Cyclopropanecarboxylic acid, [(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-[(cyclopropylcarbonyl)oxy]-1,3,4,4a,5,6,6a,12,12a,12b-decahydro-6,12-dihydroxy-4,6a,12b-trimethyl-11-oxo-9-(3-pyridinyl)-2H,11H-naphtho[2,1-b]pyrano[3,4-e]pyran-4-yl]methyl ester

Immunotoxicity

Description of key information

The test substance showed no signs of immunotoxicity when administered via the diet over a period of 4 weeks to female Wistar rats (2015/1076588). The no observed adverse effect level (NOAEL) for the immunotoxicologically relevant endpoints and for systemic toxicity was set to 4000 ppm (278 mg/kg bw/d) - the highest dose tested.

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:

From: 03 Feb 2015 To: 15 Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Lot number: 080722
Physical state/ appearance: solid, yellow
Expiration date of the lot/batch: November 01, 2015

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Supplier: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
Age at dosing: 42 ± 1 days
Body weight at dosing: 126.7 - 129.3 g
Housing: (5 animals per cage) in polysulfonate (H-Temp) cages type 2000P supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2).
Diet: Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
Water: from water bottles, ad libitum
Acclimatisation: 6 days

ENVIRONMENTAL CONDITIONS
Temperature: 20-24 °C
Humidity: 30-70 %
ventilation: 15 air changes per hour.
Photoperiod: day/night cycle was 12 hours (12 hours light from 06:00-18:00 h, 12 hours dark from 18:00-06:00 h).

IN-LIFE DATES: From: 03 Feb 2015 To: 17 Jul 2015

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF TEST DIET
For each concentration, the test-substance was weighted out and mixed with a small amount of food. Then corresponding amounts of food, depending on test group, were added to this premix to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Details of the mixers used are retained with the raw data. The test-substance preparations were mixed once before the start of the administration period.

PREPARATION OF POSITIVE CONTROL SOLUTION
To prepare the solution of Cyclophosphamide monohydrate (positive control), the appropriate amount of Cyclophosphamide monohydrate was weighed out depending on the desired concentration. Then the vehicle (drinking water) was filled up to the desired volume, subsequently mixed using a magnetic stirrer. The positive control substance preparations were prepared once, split into daily aliquots and kept frozen at -18 °C. The mixtures were administered after reaching room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the diet over a period of up to 32 days at room temperature was proven before the start of the study. The stability of Cyclophosphamide monohydrate (positive control substance) in drinking water was demonstrated at room temperature for 7 days and frozen for 32 days.

Homogeneity analyses of the test-substance preparations were performed in samples of all concentrations at the start of the study period. Considering the low standard deviation (4.6 and 5.3%) in the homogeneity analysis, it can be concluded that the test substance was distributed homogeneously in Ground Kliba maintenance diet mouse/ rat „GLP“ meal.

The concentration control analyses revealed that the determined mean values (sample 3 - 5 and samples 7 - 9 ) and single value (sample 6) of the test substancein Ground Kliba maintenance diet mouse/ rat „GLP“ meal were found to be in the range of 90 % - 110 % of the nominal concentrations. The determined concentration of the positive control substance (Cyclophosphamide monohydrate) in drinking water was slightly above the specification limit 110 % (110.7 %) of the nominal concentration.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

daily

Dose / conc.:

0 ppm

Remarks:

plain diet

Dose / conc.:

300 ppm

Remarks:

corresponding to 21 mg/kg bw/d (calculation based on LLPI food consumption HCD)

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 69 mg/kg bw/d (calculation based on LLPI food consumption HCD)

Dose / conc.:

4 000 ppm

Remarks:

corresponding to 278 mg/kg bw/d (calculation based on LLPI food consumption HCD)

No. of animals per sex per dose:

10 females/dose

Control animals:

yes, plain diet

Details on study design:

All animals were immunized 6 days before blood sampling and necropsy using 0.5 mL sheep red blood cells (4×108 SRBC/mL), administered intraperitoneally.
At the end of the administration period the animals were sacrificed after a fasting period (withdrawal of food) of at least 16-20 hours.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATION
- moribundity and mortality: twice daily on working days and once daily on saturdays, Sundays and public holidays
- clinical signs: daily

DETAILED CLINICAL OBSERVATIONS
- all animals prior to the administration period and thereafter at weekly intervals.
- The following parameters were examined: Abnormal behavior in handling, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos, Assessment of the feces discharged during the examination (appearance/ consistency), Assessment of the urine discharged during the examination, Pupil size.

