Phosphorous acid, mixed 2,4-bis(1,1-dimethylpropyl)phenyl and 4-(1,1-dimethylpropyl)phenyl triesters

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 January 2013 to 01 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with OECD & ICH test guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use (2011)

Deviations:

no

Principles of method if other than guideline:

International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline. S2(R1) document recommended for adoption at step 4 of the ICH process on 9 November 2011. Adopted at Step 5 in Europe by CHMP December 2011 (issued as EMA/CHMP/ICH/126642/2008). Adopted at Step 5 in US by FDA on June 7, 2012 (issued as 77 FR 33748 pages 33748-33749). Japan Step 5 adoption in process.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis[2,4-bis(2-methylbutan-2-yl)phenyl] 4-(2-methylbutan-2-yl)phenyl phosphite and 2,4-bis(2-methylbutan-2-yl)phenyl bis[4-(2-methylbutan-2-yl)phenyl] phosphite and tris[4-(2-methylbutan-2-yl)phenyl] phosphite.

EC Number:

700-485-5

Cas Number:

939402-02-5

Molecular formula:

Mixture of 4 components, the molecular formulae of which are: C33 H45 O3 P, C38 H55 O3 P, C43 H65 O3 P and C48 H75 O3 P

IUPAC Name:

Reaction mass of bis[2,4-bis(2-methylbutan-2-yl)phenyl] 4-(2-methylbutan-2-yl)phenyl phosphite and 2,4-bis(2-methylbutan-2-yl)phenyl bis[4-(2-methylbutan-2-yl)phenyl] phosphite and tris[4-(2-methylbutan-2-yl)phenyl] phosphite.

Test material form:

other: Clear colorless liquid

Details on test material:

Test Article I.D.: Weston 705
Test Article Batch No.: MW2K15T701
Test Article Purity: 100% (provided by Sponsor)
BioReliance Study No.: AD64PU.123012ICH.BTL
Test Article Description: Clear colorless liquid
Storage Conditions: Room temperature, stored protected from light and blanketed by nitrogen gas

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

ICR mice were obtained from Harlan, Frederick, MD and were received on 30 January 2013 for the dose range-finding assay and on 13 February 2013 for the definitive micronucleus assay. At the time of dose administration for each phase of the study, the mice were 6 weeks old.

Animal Receipt and Acclimation
Virus antibody-free (VAF) mice were obtained from a supplier that monitored mice for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were acclimated for no less than 5 days after receipt. At BioReliance, mice were observed each day for signs of illness and other conditions of poor health. All mice were judged to be healthy prior to utilization in the study.

Animal Welfare Provisions and Animal Care
This study is not duplicative or unnecessary. The number of mice and the procedures and experimental design used for this study have been reviewed and were approved by the BioReliance Institutional Animal Care and Use Committee #8 and #10. All procedures involving mice performed at BioReliance follow the specifications recommended in The Guide for the Care and Use of Laboratory Animals adopted by BioReliance.
Animals were housed in an AAALAC-accredited facility with a controlled environment of 50 ± 20% relative humidity and 72 ± 3°F temperature with a 12-hour light/dark cycle. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour.
Mice of the same sex were housed up to five per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. The purpose of this system was to supply uninterrupted positive air to each individual rodent Micro-Barrier cage and to capture the effluent air from each cage and re-filter the air (HEPA) prior to introducing the air back into the cage.
Heat-treated Sani-Chip hardwood chips were used for bedding to absorb liquids (P.J. Murphy Forest Products, Montville, NJ). Bedding was analyzed by the Manufacturer for any contaminants.
Animals were allowed free access to tap water, which meets U.S. EPA drinking water standards [water source is Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens.
A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients.
The results of bedding, food and water analyses are on file at BioReliance. There are no contaminants to the bedding, feed and water that are expected to interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

The test substance was delivered to the test system using corn oil as the vehicle. In the definitive micronucleus assay, corn oil was also used as the vehicle (negative) control substance. Corn oil (CAS No. 8001-30-7, Lot No. MKBD6671, Expiration Date: 24 September 2013) was obtained from Sigma-Aldrich and was characterized as per the Certificate of Analysis on file with the Testing Facility.

Details on exposure:

The test substance dose formulations were prepared fresh for each assay on the day of dose administration. All formulations at 200 mg/mL for the dose range-finding assay and all formulations at 50, 100 and 200 mg/mL for the definitive micronucleus assay were prepared as follows:
An appropriate amount of test substance was weighed separately for each concentration. An appropriate volume of the vehicle was added to the respective containers. Each formulation was vortexed during preparation.
All formulations appeared as yellow solutions.

