2,6-dibromo-4-cyanophenyl octanoate

Immunotoxicity

Description of key information

Key, M-410294-01-1; subacute immunotoxicity (mouse, U.S. EPA test guideline, GLP):

NOAEL (systemic): 300 ppm (corresponding to 49 mg/kg bw/day, highest dose tested)

NOAEL (immunotoxicity): 300 ppm (corresponding to 49 mg/kg bw/day, 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:

12 Jan - 31 Mar 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Version / remarks:

adopted 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

C57BL

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River laboratories, France
- Age at study initiation: 7 weeks
- Weight at study initiation: 20.3 - 24.3 g
- Housing: individually in suspended, stainless steel, wire-mesh cages
- Diet: Certified rodent powdered and irradiated diet A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 Jan 2011 To: 22 Feb 2011

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST ITEM
DIET PREPARATION
- Rate of preparation of diet (frequency): once for each concentration
- Mixing appropriate amounts with: The appropriate amount of the test item ground to a fine powder, which was added to the diet.
- Storage temperature of food: when not in use, the diet was stored at -20 °C

CYCLOPHOSPHAMIDE
PREPARATION OF DOSING SOLUTIONS:
- Lot/Batch Nr: 068K1131
- Appearance: white powder
- Purity: 98.0%
- Storage: 5 °C
- Date of Analysis: August 2008
- Expiry date: August 2011
- Route of exposure: gavage
-Time schedule: daily
- Concentration: 7.5 mg/kg bw/day
- Volume: 5 mL/kg body weight

Preparation:
- Rate of preparation (frequency): twice
Procedure: The dosing formulation of cyclophosphamide was prepared by suspending the substance in sterilized water to produce the required dosing concentration.
Storage: +5 °C in the dark when not in use

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity of the test substance in the diet was verified for the lowest and highest administered concentration before the start of the study. Dietary levels of the test substance were verified for each concentration. The homogeneity and concentration results ranged between 97 and 103% of the nominal concentration and were therefore within in-house target range of 85 and 115% for a dietary mix. Stability of the test item in the diet was analyzed for the highest and lowest dietary concentration after samples were kept at room temperature for 3 days. The test substance was stable over this period.

The homogeneity of the positive control was proven on the first formulation. Homogeneity and concentration of cyclophosphamide, formulations ranged from 99% to 101% of the nominal concentration and were therefore within in-house target range of 90 and 110% for a solution. The stability was verified in previous studies at concentrations of 0.1 to 3 g/L for a time period which covers the period of storage and usage for the current study.

Duration of treatment / exposure:

28 days

Frequency of treatment:

continuously via the diet (treatment with test substance),
daily, 7 days/week (positive control)

Dose / conc.:

30 ppm

Remarks:

corresponding to 5 mg/kg bw/day

Dose / conc.:

100 ppm

Remarks:

corresponding to 16 mg/kg bw/day

Dose / conc.:

300 ppm

Remarks:

corresponding to 49 mg/kg bw/day

No. of animals per sex per dose:

10 males

Control animals:

yes, plain diet

Details on study design:

This immunotoxicity study was performed in mice because studies in mice and rats have revealed no evidence of immunotoxicity and rats are less sensitive than mice to the general toxicological effects of the test substance. Male mice were more sensitive than female mice in the previously conducted 90-day feeding study and were therefore used for this study.

Sheep Red Blood Cell (SRBC) ADMINISTRATION
SRBC
- Supplier: BioMérieux
- Reference Number: 72141
- Storage: 2 - 8 °C
-Time schedule: Day 26
- Anesthesia: Yes (Isoflurane (Baxter, Maurepas, France))
- number of cells injected: 1 * 10^8 SRBC/animal
- Procedure: On the day of injection, Sheep Red Blood Cells were washed in PBS (Phosphate Buffered Saline), counted using a cell counting instrument (Siemens Advia 120) and diluted in PBS in order to obtain a 1 x 10^9 cells/mL preparation. SRBC preparation was kept on ice until use. All animals of all groups were injected (tail vein, 0.1 mL/animal) with SRBC preparation.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends and public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly during the treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimation and treatment and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

URINALYSIS: No

Sacrifice and pathology:

Surviving animals were sacrificed by exsanguination while under deep anesthesia (Isoflurane inhalation).

