 0.03	0.19 ± 0.07	0.04 ± 0.02**
Basophil Count	0.02 ± 0.00	0.05 ± 0.02	0.03 ± 0.01	0.01 ± 0.00	0.02 ± 0.01	0.02 ± 0.01^1	0.003 ± 0.00**
LUC Count	0.07 ± 0.02^##	0.07 ± 0.01	0.06 ± 0.01	0.03 ± 0.01**	0.04 ± 0.00*	0.05 ± 0.01*	0.03 ± 0.01*

Values represent means ± SEM.

Counts in 10³ cells/μl.

CPS: cyclophosphamide (50 mg/kg); LUC: large unstained cell.

^##Significant dose-related trend p < 0.01.

Bold values indicate significant pairwise differences when compared to the vehicle control group *p < 0.05; **p < 0.01.

¹N = 6 due to removal of outliers.

Figure 1. NK cell activity following exposure of adult female B6C3F1/N mice and F1 rats to sulfolane. Cyclophosphamide (CPS) administered via IP intraperitoneal injection at 15 mg/kg (rat) or 50 mg/kg (mice). Arrows indicate significant decreasing trend with increasing sulfolane exposure (p < 0.01 for mice and p < 0.05 for rats). Result is significantly different from vehicle control group (*p < 0.05; **p < 0.01). E:T: effector:target ratio.

Figure 2. Splenic NK cell numbers following exposure of adult female B6C3F1/N mice and F1 HSD rats to sulfolane. Cyclophosphamide (CPS) administered via IP injection at 15 mg/kg (rat) or 50 mg/kg (mice). Arrows indicate a significant decreasing trend with increasing sulfolane exposure (p < 0.05). Result is significantly different from vehicle control group (*p < 0.05; **p < 0.01).

Table 5. Splenic immunophenotyping following gavage exposure to sulfolane in female B6C3F1/N mice.

