Fatty acids, tall-oil, esters with ethylene glycol

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:

17 Oct 1989 - 09 Feb 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1983

Deviations:

yes

Remarks:

analytical purity of test substance not specified

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

1000 instead of 4000 erythrocytes counted per animal, but at three sampling time points

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

analytical purity of test substance not specified

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

151661-88-0

Test animals

Species:

mouse

Strain:

other: outbred albino mice, strain CFW 1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 20.7 - 32.8 g (males) and 19.5-24.8 g (females)
- Fasting period before study: animals were fasted overnight prior to dosing and until approximately 3-4 h after administration of test substance and control material.
- Housing: male mice were housed individually in Macrolon cages type I, female mice were housed in groups up to three in Macrolon cages type II.
- Diet: standard animal diet Altromin No. 1314 with 10 mm pellet diameter (Altrogge Spezialfutter), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days (dose range finding study) and at least 4 - 6 days (main study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
17 Oct 1989 - 30 Jan 1990

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Concentration of test material in vehicle: 300, 400 and 500 mg/mL (dose range finding study) and 500 mg/mL (main study)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in arachis oil (80°C and applied after cooling at a temperature of approx. 35°C) at an application volume of 10 mL/kg. The test substance concentrations were prepared immediately before use. Homogeneity was maintained during application using a magnetic stirrer.

Duration of treatment / exposure:

24, 48 and 72 h

Frequency of treatment:

single treatment

Post exposure period:

24, 48 and 72 h

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

2 (dose range finding study) tested at 3000, 4000 and 5000 mg/kg bw/day
6 (main study)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 10 mg/kg bw in water, application volume 10 mL/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:
As the acute oral toxicity (LD50 mouse) was determined to be higher than 5000 mg/kg bw according to acute toxicity studies, the following doses were chosen initially to determine the maximum tolerated dose: 3000, 4000 and 5000 mg/kg bw. According to the results of the dose range finding study, a dose level of 5000 mg/kg was chosen for the main study, because it is generally recommended to use the maximum tolerated dose for the micronucleus test.

TREATMENT AND SAMPLING TIMES:
The animals were sacrificed 24, 48 or 72 h after treatment.

DETAILS OF SLIDE PREPARATION:
The slides were fixed with methanol, air dried at least overnight and stained with Giemsa according to modification of Gollapudi and Kamra.

METHOD OF ANALYSIS:
Three slides per animal were prepared, one slide was randomly chosen and analysed. The slides of 5 males and 5 females per treatment group were analysed and the ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time.
The number of micronucleated cells out of 1000 polychromatic erythrocytes was determined for each animal.

Evaluation criteria:

The test is considered acceptable if the positive control substance induced statistically significant increase in polychromatic erythrocytes and the incidence of micronuclei should reasonably fall within the historical control data range of the laboratory. The test is considered positive if the test substance induced biologically as well as statistically significant (p < 0.05) increase in the frequency of micronuclei at any dose either in the male or in the female groups. The test is considered negative if the test substance did not induce any biologically as well as statistically significant (p < 0.05) increase in micronuclei at any dose either in the male or in the female groups.

Statistics:

Statistical significance of test substance values versus negative controls were calculated with the aid of tables of Kastenbaum and Bowman.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 3000, 4000 and 5000 mg/kg bw
- Solubility: soluble
- Harvest times: 24 h
- Clinical signs of toxicity in test animals: no findings were observed in any animals up to 72 h after administration.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no statistically significant induction
- Clinical signs of toxicity in test animals: signs at clinical examination were not noticed
- Ratio of PCE/NCE (for Micronucleus assay): no statistically significant changes were observed
- Statistical evaluation: the investigated sample did not induce a statistically significant (time dependent) increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of male or female mice.

Any other information on results incl. tables

Table 1: Mean values per group of PCE and PCE/NCE

Treatment group (sampling time	Species and sex	Dose	Mean of n=5 animals per group
			Micronucleated cells/ 1000 PCE	Ratio of PCE/NCE
Negative control (vehicle) (24 h)	5 male mice	10 mL/kg	1.6	1.0
	5 female mice		1.8	0.9
Positive control (24 h)	5 male mice	10 mg/kg	9.8	1.1
	5 female mice		7.4	1.0
Test substance
24 h	5 male mice	5000 mg/kg	2.4	1.1
	5 female mice		1.6	1.0
48 h	5 male mice	5000 mg/kg	1.2	1.0
	5 female mice		1.4	1.0
72 h	5 male mice	5000 mg/kg	1.2	1.3
	5 female mice		1.4	1.2

PCE: Polychromatic erythrocytes

NCE: Nonchromatic erythrocytes

Applicant's summary and conclusion

Conclusions:

No cytogenic potential was found in vivo.