N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Immunotoxicity

Administrative data

Endpoint:

immunotoxicity

Remarks:

acute

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From Feb. 16, 1993 to Jul. 5, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable study report which meets basic scientific principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was assessed for its ability to induce immunomodulatory effects using a murine model (C3H mouse) where a dispersion of material is injected intraperitoneally and cell populations are enumerated and stained after 4 hours and after 48 hours. In addition, the isolated peritoneal cells were analysed for the expression of mRNA species for defined immunomodulatory cytokines.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

EC Number:

401-990-0

EC Name:

N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Cas Number:

106990-43-6

Molecular formula:

C132 H250 N32

IUPAC Name:

N2-[2-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}[3-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}amino)propyl]amino)ethyl]-N2-[3-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}amino)propyl]-N4,N6-dibutyl-N4,N6-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine

Details on test material:

- Physical state: white powder
- Storage condition of test material: Stored at room temperature in a dry environment.

Test animals

Species:

mouse

Strain:

C3H

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: From a specific pathogen-free colony (Olac-Harlan).
- Age at study initiation: 6-8 weeks.
- Weight at study initiation: No data.
- Fasting period before study: No data.
- Housing: During acclimatisation and treatment the mice were housed in cages of five using polypropylene cages with stainless steel tops and grid floors. These cages were suspended on racks over paper to collect the excreta. During treatment the animals in any cage were of the same treatment group. Excess animals were removed from the study room immediately after treatment had commenced.
- Diet (e.g. ad libitum): Rat and Mouse No.l maintenance diet (R/M1(E) SQC) supplied by Special Diets Services, Witham, Essex
- Water (e.g. ad libitum): tap water
- Acclimation period: A minimum of 6 days after receipt.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24°C.
- Humidity (%): 45-70%.
- Air changes (per hr): Minimum 15/hr with no recirculation, using high efficiency filters.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

physiological saline

Remarks:

(0.9% sodium chloride)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The suspension was homogenized for approximately 20 seconds immediately prior to use to ensure there was an even suspension. The resulting suspension was administered by intraperitoneal injection at 0.1 mL per 10 g body weight.


VEHICLE
- Amount of vehicle (if gavage): 0.1 mL per 10 g body weight.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

once

Frequency of treatment:

Single exposure.

Doses / concentrations

Remarks:

Doses / Concentrations:
5 or 50 mg/kg body weight
Basis:
other: Intraperitoneal injection

No. of animals per sex per dose:

5 mice per dose per time-point.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a preliminary study, a group of 2 mice were injected with a dose equivalent to 50% of the reported rat oral LD50 (< 5000 mg/kg bw) and assessed regularly until 48 hours had elapsed for any signs of morbidity. The dosing solution for 50% LD50 was 250 mg/ml (0.1 ml/10 g bw). If any animals died then the experiment was repeated with lower doses of test article. Test article caused a marked increase in cell numbers recovered by lavage at 48 hours post dose, which was mainly due to an increase in the number of neutrophils. Due to the mortality experienced in the preliminary study the experiment was repeated at 10% of the quoted rat oral LD50 for the test article. No animals showed any signs of ill health in the 72 hours between dosing and post mortem. There was an increase in cell numbers seen in test article dosed mice, again due mainly to an increase in neutrophils. From the results of this study, the maximum tolerated dose was set at 10% of the quoted rat oral LD50 and the low dose at 1% of the quoted rat oral LD50 (50 and 5 mg/kg bw).

Examinations

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes, clinical signs and mortalities

DETAILED CLINICAL OBSERVATIONS: No.

BODY WEIGHT: No.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: No.

CLINICAL CHEMISTRY: No.

URINALYSIS: No.

Sacrifice and pathology:

GROSS PATHOLOGY: No.
HISTOPATHOLOGY: No.

Cell viabilities:

SPLEEN: No.
THYMUS: No.
BONE MARROW: No.

Humoral immunity examinations:

ANTIBODY PLAQUE FORMING CELLS (PFC) ASSAY: No.
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): No.

Specific cell-mediated immunity:

ONE-WAY MIXED LYMPHOCYTE CULTURE (MLC) ASSAY: No.
DELAYED-TYPE HYPERSENSITIVITY (DTH) REACTION: No.
CYTOTOXIC T-LYMPHOCYTE (CTL) ASSAY: No.

Non-specific cell-mediated immunity:

NATURAL KILLER (NK) CELL ACTIVITY: No.

