O,O'-dioctadecylpentaerythritol bis(phosphite)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Bacterial Gene Mutation: Negative. Mammalian Gene Mutation: Negative. in vivo Cytogenicity Study (Micronucleus): Negative

Link to relevant study records

Reference

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:

28 July 2003 to 1 September 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD, USA
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: 28.5 - 32.5 g (males); 20.5 - 25.7 g (females)
- Housing: 5 animals per cage (by sex)
- Diet: certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Chow) ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days minimum

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 °F
- Humidity: 50 ± 20 %
- Photoperiod: 12 hours light / 12 hours dark

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: based on a solubility determination of the test material and compatibility of the vehicle with the test system. Corn oil was selected as the vehicle of choice

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was prepared in a workable stock solution in corn oil up to 200 mg/L.

Duration of treatment / exposure:

One IP injection at a constant volume of 20 mL/kg bw.

Frequency of treatment:

Single injection

Post exposure period:

Mice were sacrificed 24 (Positive control and 500 and 100 mg/kg groups) and 48 (Vehicle control and 2000 mg/kg group) hours after treatment.

Remarks:

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

No. of animals per sex per dose:

Vehicle control: 10 per sex
500 mg/kg treatment group: 5 per sex
1000 mg/kg treatment group: 5 per sex
2000 mg/kg treatment group: 15 (Including 5 replacement animals per sex to ensure the availability of 5 animals in the micronucleus ass)
Positive control: 5 per sex

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route Intraperitoneal injection
- Doses / concentrations: 50 mg/kg (one IP injection at a constant volume of 20 mL/kg bw) prepared in distilled water

Tissues and cell types examined:

Femur bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Doses were selected based on a pilot toxicity study performed prior to the definitive test. Five male and five female mice were exposed to 2000 mg/kg and two mice of each sex were exposed to concentrations of 1, 10, 100 and 1000 mg/kg of the test material. All injections were administered as a single dose at a volume of 20 mL/kg. Mice were observed immediately after dosing, and daily thereafter for 3 days for clinical signs of toxicity. Bodyweights were recorded prior to dosing and again at days 1 and 3 post dosing.

DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice time, five animals per sex per treatment were sacrificed and, immediately after sacrifice, the femurs exposed and the bone marrow aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred into a centrifuge tube containing 1 mL foetal bovine serum and pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant drawn off. The cells were resuspended by aspiration and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each animal. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Slides were assessed visually, using medium magnification (10 x 40), a well spread area with clear staining of each slide was selected for evaluation. 2000 erythrocytes were scored under oil immersion (10 x 100) for micronuclei.
2000 polychromatic erythrocytes were scored for the presence of micronuclei (defined as round, darkly staining nuclear fragments having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte). The number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes counted was also recorded per 1000 erythrocytes.
Polychromatic erythrocytes (immature erythrocytes) stain bluish, normochromatic erythrocytes (normocytes, mature erythrocytes) stain pink.

The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each animal and treatment group. In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.

Evaluation criteria:

The test material was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time and there was no evidence of a dose-response relationship. The test material was considered to be positive if a statistically significant treatment-related increase (p ≤ 0.05) in micronucleated polychromatic erythrocytes was observed compared to the vehicle control. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, this assay was considered a suspect or unconfirmed positive. Values that were statistically significant but did not exceed the range of the historical negative or vehicle controls were judged to be not biologically significant.

