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:
from 16 October 2008 to 11 December 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study but there was no data on the test substance (purity)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009
Reference Type:
other: Statement of purity for Amber core
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
: no certificate of analysis, few data on the test substance.
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
2007-10-15, Department of health of the government of the United kingdom
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Amber Core (P#620), 1-(2-tert-Butylcyclohexyloxy)-2-butanol

Test animals

Species:
mouse
Strain:
other: albino Hsd:ICR (CD-1)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK
- Age at study initiation: 5-8 weeks old
- Weight at study initiation: 22 to 30g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: in groups of up to 7 in solid-floor polypropylene cages with wood-flake bedding.
- Diet (e.g. ad libitum): free access to Harlan Teklad 2014 Rodent Pelleted Diet
- Water (e.g. ad libitum): free access
- Acclimation period: Yes, for 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: no data

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Sodium chloride 0.9% w/v
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 100 or 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): 300407503
- Purity: no data
Details on exposure:
See details in Table 7.6.2/2.
Duration of treatment / exposure:
the test subtance was injected, and 24 or 48 after this administration, the animals were killed.
Frequency of treatment:
once only.
Post exposure period:
One group of mice from each dose level was killed 24 hours following treatment and a second group dosed with test material at 2000 mg/kg bw was killet at 48 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
500; 100; 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Main test: 7 male mice/dose group
Positive control(s):
cyclophosphamide (Acros Organics, batch no. A0164185)
- Justification for choice of positive control(s): Cyclophosphamide is known to produce micronuclei under the conditions of the test.
- Route of administration: oral route
- Doses / concentrations: the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water.

Examinations

Tissues and cell types examined:
All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: the doses were selected in accordance with the results obtained in the range-finding study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): no additionnal information

DETAILS OF SLIDE PREPARATION: Immediately after termination, both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. These smears were air-dry and cover-slipped using mounting medium.

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification.

OTHER: 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 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 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 and standard deviations.
Evaluation criteria:
A comparison was made between the number of micronucleated PCE occuring in each of the test material groups and the number occuring in the corresponding vehicle control group. A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the nhumber of micronucleated PCE was observed for either the 24 or 48-hour kill times when compared to their corresponding group.
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:
Student's t test following a ѵ(x+1) transformation, ANOVA 1

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000-2000 mg/kg bw
- Solubility: no data
- Clinical signs of toxicity in test animals: no death occured at any dose level and at any exposure mode (oral or intraperitoneal). In animals dosed with the test material via the intraperitoneal route, clinical signs were observed at 2000 mg/kg bw (Huched posture and ptosis).
- Evidence of cytotoxicity in tissue analyzed: not examined
- 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 test. No evidence of toxicity was observed in animals dosed with test material via the oral route and, therefore systemic absorption could not be confirmed using this dose route. Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration, therefore this was selected for use in the main test. The maximum recommended dose of the test material, 2000 mg/kg bw, was selected for use in the main test, with 1000 and 500 mg/kg bw as the lower dose levels.
- Harvest times: not applicable
- High dose with and without activation: not applicable

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay): there was no statistically significant increase in the incidence of micronucleated PCE (see details in table 7.6.2/3).
- Ratio of PCE/NCE (for Micronucleus assay): modest decrease (but not statistically significant) in the PCE/NCE ratio in both the 24 and 48h test material dose groups when compared to their concurrent control groups. This, together with the observation of clinical signs (Hunched posture, ataxia, lethargy, ptosis and splayed gait) was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved (see details in table 7.6.2/3).
- Appropriateness of dose levels and route: see above.
- Statistical evaluation: the number of micronucleated PCE was statistically increased only for the positive control (P<0.001)

Any other information on results incl. tables

Table 7.6.2/3:Results obtained in the main test

Treatment group

Dose level (mg/kg bw)

Kill time (hours after dosing)

Number of PCE with micronuclei per 2000 PCE

PCE/NCE ratio

Group Mean

SD

Group Mean

SD

Vehicle control

0

24

1.3

1.5

0.65

0.11

Amber Core (P#620)

500

0.1

0.4

0.51

0.5

1000

0.9

1.6

0.65

0.18

2000

0.3

0.5

0.57

0.18

Positive control

50

23.0***

5.4

0.72

0.17

Vehicle control

0

48

0.9

1.2

0.80

0.30

Amber Core (P#620)

2000

0.6

1.1

0.53

0.17

PCE: Polychromatic erythrocytes

NCE: Normochromatic erythrocytes

SD: Standard deviation

*** : P<0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the test conditions, Amber Core (P#620) is not considered as genotoxic in this micronucleus test.
Executive summary:

In an in vivo micronucleus assay performed according to the OECD guideline No. 474, and in compliance with the GLP, Amber core (P#620) (purity unknown) was administratedviaintraperitoneal route to male albino Hsd:ICR (CD-1) mice (7 animals/dose level). In a preliminary test, two mice (one female, one male) were dosed once only at the appropriate dose level by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe. In the preliminary test, 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 test. No evidence of toxicity was observed in animals dosed with test materialviathe oral route and, therefore systemic absorption could not be confirmed using this dose route. Adequate evidence of test material toxicity was demonstratedviathe intraperitoneal route of administration, therefore this was selected for use in the main test. The maximum recommended dose of the test material, 2000 mg/kg bw, was selected for use in the main test, with 1000 and 500 mg/kg bw as the lower dose levels.

In the main, test, animals were therefore treated with Amber Core via one intraperitoneal injection. 24 or 48 Hours after the treatment, animals were killed and bone marrow was collected for further analysis. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated in order to determine the toxicity of the test material.

A modest decrease (but not statistically significant) was observed in the PCE/NCE ratio in both the 24 and 48h test material dose groups when compared to their concurrent control groups. This, together with the observation of clinical signs (Hunched posture, ataxia, lethargy, ptosis and splayed gait) was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved. Furthermore, there was no statistically significant increase in the incidence of micronucleated PCE.

In conclusion, under the test conditions, Amber Core (P#620) is not considered as genotoxic in thisin vivomicronucleus test. Therefore, Amber Core is not classified as genotoxic according to the criteria of the Annex VI of the of the Regulation (EC) No 1272/2008 (CLP) and the Annex VI of the Directive 67/548/EC.

This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 474.