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:
29 January 2014 to 7 April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant study, performed following an appropriate international test guideline (OECD 474).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
in rats
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
Batch: 99225501
Purity: 99.3 %
Expiry Date: 26 March 2014

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
The test organisms were supplied by Charles River UK Limited, Margate, Kent, England. For the preliminary toxicity tests both male (26.3 to 31.2 g) and female (21.2 to 25.3 g) animals were tested. During the definitive stage of the study male mice 35 to 45 days old and weighting 22.2 g and 27.6 g were used. Upon arrival at the test facility the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups and given a unique tail tattoo. Each group was kept with the sexes separated in cages. The animals were kept in a controlled environment with the thermostat and relative humidity within target ranges of 19 to 23°C and 40 to 70% respectively, throughout the study. 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. Animals were acclimatised to laboratory conditions for at least 5 days before testing.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Suspensions of the test substance were prepared in 1% methylcellulose in purified water. Methylcelluose was obtained from Sigma Aldrich batch number SLBG2263V.
Details on exposure:
From the results obtained in the preliminary toxicity test, dose levels of 18.75, 37.5 and 75 mg/kg/day were used for the micronucleus test. Refer to Table 1 for details of the study design. All animals in the vehicle control, test substance dose groups were dosed orally by gavage, on two occasions approximately 24 hours apart, using a dose volume of 10 mL/kg. The positive control group were dosed at 20 mL/kg via oral gavage.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
Animals were treated with allylamine orally by gavage on two occasions approximately 24 hours apart.
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
18.75, 37.5 and 75 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
The definitive study was performed on males only, as no significant differences in toxicity were observed between the sexs, during the preliminary toxicity study. Please refer toTable 1 for numbers of animals per treatment.
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C obtained from Sigma Aldrich, batch number SLBD1982V was used as the positive control compound. A solution was prepared using purified water at a concentration of 0.6 mg/mL just prior to administration. Animals in the positive control group were dosed on a single occasion at 20 mL/kg via oral gavage.

Examinations

Tissues and cell types examined:
Slides prepared from the bone marrow of both femurs of each animal were evaluated for presence of micronucleated polychromatic and normochromatic erythrocytes.
Details of tissue and slide preparation:
The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration. The resulting cell suspensions were centrifuged at 1000 rpm (150 x g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum and the following fixation and staining procedure was followed; The suspensions were firstly fixed for a minimum of 10 minutes in methanol and allowed to air-dry. After which they were rinsed in purified water and stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes. After staining the suspensions were washed in purified water for 5 minutes, rinsed in cold tap water for 2 minutes and stored at room temperature until required. Immediately prior to scoring, slides are wet mounted with coverslips using purified water.
Evaluation criteria:
Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.
Statistics:
For the proportion of polychromatic erythrocytes at 24 hours after dosing, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.

For incidences of micronucleated polychromatic erythrocytes at 24 hours after dosing, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to 3. Exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No clinical signs of toxicity were observed for the vehicle control, positive control or animals administered Allylamine at any treatment level over the duration of the test. Some small incidences of bodyweight loss were observed throughout the micronucleus test.

Allylamine did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes in male CD1 mice. Allylamine did not cause any significant increases in the incidence of micronucleated normochromatic erythrocytes in male CD1 mice. Allylamine did not cause any statistically significant decreases in the proportion of polychromatic erythrocytes in male CD1 mice.

Any other information on results incl. tables

Table 2: Individual animal data

 

Treatment              (mg/kg/day)

Animal number

Proportion PCE (%)

MPCE

PCE

NCE

MNCE

Vehicle

201

54.3

3

546

459

0

 

202

37.1

1

380

644

0

 

203

39.3

1

393

607

0

 

204

49.0

1

491

512

0

 

205

43.0

2

432

573

0

 

206

61.0

0

662

423

0

Allylamine

211

45.0

1

452

553

0

(18.75)

212

49.0

1

490

511

0

 

213

49.0

2

490

511

0

 

214

58.9

2

594

415

0

 

215

62.9

0

633

374

0

 

216

47.7

1

480

527

0

Allylamine

221

54.7

0

551

456

0

(37.5)

222

43.2

1

440

578

0

 

223

49.1

1

518

538

0

 

224

51.3

1

520

494

0

 

225

44.2

2

445

562

0

 

226

43.8

2

440

564

0

Allylamine

231

48.6

2

487

515

0

(75)

232

41.4

2

415

587

0

 

233

47.1

2

473

531

0

 

234

45.7

2

461

548

0

 

235

48.0

1

481

522

0

 

236

50.3

1

505

499

0

 

237

47.4

1

479

531

0

 

238

49.9

2

508

510

0

MitomycinCa

 

241

54.0

32

554

471

0

(12)

242

37.0

47

372

633

0

 

243

45.2

43

457

554

0

 

244

49.4

37

495

508

0

 

245

56.3

39

578

448

0

 

Vehicle

1% methylcellulose in purified water.

PCE

Polychromatic erythrocytes

MPCE

Number of micronucleated cells observed per 2000 polychromatic erythrocytes examined

NCE

Total number of normochromatic erythrocytes examined for micronuclei

MNCE

Number of micronucleated normochromatic erythrocytes observed

a

Positive control dosed once only 24 hours prior to termination

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conidtions of this study, orally dosed allylamine did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male CD1 mice.
Executive summary:

A study was performed to assess the potential induction of micronuclei by allylamine in bone marrow cells of CD1 mice. Animals were treated with allylamine orally by gavage on two occasions approximately 24 hours apart. On the basis of results a preliminary toxicity test, dose levels of 18.75, 37.5 and 75 mg/kg/day were selected for the micronucleus test. No substantial differences in toxicity were observed between the sexes in the preliminary toxicity test, therefore, the micronucleus test was performed using male animals only. Analysis of bone smears collected 24 hours after the second treatment revealed no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistically significant decreases in the proportion of polychromatic erythrocytes at any dose level, compared to vehicle control values.The positive control compound, Mitomycin C, produced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (p<0.01).