MACROPHAGE NUMBER AND FUNCTION: Yes.
- Method: Four and forty eight hours later, groups of mice were killed and the peritoneal cells were harvested by lavage with 0.9% saline solution. The cells from individual animals were counted and samples removed for a differential count using cytocentrifuge preparations. Total cell numbers and changes in the sub-population distribution of activated macrophage as characterised by the markers MAC-1, MOMA-2, CR3, and Class II were observed.
- Dose groups: 5 and 50 mg/kg groups at 4 and 48 hours.
- No. of animals: 5 mice per dose group per time-point.

Other functional activity assays:

SPLEEN CELL PROLIFERATION ASSAY (ANTI-CD3 MEDIATED T CELL PROLIFERATION): No.
ENUMERATION TOTAL B CELLS, TOTAL T CELLS AND T CELL SUBPOPULATIONS: No.

Other examinations:

Analysis for cytokine mRNA expression (IL-1a, IL-1ß, IL-6, TNFa and a-actin) in the cells recovered from the pertitoneal cavity was conducted.

Positive control:

The positive control group were injected with 1.5 mL Thioglycollate broth per mouse intraperitoneally.

Statistics:

No data.

Results and discussion

Results of examinations

Food efficiency:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY: No animals showed any signs of ill health in the 72 hours between dosing and post mortem. There was an dose related increase in cell numbers seen in test article dosed mice, again due mainly to an increase in neutrophils.

NON-SPECIFIC CELL-MEDIATED IMMUNITY
-Increase in total cell numbers and in total macrophages.
-No major changes in the sub-population distribution of activated macrophages.

OTHER FINDINGS
- In the saline (negative control) groups there was no expression of mRNAs for IL-1a, IL-1ß, TNFa, IL-6 although expression of the house-keeping gene, actin was detected.
- In the positive control group (thioglycollate broth) mRNAs for IL-1a and IL-1ß were highly induced in the 4 hour treatment groups and TNFa and IL-6 mRNAs were slightly induced. By 48 hours after thioglycollate administration the steady state levels of these mRNAs had declined to background levels.
-Administration of high dose test article (50 mg/kg) induced increased expression of TNFa mRNA, where as at the lower dose level (5 mg/kg bw), there was little effect on TNFa but increased expression of IL-1β compared to the positive control. The high dose caused a slight elevation of IL-1ß mRNA levels but was most noticeable for eliciting a large and sustained increase in the steady state levels of TNFa mRNA; this effect was not seen at the lower dose. The high dose of test substance caused total inhibition of the expression of all mRNA including actin; this could be attributed to a failure of the cells to attach and spread onto the plastic substrate due to the toxic effect of the compound. This effect was dose dependent since limited cytokine expression was evident in the lower dose of test substance and a small percentage of cells could be observed spreading onto the plastic substrate. The high dose of caused total inhibition of the expression of all mRNA including actin; this could be attributed to a failure to attach and spread onto the plastic substrate due to the toxic effect of the compound, a similar situation to the cells from the 4 hour group. This effect was dose dependent since limited cytokine expression was evident in the lower dose, and TNFα mRNA expression was similar to controls after 3 hours stimulation and slightly enhanced after 24 hours.
- After 48 hours exposure there were no significant effects on cytokine expression.
- When cells from the peritoneal cavity of 4 hours exposed mice were cultured and stimulated with lipopolysaccharide, cells from the high dose group failed to adhere properly to substrate and exhibited reduced Cytokine expression. At the lower dose, cytokine gene expression was significantly inhibited for the test substance.

Effect levels

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Any other information on results incl. tables

Effect of intraperitoneal administration of test articles on cytokine mRNA expression in peritoneal cells recovered by lavage

Test article	Cytokine mRNA levels
	IL1a	IL1ß	IL6	TNFa	Actin
4 h post dosing
Positive control	++	+++	+	+	++
TS, 50 mg/kg bw	±	+	±	+++	+
TS, 5 mg/kg bw	+	++	±	±	++
Saline control	±	-	±	±	++
48 h post dosing
Positive control	±	±	-	+	++
TS, 50 mg/kg bw	-	+	-	+	++
TS, 5 mg/kg bw	-	±	-	±	+++
Saline control	-	-	-	±	++
4 hours post dosing and cultured with LPS for 3 hours
Positive control	++	++	+	++	+++
TS, 50 mg/kg bw	-	-	-	-	-
TS, 5 mg/kg bw	-	±	-	+	±
Saline control	+++	+++	++	+++	+++
48 hours post dosing and cultured with LPS for 3 hours
Positive control	+++	+++	++	+++	+++
TS, 50 mg/kg bw	-	-	-	-	-
TS, 5 mg/kg bw	+	±	-	+++	++
Saline control	++	++	++	+++	+++

TS: test substance