	Sulfolane (mg/kg)						CPS
	0	1	10	30	100	300
Total spleen cells	73.7 ± 6.7	67.7 ± 3.8	66.3 ± 5.7	67.0 ± 4.8	59.5 ± 2.4	69.3 ± 6.3	16.3 ± 1.5**
Total lymphocytes	73.1 ± 6.6	66.9 ± 3.8	65.3 ± 5.6	65.7 ± 4.8	58.2 ± 2.3	68.0 ± 6.1	15.9 ± 1.5**
Total lymphocytes (%)	99.3 ± 0.1^##	98.9 ± 0.3	98.6 ± 0.3	98.0 ± 0.4**	97.8 ± 0.5*	98.2 ± 0.2**	97.3 ± 0.6**
Total T cells	23.7 ± 2.3	19.9 ± 1.2	19.2 ± 2.1	20.0 ± 1.9	18.4 ± 0.8	21.1 ± 2.0	7.69 ± 0.72**
Total T cells (%)	32.3 ± 0.8	29.8 ± 0.4	29.3 ± 1.3	30.3 ± 1.0	31.3 ± 0.5	31.2 ± 1.2	48.4 ± 0.8**
CD4^+ T cells	14.2 ± 1.5	11.7 ± 0.7	11.4 ± 1.1	12.2 ± 1.1	10.6 ± 0.5	11.9 ± 1.1	3.87 ± 0.42**
CD4^+ T cells (%)	59.6 ± 1.0	58.6 ± 0.4	59.9 ± 1.6	61.1 ± 1.7	57.8 ± 0.6	56.6 ± 1.0	50.0 ± 1.0**
CD8^+ T cells	8.56 ± 0.87	7.50 ± 0.56	7.11 ± 0.86	7.30 ± 0.58	6.63 ± 0.33	7.33 ± 0.79	2.90 ± 0.25**
CD8^+ T cells (%)	36.0 ± 0.6	37.4 ± 0.6	36.7 ± 0.9	36.9 ± 1.1	35.6 ± 0.6	34.5 ± 0.9	38.0 ± 0.7*
B cells	43.5 ± 3.8	41.6 ± 2.2	40.8 ± 3.4	39.7 ± 2.9	35.4 ± 1.4	42.0 ± 3.9	7.36 ± 0.69**
B cells (%)	59.7 ± 0.8	62.3 ± 0.5*	62.6 ± 1.2	60.6 ± 0.5	60.9 ± 0.3	61.6 ± 1.0	46.3 ± 0.5**
NK cells	1.81 ± 0.14^##	1.69 ± 0.13	1.41 ± 0.14	1.64 ± 0.14	1.33 ± 0.11*	1.39 ± 0.14*	0.30 ± 0.03**
NK cells (%)	2.51 ± 0.09^##	2.51 ± 0.10	2.21 ± 0.19	2.49 ± 0.10	2.28 ± 0.14	2.03 ± 0.08**	1.92 ± 0.06**
Monocytes/ macrophages	1.57 ± 0.19	1.39 ± 0.11	1.48 ± 0.15	1.53 ± 0.10	1.32 ± 0.07	1.48 ± 0.18	0.26 ± 0.03**
Monocytes/ macrophages (%)	2.12 ± 0.09	2.04 ± 0.08	2.24 ± 0.14	2.29 ± 0.20	2.32 ± 0.15	2.11 ± 0.13	1.64 ± 0.16*
Neutrophils	0.76 ± 0.12	0.66 ± 0.07	0.91 ± 0.22	0.82 ± 0.22	0.53 ± 0.04	0.65 ± 0.08	0.05 ± 0.01**
Neutrophils (%)	1.00 ± 0.09	0.97 ± 0.06	1.40 ± 0.32	1.29 ± 0.40	1.01 ± 0.12	0.95 ± 0.09	0.30 ± 0.03**
Eosinophils	0.40 ± 0.07	0.28 ± 0.02	0.33 ± 0.05	0.31 ± 0.03	0.23 ± 0.02	0.28 ± 0.03	0.06 ± 0.01*
Eosinophils (%)	0.53 ± 0.06	0.42 ± 0.04	0.49 ± 0.07	0.46 ± 0.02	0.40 ± 0.03	0.42 ± 0.05	0.42 ± 0.07
Total T cell:B cell ratio	0.54 ± 0.02	0.48 ± 0.01	0.47 ± 0.03	0.50 ± 0.02	0.51 ± 0.01	0.51 ± 0.03	1.05 ± 0.03**
CD4^+:CD8^+ T cell ratio	1.67 ± 0.05	1.57 ± 0.03	1.64 ± 0.08	1.66 ± 0.04	1.63 ± 0.03	1.65 ± 0.05	1.32 ± 0.05**

Mean ± SEM (N = 7–8/treatment group).

Counts in 1 × 10⁶ cells/spleen.

CPS: cyclophosphamide (50 mg/kg); LUC: large unstained cell.

^##Significant dose-related trend p < 0.01.

Bold values indicate significant pairwise differences when compared to the vehicle control group *p < 0.05; **p < 0.01.

Table 6. Splenic immunophenotypes following drinking water exposure to sulfolane in male and female F1 HSD rats.