GROSS PATHOLOGY: Yes
The necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded but not sampled.
Organ weight was recorded for: Spleen and thymus.

HISTOPATHOLOGY: No

Cell viabilities:

No data

Humoral immunity examinations:

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes
- Method: Rat Anti-Sheep Red Blood Cell IgM ELISA kits from Life Diagnostics (West Chester, PA 19380 – USA)
- Dose groups: all
- No. of animals: 10/dose group (all animals)

Specific cell-mediated immunity:

No data

Non-specific cell-mediated immunity:

No data

Other functional activity assays:

None

Other examinations:

None

Positive control:

Yes, one group of animals received 7.5 mg/kg bw/day cyclophosphamide (immunosuppressive agent) in sterilized water by gavage.
Cyclophosphamide: batch number 068K1131: a white powder, 98.0% purity

Statistics:

Mean and standard deviation were calculated for each group and time period.

Body weight change, terminal body weight, absolute and relative organ weight parameters:
To analyze the differences between the control and the positive group cyclophosphamide, an F test was performed. If this showed significant differences, a 2-sided modified t-test followed. If no significance was found, a 2-sided T-test was performed.
To compare control and test substance groups, group variances were analyzed with the Bartlett test followed with an Analysis of Variance (ANOVA) and a 2-sided
Dunnett's test (if needed) when variance was equal and a Kruskal-Wallis and 2-sided Dunn test (if needed) when variance differed.

Body weight and average food consumption/day parameters:
The comparison between the control and positive group was similar to the above described, only that the data was transformed using the log transformation if the F test showed a significant difference.
Then, the procedure was repeated on the transformed data.

The comparison between the control and test substance groups was also similar to the above described, only that the data was transformed using the log transformation if the Bartlett test showed a significant difference. Then, the procedure was repeated on the transformed data and followed as described above.

If one or more group variance (s) equaled 0, means were compared using non-parametric procedures.

Immunological parameters:
The control and positive group were compared using the 2-sided Mann-Whitney test.
The control and test substance group were compared with the Kruskal-Wallis-test. If a significant difference was found, the 2-sided Dunn Test was used.

Significance in all of the tests was defined as p ≤ 0.05.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related signs observed in the treatment groups and positive control group.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

not applicable

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no differences regarding body weight and body weight changes in the treatment groups and positive control group compared to the control group.

One animal in the 30 ppm group showed body weight loss between study days 22 and 29. This was considered not to be treatment-related as it was not found in any other animal.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences regarding food consumption in the treatment groups and positive control group compared to the control group.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

no effects observed

Description (incidence and severity):

Please refer to 'Humoral immunity examinations'.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no differences regarding terminal body weight and organ weights in the treatment groups and positive control group compared to the control group.

Positive control (Cyclophosphamide)
Mean absolute and relative spleen (-23 to -25%) and thymus (-24% and -26%) weights were decreased compared to controls.

Summarized results can be found in Attachment 2 in the attached background material.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no differences between control and treatment groups. In the positive control group, atrophic/small spleen and/or thymus were noted in 7/10 and 8/10 animals, respectively.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Cell viabilities:

not examined

Description (incidence and severity):

not applicable

Humoral immunity examinations:

no effects observed

Description (incidence and severity):

For the test substance, no statistically significant differences were noticed as compared to the concurrent control group. Slightly lower mean anti-SRBC IgM concentrations as compared to the control group were observed at 300 (-36%) and 100 ppm (-23%). However, only few values (4/10 and 1/10, respectively) were lower than the lowest individual value of the control group. Therefore these changes were considered not to be relevant.

