Oxidised Crude Tall Oil

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2015 to 28 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This testing was performed for the purpose of chemical notification in China. Therefore no testing proposal was included.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2014

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

other: in vivo micronucleus teste

Test material

Test material information

Constituent 1

Reference substance name:

Oxidised Crude Tall Oil

EC Number:

938-639-0

Molecular formula:

C17-20H29-35CO2H. O6-10

IUPAC Name:

Oxidised Crude Tall Oil

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): EnvaMul™200
- Substance type: UVCB

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

recommended by international guidelines

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: 31-40 g
- Assigned to test groups randomly: yes
- Fasting period: withheld 3 - 4 hours prior to dosing until 3 - 4 hours after administration
- Housing:
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH,
Soest, Germany) ad libitum)
- Water : tap water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 22.6°C
- Humidity (%): 38-73%
- Air changes (per hr): ca 10/h
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility/stability of the substance
- Amount of vehicle (if gavage or dermal): 10 mL

Details on exposure:

dosing solutions prepared on day of treatment (doses based on the outcome of a pre-test in 3 males and 3 females at 2000 mg/kg bw)

Duration of treatment / exposure:

2 days

Frequency of treatment:

once at 0 and 24 h

Post exposure period:

24 hour after last dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5 males/ dose + additional 18 animals at 2000 mg/kg for blood sampling at 0.5, 1, 2, 4, 6 and 8 hours after the second dosing

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide;
- Justification for choice of positive control(s): according to the guideline
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow for normo- and poly-chromated erithocytes

Details of tissue and slide preparation:

Bone marrow was sampled 48 hours after the first dosing from both femurs, flushed withetal calf serum and the cell suspension was centrifuged at 216 g for 5 min. Smears were prepared after removal of the supernatant (one drop left behind) and suspended on a clean slide that was coloured by Giemsa based staining (Wright-stain-procedure)

Evaluation criteria:

A test substance is considered positive in the micronucleus test if:
It induces a biologically as well as a statistically significant (Dunnett’s test, one-sided, p < 0.05)
increase in the frequency of micronucleated polychromatic erythrocytes and the number of
micronucleated polychromatic erythrocytes in the animals should be above the historical control
data range.

A test substance is considered negative in the micronucleus test if:
None of the tested concentrations show a statistically significant (Dunnett’s test, one-sided,
p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number
of micronucleated polychromatic erythrocytes in the animals should be within the historical control
data range.

Statistics:

ToxRat Professional version 3.0

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

One animal dosed with 1000 mg/kg test substance died immediately after the second dosing due to a technical error while dosing and one animal dosed with 500 mg/kg showed lethargy and a rough coat after the second dosing but recovered within 23 hours after second dosing. No treatment-related clinical signs or mortality were noted in the other animals.

Analyzed blood concentrations of the substance (from the 2000 mg/kg bw additional mice) were above the upper limit of quantification as validated in the analytical method (> 10000 ng/L).

Any other information on results incl. tables

 

GroupTreatment	Dose (mg/kg bw		Number of micronucleatedpolychromatic erythrocytes (mean ± S.D.)^(1,2	Ratio polychromatic/ normochromatic erythrocytes (mean ± S.D.)^(1,2
Vehicle control		0	3.6 ±0.9	0.95 ±0.07
Test substance		2000	4.0 ±1.0	0.90 ±0.08
Test substance		1000	4.5 ±1.7	0.94 ±0.05
Test substance		500	3.4 ±1.1	0.91 ±0.06
CP		40	38.2 ±  6.8^(4)	0.54 ±0.12

Vehicle control = Corn oil

CP = Cyclophosphamide.

(1) Four to five animals per treatment group. 

(2) At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.

(3) The ratio was determined from at least the first 1000 erythrocytes counted.

(4) Significantly different from corresponding control group (Studentsttest, P = 0.01).

 

Applicant's summary and conclusion

Conclusions:

The substance is not clastogenic in the bone marrow micronucleus test of mice up to a dose of 2000 mg/kg

Executive summary:

In a study according to OECD 474 5 male mice/groupwere dosed twice via oral gavage with vehicle (corn oil), the substance at 2000, 1000 and 500 mg /kg bw or with 40 mg/kg cyclophosphamide (dosed only once). One animal dosed at 1000 mg/kg bw died and one at 500 mg/kg bw showed lethargy and a rough coat after the second dosing but recovered within 23 hours. No treatment related clinical signs or mortality were noted in the other animals. Blood samples collected at 0.5, 1, 2, 4, 6 and 8 hours after dosing from three satellite animals (dosed at 2000 mg/kg bw) per time point treated with 2000 mg showed concentrations of the substance above the upper limit of quantification (> 10000 ng/L), which confirms exposure.

Bone marrow was sampled 48 hours after the first dosing. No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the substance compared to the vehicle treated animals. No toxicity on erythropoiesis of the substance was observed (expressed as the ratio of polychromatic to normochromatic erythrocytes).

The substance is not clastogenic in the bone marrow micronucleus test of mice up to a dose of 2000 mg/kg.