Preparation of Positive Control Substance Formulation
An aqueous dosing formulation of CP at a concentration of 5 mg/mL was prepared fresh on the day of dose administration. An appropriate amount of CP was dissolved in an appropriate volume of sterile water for injection (B. Braun Medical, Inc., CAS No. 7732-18-5, Lot No. J1L003, Expiration Date: September 2013). The accuracy of preparation and stability of the CP formulation was demonstrated by acceptable results that met the criteria for a valid test.

Duration of treatment / exposure:

2 days

Frequency of treatment:

Single dose

Post exposure period:

48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500, 1000 & 2000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

Dose range finding assay: 3 mice/sex
Definitive assay: 5 males per group

Control animals:

yes, concurrent vehicle

Positive control(s):

Characterization of Positive Control Substance
Cyclophosphamide monohydrate (CP, CAS No. 6055-19-2, Lot No. SLBC0666V, Expiration Date: 31 March 2015), was obtained from Sigma-Aldrich. Neat CP was characterized as per the Certificate of Analysis on file with the Testing Facility.

Preparation of Positive Control Substance Formulation
An aqueous dosing formulation of CP at a concentration of 5 mg/mL was prepared fresh on the day of dose administration. An appropriate amount of CP was dissolved in an appropriate volume of sterile water for injection (B. Braun Medical, Inc., CAS No. 7732-18-5, Lot No. J1L003, Expiration Date: September 2013). The accuracy of preparation and stability of the CP formulation was demonstrated by acceptable results that met the criteria for a valid test.

Dose Administration Procedure
All dose formulations were administered at a dose volume of 10 mL/kg by single oral administration using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The oral route of administration has been routinely used, validated and is widely-accepted for use in the mammalian bone marrow erythrocyte micronucleus assay. All mice in the experimental and control groups were weighed immediately before dose administration and the administered volume was based on individual body weight. Mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity.

Examinations

Tissues and cell types examined:

Weston 705, was evaluated for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (mnPCEs) cells in mouse bone marrow.

Details of tissue and slide preparation:

Bone Marrow Collection and Slide Preparation
At the scheduled bone marrow collection time, five mice per sex per treatment were euthanized by CO2 asphyxiation verified by toe pinch reflex. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a labeled centrifuge tube containing approximately 1 mL of fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100xg for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were air dried and fixed in methanol. One set of slides was stained with a nucleic acid-specific stain, acridine orange, and was used in microscopic evaluation. The second set of slides was stored at BioReliance as a backup.

Scoring for Micronuclei (Bone Marrow Evaluation)
To control for bias, bone marrow slides were coded using a random number table by an individual not involved with the scoring process. Bone marrow was evaluated by fluorescent microscopy. The staining procedure permits the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange PCEs and ghost-like, dark green NCEs, respectively). Slides initially were scanned using medium magnification to locate suitable areas where the cells are well spread and stained. Next, cells were scored using a high power oil immersion lens as follows.
The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring was based upon the micronucleated cell, not the micronucleus; thus, occasional cells with more than one micronucleus are counted as one micronucleated PCE (mnPCE), not two (or more) micronuclei.
At least 2000 PCEs/animal were scored for the presence of micronuclei (mnPCEs) whenever possible. In addition, at least 1000 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity.

Evaluation criteria:

Evaluation of Test Results
Once the criteria for a valid assay have been met, the results are evaluated as follows:
-The test substance is considered to be positive if it induces a significant increase in mnPCE frequency (p≤ 0.05) at any dose level or sampling time compared to the concurrent vehicle control.
-The test substance is considered to be negative if no significant increase in mnPCE frequency is observed (p > 0.05) compared to the concurrent vehicle control.
-Other criteria may be used in reaching a conclusion about the study results (e.g., magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.). In such cases, the Study Director uses sound scientific judgment to clearly report and describe any such considerations.

Statistics:

The frequency of mnPCEs and the proportion of PCEs to total erythrocytes were determined for each animal and treatment group. Statistical significance (p ≤ 0.05) was determined using binomial distribution (Kastenbaum-Bowman tables).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Dose Range-Finding Assay
No mortality occurred at 2000 mg/kg during the course of the dose range-finding assay. Piloerection was noted in all of the male mice. No appreciable reductions in the mean group body weight were observed in any of the treatment groups.