Statistics:

The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman table which are based on binomial distribution. All analyses were performed separately for each sex.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1, 10, 100, 1000 and 2000 mg/kg
- Solubility: The solubility of the test material in the vehicle corn oil was confirmed in a solubility test. The test material was a workable suspension at 200 mg/mL, the maximum concentration tested.
- Clinical signs of toxicity in test animals: No mortality occurred at any doses during the course of the study. Piloerection was seen in male mice at 100 and 1000 mg/kg and in male and female mice at 2000 mg/kg and lethargy in males at 1000 mg/kg and in males and females at 2000 mg/kg. In the absence of mortality in the pilot study, the highest dose for the definitive study was set at 2000 mg/kg.
- Rationale for exposure: Intraperitoneal injection was selected to maximise exposure.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: No mortality occurred at any dose level during the course of the study. Lethargy was observed in male and female mice at 1000 and 2000 mg/kg and piloerection in males and females at all doses tested. All other mice treated with test or control articles appeared normal during the course of the study.
- Induction of micronuclei (for Micronucleus assay): The test material did not increase the number of micronuclei at any dose level.
- Ratio of PCE/NCE (for Micronucleus assay): Bone marrow cells collected 24 and 48 hours after treatment showed no appreciable changes in the ratio of polychromatic erythrocytes to total erythrocytes relative to the vehicle control in the test material treated groups suggesting that the test material did not inhibit erythropoiesis.
- Statistical evaluation: The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in test material treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time.

Table 2: Clinical Signs and Mortality - definitive study

Treatment (20 mL/kg)	Observation	No. mice with clinical signs/total		No. or mice dead/total
		males	females	males	females
Vehicle control*	Normal	10/10	10/10	0/10	0/10
Test material 500 mg/kg	Piloerection	5/5	5/5	0/5	0/5
Test material 1000 mg/kg	Lethargy	5/5	5/5	0/5	0/5
	Piloerection	5/5	5/5
Test material 2000 mg/kg	Lethargy	15/15	15/15	0/15	0/15
	Piloerection	15/15	15/15
Positive control*	Normal	5/5	5/5	0/5	0/5

* Vehicle control = corn oil 20 mL/kg; Positive control = 50 mg/kg cyclophosphamide monohydrate (CP) at 20 mL/kg in distilled water

 

Table 3: Summary of Bone Marrow Micronucleus Analysis

Treatment (20 mL/kg)	Sex	Time (hr)	No. of mice	PCE/total erythrocytes (mean ± SD)	Changes from control (%)	Micronucleated polychromatic erythrocytes
						No. per 1000 PCEs (mean ± SD)	No. per PCEs scored
Vehicle control*	M	24	5	0.456 ± 0.07	-	0.6 ± 0.22	6/1000
	F	24	5	0.526 ± 0.09	-	0.5 ± 0.35	5/1000
Test material 500 mg/kg	M	24	5	0.451 ± 0.03	-1	0.6 ± 0.22	6/1000
	F	24	5	0.465 ± 0.02	-12	0.5 ± 0.50	5/1000
Test material 1000 mg/kg	M	24	5	0.473 ± 0.04	4	0.5 ± 0.35	5/1000
	F	24	5	0.479 ± 0.05	-9	0.5 ± 0.35	5/1000
Test material 2000 mg/kg	M	24	5	0.447 ± 0.03	-2	0.5 ± 0.35	5/1000
	F	24	5	0.485 ± 0.06	-8	0.7 ± 0.27	7/1000
Positive control*	M	24	5	0.335 ± 0.03	-27	22.2 ± 2.20	222/1000¹
	F	24	5	0.325 ± 0.01	-38	20.4 ± 2.43	204/1000¹
Vehicle control	M	48	5	0.502 ± 0.06	-	0.3 ± 0.27	3/1000
	F	48	5	0.483 ± 0.05	-	0.6 ± 0.22	6/1000
Test material 2000 mg/kg	M	48	5	0.471 ± 0.05	-6	0.6 ± 0.22	6/1000
	F	48	5	0.467 ± 0.03	-3	0.8 ± 0.27	8/1000

* Statistically significant (p≤0.05)

* Vehicle control = corn oil 20 mL/kg; Positive control = 50 mg/kg cyclophosphamide monohydrate (CP) at 20 mL/kg in distilled water

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of the study, a single intraperitoneal administration of test material at doses up to 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone barrow. Therefore, the test material was considered to be negative in the micronucleus test using male and female ICR mice.

