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Description of key information

ORAL
NOAEL (immunotoxicity) = 1000 mg/kg bw/day, rat (male), US EPA OPPTS 870.7800, Boverhof et al. (2010)

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
immunotoxicity: short-term oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 May 2010 to 11 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.7800
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Specific details on test material used for the study:
Purity: 94.5%
Species:
rat
Strain:
other: F344/DuCrl
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: ca. 7 weeks
- Weight at study initiation (group means on test day 1): 146.7 - 147.5 g
- Housing: Animals were housed one per cage in stainless steel cages. Cages had wire mesh floors and were suspended above absorbent paper. Non-woven gauze was placed in the cages to provide a cushion from the flooring for rodent feet. The gauze provided environmental enrichment. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
- Diet: ad libitum
- Water: municipal water, ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIETARY EXPOSURE
Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Premixes and diets were mixed weekly and dietary concentrations adjusted based upon the most recent body weight and feed consumption data. Initial concentrations of test material in the diet were calculated from historical body weights and feed consumption data.
Positive control solutions of cyclophosphamide monohydrate (CP) were prepared in sterile saline at a concentration of 20 mg/mL and administered at a dose volume of 1 mL/kg body weight in order to achieve the dose of 20 mg/kg. Daily aliquots were stored at -4º C and thawed as needed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Dose Confirmation and Homogeneity
Dose confirmation analyses of all dose levels, plus control and premix, were determined pre-exposure. The homogeneity of the low- and high-dose test diets was determined concurrent with dose confirmation. The method used for analysing the test material in the diet was a solvent extraction method followed by analysis using liquid chromatography-mass spectrometry (LC/MS).
These analyses indicated the mean concentration ranged from 93.4 to 102.7 % indicating acceptable concentrations of test material. The relative standard deviations were 1.8 and 4.7 %, respectively, indicating that the premix and diet were homogeneously mixed.

- Stability
The test material was previously determined to be stable in rodent chow for 35 days at concentrations of 0.00258 to 5 % (w/w). The established concentration range and duration spanned those used in this study. Therefore, additional stability analyses were not conducted.
Duration of treatment / exposure:
29 days
Frequency of treatment:
continuous (in diet)
Remarks:
Doses / Concentrations:
0, 1000 mg/kg/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 1038 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose level was selected based on a review of existing repeat-dose toxicity data. Due to the low toxicity of the test material in previous repeat dose toxicity studies, a limit dose study was conducted in which only the limit dose of 1000 mg/kg/day was evaluated as per the EPA Health Effects Test Guideline OPPTS 870.7800 paragraph (e). The only treatment-related effect that was expected at this dose was an increase in cecum weight.
- Rationale for selecting male animals only: Males appear to be more sensitive for target-organ (cecum) effects as the NOAEL was lower for males in the 90-day toxicity study due to more pronounced effects on the cecum.
- Rationale for animal assignment: Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study.
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted at least once a day. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could have been observed include, but were not limited to: decreased/increased activity, repetitive behaviour, vocalisation, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in faecal consistency, and faecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were conducted on all animals pre-exposure and once per week throughout the study. The examination included cage-side, hand-held and open-field observations, which are recorded categorically or using explicitly defined scales

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed pre-exposure, twice weekly throughout the study (excluding IPC group), and at necropsy (terminal). Body weight gains were calculated relative to test day one.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: Feed consumed was determined pre-exposure, twice during the first week and at least weekly for all animals (excluding IPC group) by weighing feed containers at the start and end of a measurement cycle. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feeder - final weight of feeder) / (number of days in measurement cycle x number of animals per cage)
The test material intake (TMI) was calculated using feed concentrations, body weights and feed consumption in the following equation:
TMI = [feed consumption (g/day) x 1000 mg/g x (% of test material in feed / 100)] / [((current body weight (g) + previous body weight (g)) / 2) / 1000 g/kg]

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the scheduled necropsy
- Anaesthetic used for blood collection: Yes (CO₂/O₂)
- Animals fasted: No
- How many animals: All animals (vehicle control and test material animals only)
- Parameters checked included: Haematocrit (HCT), Haemoglobin (HGB) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Differential WBC count, Platelet (PLT) count, Reticulocyte (RET) count, Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin Concentration (MCHC)

CLINICAL CHEMISTRY: No

URINALYSIS: No
Sacrifice and pathology:
SACRIFICE
Non-fasted rats submitted alive for necropsy were anaesthetised by the inhalation of CO₂/O₂, weighed, and blood obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanised by decapitation.

GROSS PATHOLOGY: Yes
A limited gross necropsy was conducted on all animals. The necropsy included an examination of the external tissues and all orifices. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The head was removed, the skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised.
For the vehicle control, test material, and positive control group animals, the spleen and thymus were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated.

