Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1998-08-04 to 1998-10-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study conducted with the read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: the USA EPA, TSCA and FIFRA guidelines and the Japanese Ministry of International Trade and Industry guidelines for testing of new chemical substances.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: viscous liquid
Details on test material:
- Molecular formula (if other than submission substance): C28H62NO4P (for a representative structure: Phosphoric acid, di(C8)ester, compds with C12 amine)
- Molecular weight (if other than submission substance): 507.76
- Smiles notation (if other than submission substance): CCCCCCCCCCCCN.O=P(O)(OCCCCCCCC)OCCCCCCCC
- InChl (if other than submission substance): InChI=1/C12H27N/c1-2-3-4-5-6-7-8-9-10-11-12-13/h2-13H2,1H3
- Substance type: organic (Alkylamine salt of alkyl phosphoric acid)
- Physical state: extremely pale straw coloured viscous liquid (Sponsor’s description: clear yellow liquid)
- Composition of test material, percentage of components: Data relating to the identity, purity and stability of the test material are the responsibility of the Sponsor
- Storage condition of test material: room temperature in the dark

Test animals

Species:
mouse
Strain:
other: Crl:CD-1 TM(1CR)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: five to eight weeks
- Weight at study initiation: 24 to 29 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in groups of up to seven in solid-floor polypropylene cages with woodflakes bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23
- Humidity (%): 54 to 62
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: intraperitoneal (main study), oral and intraperitoneal (range-finding studies)
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: the substance is well soluble in arachis oil
- Concentration of test material in vehicle: 12.5, 6.25 and 3.125 mg/mL
- Lot/batch no. (if required): 1
Details on exposure:
Test substance, vehicle and positive control substance were administered at dose volume of 10 mL/kg bw.
Duration of treatment / exposure:
single i.p. administration
Frequency of treatment:
not applicable
Post exposure period:
24 hours:
- test material (3 groups of animals);
- vehicle control (1 group of animals);
- positive control (1 group of animals).
48 hours:
- test material (1 group of animals, the highest dose)
- vehicle control (1 group of animals)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
31.25, 62.5 and 125 mg/kg bw
Basis:
other: i.p. administration (main study)
Remarks:
Doses / Concentrations:
1000 and 2000 mg/kg bw
Basis:
actual ingested
range-finding study
Remarks:
Doses / Concentrations:
125, 250, 500, 750, 1000 and 2000 mg/kg bw
Basis:
other: i.p. administration (range-finding study)
No. of animals per sex per dose:
7 (test material and vehicle controls)
5 (positive control)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water. The concentration, homogeneity and stability of the positive control material and its preparation were not determined by analysis.
- Justification for choice of positive control(s): as recommended by OECD guideline 474
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw / 5 mg/mL

Examinations

Tissues and cell types examined:
bone marrow/erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: range-finding toxicity study (please see below in "Any other information on materials and methods".

TREATMENT AND SAMPLING TIMES: sampling after 24 and 48 hours (see below).

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice (ie. 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, allowed to air-dry and coverslipped using mounting medium.

METHOD OF ANALYSIS:
Slide Evaluation
Stained bone marrow smears were coded and examined blind using light microscopy at xl000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although ocasionally 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 erythrocytes were 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.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group. A positive mutagenic response was demonstrated when a statistically significant dose-responsive increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group. If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test. A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was 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) 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
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs and deaths
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 and 2000 mg/kg bw (oral route), 125-2000 mg/kg bw (i.p. administration).

- Clinical signs of toxicity in test animals: In animals dosed with test material via the oral route, no premature deaths occurred up to the maximum recommended dose of 2000 mg/kg, and clinical signs were observed in animals dosed at 1000 mg/kg as follows: hunched posture, ptosis and increased salivation. In animals dosed with the test material via the intraperitoneal route, premature deaths occurred at and above 250 mg/kg, and clinical signs were observed at and above 125 mg/kg as follows: hunched posture, pilo-erection, lethargy, pallor of the extremities, distended abdomen, decreased respiratory rate, laboured respiration, ptosis, ataxia and diuresis.

