Reaction Products of Diphosphorus Pentaoxide with n-Alcohols, C8-10 (even), salted with Amines, C12-14, Tert-alkyl

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:

1995-05-09 - 1995-06-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Scientifically valid, well documented GLP study according to EU method B.12 on the registered substance itself.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Products of Diphosphorus Pentaoxide with n-Alcohols, C8-10 (even), salted with Amines, C12-14, Tert-alkyl

EC Number:

943-016-1

Molecular formula:

Too complex

IUPAC Name:

Reaction Products of Diphosphorus Pentaoxide with n-Alcohols, C8-10 (even), salted with Amines, C12-14, Tert-alkyl

Test material form:

liquid: viscous

Details on test material:

- Substance type: pure substance
- Physical state: liquid
- Storage condition of test material: room temperature

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Manston, Kent
- Age at study initiation: 5-7 weeks
- Weight at study initiation: 24-29 g (males), 20-25 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: none
- Housing: in groups of up to 5 in solid-floor polypropylene cages with woodflake bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 49-60%
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Concentration of test material in vehicle: 0.1 - 200 mg/mL (range-finding study); 3.75 - 15 mg/mL (micronucleus study)
- Amount of vehicle: 10 mL/kg
- Lot/batch no. (if required): 014851

Details on exposure:

All animals were dosed once only via the intraperitoneal route at the appropriate dose level using a hypodermic needle attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.

Duration of treatment / exposure:

single exposure, sacrifice after 24h and 48h

Frequency of treatment:

single administration

Post exposure period:

24h and 48h

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1, 10, 100, 150, 200, 500, 1000, 2000 mg/kg
Basis:
nominal conc.
in range-finding toxicity study

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 37.5, 75, 150 mg/kg bw
Basis:
nominal conc.
in micronucleus test

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow / erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Doses were selected based on the findings of the Range-finding Toxicity Study

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and resuspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, dried and coverslipped using mounting medium.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined 'blind' using light microscopy at x1000 magnification. The incidence of micronucleated cells per 1000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 polychromatic erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values for males and females separately and combined.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the three test material groups and the number occurring in the corresponding vehicle control groups.
A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
If these criteria are not demonstrated, then the test material is considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio is shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a (x + 1)exp(1/2) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Highest dose (150 mg/kg) affected

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1 - 2000 mg/kg
- Clinical signs of toxicity in test animals: Premature deaths were seen in animals dosed with the test material at and above 200 mg/kg. Clinical signs were observed in animals dosed with test material at 150 mg/kg, and were as follows: hunched posture, lethargy, decreased respiration, ataxia, piloerection, ptosis, tiptoe gait and distended abdomen. With premature deaths occurring at and above 200 mg/kg and no clinical signs seen below 150 mg/kg, the maximum tolerated dose selected for the main study was 150 mg/kg with 75 and 37.5 mg/kg as the lower dose levels.
- Evidence of cytotoxicity in tissue analyzed: No necropsies were performed.
- Harvest times: Animals were observed 1 hour after dosing and subsequently once daily for 3 days.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Only the positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes. There was no significant increase in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups. The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.
- Ratio of PCE/NCE (for Micronucleus assay): There was a statistically significant change in the PCE/NCE ratio in the 24-hour test material 150 mg/kg dose group when compared to the concurrent vehicle control groups indicative of a cytotoxic response in the bone marrow.
- Appropriateness of dose levels and route: The statistically significant change in the PCE/NCE ratio in the 24-hour test material 150 mg/kg dose group is indicative of a cytotoxic response in the bone marrow and thus confirming systemic absorption and exposure of the target tissue. Hence, the route is appropriate as target tissue exposure is confirmed. Also, one premature death and clinical signs were observed additionally to the bone marrow toxicity, which is stipulated in the guideline.
- Statistical evaluation: Performed, results see above.

Any other information on results incl. tables

Table 3: Micronucleus study – Summary of group mean data

Treatment group	Number of PCE with micronuclei per 1000 PCE		PCE/NCE ratio
	Group mean	SD	Group mean	SD
Vehicle Control 48h sampling time	1.5	1.1	1.25	0.31
Vehicle Control 24 sampling time	0.6	1.1	1.23	0.27
Positive Control 24 sampling time	23.9***	7.6	1.40	0.37
Test item 150 mg/kg 48h sampling time	1.3	2.1	0.95	0.42
Test item 75 mg/kg 48-hour sampling time	1.1	1.0	1.16	0.37
Test item 37.5 mg/kg 48-hour sampling time	2.3	1.9	1.39	0.66
Test item 150 mg/kg 24-hour sampling time	1.6	1.6	0.84*	0.36
Test item 75 mg/kg 24-hour sample time	0.8	1.0	1.21	0.30
Test item 37.5 mg/kg 24-hour sampling time	0.7	0.7	1.77	1.57

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

SD = standard deviation

*** = p < 0.001

* = p < 0.05

Tables containing the individual and group mean data can be found as attachment.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The study is well-documented and was performed according to EU Method B.12 under GLP conditions. Hence, the information provided in the report can be considered reliable. Both positive and negative controls gave the appropriate response, so the test system and the obtained results can be regarded as valid. In consequence, the results are suitable for the assessment of the capability of the test item to induce micronuclei in mammalian cells in vivo. The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test. So the test item can be considered to be non-genotoxic under the conditions of the test.

Executive summary:

A study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered via the intraperitoneal route to male and female albino CD-1 mice. The method followed has been designed to comply with Method B12 of the EEC Commission Directive 92/69/EEC which constitutes Annex V of Council Directive 57/548/EEC

Following a preliminary range-finding study to confirm the toxicity of the test material, the micronucleus study was conducted using the test material at the maximum tolerated dose level of 150 mg/kg with 37.5 and 75 mg/kg as the lower two dose levels.

In the micronucleus study, groups often mice (five males and five females) were given a single intraperitoneal dose of the test material at 37.5, 75 or 150 mg/kg. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

Further groups of mice were dosed via the intraperitoneal route with arachis oil or orally with 50 mg/kg bw cyclophosphamide, to serve as vehicle and positive controls respectively.

There was no evidence of a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the concurrent vehicle control groups. A statistically significant change in the PCE/NCE ratio was observed after dosing with the test material at 150 mg/kg in the 24-hour group indicative of a cytotoxic response in the bone marrow and thus confirming systemic absorption and exposure of the target tissue.

The positive control material produced a significant increase in the frequency of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the concurrent vehicle control groups.

The test material was considered to be non-genotoxic under the conditions of the test.

The study is classified as acceptable and satisfies the requirement for EU method B.12 for in vivo cytogenetic mutagenicity data.