Registration Dossier

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:
18 August 1999 and 23 September 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD, EU and US EPA test guidelines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identity: HP-10
Chemical name: 2,4,8,10-tetrakis(1,1-dimethylethyl)-6-(2-ethylhexyoxy)-12H-dibenzo[ d.g][1,3,2]dioxaphosphocin
CAS registry number: 126050-54-2
Appearance: White powder
Storage conditions: Room temperature/dark
Batch number: 10549
Expiry date: 27 January 2000
Purity: 100%
Date received: 3 August 1999

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
All animals in this study were Specific Pathogen Free CD-1 outbred albino mice of Swiss origin.
Males weighed between 28 and 30 grams and females weighed between 22 and 24 grams on despatch from Charles River UK Limited, Margate, Kent, England.
On arrival the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups and tail marked. Each group was kept, with the sexes separated, in disposable cages and maintained in a controlled environment, with the thermostat set at 22°C and relative humidity set at 50%. The room was illuminated by artificial light for 12 hours per day. All animals were allowed free access to pelleted expanded rat and mouse No.1 maintenance diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) and tap water ad libitum. Food and tap water are routinely analysed for quality at source. Dietary contaminants are not suspected of having any significant effect on parameters measured in this test in this laboratory at any time over the last ten years. All animals were acclimatised for a minimum of 5 days, examined daily and weighed prior to dosing.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Suspensions of the test substance were freshly prepared on the morning of the test (using identical methods for each phase of the test) and were individually formulated to the concentrations in corn oil.
Details on exposure:
All animals in all groups were dosed with the standard volume of 20 ml/kg bodyweight. The test substance and negative control were dosed by intraperitoneal injection, and mitomycin C, the positive control compound, was administered orally by intragastric gavage.
Duration of treatment / exposure:
24 & 48 hours exposure
Frequency of treatment:
Single treatment
Post exposure period:
up to 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 males & females for 500, 1000 & positive control.
10 males & 10 females for vehicle control & 2000 dose groups.
Control animals:
yes
Positive control(s):
Mitomycin C, obtained from Sigma Chemical Company, batch number 118H2500; was used as the positive control compound. It was prepared as a solution in purified water, at a concentration of 0.6 mg/ml, just prior to administration.

Examinations

Tissues and cell types examined:
erythrocytes
Details of tissue and slide preparation:
The animals were killed by cervical dislocation following carbon dioxide inhalation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 ml of pre-filtered foetal calf serum using a 2 ml disposable syringe fitted with a 21 gauge needle. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner (Schmid 1976). At least three smears were made from each animal. The prepared smears were fixed in methanol(> 10 minutes). After air-drying the smears were stained for 10 minutes in 10% Giemsa (prepared by 1:9 dilution of Gurr's improved R66 Giemsa (BDH) with purified water).
Following rinsing in purified water and differentiation in buffered purified water, the smears were rinsed in purified water, air-dried and mounted with covers lips using DPX.
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Usually only one smear per animal was examined. The remaining smears were held temporarily in reserve in case of technical problems with the first smear.

Micronuclei are identified by the following criteria:
• Large enough to discern morphological characteristics
• Should possess a generally rounded shape with a clearly defined outline
• Should be deeply stained and similar in colour to the nuclei of other cells - not black
• Should lie in the same focal plane as the cell
• Lack internal structure, ie they are pyknotic
• There should be no micronucleus-like debris in the area surrounding the cell.
The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept as recommended by Schmid (1976).
Evaluation criteria:
A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (P<0.01); individual and/or group mean values should exceed the laboratory historical control range (Morrison and Ashby 1995). A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P>0.01) and where these values fall within the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.
Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related decrease in the proportion of immature erythrocytes (P<0.01). This decrease would normally be evident at the 48 hour sampling time; a decrease at the 24 hour sampling time is not necessarily expected because of the relatively long transition time of erythroid cells [late normoblast -> immature erythrocyte (approximately 6 hours) -> mature erythrocyte (approximately 30 hours)].
Statistics:
The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. Non-parametric statistical methods were chosen for analysis of results because:
• They are suited to analysis of data consisting of discrete/integer values with ties such as the incidence of micronucleated immature erythrocytes
• The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of nonparametric analysis and classical analysis of variance are very similar)
• 'Outliers' are frequently found in the proportion of immature erythrocytes for both control and treated animals; non-parametric analysis based on rank does not give these values an undue weighting.
Unless there is a substantial difference in response between sexes (which occurs only rarely) results for the two sexes are combined to facilitate interpretation and maximise the power, of statistical analysis.
For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected (Agresti et al. 1990); for individual intergroup comparisons (ie the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Results and discussion