FOOD CONSUMPTION
Food consumption was determined weekly (as representative value over 7 days) for each cage. The average food consumption per cage was used to estimate the mean food consumption in grams per animal per day.

WATER CONSUMPTION
Drinking water consumption was observed by daily visual inspection of the water bottles for any overt changes in volume.

BODY WEIGHT
During the administration period the body weight was determined on study day 0 (start of the administration period) and weekly, thereafter. The body weight change of the animals was calculated from these results.

The mean daily intake of test substance (test group means) was calculated based upon individual values for body weight and food consumption.

FCx x C/BWx = test-substance intake for day x

BWx =body weight on study day x [g]
FCx =mean daily food consumption on study day x [g]
C =concentration in the food on study day x [mg/kg]

Sacrifice and pathology:

NECROPSY
The animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS
- Organ weights were measured for liver, spleen, thymus.
- The following organs or tissues were fixed in 4 % buffered formaldehyde solution: all gross lesions, liver, spleen, thymus. From the liver, each one slice of the Lobus dexter medialis and the Lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast.

HISTOPATHOLOGY: NO

BLOOD SAMPLING
Blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The results of clinical pathology examinations (10 animals per test group) were expressed in International System (SI) units.

Humoral immunity examinations:

The Anti SRBC-IgM ELISA of Life Diagnostics Inc, West Chester, USA (cat. no. 4200-2), was performed. Each sample was diluted 1:501. SRBC-IgM concentrations outside the standard curve range were measured in a second test run with an appropriate dilution. Generally, two in-house controls were measured with each test run. The ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Positive control:

Cyclophosphamide monohydrate: 4.5 mg/kg bw/d
Administered daily by gavage for 4 weeks, administration volume 10 mL/kg bw/d

Statistics:

Means and standard deviations of each test group were calculated for several parameters.
Body weight, body weight change (groups 0, 1, 2 and 3): A comparison of each test group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
Body weight, body weight change (groups 0 and 4): Comparison of the dose group with the control group was performed using the STUDENT’S t-test (two-sided) for the hypothesis of equal means.
Clinical pathology parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians.
Weight parameters: Test groups 1, 2 and 3: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each test group with the control group was performed using the WILCOXON test (two-sided) for the hypothesis of equal medians.
Test group 4: Comparison of test group 4 with the control group was performed using the WILCOXON test (two-sided) for the hypothesis of equal medians.

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical signs were observed in all rats treated with the test substance or with Cyclophosphamide monohydrate.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was observed.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No significant deviations from control group regarding body weights of test groups 1, 2 and 3 (300, 1000 and 4000 ppm test substance) as well as from the positive control group (test group 4 (4.5 mg/kg bw/d), Cyclophosphamide monohydrate) were determined. Moderate changes that were observed were considered non-adverse.

Body weight changes were not significantly decreased in animals of test group 3 (4000 ppm) from study day 0 to 7 (-24%) and in overall interval (-11 %) as well as in animals of test group 2 (1000 ppm) from study day 21 to 28 (-20 %).

The body weight change values of test group 4 (4.5 mg/kg bw/d, Cyclophosphamide monohydrate) were significantly decreased from day 14 to 21 (-29 %). Additionally, body weight changes were decreased not significantly from study day 7 to 14 (-15 %) and in overall value (-13 %).

It cannot be excluded that the minor alterations of body weight change observed after the exposure to the test substance, and the positive control, Cyclophosphamide monohydrate, were treatment-related, but they did not manifest in significant differences in the body weight of exposed animals in comparison to control. These findings were considered being not adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

From study day 0 to 7 the food consumption of test group 3 (4000 ppm) was increased noticeable (85.6%) measured by food amount decreased in the fodder rack. This was considered to be non-realistic because of the animals spreading the feed out of the fodder rack. Based on the body weight changes animals of test group 3 (4000 ppm) must have had food consumption in a normal range. From day 7 onwards no food consumption was calculated because of spilled feed. As a result determination of test substance intake is affected for this test group.