	Sulfolane (mg/kg)					CPS (N = 8)
	0	30	100	300	1000
F1 MALES
Total spleen cells	510 ± 30.5^#	509 ± 32.6	419 ± 23.0	511 ± 35.6	394 ± 37.9	94.4 ± 9.8**
Total Lymphocytes	482 ± 29.0	485 ± 31.3	398 ± 21.6	484 ± 33.9	373 ± 36.4	91.4 ± 9.6**
Total Lymphocytes (%)	94.6 ± 0.2	95.3 ± 0.3	95.0 ± 0.2	94.9 ± 0.2	94.6 ± 0.3	96.8 ± 0.2**
Total T cells	86.2 ± 5.6	92.6 ± 5.3	73.2 ± 4.0	93.4 ± 7.7	74.1 ± 8.4	27.8 ± 3.2**
Total T cells (%)	17.8 ± 0.5	19.2 ± 0.5	18.5 ± 0.4	19.2 ± 0.4	19.4 ± 0.5	30.3 ± 0.7**
CD4^+ T cells	45.0 ± 2.5	49.3 ± 3.0	38.8 ± 2.2	49.1 ± 3.7	40.1 ± 4.7	13.3 ± 1.4**
CD4^+ T cells (%)	52.7 ± 1.5	53.2 ± 1.3	53.2 ± 1.4	53.3 ± 1.8	54.2 ± 1.8	48.5 ± 2.3
CD8^+ T cells	28.6 ± 2.7	32.0 ± 2.3	24.5 ± 1.6	32.8 ± 3.7	26.5 ± 3.4	12.6 ± 1.7**
CD8^+ T cells (%)	32.8 ± 1.5	34.6 ± 1.5	33.6 ± 1.4	34.4 ± 1.6	35.6 ± 1.7	44.9 ± 2.4**
B cells	92.1 ± 9.3	90.7 ± 6.8	80.1 ± 5.7	98.1 ± 7.3	74.8 ± 8.6	10.8 ± 1.2**
B cells (%)	18.7 ± 0.7^#	18.6 ± 0.5	20.0 ± 0.7	20.2 ± 0.7	19.8 ± 0.7	11.7 ± 0.3**
NK cells	44.6 ± 5.6^#	42.1 ± 4.2	33.2 ± 2.4	37.8 ± 4.6	29.8 ± 3.5	6.70 ± 0.73**
NK cells (%)	8.99 ± 0.77	8.66 ± 0.63	8.42 ± 0.52	7.69 ± 0.62	8.11 ± 0.64	7.44 ± 0.63
Monocytes/ macrophages	15.1 ± 0.8^##	15.1 ± 1.0	11.6 ± 0.7	15.5 ± 1.5	10.4 ± 1.2**	1.80 ± 0.23**
Monocytes/ macrophages (%)	3.02 ± 0.14	2.97 ± 0.07	2.78 ± 0.11	3.01 ± 0.15	2.67 ± 0.15	1.87 ± 0.10**
Neutrophils	13.9 ± 0.9^##	13.2 ± 0.9	10.6 ± 0.8	12.4 ± 1.0	9.39 ± 1.07**	4.59 ± 0.58**
Neutrophils (%)	2.76 ± 0.14^#	2.59 ± 0.07	2.51 ± 0.09	2.44 ± 0.15	2.37 ± 0.11	4.87 ± 0.32**
Eosinophils	1.90 ± 0.17	1.58 ± 0.12	1.38 ± 0.09	1.93 ± 0.17	1.37 ± 0.14	0.64 ± 0.10**
Eosinophils (%)	0.37 ± 0.02	0.32 ± 0.03	0.33 ± 0.02	0.38 ± 0.02	0.36 ± 0.02	0.68 ± 0.08**
Total T cell:B cell ratio	0.97 ± 0.05	1.04 ± 0.04	0.94 ± 0.04	0.96 ± 0.04	0.99 ± 0.04	2.61 ± 0.11**
CD4^+:CD8^+ T cell ratio	1.68 ± 0.15	1.59 ± 0.11	1.63 ± 0.10	1.62 ± 0.14	1.60 ± 0.14	1.12 ± 0.12*
F1 FEMALES
Total spleen cells	364 ± 23.5	344 ± 19.4	308 ± 6.1	291 ± 15.1	384 ± 30.0	65.5 ± 4.4**
