Hydrocarbons, C12-C16, n-alkanes, isolkanes, alkenes

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

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

Test material information

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.