Food consumption of test group 2 (1000 ppm) was increased from day 7 to 28 with a maximum of 33% (day 14 to 21). Therefore, overall food consumption in test group 2 (1000 ppm) was increased (18.6%). These findings were assessed as being related to treatment, in the meaning of potential spilling the feed but not as an increased food consumption.
Calculated food consumption values above 25 g/animal/d were excluded from calculation in the summary tables.
No significant different body weight gains were observed for females in all test groups it can be assumed that in all test groups the animals were eating the normal amount of diet to support the observed growth of the animals in the physiological range of this rat strain and comparable to the current control.
Regarding food consumption no significant deviations were determined for animals of positive control group (test group 4 (4.5 mg/kg bw/d), Cyclophosphamide monohydrate) in comparison to control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No test substance-related findings with respect to water consumption were observed in animals treated with the test substance or with Cyclophosphamide
monohydrate.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Description (incidence and severity):

Primary T-cell dependent antibody response (anti-SRBC IgM ELISA)
Six days after immunization, no changes in the SRBC IgM titers were found in female rats dosed with the test substance. SRBC titers were lower in rats of the positive control group although not statistically significantly due to the high individual variation of SRBC titers. The SRBC titer ratio between the negative and the positive control group in the present study is similar to that of other studies with immunized rats (means 2.2-6.2; medians 1.6-3.6).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

All mean absolute weight parameters did not show significant differences when compared to the control group 0.

When compared to the control group 0 (set to 100%), the mean relative weights of the liver (114%) and the thymus (122%) were significantly increased in test group 3. All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The increased mean relative liver weight (3.094%) in females of test group 3 (4000 ppm test substance) was above the historical control data (2.496% - 2.918%) and was therefore considered to be treatment-related. The increased mean relative thymus weight (0.315%) in females of test group 3 (4000 ppm test substance) lay within the historical control range (0.208% - 0.315%) and was regarded to be incidental.

Cyclophosphamide monohydrate
When compared to the control group 0 (set to 100%), the mean absolute and relative weights of the spleen (66% and 68%, respectively) and the thymus (60% and 61%, respectively) were significantly decreased in test group 4. All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
The decreased absolute and relative spleen (0.25 g / 0.142%) and thymus (281.1 mg / 0.158%) weights in females of test group 4 (4.5 mg/kg bw/d Cyclophosphamide monohydrate) lay clearly below the range of the historical control data (spleen: 0.326 g - 0.464 g / 0.195% - 0.255%; thymus: 352.6 g - 577.875 mg / 0.208% - 0.315%). These weight changes were regarded to be treatment-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Test substance: There were no treatment-related gross lesions.
Cyclophosphamide monohydrate: A reduced size of the thymus was observed in two (out of 10) females of test group 4 (4.5 mg/kg bw/d).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Cell viabilities:

not examined

Humoral immunity examinations:

not examined

Specific cell-mediated immunity:

not examined

Non-specific cell-mediated immunity:

not examined

Other functional activity assays:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Immunotoxicity, systemic toxicity

Effect level:

278 mg/kg bw/day

Based on:

test mat.

Sex:

female

Remarks on result:

other: Highest dose tested

Remarks:

corresponding to 4000 ppm

Key result

Critical effects observed:

no

System:

immune system

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

278 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline compliant

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

Key study: Immunotoxicity 2015/1076588

The test substance was administered to groups of 10 female Wistar rats at dose levels of 0 ppm, 300 ppm, 1000 ppm and 4000 ppm via the diet over a period of 4 weeks. At the same time, a positive control group for immunotoxicity consisting of 4 female Wistar rats received 4.5 mg/kg body weight/day (mg/kg bw/d) Cyclophosphamide monohydrate by gavage.

All animals were immunized 6 days before blood sampling and necropsy using 0.5 mL sheep red blood cells (4×108 SRBC/mL), administered intraperitoneally. At the end of the administration period different end points for the detection of an immunotoxic potential were determined.

Under the conditions of the study the test substance did not reveal any signs of immunotoxicity when administered via the diet over a period of 4 weeks to female Wistar rats. The no observed adverse effect level (NOAEL) for the immunotoxicologically relevant endpoints and for systemic toxicity was set to 4000 ppm (278 mg/kg bw/d) - the highest dose tested.

The oral administration of the positive control substance Cyclophosphamide monohydrate (4.5 mg/kg bw/d) led to findings indicative of immunotoxicity. This was represented by significantly lower SRBC IgM antibody titres as well as reduced spleen and thymus weights. Thus, assay sensitivity was verified in the present immunotoxicity study performed in female Wistar rats.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

No immunotoxicity was observed up to a concentration of 4000 ppm (278 mg/kg bw/d). As a result the substance is not considered to be classified for immunotoxiciity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/918.