Total Lymphocytes	345 ± 23.0	329 ± 19.1	294 ± 5.7	276 ± 15.1	370 ± 29.3	61.5 ± 4.2**
Total Lymphocytes (%)	94.7 ± 0.9	95.5 ± 0.6	95.3 ± 0.4	94.8 ± 0.6	96.3 ± 0.4	93.9 ± 1.1
Total T cells	51.0 ± 3.0	50.4 ± 3.2	47.8 ± 1.4	43.7 ± 2.3	61.0 ± 5.2	18.1 ± 1.2**
Total T cells (%)	14.9 ± 0.4^##	15.3 ± 0.4	16.2 ± 0.3*	15.9 ± 0.3*	16.5 ± 0.5*	29.5 ± 0.9**
CD4^+ T cells	29.3 ± 2.2	29.3 ± 1.5	26.3 ± 0.8	24.6 ± 1.3	34.6 ± 3.5	9.42 ± 0.61**
CD4^+ T cells (%)	57.0 ± 1.4	58.7 ± 1.4	55.1 ± 0.7	56.6 ± 1.6	56.2 ± 1.3	52.5 ± 2.2
CD8^+ T cells	18.5 ± 1.0	17.8 ± 1.7	18.2 ± 0.9	15.9 ± 1.2	22.3 ± 1.8	7.85 ± 0.77**
CD8^+ T cells (%)	36.6 ± 1.5	34.7 ± 1.4	37.9 ± 0.9	36.2 ± 1.4	36.9 ± 1.2	43.0 ± 2.3*
B cells	59.3 ± 4.9	55.8 ± 3.8	51.2 ± 2.0	48.6 ± 3.3	70.3 ± 7.1	6.08 ± 0.66**
B cells (%)	17.1 ± 0.6^#	17.0 ± 0.7	17.4 ± 0.6	17.5 ± 0.6	18.8 ± 0.6	9.81 ± 0.67**
NK cells	40.7 ± 2.4	32.3 ± 2.7	34.4 ± 2.5	30.6 ± 2.6*	43.4 ± 4.5	4.77 ± 0.63**
NK cells (%)	12.1 ± 0.7	10.0 ± 0.7	11.7 ± 0.8	11.1 ± 0.8	11.8 ± 0.9	7.61 ± 0.71**
Monocytes/ macrophages	9.72 ± 0.72	9.21 ± 0.72	9.18 ± 0.36	8.78 ± 0.69	9.94 ± 1.24	1.29 ± 0.14**
Monocytes/ macrophages (%)	2.68 ± 0.13	2.67 ± 0.12	2.99 ± 0.13	3.02 ± 0.19	2.54 ± 0.13	1.96 ± 0.12**
Neutrophils	6.31 ± 0.50	5.62 ± 0.48	5.69 ± 0.35	5.66 ± 0.46	6.85 ± 0.64	2.85 ± 0.46**
Neutrophils (%)	1.76 ± 0.13	1.64 ± 0.10	1.85 ± 0.12	1.94 ± 0.09	1.79 ± 0.08	4.37 ± 0.69**
Eosinophils	1.17 ± 0.11	1.00 ± 0.07	0.95 ± 0.05	0.87 ± 0.07	1.15 ± 0.08	0.46 ± 0.08**
Eosinophils (%)	0.32 ± 0.02	0.29 ± 0.01	0.31 ± 0.01	0.30 ± 0.02	0.31 ± 0.02	0.73 ± 0.12**
Total T cell:B cell ratio	0.89 ± 0.05	0.92 ± 0.05	0.94 ± 0.04	0.92 ± 0.04	0.89 ± 0.04	3.15 ± 0.29**
CD4^+:CD8^+ T cell ratio	1.61 ± 0.11	1.74 ± 0.11	1.47 ± 0.05	1.61 ± 0.11	1.56 ± 0.09	1.27 ± 0.14

Mean ± SEM (N = 11–12/treatment group)

Counts in 1 × 10⁶ cells/spleen.

CPS, cyclophosphamide (15 mg/kg)

^#Significant dose-related trend p < 0.05, ^##p < 0.01.

Bold values indicate significant pairwise differences when compared to the vehicle control group *p < 0.05; **p < 0.01.