Test results
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:
PRELIMINARY TOXICITY TEST: One female animal dosed with HP-10 at 2000 mg/kg had convulsions and died immediately after dosing. This death was considered to be due to mis-dosing. Results from the three remaining animals in this dose group showed that a dose level of 2000 mg/kg was tolerated, and this level was considered to be an appropriate maximum for use in the micronucleus test. Dose levels of 500, 1000 and 2000 mg/kg bodyweight were chosen for use in the main test.

MICRONUCLEUS TEST
Clinical signs and mortalities: No mortalities were obtained in the micronucleus test. No adverse clinical signs were obtained for the vehicle control or positive control treated animals over the duration of the test.
Micronucleated immature erythrocyte counts (mie): The test substance did not cause any statistically significant increases m the number of micronucleated immature erythrocytes at either sampling time (P>0.01).
Mitomycin C caused large, highly significant increases (P<0.001) in the frequency of micronucleated immature erythrocytes.
Micronncleated mature erythrocytes (mme): The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time.
Proportion of immature erythrocytes (% ie/ie +me): The test substance failed to cause any significant decreases in the proportion of immature erythrocytes (P>0.01).
Mitomycin C did not cause any statistically significant decreases in the proportion (P>0.01) – it should be noted that even very cytotoxic compounds such as mitomycin C do not always produce a substantial decrease in this proportion as early as the 24 hour sampling time because of the lag caused by erythrocyte maturation.

Any other information on results incl. tables

TABLE 1 – Summary of results and statistical analysis

Sampling time

Treatment

Dose (mg/kg)

% ie/(ie+me) (mean)

Incidence mie (mean)

Incidence mme (group mean)

24 Hours

Vehicle control

-

43

0.9

0.7

HP-10

500

45

0.6

0.0

HP-10

1000

49

0.3

0.8

HP-10

2000

49

1.2

0.0

Mitomycin C

12

49

48.6***

1.5

48 Hours

Vehicle control

-

44

0.5

1.1

HP-10

2000

36

0.4

0.3

% ie/(ie+me): Proportion of immature erythrocytes

mie: Number of micronucleated cells observed per 2000 immature erythrocytes examined

mme: Number of micronucleated cells calculated per 2000 mature erythrocytes examined

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-side probabilities):

*** P<0.001 (highly significant)

otherwise P>0.01 (not significant)

† Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table.

 

TABLE 2 – Results for individual animals – 24 hour sampling time

Treatment

Dose (mg/kg)

Animal number

% ie/(ie+me)

Incidence mie

me

Incidence mme

Vehicle control

-

M 201

54

0

470

0

M 202

44

1

559

0

M 203

35

0

650

0

M 204

32

2

678

1

M 205

32

2

686

1

F 206

51

0

491

0

F 207

58

0

416

0

F 208

57

2

429

0

F 209

28

2

718

0

F 210

40

0

624

0

HP-10

500

M 211

46

0

540

0

M 212

39

0

613

0

M 213

44

1

558

0

M 214

39

0

624

0

M 215

36

0

662

0

F 216

55

2

448

0

F 217

35

1

648

0

F 218

41

1

590

0

F 219

43

1

572

0

F 220

71

0

288

0

HP-10

1000

M 221

43

2

575

1

M 222

54

0

458

0

M 223

58

0

417

1

M 224

50

0

503

0

M 225

38

0

618

0

F 226

52

0

476

0

F 227

68

1

328

0

F 228

28

0

724

0

F 229

52

0

477

0

F 230

49

0

510

0

HP-10

2000

M 231

53

4

468

0

M 232

38

0

620

0

M 233

50

0

497

0

M 234

48

2

516

0

M 235

48

0

523

0

F 236

50

2

500

0

F 237

34

2

656

0

F 238

46

0

541

0

F 239

72

2

272

0

F 240

54

0

465

0

Mitomycin C

12

M 241

59

32

433

0

M 242

50

54

507

0

M 243

61

47

393

1

M 244

35

35

738

0

M 245

42

58

584

0

F 246

58

60

422

0

F 247

49

25

508

1

F 248

47

30

531

2

F 249

35

66

654

0

F 250

59

79

415

0

%ie/(ie+me): Proportion of immature erythrocytes

mie: Number of micronucleated cells observed per 2000 immature erythrocytes

me: Total number of mature erythrocytes examined for micronuclei

mme: Number of mironucleated mature erythrocytes observed

 

TABLE 3 – Results for individual animals – 48 hour sampling time

Treatment

Dose (mg/kg)

Animal number

% ie/(ie+me)

Incidence mie

me

Incidence mme

Vehicle control

-

M 301

24

0

765

0

M 302

30

0

707

0

M 303

38

0

624

0

M 304

34

2

658

1

M 305

47

1

527

0

F 306

65

1

348

1

F 307

47

0

530

1

F 308

61

0

391

0

F 309

52

1

481

0

F 310

42

0

578

0

HP-10

2000

M 331

26

0

739

0

M 332

32

0

696

0

M 333

16

0

842

1

M 335

27

0

735

0

M 336

48

1

525

0

F 337

40

0

601

0

F 338

39

1

606

0

F 338

38

0

625

0

F 339

56

0

447

0

F 340

38

2

617

0

% ie/(ie+me): Proportion of immature erythrocytes

mie: Number of micronucleated cells observed per 2000 immature erythrocytes

me: Total number of mature erythrocytes examined for micronuclei

mme: Number of micronucleated mature erythrocytes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in mice treated with HP-10 and killed 24 or 48 hours later, compared to vehicle control values (p>0.01 in each case).
It is concluded that HP-10 did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by intraperitoneal injection in this in vivo test procedure.
Executive summary:

The purpose of this study was to assess the potential of HP-10 to induce mutagenic effects in mice following acute intraperitonal administration using an in vivo cytogenetic system (Boller and Schmid 1970, MacGregor et a/1987, Mavoumin et al 1990). The intraperitoneal route was chosen for this particular study to maximise potential absorption of the test substance.

The procedures used were based on the recommendations of the following guidelines:

-OECD Guideline for the Testing of Chemicals. (1997) Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test, Guideline 474.

-EEC Annex to directive 92/69/EEC (1992) Part B: Methods for determination of toxicity, B.12. Mutagenicity (Micronucleus test). Official Journal No. L 383 A, 35, 154.

-US EPA (1998) Health Effects Test Guidelines; OPPTS 870.5395 Mammalian erythrocyte micronucleus test. EPA 712-C-98-226.

 

This study was designed to assess the potential induction of micronuclei by HP-10 in bone marrow cells of mice. Mice were treated with a single intraperitoneal administration of the test substance at dose levels of 500, 1000 and 2000 mg/kg bodyweight. A preliminary toxicity test had previously shown that a dose of 2000 mg/kg (the standard limit dose for the micronucleus test) was expected to be tolerated; this level was therefore selected as an appropriate maximum for use in the micronucleus test.

The test substance and negative control were administered by intraperitoneal injection. The negative control group received the vehicle, corn oil. A positive control group was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bodyweight.

Bone marrow smears were obtained from five male and five female animals in the negative control, each of the test substance groups and the positive control group 24 hours after dosing. In addition bone marrow smears were obtained from five male and five female animals in the negative control and high level treatment groups 48 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in mice treated with HP-10 and killed 24 or 48 hours later, compared to vehicle control values (p>0.01 in each case).

The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated immature erythrocytes (p<0.001).

It is concluded that HP-10 did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by intraperitoneal injection in this in vivo test procedure.