Executive summary:

Male and female ICR mice were exposed to 100, 500, 1000 and 2000 mg/kg bw of the test material which was administered in a total volume of 20 mL/kg as a single IP injection. Corn oil was used as the vehicle. Under the conditions of the study, no mortality was observed. Clinical signs following dose administration included lethargy in mice treated with 1000 and 2000 mg/kg and piloerection in all mice treated. Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronculeated polychromatic erythrocytes. No appreciable changes in the ratio of polychromatic erythrocytes to total erythrocytes relative to the vehicle control were apparent in the test material treated groups suggesting that the test material did not inhibit erythropoiesis. The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in test material treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time. The results of the assay indicate that under the conditions of the test, the test material did not induce a significant increase in micronucelated polychromatic erythrocytes in either male or female ICR mice. The test material was determined to be negative in the mouse micronculeus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

IN VITRO BACTERIAL GENE MUTATION

The potential for the test material to cause gene mutation in bacterial strains was determined in an Ames test. During the study, three strains of Salmonella typhimurium (TA97, TA98 and TA100) and one Escherichia coli strain (WP2) were treated in the presence and absence S9 mix. Test material was dispersed in acetone/Tween-80 and dosed at 0, 0.05, 0.1, 0.2 and 0.5 mg/plate. Under the conditions of the test neither an increase in revertant number nor observation of toxicity in cells were noted. However, because the test material appeared cloudy when mixed with the acetone/Tween-80 solution it was not known whether the test material was available to the bacterial cells to cause an effect.

The study was presented with limited information on methods employed and very limited information on results. It is therefore not possible to assess the reliability of the presented data. No positive control was included in the study, and the study only used four bacterial strains, the study did however include a strain capable of detecting cross-linking mutagens. The study was assigned a reliability score of 4 in accordance with the principles of assessing data quality, described in Klimisch et al (1997).

IN VITRO GENE MUTATION IN MAMMALIAN CELLS

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of the test item was selected following the results of a preliminary toxicity test and was 19.53 to 625 μg/ml in both the absence and presence of metabolic activation in Experiment 1. In Experiment 2 the dose range was 1.22 to 156.25 μg/ml in the absence of metabolic activation, and 19.53 to 625 μg/ml in the presence of metabolic activation.

Results

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity and by the incidence of a test item precipitate effectively reducing exposure to the cultures. A precipitate of the test item was observed at and above 78.13 μg/ml in the absence of metabolic activation and at and above 156.25 μg/ml in the presence of metabolic activation in Experiment 1. In Experiment 2 a precipitate of the test item was observed at and above 39.06 μg/ml in the absence of metabolic activation and at and above 156.25 μg/ml in the presence of metabolic activation. The negative (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

 

Conclusion

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

IN VIVO CYTOGENICITY (MICRONUCLEUS)

Male and female ICR mice were exposed to 100, 500, 1000 and 2000 mg/kg bw of the test material which was administered in a total volume of 20 mL/kg as a single IP injection. Corn oil was used as the vehicle. Under the conditions of the study, no mortality was observed. Clinical signs following dose administration included lethargy in mice treated with 1000 and 2000 mg/kg and piloerection in all mice treated. Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronculeated polychromatic erythrocytes. No appreciable changes in the ratio of polychromatic erythrocytes to total erythrocytes relative to the vehicle control were apparent in the test material treated groups suggesting that the test material did not inhibit erythropoiesis. The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in test material treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time. The results of the assay indicate that under the conditions of the test, the test material did not induce a significant increase in micronucelated polychromatic erythrocytes in either male or female ICR mice. The test material was determined to be negative in the mouse micronculeus assay.

The study was performed in accordance with the standardised guideline OECD 474 and under GLP conditions. The study was reported to a high standard, and was therefore assigned a reliability score of 1 in accordance with the principles of assessing data quality as defined in Klimisch et al (1997).

Justification for selection of genetic toxicity endpoint
In vivo study

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for germ cell mutagenicity.