Registration Dossier

Administrative data

Endpoint:
immunotoxicity: short-term inhalation
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported study conducted under GLP and in compliance with relevant US EPA test guideline. Although study conclusions are clear, questionable relevance to toxicity assessment of MTBE makes Klimisch 2 appropriate.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Health assessment of gasoline and fuel oxygenate vapors: Immunotoxicity evaluation
Author:
White Jr., K L et al
Year:
2014
Bibliographic source:
Regulatory Toxicology and Pharmacology 70 (2014) S43–S47

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.7800
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: vapours collected as condensates
Details on test material:
BGVC = vapour condensate collected from baseline gasoline (reformulated gasoline Summer baseline fuel meeting CAA section 211(k)(10)(B)(i) specification (40 CFR 79.55)).
G/MTBE = vapour condensate collected from baseline gasoline with MTBE added to create 2.7 wt% O2 content (corresponding to 14.9 vol% MTBE)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
Animals used in this investigation were satellite groups taken (after 4 weeks of exposure) from the subchronic inhalation toxicity study of Clark et al, Health assessment of gasoline and fuel oxygenate vapors: Subchronic inhalation toxicity. Regulatory Toxicology and Pharmacology 70 (2014) S18–S28 (paper summarised at section 7.5.2 of this dossier).

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
other: air + nitrogen
Details on exposure:
Nitrogen was applied to the supplied containers of BGVC and G/MTBE vapour condensates to generate stable exposure concentrations of the test vapours (using a 3-way split flow to give test substance pressurisation, purge flow and vapourisation via a volatilisation chamber).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were monitored:
- gravimetrically by measurement of chamber airflow and vapour condensate consumption
- during each exposure (4 times) by IR spectrophotometry of breathing zone samples
- by weekly GC analysis of charcoal tube collected control and test samples for determination of major components (at least 18) comprising 80 wt% or more of the tested vapour condensates
- by weekly particle size sampling and measurement to identify any aerosol present.
Duration of treatment / exposure:
6h/day, 5 days/week for 4 weeks (giving 20 exposures in total)
Frequency of treatment:
Daily as specified
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/cu.m vapour condensate (air control)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2050, 10148, 20234 mg/cum BGVC
Basis:
analytical conc.
Nominals 2000, 10000, 20000 mg/cu.m
Remarks:
Doses / Concentrations:
2029, 10099, 20242 mg/cu.m G/MTBE
Basis:
analytical conc.
Nominals 2000, 10000, 20000 mg/cu.m
No. of animals per sex per dose:
10 females/group
Control animals:
yes, sham-exposed
Details on study design:
Nitrogen was applied to the supplied containers of BGVC and G/MTBE vapour condensates to generate stable exposure concentrations of the test vapours (using a 3-way split flow to give test substance pressurisation, purge flow and vapourisation via a volatilisation chamber).
Chamber concentrations were monitored:
- gravimetrically by measurement of chamber airflow and vapour condensate consumption
- during each exposure (4 times) by IR spectrophotometry of breathing zone samples
- by weekly GC analysis of charcoal tube collected control and test sample for determination of major components of the tested vapour condensates
- by weekly particle size sampling and measurement to identify any aerosol present.

Examinations

Observations and clinical examinations performed and frequency:
Observations related to immunotoxicology investigations only are detailed in the published paper.
Sacrifice and pathology:
Blood samples were taken for serum collection at termination (by CO2 inhalation). Thymuses were excised, weighed and preserved in addition to spleen collection.

4 days prior to termination, rats were given iv injections of sheep red blood cells (sRBC: 2 x 10E+08) for antigen sensitisation. Termination (1 day after last exposure) was followed by removal and weighing of spleens. These were then placed in balanced salt solution (EBSS with HEPES and gentamicin) and kept on ice for further processing. Single-cell (splenocyte) preparations were prepared using a laboratory blender, resuspended in balanced salts solution as before and diluted to the required cell densities.
Cell viabilities:
Splenocyte viability was determined using propidium iodide and a flow cytometer.
Humoral immunity examinations:
Primary IgM response to sRBC was measured by the plaque assay (IgM antibody-forming cell response to the T-dependent antigen sheep erythrocytes) using the Jerne technique.