Definitive Micronucleus Study
Clinical Signs
No mortality occurred at any dose level during the course of the micronucleus assay. Piloerection was noted in some of the mice at 1000 mg/kg and all of the mice at 2000 mg/kg. All other mice dosed with test and control substances appeared normal during the course of the study.

Bone Marrow Evaluation
-No reductions were observed in the PCEs/EC ratio with Weston 705 at any dose level relative to the respective vehicle control groups.
-No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance-treated groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman tables).
-CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p ≤ 0.05, Kastenbaum-Bowman tables). The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

Any other information on results incl. tables

Dose Range-Finding Assay – Clinical Signs of Toxicity Following a Single Oral Administration of Weston 705 in ICR MIce

Treatment (10 mL/kg)	Observation	Number of Animals With Observed Signs/Number of Surviving Animals						Number of Animals Found Dead/Total Number of Animals Dosed
		Males			Females
		Day 0	Day 1	Day 2	Day 0	Day 1	Day 2	Males	Females
Weston 705	Normal	0/3	0/3	0/3	3/3	3/3	3/3	0/3	0/3
2000 mg/kg	Piloerection	3/3	3/3	3/3	0/3	0/3	0/3

 

Dose Range-Finding Assay – Body Weight and Mortality Data Following a Single Oral Administration

of Weston 705 in ICR Mice

Treatment (10 mL/kg)	Sex	Group Mean Body Weights (g ± SD)			%Change^1
		Day 0	Day 1	Day 2	Day 1	Day 2	Mortality^2
Weston 705 2000 mg/kg	M	32.6 ±1.3	30.3 ±0.8	31.0 ±1.6	-7.1%	-4.9%	0/3
	F	25.3 ±0.4	25.9 ±0.7	25.3 ±0.8	2.4%	0.0%	0.3

¹%Change = (Post-treatment weight – Pre-treatment weight) x 100

                                               Pre-treatment weight

²Reported as number of mice found dead after dose administration/total number tested

 

Definitive Micronucleus Assay – Clinical Signs of Toxicity Following a Single Oral Administration

of Weston 705 in ICR Mice

Treatment (10 mL/kg)	Observation	Number of Animals With Observed Signs/Number of Surviving Animals			Number of Animals Found Dead/Total Number of Animals Dosed
		Males
		Day 0	Day 1	Day 2	Males
Corn oil	Normal	10/10	10/10	5/5	0/10
Weston 705 400 mg/kg	Normal	5/5	5/5	N/A	0/5
1000 mg/kg	Normal Piloerection	5/5 0/5	2/5 3/5	N/A	0/5
2000 mg/kg	Piloerection	10/10	10/10	5/5	0/10
Cyclophosphamide 50 mg/kg	Normal	5/5	5/5	N/A	0/5

N/A = No observations were applicable since these animals were sacrificed for 24-hour bone marrow collection.

 

Summary of Bone Marrow Micronucleus Analysis Following a Single Oral Administration of Weston 705 in ICR Mice

Treatment (10 mL/kg)	Sex	Time (hr)	Number of Animals	PCE/Total Erythrocytes (Mean +/- SD)	Change from Control (%)	Number of mmPCE/1000 PCE (Mean +/- SD)	Number of mmPCE/PCE Scored
Corn oil	M	24	5	0.508 ± 0.03	---	0.1 ± 0.22	1 / 10000
Weston 705 500 mg/kg	M	24	5	0.518 ± 0.06	2	0.3 ± 0.45	3 / 10000
1000 mg/kg	M	24	5	0.561 ± 0.03	10	0.0 ± 0.00	0 / 10000
2000 mg/kg	M	24	5	0.514 ± 0.03	1	0.3 ± 0.27	3 / 10000
Cyclophosphamide 50 mg/kg	M	24	5	0.466 ± 0.3	-8	34.1 ± 4.34	*341 / 10000
Corn oil	M	48	5	0.550 ± 0.02	---	0.1    0.22	1 / 10000
Weston 705 2000 mg/kg	M	48	5	0.583 ± 0.05	6	0.3 ± 0.27	3 / 10000

PCE: Polychormatic Erythrocytes; mmPCE: MIcronucelated Polychormatic Erythrocytes

*Statistically significant increase compared to vehicle control, p≤0.05 (Kastenbaum-Bowman tables)

 