- Rationale for exposure: The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main study. Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration, therefore, this was selected for use in the main study. The maximum tolerated dose (MTD) of the test material, 125 mg/kg, was selected for use in the main study, with 31.25 and 62.5 mg/kg as the lower dose levels.

- Harvest times: 48 hours after dosing

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.
- Ratio of PCE/NCE (for Micronucleus assay): There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups.
- Appropriateness of dose levels and route: yes.
- Statistical evaluation: no statistically significant differences in analysed parameters between test and vehicle control groups.

Any other information on results incl. tables

Range-finding toxicity study

The mortality data are summarised as follows:

DOSE LEVEL mg/kg SEX NUMBER OF ANIMALS TREATED ROUTE DEATHS ON DAY TOTAL DEATHS
0 1 2
1000 Male 2 oral 0 0 0 0/4
Female 2 oral 0 0 0
2000 Male 2 oral 0 0 0 0/4
Female 2 oral 0 0 0
125 Male 2 ip 0 0 0 0/2
250 Male 2 ip 0 0 1 1/2
500 Male 2 ip 2 - - 2/2
750 Male 2 ip 2 - - 2/2
1000 Male 2 ip 2 - - 3/4
Female 2 ip 1 - -
2000 Male 2 ip 2 - - 4/4
Female 2 ip 2 - -

i.p. = intraperitoneal

- = no data

Micronucleus Study

Mortality Data and Clinical Observations

There were premature deaths seen in the 48-hour 125 mg/kg test material dose group. Clinical signs were observed in animals dosed with the test material at and above 62.5 mg/kg bw in both the 24 and 48-hour groups where applicable, these were as follows: hunched posture, lethargy, ataxia, ptosis, emaciation, decreased respiratory rate, laboured respiration, dehydration, increased lacrimation, distended abdomen, pilo-erection, increased salivation and pallor ogf the extremities. It was considered that the loss of animals due to premature death did not affect the integrity of the study. Whilst only four animals survived, one less than the five per group recommended in the OECD guidelines, they were considered to be an adequate number on the grounds of animal welfare. There was no evidence of a mutagenic response in the 24-hour groups, therefore, it was considered unnecessary to repeat the 48-hour dose group.

Evaluation of Bone Marrow Slides

A summary of the results of the micronucleus study is summarised as follows:

TREATMENT GROUP NUMBER OF PCE WITH MICRONUCLEI PER 2000 PCE PCE/NCE RATIO
GROUP MEAN SD GROUP MEAN SD

1. VEHICLE CONTROL

48-hour sampling time

1.9 1.2 1.04 0.34

2. VEHICLE CONTROL

24-hour sampling time

1.0 1.0 1.06 0.32

3. POSITIVE CONTROL

24-hour sampling time

69.0*** 32.1 1.26 0.52

4. Test materiala
125 mg/kg
(48-hour sampling time)

2.0 1.2 1.13 0.28

5. Test material
125 mg/kg
(24-hour sampling time)

2.0 1.8 0.79 0.43

6. Test material
62.5 mg/kg
(24-hour sampling time)

2.0 2.3 0.99 0.47

7. Test material
31.25 mg/kg
(24-hour sampling time)

2.0 1.7 0.91 0.33

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

SD = standard deviation

*** = p < 0.001

a = data from four animals

There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups. There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups. However, the presence of premature deaths and clinical observations was taken to indicate that systemic absorption had occurred. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material was 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 to mice. The method used has been designed to comply with the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990). The study design also complies with the revised OECD Guidelines for Testing of Chemicals No.474 "Micronucleus Test", Method B12 of the EEC Commission Directive 92/69/EEC, the USA EPA, TSCA and FIFRA guidelines and the Japanese Ministry of International Trade and Industry guidelines for testing of new chemical substances.

A range-finding study was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes, therefore the main study was performed using only male mice. The micronucleus study was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) of 125 mg/kg with 31.25 and 62.5 mg/kg as the two lower dose levels. 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 given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. However, the presence of premature deaths and clinical signs was taken to indicate that systemic absorption had occurred. It was considered that the premature loss of three animals in the 48-hour 125 mg/kg test material dose group did not affect the integrity of the study.

The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

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