Spleen cell suspension (0.1 ml) was added to Guinea pig complement + sRBc (each 25 microlitres) in 0.5 ml warm agar, plated and incubated (36-38C, 3h). Plaques were counted, taking counts from tested dilutions giving 100-300 plaques/plate. Splenocyte counts , cells/spleen and Antibody-Forming Cells/10E+06 spleen cells were determined. Since each observed plaque is generated from a single IgM antibody-producing B cell, AFC/spleen can be calculated. Resultant data were expressed as specific activity (AFC/10E+06 spleen cells) and total
spleen activity (AFC/spleen); following US NTP immunotoxicology assay practice, the plaque-forming assay results were not adjusted for spleen cell viability.
Specific cell-mediated immunity:
Not directly evaluated
Non-specific cell-mediated immunity:
Not directly evaluated
Positive control:
Rats of the positive control group were injected intraperitoneally with cyclophosphamide (50 mg/kg) daily for 4 days prior to termination.
Statistics:
Data were tested for homogeneity of variance (Bartlett’s Chi Square Test), then homogeneous data were analysed by parametric 1-way ANOVAR). When significant differences were seen, test groups were compared to air controls using Dunnett’s t Test. Nonhomogeneous data were evaluated using non-parametric ANOVAR. When significant differences were seen, test groups and controls were compared using the Gehan-Wilcoxon Test. Jonckheere’s Test was used to test for exposure-related trends across control and test groups. Positive controls were compared to the air control group using Student’s t Test (evidence of decreased immune response, p<0.05 being required for study validity).

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
terminal bodyweights unaffected by treatment
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Gross pathological findings:
not specified
Details on results:
Results of the plaque assays revealed no effect of inhalation exposure to BGVC or G/MTBE on IgM antibody-forming cell response .

Specific immunotoxic examinations

Cell viabilities:
not specified
Humoral immunity examinations:
no effects observed
Description (incidence and severity):
IgM antibody-forming cell responses did not differ between rats exposed to BGVC or G/MTBE and the air controls, whether evaluated as specific (AFC/10E+06 spleen cells) or as total spleen activity (AFC/spleen). No treatment-related trend was seen.
Specific cell-mediated immunity:
not examined
Non-specific cell-mediated immunity:
not examined
Other functional activity assays:
not examined
Other findings:
no effects observed
Description (incidence and severity):
Terminal spleen and thymus weights unaffected, except in positive control group

Any other information on results incl. tables

Plaque assay (IgM AFC response to T-dependent sheep erythrocyte antigen) and body/organ weight results

 

BGVC (mg/cu.m)

G/MTBE (mg/cu.m)

 

0 (Air

control)

2000

10000

20000

CP pos

control

0 (Air

control)

2000

10000

20000

CP pos

control

Body

weight

 (g)

247.8

258

250

245.7

224.4*

248.3

265.9

242.5

254.8

231.3

Spleen

weight

 (mg)

615

647

600

675

265*

646

637

574

651

283*

Spleen

 cells

(x10E+07)

53.18

62.26

55.82

57.96

9.69*

72.09

76.52

65.13

76.28

10.65*

IgM AFC

(1)

1639

1540

1687

1175

3*

1646

1128

1490

1680

0*

IgM AFC

(2)

880

980

903

685

(3)

0*

1162

887

966

1245

0*

 (1) IgM antibody-forming cell response/10E+06 spleen cells

 (2) IgM antibody-forming cell response/spleen x10E+03

 (3) Group mean excludes a single, rejected value (outlier)

 CP pos control = cyclophosphamide positive control group (dose 50 mg/kg/day for last 4 days)

 * significantly different from air control, p<0.01

Applicant's summary and conclusion

Conclusions:
In this study, subacute inhalation exposure of female rats to BGVC or G/MTBE vapour condensates did not significantly change their humoral immune response, as determined by the plaque assay (IgM antibody-forming cell response to T-dependent antigen sheep erythrocytes). Spleen weight, spleen cell number and IgM antibody production (expressed in terms of specific activity or total spleen activity) were unaffected by these exposures, at concentrations up to 50% of the lower explosion limits of the test materials. Also, the presence of MTBE in G/MTBE did not significantly alter the measured humoral immune response to the baseline gasoline vapour condensate (BGVC) alone.
Executive summary:

Exposure of female rats to BGVC or G/MTBE vapour condensates (6 h/day, 5 days/ week over 4 weeks) did not alter humoral immune response as determined by IgM antibody-forming cell response to T-dependent antigen sheep erythrocytes. Spleen weight, spleen cell number and IgM antibody production (expressed in terms of specific activity or total spleen activity) were unaffected. Inclusion of the MTBE in G/MTBE did not significantly alter the measured humoral immune response to the baseline gasoline vapour condensate (BGVC) alone.