Induction of Micronucelated Polychromatic Erythrocytes in Bone Marrow Collected

24 Hours Following a Single Oral Administration of Weston 705 in ICR Mice

Treatment (10 mL/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Corn oil	M	101 102 103 104 105	0.508 0.520 0.551 0.469 0.493	0 / 2000 0 / 2000 1 / 2000 0 / 2000 0 / 2000
Weston 705 500 mg/kg	M	106 107 108 109 110	0.539 0.460 0.507 0.612 0.470	1 / 2000 0 / 2000 0 / 2000 2 / 2000 0 / 2000
1000 mg/kg	M	111 112 113 114 115	0.550 0.572 0.524 0.557 0.600	0 / 2000 0 / 2000 0 / 2000 0 / 2000 0 / 2000
2000 mg/kg	M	116 117 118 119 120	0.528 0.533 0.540 0.480 0.488	0 / 2000 1 / 2000 1 / 2000 0 / 2000 1 / 2000
Cyclophosphamide 50 mg/kg	M	121 122 123 124 125	0.447 0.447 0.513 0.490 0.432	60 / 2000 64 / 2000 82 / 2000 71 / 2000 64 / 2000

PCE: Polychromatic Erythrocytes

 

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Collected

48 Hours Following a Single Oral Administration of Weston 705 in ICR Mice

Treatment (10 mL/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Corn oil	M	126 127 128 129 130	0.576 0.563 0.556 0.531 0.526	0 / 2000 0 / 2000 0 / 2000 0 / 2000 1 / 2000
Weston 705 2000 mg/kg	M	131 132 133 134 135	0.649 0.598 0.508 0.561 0.599	0 / 2000 1 / 2000 0 / 2000 1 / 2000 1 / 2000

PCE: Polychromatic Erythrocytes

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of the study conduct as described in this report, a single oral administration of Weston 705 at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of male ICR mice. Therefore, Weston 705 was concluded to be negative in the mouse micronucleus assay.

Executive summary:

The test substance, Weston 705, was evaluated for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (mnPCEs) cells in mouse bone marrow.

 

The study was conducted in accordance with the following guideline:

-OECD Guidelines for the Testing of Chemicals: Health Effects, No. 474 (1997)

-International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use (2011)

 

The study was conducted in two phases: a dose range-finding assay that evaluated the toxicity of the test substance and a definitive micronucleus assay that evaluated the genotoxic potential of the test substance.

 

Weston 705 was formulated using corn oil as the vehicle based on the solubility of the test substance in the vehicle and compatibility with the test system and route of administration. The test substance was soluble in corn oil at approximately 200 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance. Corn oil was also used as the negative (vehicle) control in the definitive micronucleus assay. Cyclophosphamide monohydrate (CP), at a dose of 50 mg/kg, was used as the positive control substance in the definitive micronucleus assay. In both assays, test or control substances were administered by single oral gavage at a dose volume of 10 mL/kg body weight. Animals were observed for clinical signs of toxicity following dose administration and at least once daily thereafter.

 

In the dose range-finding assay, three mice/sex/group were exposed to Weston 705 at the maximum OECD guideline recommended dose level of 2000 mg/kg. No mortality was observed. Piloerection was noted in male mice only. No appreciable changes in the mean group body weight were observed during the study period.

 

Due to the absence of mortality at 2000 mg/kg in the dose range-finding assay, the definitive micronucleus assay was conducted exposing the animals to Weston 705 at dose levels of 500, 1000 and 2000 mg/kg. The vehicle and positive control substances were tested concurrently. Since no substantial differences in the clinical signs of toxicity between the sexes were observed in the dose range-finding assay, only male mice were used in the definitive micronucleus assay.

 

At the scheduled time point, animals were euthanized and the femoral bone marrow was collected. Bone marrow smears (slides) were prepared and stained with acridine orange, a nucleic acid specific stain and were examined microscopically for the presence of micronuclei (mnPCEs). A statistical analysis of the data was performed using the Kastenbaum-Bowman tables (binomial distribution, p ≤ 0.05). In addition, the ratio of PCEs to total erythrocytes (PCEs/EC ratio) was also evaluated as an indication of bone marrow toxicity.

 

In the definitive micronucleus assay,

-No mortality was observed in any of the treatment groups. All mice in the control substance (vehicle or positive) groups and all mice in the 500 mg/kg treatment group appeared normal during the study period. Piloerection was noted at both 1000 and 2000 mg/kg.

-No reductions were observed in the PCEs/EC ratio with Weston 705 at any dose level relative to the respective vehicle control groups.

-No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance-treated groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman tables).

-CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p ≤ 0.05, Kastenbaum-Bowman tables). The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

 

Under the conditions of the study as described in this report, a single oral administration of Weston 705 at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of male ICR (CD-1) mice. Therefore, Weston 705 was concluded to be negative in the mouse micronucleus assay.