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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Hydrocarbons, C12-C16, n-alkanes, isolkanes, alkenes
EC Number:
810-258-3
Molecular formula:
not applicable; UVCB
IUPAC Name:
Hydrocarbons, C12-C16, n-alkanes, isolkanes, alkenes
Constituent 2
Reference substance name:
Alcohols, C2-33, manuf. of, by-products from overheads
IUPAC Name:
Alcohols, C2-33, manuf. of, by-products from overheads
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material: Alcohols, C2-33, manuf. of, by-products from overheads
- Substance type: Product, HF-1000
- Physical state: clear yellow liquid
- Odour: oily/solvent
- Purity test date: 2010/10/24
- Lot/batch No.: 68310

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males: 217-265 g; females: 146-184 g
- Assigned to test groups randomly: each animal was assigned a unique coded number from a computer numbered sequence ranging from 201 to 243
- Fasting period before study: no
- Housing: 5 animals per cage, gender separated, polycarbonate/stainless steel fgrid tops (61x43.5x24 cm)
- Diet : Food was freely available to the rats all times; International certified rodent chow supplied by IPS Ltd., UK
- Water: Tap water (ad libitum)
- Acclimation period: not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9-22.5
- Humidity (%): 47-79
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% w/v carboxymethyl cellulose with 0.1% w/v Tween 80 in water
- Justification for choice of solvent/vehicle: not mentioned
Details on exposure:
All animals were exposed to test or control materials via the intraperitoneal dose route.
Frequency of treatment:
single application
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
10 ml/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Vehicle: 5m + 5f
100 mg/kg bw: 5m
200 mg/kg bw: 5m
400 mg/kg bw: 10 m + 10 f
positive control: 3m
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: intraperitoneal
- Dose: 50 mg/kg bw
- Vehicle: see description
- Total application volume: 10 ml/kg bw
- post exposure period: 24 hours

Examinations

Tissues and cell types examined:
bone marrow; polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: preliminary range finding test were undertaken prior to the micronucleus test.
Range finding study: 400, 600, 800 and 1000 mg/kg (for each doses 1m+1f)
The rats were observed for clinical signs or motality.

DETAILS OF SLIDE PREPARATION:
Rats were killed by CO2 asphyxiation. One femur of each rat was promptly removed and freed adherent tissue. A small hole was made in the neck of one femur and the bone marrow flushed with.The tubes were centrifuged to pellet the cells. All but a few drops of supernatant fluid were discarded.
Two slides were prepared from each tube per animal. The smears were left to air-dry. They were then fixed in methanol and immeresd in Giemsa stain solution.

METHOD OF ANALYSIS:
One of the two prepared slides was selected for examination. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micronucleated cells (MN-NCE).
Evaluation criteria:
The mean micronucleus incidence in vehicle control-dosed and untreated CD rats has, in this laboratory, been determined as 0.04 ± 0.05%: a range of 0.01-0.13% per group of 5-7 rats and 0.02-0.11% per group of 10-12 rats. This frequency is an agreement with published data for miccronucleus tests with CD rats (Tamura et al, 1990; Salmone and Mavourin, 1994). These historical data have been used in the evaluation of the response in this test.
Statistics:
No statistical analysis was performed as the levels of MN-PCE induction fell within the determined historical control frequencies.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:

200, 400 and 600 mg/kg bw no deaths; 1000 mg/kg two deaths

- Clinical signs of toxicity in test animals:
Range-finding study:Clinical signs affecting the rats behaviour, breathing and posture were observed in the range finding tests. Based on these toxicity observations, the maximum tolerated dose of HF-1000 was judged to be in the region of 400 mg/kg bw.

Test itemgroup: There was no indication of bone marrow toxicity in any of the test item dose groups.
RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals:
No animal deaths occured in the main micronucleus test. No animals displayed signs of abnormality.

- Induction of micronuclei:
Positive control: Exposure of rats to the positive control agent, cyclophosphamide, induced large increases in bone marrow micronuclei. The mean MN-PCE frequency for the rats was 2.68%. An evident increase in the number of MN-NCE was also observed.
Test substance: There was no indication that the test substance induced bone marrow micronuclei in the treated rats. The highest MN-PCE frequency recorded for the test item was in the high dose females, where an incidence of 0.03% was observed.

Any other information on results incl. tables

Treatment Dose (h) Sex No. of rats scored Erythrocytes
NCE PCE PCE/NCE Mean ± SD
No of MN-NCE PCE analysed No of MN-PCE MN-PCE [%]
Vehicle 0 + 24 m 5 2 10006 3 0.03 0.68 ± 0.16
f 5 2 10001 3 0.03 0.72 ± 0.10
m+f 10 4 20007 6 0.03 0.70 ± 0.13
100 mg/kg 0 + 24 m 5 0 10000 2 0.02 0.60 ± 0.07
200 mg/kg 0 + 24 m 5 0 10002 2 0.02 0.65 ± 0.14
400 mg/kg 0 + 24 h m 10 1 20002 2 0.01 0.66 ± 0.14
f 10 8 20002 7 0.03 0.61 ± 0.11
m+f 20 9 40004 9 0.02 0.63 ± 0.11
positive control 0 + 24 h m 3 89 α 6001 161 2.68 φ 0.31 ± 0.01

α = Evident response in NCE

φ = Positive response in PCE

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It was conducted that Hydrocarbons, C12-C16, n-alkanes, isoalkanes, alkenes did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 400 mg/kg bw in male and female CD rats using 0h + 24 h intraperitoneal and 48 sampling regimen.