HISTOPATHOLOGY: Yes
For the vehicle control and test material group animals, representative samples of the spleen, cecum, thymus, sternum, mesenteric lymph node, Peyer’s patch (GALT), and gross lesions were collected and preserved in neutral, phosphate buffered 10 % formalin. No tissues were preserved for the positive control animals.
Humoral immunity examinations:
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes
- Method: On days 24 - 28, animals in the positive control group were injected i.p. with cyclophosphamide (CP) at a dose of 20 mg/kg in isotonic sterile saline. This dose of CP was expected to result in approximately 90 % reduction in the anti-SRBC antibody response.
Five days prior to sacrifice, rats were immunised with a single 0.5 mL i.v. injection of 2 x 10⁸ SRBC in isotonic sterile saline, via the lateral tail vein. On day 29 rats were anesthetised via CO₂/O₂, weighed, and blood samples obtained from the orbital sinus. Blood samples designated for assessment of anti-SRBC IgM were collected in labelled serum separator tubes, allowed to clot, separated from blood cells via centrifugation and stored at -20 °C until conduct of the ELISA.
Serum samples were analysed for anti-SRBC IgM using a commercially available ELISA kit. This assay used detergent solubilised SRBC ghosts for solid phase (microtiter wells) immobilisation and horseradish peroxidase (HRP) conjugated anti-rat IgM antibodies for detection. Test serum or plasma samples were diluted and incubated in the microtiter wells for 45 minutes. The microtiter wells were subsequently washed and HRP conjugate was added and incubated for 45 minutes. Anti-SRBC IgM molecules were thus sandwiched between immobilised SRBC antigens and the detection antibody conjugate. The wells were then washed to remove unbound HRP-labelled antibodies and tetramethylbenzidine peroxidase (TMB) reagent was added and incubated for 20 minutes at room temperature. This resulted in the development of a blue colour. Colour development was stopped by the addition of Stop Solution, changing the colour to yellow, and optical density was measured spectrophotometrically at 450 nm. The concentration of anti-SRBC IgM was proportional to the optical density of the test sample. Concentration of the anti-SRBC IgM in the test samples was determined through the use of a standard curve prepared by serial dilution of lyophilised rat anti-SRBC standard stock provided by the manufacturer. The standard curve consisted of units of anti-SRBC IgM (u/mL) plotted versus absorbance values at 450 nm. Only the linear portion of the resultant curve was used for the assessment of anti-SRBC IgM concentrations in the test samples. Test samples were run at multiple dilutions and only dilutions falling within the linear region of the standard curve were used to determine the concentration of anti-SRBC IgM in each of the samples. In cases where multiple test sample dilutions fell within the linear range of the standard curve, an average value was determined to result in a single reportable concentration in u/mL for each test serum sample. A 25-fold dilution was the minimum dilution examined as specified by the manufacturer.
Positive control:
Cyclophosphamide (CP), dosed at 20 mg/kg via intraperitoneal injection on study days 24-28.
Statistics:
Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, organ weights, appropriate haematologic data and the SRBC ELISA data was evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA). If significant at alpha = 0.05, the ANOVA was followed respectively by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control. The experiment-wise alpha level was reported for these two tests. For ELISA data the positive control data was compared to the negative control data in an analysis that was separate from the treated groups and negative control. Detailed clinical observations incidence data was statistically analysed by a z-test of proportions comparing each treated group to the control group (alpha = 0.05; Bruning and Kintz, 1987). Descriptive statistics only (means and standard deviations) were reported for body weight gains, RBC indices and differential WBC counts. Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969), but routinely excluded only from feed consumption statistics. Outliers may have been excluded from other analyses only for documented, scientifically sound reasons.
As numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.
Clinical signs:
no effects observed
Description (incidence and severity):
Examinations performed on all animals showed no treatment-related findings.
Mortality:
no mortality observed
Description (incidence):
All rodents survived to scheduled necropsy
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically identified differences in the body weights or body weight gains when compared to controls and no differences related to treatment.
On test day 29 the body weight and body weight gain of animals in the positive control group were decreased 5.5 and 14.5 %, respectively, when compared to control animals. Body weight and body weight gains were similar to control through test day 22. These differences were attributed to the daily IP injections of CP on test days 24-28.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Feed consumption for animals in the treated group was similar to controls throughout the duration of the study.
Rats were given 0 or 1000 mg/kg/day, which corresponded to time-weighted average doses of 0 or 1038 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in any of the haematologic parameters.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in the organ weights.
As expected, the positive control animals had statistically identified, CP-induced decreases in absolute (45.9 %) and relative (42.8 %) spleen weights when compared to control animals. CP also induced decreases in absolute (75.7 %) and relative (74.0 %) thymus weights when compared to control animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The only treatment-related gross observation was an increased size of the cecum, noted in all males given 1000 mg/kg/day. All remaining gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material.
As expected, the positive control animals had CP-induced decreases in spleen and thymus size.
Cell viabilities:
no effects observed
Humoral immunity examinations:
no effects observed
Specific cell-mediated immunity:
not examined
Description (incidence and severity):
SRBC ANTIBODY RESPONSE (ANTI-SRBC ELISA)
Analysis of the data did not show a statistically significant difference between the test material dose group (1000 mg/kg/day) and the vehicle control. The positive control cyclophosphamide produced a statistically significant and greater than 99 % reduction in the anti-SRBC IgM response
Non-specific cell-mediated immunity:
not examined
Other functional activity assays:
not examined
Other findings:
not specified
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no test substance related effects at the highest dose tested
Conclusions:
Under the conditions of the study, the test material did not exhibit evidence of immunotoxicity as it did not result in a treatment related effect on the primary immune response to SRBCs in male rats.
Executive summary:

The immunotoxic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline EPA OPPTS 870.7800.

During the study ten male F344/DuCrl rats per group were given test diets formulated to supply 0 or 1000 mg test material / kg body weight /day (mg/kg/day) for 29 days. Actual time-weighted average doses were 0 or 1038 mg/kg/day, respectively. An additional group of ten male F344/DuCrl rats exposed to 0 mg/kg/day were included to serve as positive immunosuppressive controls and received intraperitoneal (i.p.) doses of 20 mg/kg cyclophosphamide (CP) on study days 24 - 28. The immunotoxic potential of the test material was assessed through the evaluation of the primary antibody response to sheep red blood cells (SRBC) using an enzyme-linked immunosorbant assay (ELISA) approach that measured the concentration of serum anti-SRBC IgM. Additional parameters evaluated included daily cage-side observations, weekly detailed clinical observations, body weights, feed consumption, haematology, selected organ weights, and gross examinations of selected tissues.

The only treatment-related effect in the study was the gross pathological observation of an increased size of the cecum, noted in all rats given 1000 mg/kg/day. There were no treatment-related effects on clinical signs, body weight, feed consumption, haematology parameters or spleen and thymus weights, when compared to control animals.

Analysis of the anti-SRBC IgM ELISA data did not show a statistically significant difference between the test material dose group (1000 mg/kg/day) and the vehicle control. The positive control cyclophosphamide produced a statistically significant and greater than 99 % reduction in the anti-SRBC IgM response.

Based on the results from this study, the test material did not exhibit evidence of immunotoxicity as it did not result in a treatment-related decrease in the primary immune response to SRBCs in male rats. Furthermore, no treatment-related effects were observed on spleen and thymus weights or haematological parameters. The no-observed-effect level (NOEL) for immunotoxicity was the limit dose of 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available study was conducted under GLP conditions and in accordance with a standardised guideline. The study was therefore assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997). The quality of the database is good.

Effect on immunotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The immunotoxic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline EPA OPPTS 870.7800.

During the study ten male F344/DuCrl rats per group were given test diets formulated to supply 0 or 1000 mg test material / kg body weight /day (mg/kg/day) for 29 days. Actual time-weighted average doses were 0 or 1038 mg/kg/day, respectively. An additional group of ten male F344/DuCrl rats exposed to 0 mg/kg/day were included to serve as positive immunosuppressive controls and received intraperitoneal (i.p.) doses of 20 mg/kg cyclophosphamide (CP) on study days 24 - 28. The immunotoxic potential of the test material was assessed through the evaluation of the primary antibody response to sheep red blood cells (SRBC) using an enzyme-linked immunosorbant assay (ELISA) approach that measured the concentration of serum anti-SRBC IgM. Additional parameters evaluated included daily cage-side observations, weekly detailed clinical observations, body weights, feed consumption, haematology, selected organ weights, and gross examinations of selected tissues.

The only treatment-related effect in the study was the gross pathological observation of an increased size of the cecum, noted in all rats given 1000 mg/kg/day. There were no treatment-related effects on clinical signs, body weight, feed consumption, haematology parameters or spleen and thymus weights, when compared to control animals.

Analysis of the anti-SRBC IgM ELISA data did not show a statistically significant difference between the test material dose group (1000 mg/kg/day) and the vehicle control. The positive control cyclophosphamide produced a statistically significant and greater than 99 % reduction in the anti-SRBC IgM response.

Based on the results from this study, the test material did not exhibit evidence of immunotoxicity as it did not result in a treatment-related decrease in the primary immune response to SRBCs in male rats. Furthermore, no treatment-related effects were observed on spleen and thymus weights or haematological parameters. The no-observed-effect level (NOEL) for immunotoxicity was the limit dose of 1000 mg/kg/day.


Justification for selection of effect on immunotoxicity via oral route endpoint:
Only one study is available.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to immunotoxicity

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to immunotoxicity.