Oral subchronic immunotoxicity study of ethyl tertiary butyl ether in the rat

Figures & data

Table 1.  Body weight (g) and organ weights (mg) in female Crl:CD(SD) rats exposed to ethyl tertiary butyl ether (ETBE) for 28 days.

Parameter	Vehicle (10)	ETBE (mg/kg/dav)			CPS 50 mg/kg (10)	H/NH	Trend analysis
		250 (10)	500 (10)	1000 (10)
Body weight (g)	236 ± 4	243 ± 6	247 ± 5	233 ± 5	224 ± 5	H	NS
Spleen weight (mg)	266 ± 11	257 ± 8	227 ± 13	242 ± 13	114 ± 4**	H	p < 0.05
% Body weight	0.11 ± 0.01	0.11 ± 0.01	0.09 ± 0.01	0.11 ± 0.01	0.05 ± 0.01**	H	p < 0.01
Thymus weight (mg)	170 ± 8	178 ± 13	183 ± 8	180 ± 16	40 ± 2**	H	NS
% Body weight	0.07 ± 0.01	0.07 ± 0.01	0.07 ± 0.01	0.08 ± 0.01	0.02 ± 0.01**	H	NS

Female Crl:CD (SD) rats were administered the vehicle (corn oil) or ETBE via oral gavage daily for 28 days. The positive control, cyclophosphamide (CPS), was administered i.p. to the positive control group only on Days 24 through 27 of exposure. On the day of euthanasia, the rats were necropsied and indicated organs weighed. Values represent the mean ± SE (standard error) derived from the number of animals indicated in parentheses. H = homogeneous data and NH = non-homogeneous data were evaluated using the Bartlett’s Test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. When significant differences occurred, exposed groups were compared to the vehicle using the Dunnett’s Test. Non-homogeneous data were evaluated using a non-parametric analysis of variance. When significant differences occurred, exposed groups were compared to the vehicle using the Wilcoxon Rank Test. The positive control was compared to the vehicle using the Student’s t-test. *Values significantly different from the vehicle at p < 0.05; **significant at p < 0.01. The Jonckheere’s Test was used to test for exposure level-related trends among the vehicle and exposed groups. NS, not significant.

Table 2.  Spleen antibody-forming cell response to T-dependent antigen sheep erythrocytes (SRBC) in female Crl:CD(SD) rats exposed to ethyl tertiary butyl ether (ETBE) for 28 days.

Exposure	Body weight (g)	Spleen weight (mg)	Spleen cells (× 10^7)	IgM AFC/10^6 spleen cells	IgM AFC/spleen (× 10^3)
Vehicle	236 ± 4 (10)	266 ± 11 (10)	70.01 ± 5.09 (10)	2088 ± 326 (10)	1418 ± 206 (10)
ETBE
250 mg/kg/day	243 ± 6 (10)	257 ± 8 (10)	69.46 ± 3.40 (10)	1641 ± 163 (10)	1133 ± 109 (10)
5 00 mg/kg/day	247 ± 5 (10)	227 ± 13 (10)	63.28 ± 3.81 (10)	2964 ± 453 (10)	1935 ± 354 (10)
1000 mg/kg/day	233 ± 5 (10)	242 ± 13 (10)	64.60 ± 4.60 (10)	2251 ± 363 (10)	1532 ± 295 (10)
Cyclophosphamide 5 0 mg/kg	224 ± 5 (10)	114 ± 4** (10)	9.87 ± 0.81** (10)	0 ± 0** (10)	0 ± 0** (10)
H/NH	H	H	H	NH	NH
Trend Analysis	NS	p < 0.05	NS	NS	NS

Female Crl:CD(SD) rats were administered the vehicle (corn oil) or ETBE via oral gavage daily for 28 days. The positive control, cyclophosphamide, was administered i.p. to the positive control group only on days 24 through 27 of exposure. Four days prior to sacrifice, all rats were immunized (i.v.) with 2 × 10⁸ SRBC. On the day of sacrifice (Day 28), spleens were removed and placed into tubes containing EBSS with 15 mM HEPES and supplemented with gentamicin. Spleens were prepared into single cell suspensions and the number of IgM SRBC antibody-forming cells was determined. Values represent the mean ± SE (standard error) derived from the number of animals indicated in parentheses. H = homogeneous data and NH = non-homogeneous data were evaluated using the Bartlett’s Test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. When significant differences occurred, exposed groups were compared to the vehicle using the Dunnett’s Test. Non-homogeneous data were evaluated using a non-parametric analysis of variance. When significant differences occurred, exposed groups were compared to the vehicle using the Wilcoxon Rank Test. The positive control was compared to the vehicle using the Student’s t-test. *Values significantly different from the vehicle at p < 0.05; **significant at p < 0.01. The Jonckheere’s Test was used to test for exposure level-related trends among the vehicle and exposed groups. NS, not significant.

Figure 1.  Individual IgM antibody-forming cell responses to sheep erythrocytes in female Crl:CD (SD) rats exposed to ETBE via oral gavage for 28 days—Day 4 response. Results of AFC response expressed as individual animal values for specific activity (AFC/10⁶ spleen cells; left panel) or as total spleen activity (AFC/spleen; right panel). N = 10 rats/exposure level for 0, 250, 500, and 1000 mg ETBE/kg/day and 50 mg CPS/kg/day (study Days 24–27).