Positive control (Cyclophosphamide)
Anti-SRBC IgM mean concentration was statistically significantly lower as compared to the control group (-76%, p =<0.01), proving the sensitivity of the assay to identify an immunosuppressive agent.

Summarized results can be found in Attachment 1 in the attached background material.

Specific cell-mediated immunity:

not examined

Description (incidence and severity):

not applicable

Non-specific cell-mediated immunity:

not examined

Description (incidence and severity):

not applicable

Other functional activity assays:

not examined

Description (incidence and severity):

not applicable

Other findings:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

Immunotoxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects were observed up to the highest dose level.

Remarks on result:

other: corresponding to an actual dose ingested of 49 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed up to the highest dose level.

Remarks on result:

other: corresponding to an actual dose ingested of 49 mg/kg bw/day.

Key result

Critical effects observed:

no

Conclusions:

The study assessed the effects of the test item on the immune system in mice after dietary administration of the test substance for 28 days. The study is considered valid and in compliance with GLP and the guideline US-EPA-OPPTS Series 870, Health Effects Testing Guidelines, N°870.6300.
The administered doses were 30, 100 and 300 ppm. Cyclophosphamide served as a positive control and proved the sensibility of the test system. Under the conditions of the study, the dietary NOAEL for systemic toxicity and immunotoxicity was set at 300 ppm (49 mg/kg bw/day), the highest dose level tested, since no adverse effects were observed up to and including this dose level.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

49 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

U.S. EPA test guideline and GLP study, fulfilling the criteria of suitability as key study.

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

Mode of Action Analysis / Human Relevance Framework

No data available.

Additional information

The potential immunotoxicity of the test substance was assessed in male C57BL mice according to an U.S. EPA test guideline, under GLP conditions. This study is considered suitable as key study (M-410294-01-1, 2011). Groups of 10 C57BL mice rats/per dose were exposed daily via the diet to 30, 100 and 300 ppm test item, corresponding to 5, 16 and 49 mg/kg bw/day (mean ratios) of the test substance for at least 28 days. The dosing solutions were prepared by mixing the appropriate amounts of the test substance with the diet. The control group received the plain diet only. One group of rats received 7.5 mg/kg bw Cyclophosphamide as a positive control for immunosuppression. Cyclophosphamide was administered solubilized in deionized water via gavage. Both the concentration and homogeneity of the test substance in the diet and the positive control in the vehicle were analyzed and considered valid. Clinical signs were recorded daily and body weight and food consumption were measured weekly. A detailed physical examination was performed weekly throughout the study. On Day 26, rats were injected in the tail vein with a 0.1 mL preparation of Sheep Red Blood Cells (SRBC), containing 1 x 10^9 cells/mL. Four days after SRBC immunization, blood was taken from all animals (non-fasted) and analyzed for anti SRBC- IgM using an ELISA kit. When animals were sacrificed on Day 30, the gross necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded but not sampled. The organ weight was recorded for spleen and thymus. No mortality was observed in the study. There were no treatment-related signs observed in the treatment groups and positive control group. Body weight and feed consumption were not affected by treatment with the test substance or the positive control as compared to the vehicle control. One animal in the 30 ppm group showed body weight loss between study days 22 and 29. This was considered not to be treatment-related as it was not found in any other animal. No differences regarding gross pathology or organ weights were found between treatment and control groups at necropsy. The SRBC response was unchanged in treatment groups compared to control groups. In the positive control group, the concentration of anti-SRBC IgM was 76% lower than the controls. Spleen and thymus were smaller and lighter than in vehicle control animals and atrophic/small spleen and/or thymus were noted (7/10 and 8/10 animals, respectively). With these results, the positive control cyclophosphamide confirmed the sensitivity of the test system. No immunotoxic properties were noted in male mice at doses of test item up to and including 300 ppm, corresponding to 49 mg/kg bw/day.

Under the conditions of the study, the NOAEL for immunotoxicity and systemic toxicity was set to 49 mg/kg bw/day (the highest administered dose).

Justification for classification or non-classification

The available data on immunotoxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.