[No public or meaningful name is available]

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:

23 Sep - 05 Dec 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

(1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C18, branched and linear

Molecular formula:

Not applicable as UVCB

IUPAC Name:

Amides, C18, branched and linear

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Amides, C18, branched and linear
- Physical state: extremely pale yellow granular solid
- Analytical purity: 99.3%
- Lot/batch No.: 202637
- Expiration date of the lot/batch: 20 Apr 2016
- Storage condition of test material: room temperature in the dark

Test animals

Species:

mouse

Strain:

other: Albino Hsd: ICR(CD-1)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 6 – 10 weeks
- Weight at study initiation: 22.7 – 30.4 g
- Assigned to test groups randomly: yes
- Housing: The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding.
- Diet: Harlan Teklad 2014C Global Certified Rodent Diet (Harlan Laboratories UK, Ltd., Oxon, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 – 25
- 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: arachis oil
- Justification for choice of solvent/vehicle: Due to the chemical characteristics of the test item and its limited solubility in organic and aqueous media, arachis oil was chosen as the vehicle.
- Concentration of test material in vehicle: 10, 20 and 40 mg/mL
- Amount of vehicle: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of the study the test item was prepared as required as a suspension at the appropriate concentration in arachis oil. No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours or if being applied to the test system; it Is assumed that the formulation was stable for this duration.

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single treatment

Post exposure period:

24 h after treatment (100, 200, 400 mg/kg bw )
48 h after treatment (400 mg/kg bw)

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 200, 400
Basis:
other: nominal dose

No. of animals per sex per dose:

7 (control), 7 (dose group), 5 (positive control)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test.
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:

Tissue: bone marrow
Cell type: bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A range-finding toxicity study was performed to determine a suitable dose level and route of administration for the main test. The range-finding toxicity test was also used to determine if the main test was to be performed using both sexes or males only.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with the test item at 400 mg/kg bw was killed after 48 hours. In addition, the negative and positive control groups were dosed orally and killed 24 hours following dosing.

DETAILS OF SLIDE PREPARATION: Immediately following termination (24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with fetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and a cover slip applied 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 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 together with appropriate group mean values and standard deviations.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group. A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to the vehicle control group. If these criteria were not fulfilled, then the test item 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 vehicle control group.

Statistics:

All data were statistically analyzed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analyzed 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:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 400 – 2000 mg/kg bw
- Clinical signs of toxicity in test animals: Clinical signs were not observed in any of the animals dosed with the test item at 2000 mg/kg bw via the oral route. Therefore, with no evidence of exposure to the target tissue, the intraperitoneal route was investigated to maximize the potential exposure of the test item to the animals. The clinical signs observed in animals dosed with the test item at 1000 mg/kg bw via the intraperitoneal route were as follows: hunched posture, ptosis and emaciation. Due to the severity of the clinical signs, one of the animals was killed in extremis. In animals dosed with the test item at 400 and 600 mg/kg bw via the intraperitoneal route, the following clinical signs were observed: hunched posture, ptosis, lethargy and pilo-erection. Confirmatory animals were dosed with the test item at 400 mg/kg bw and the clinical signs of hunched posture and ptosis were observed. Qualitative analysis of the slides prepared from the confirmatory animals indicated modest but acceptable levels of toxicity to the bone marrow. The test item 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. With evidence of acceptable toxicity via the intraperitoneal route, the considered maximum tolerated dose (MTD) of the test item, 400 mg/kg bw, was selected for use in the main test with 200 and 100 mg/kg bw as the lower dose levels.
- Evidence of cytotoxicity in tissue analyzed: yes, bone marrow samples were taken from animals dosed with the confirmatory dose level of 400 mg/kg bw 48 hours after dosing. Slides were then prepared and qualitatively assessed to ensure that any bone marrow toxicity observed was within acceptable limits for the main test.
- 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 the 48 or 24-hour 400 mg/kg bw test item dose groups, or the 24-hour 200 mg/kg bw dose group. A very modest, but statistically significant, increase was observed in the 24-hour 100 mg/kg bw test item dose group when compared to the vehicle control group. However, the group mean for the vehicle control was very low, none of the individual animals had any marked increases in the number of micronucleated PCE, and the group mean value was well within the historical vehicle control range. The response was therefore considered to be artefactual and of no toxicological significance.
- Ratio of PCE/NCE (for Micronucleus assay): Modest, but statistically significant, decreases in the PCE/NCE ratio were observed in the 48 and 24-hour 400 mg/kg bw test item dose groups, and also the 24-hour 200 mg/kg bw test item dose group, when compared to the vehicle control. These decreases, together with the observation of clinical signs, were taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

Any other information on results incl. tables

Table 1: Micronucleus Test – Summary of Group Mean Data

Treatment group	Dose [mg/kg]	Sampling time [h]	Mean frequency of PCE with Micronuclei per 2000 PCE		PCE/NCE ratio per 1000 erythrocytes
			Group mean	SD	Group mean	SD
Vehicle control	0	48	0.6	1.1	0.95	0.18
Test substance	400	48	2.1	3.2	0.70*	0.22
	400	24	1.1	1.1	0.65**	0.14
	200	24	0.9	1.2	0.64**	0.19
	100	24	2.3*	1.8	1.15	0.67
Positive control	50	24	64.0**	13.5	0.69*	0.18

*: p< 0.05

**: p< 0.01

PCE: polychromatic erythrocytes

NCE normochromatic erythrocytes

SD: standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative