Fatty acids, tall-oil, magnesium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The hazard information on the substance is based on read across from Tall oil and 2-ethylhexanoic acid and reveals low toxicity. Furthermore, the source substances contain higher or similar concentrations of the constituents used for read across compared to the target substance. There are no scientific reasons indicating that the constituents of the substance can interact in a way that will influence the toxicological/ecotoxicological properties of the substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Salmonella Typhimurium Reverse Mutation Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-08-22 - 2005-09-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read-across, see attached file.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 july, 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA153

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Test substance (all strains: 1st experiment; TA98, TA100, TA102 and TA1535: 2nd experiment)

Dose: 5000, 1667, 556, 185 and 62 µg/plate

Number of samples: 3 samples

Test substance (TA97a: 2nd experiment)

Dose: 556, 185, 62, 21, 7 and 2.3 µg/plate

Number of samples: 3 samples

Justification: The concentrations for the first experiment were set according to a preliminary toxicity test, see 3.2.
The test substance was not toxic up to 5000 µg/petri dish.

It was therefore decided to use 5000 µg/plate as the highest concentration which is the limit concentration according to the guidelines. Each of the other 4 concentrations was 1/3 of the preceding one.

For the second experiment the concentrations were changed only for strain TA97a according to the first experiment. The test substance was toxic to strain TA97a at 5000 and 1667 µg/plate. Thus it was decided to use 556 µg/plate as the highest concentration for the second experiment, which could be in the toxic range and to use a total of 6 concentrations.

The number and intervals of the concentrations are in accordance with the guidelines (at least 5 concentrations with intervals of about 10 ).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylene-diamine, t-Butyl-hydroperoxide, 2- Aminoanthracene, 1,8-Dihydroxy-anthraquinone, 7,12-Dimethylbenz[a]anthracene, 2-Nitrofluorene

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the preliminary test with strain TA100 no toxicity was seen up to 5000 µg/plate.

In the main test the test substance was only toxic to strain TA97a, resulting in a completely or almost missing bacterial background lawn at 5000 and 1667 µg/plate. At 556 µg/plate and beneath the bacterial background was normal.

Conclusions:

According to these results, "CRUDE TALL OIL" is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 5000 µg/plate.

Executive summary:

Method

"CRUDE TALL OIL" was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part B.13/14.

The test substance was dissolved in DMSO. The following concentrations were tested:

62, 185, 556, 1667 and 5000 µg per plate without external metabolisation, and

62, 185, 556, 1667 and 5000 µg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.

In the second experiment the concentrations were changed for strain TA97a as follows:  

2.3, 7, 21, 62, 185 and 556 µg per plate without external metabolisation, and

2.3, 7, 21, 62, 185 and 556 µg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.

The test was performed according to the "direct plate incorporation method". As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed.  

Results

Positive controls:

All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.  

Test substance:

Toxicity:

The test substance was toxic only to strain TA97a, resulting in a completely or almost missing bacterial background lawn at 5000 and 1667 µg/plate. At 556 µg/plate and beneath the bacterial background was normal. No toxicity was noted in the other strains.

Solubility:

At the 5000, 1667 and 556 µg/plate samples a precipitate was visible when the test substance was mixed with the agar. At 5000 µg/plate the precipitate was still visible when the colonies were counted but did not impede the counting.

Mutagenicity:

In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.  

Conclusion

According to the results obtained in this study, "CRUDE TALL OIL" is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 5000 µg/plate.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: Chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June 2001 - 26 September 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read-across, see attached file.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Testing and evaluation of clastogenic potential - chromosomal aberrations assays.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The cell line used was Chinese hamster ovary (CHO) cells, obtained from the University of Leiden in 1987.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced S9 enzymes (supernatant post-mitochondrial fraction obtained after centrifugation at 9000 G) were prepared from the livers of adult, male Fischer rats, as described by Ames et al (1975).

Test concentrations with justification for top dose:

For the first experiment, 9 dose levels, covering a wide concentration range, were tested. The highest dose was 5000 µg.ml-1, the maximum allowable concentration and subsequent dose levels were halving dilutions.

The presence of S9 mix had to be repeated due to toxicity and the following concentrations were tested:

in Test 1: 10, 20, 40, 60 and 80 µg.ml-1

In Test 2, the dose levels selected were:
Presence of S9 mix: 5, 10, 20, 30, 40, 50 and 60 µg.ml-1
Absence of S9 mix: 50, 55, 60, 62.5, 65, 67.5 and 70 µg.ml-1

Vehicle / solvent:

Dimethylsulphoxide

Untreated negative controls:

other: Historically negative control data

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

other: weakly clastogenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: Historical negative control

Positive controls validity:

valid

Additional information on results:

Tall Oil, CAS No. 8002-26-4, was toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix. It was tested to the maximum permitted concentration of 5000 µg.ml-1 in the absence of S9 mix and up to 80 µg.ml-1 in the presence of S9 mix (Test 1). Toxicity was noted at 60 and 80 µg.ml-1 in the presence of S9 mix and at 156-5000 µg.ml-1 in the absence of S9 mix. In Test 2, Tall Oil, CAS No. 8002-26-4, was tested up to 60 µg.ml-1 in the presence of S9 mix and up to 70 µg.ml-1 in the absence of S9 mix. Toxicity was noted in cultures treated with 30-60 µg.ml-1 (presence of S9 mix) and in cultures treated with 70 µg.ml-1 (absence of S9 mix).
It was concluded that Tall Oil, CAS No. 8002-26-4, was weakly clastogenic when tested with Chinese hamster ovary cell in vitro in the presence of S9 mix. The response was observed only at a concentration level deemed overtly toxic to the cells.

Conclusions:

It was concluded that Tall Oil, CAS No. 8002-26-4, was weakly clastogenic when tested with Chinese hamster ovary cell in vitro in the presence of S9 mix.  The response was observed only at a concentration level deemed overtly toxic to the cells.

Executive summary:

Aim:

Tall Oil, CAS No. 8002-26-4, was submitted for testing and evaluation of clastogenic potential.  

Method:

Chromosomal aberrations assays were performed with duplicate, Chinese hamster ovary (CHO) cell cultures.  This study was conducted incorporating two independent tests.  Dimethylsulphoxide was the vehicle and cyclophosphamide and methyl methanesulphonate were the positive controls used in both tests. Both tests were conducted in the presence and absence of a postmitochondrial supernatant fraction obtained from the livers of adult, male rats treated with Aroclor 1254 (S9) and a NADPH-generating system.

Cultures, established approximately 20 hours before testing, were treated for 6 h in the presence or 6 and 22 hours in the absence of S9 mix.  Cultures were harvested at 24 h (Test 1 and 2) or 48 h (Test 2) post treatment.

Results/Conclusion

Tall Oil, CAS No. 8002-26-4, was toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix.  It was tested to the maximum permitted concentration of 5000 µg.ml-1 in the absence of S9 mix and up to 80 µg.ml-1 in the presence of S9 mix (Test 1).  Toxicity was noted at 60 and 80 µg.ml-1 in the presence of S9 mix and at 156-5000 µg.ml-1 in the absence of S9 mix.  In Test 2, Tall Oil, CAS No. 8002-26-4, was tested up to 60 µg.ml-1 in the presence of S9 mix and up to 70 µg.ml-1 in the absence of S9 mix. Toxicity was noted in cultures treated with 30-60 µg.ml-1 (presence of S9 mix) and in cultures treated with 70 µg.ml-1 (absence of S9 mix).

It was concluded that Tall Oil, CAS No. 8002-26-4, was weakly clastogenic when tested with Chinese hamster ovary cell in vitro in the presence of S9 mix.  The response was observed only at a concentration level deemed overtly toxic to the cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-01 - 2010-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read-across, see attached file.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The dose range of the test material used in the preliminary toxicity test was 19.53 to 5000 µg/ml.

Doses: 0 19.53, 39.06, 78.1, 156.25, 312.5, 625, 1250, 2500, 5000

Results from the preliminary toxicity test were used to set the test material dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 µg/ml or 10 mM.
ii) The presence of excessive precipitate where no test material-induced toxicity was observed.
iii) Test material-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Vehicle and positive controls were used in parallel with the test material. Vehicle (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batch 0001423147 at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.
level, either with or without metabolic activation, in either the first or the second experiment.

Executive summary:

Introduction.

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method 817 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Methods.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1 % S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 5 to 70 µg/ml in the absence of metabolic activation, and 6.25 to 150 µg/ml in the presence of metabolic activation for the first experiment. For the second experiment the dose range was 10 to 160 µg/ml in the absence of metabolic activation, and 20 to 100 µg/ml in the presence of metabolic activation.

Results.

The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose

Conclusion.

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

level, either with or without metabolic activation, in either the first or the second experiment.

Reference 4

Endpoint:

genetic toxicity in vitro, other

Remarks:

see the read across studies that are cross linked in this chapter

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attached file for justification for read across.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA153

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

60 micrograms/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Tall oil was negative in Ames test in TA97a, TA98, TA100, TA102 and TA153 up to 5000microgram/plate. Tall oil was also negative in mouse lymphoma L5178Y cells, however slightly claustragenic in CHO cells at toxic doses. 2-ethanoic acid was nehgatiove in CHO cells.

Remarks on result:

other: 2-EHA

Executive summary:

Tall oil was negative in Ames test in TA97a, TA98, TA100, TA102 and TA153 up to 5000microgram/plate. Tall oil was also negative in mouse lymphoma L5178Y cells, however slightly claustragenic in CHO cells at toxic doses. 2-ethanoic acid was negative in CHO cells. Thus, reaction mass of Fatty acids, tall oil, 2-ethylhexanoic acid and magnesium salts does not require classification for genetic toxicity.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-08-30 till 2007-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no rescrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO (Chinese hamster ovary) cell line (substrain K3) (1, 2) is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254

Test concentrations with justification for top dose:

1 - 10 - 50 - 100 - 250 - 500 - 750 - 1000 - 1500 μg/mL

Vehicle / solvent:

Due to the limited solubility of the test substance in water, dimethylsulfoxide (DMSO) was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT test and for which historical data are available.
The final concentration of the vehicle DMSO in the culture medium was 1% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

without metabolic activation: positive control substance; ethylmethanesulphonate. With metabolic activation: positive control substance: methylcholanthrene.

Details on test system and experimental conditions:

Day 1: Seeding of the cells pretreated with "HAT" medium: in 175 cm² flasks (1x106 cells in 20 mL) and in 25 cm² flasks (200 cells in 5 mL)
Day 2: Test substance incubation (20 – 24 hours after seeding); exposure period (4-hour and 24-hour); washing of the cultures (4-hour exposure);
1st cytotoxicity determination (cloning efficiency 1: survival)
Day 3: Washing of the cultures (24-hour exposure)
Day 5: 1st passage of the treated cells
Day 9: 2nd passage of the treated cells with seeding in the selection medium ("TG" medium); 2nd cytotoxicity determination (cloning efficiency 2: viability)
From day 16: Drying, fixation, staining and counting of the selected colonies
In this study all incubations were performed at 37°C with a relative humidity of > 90% in a 5% (v/v) CO2 atmosphere.

Evaluation criteria:

• The absolute cloning efficiencies of the negative controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative controls should fall within our historical negative control data range of 0 – 15 mutants per 106 clonable cells.
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies.
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Statistics:

no

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 10 μg/mL) induced clearly increased mutant frequencies as expected. The values of the corrected mutant frequencies (MFcorr.: 91.24 – 553.18 per 106 cells) were clearly within our historical positive control data range (without S9 mix 4 hours treatment: MFcorr.: 48.83 –587.77 per 106 cells; without S9 mix 24 hours treatment: MFcorr.: 272.94 – 331.63 per 106 cells; with S9 mix: MFcorr.: 29.06 – 413.54 per 106 cells.

The osmolarity was not influenced by test substance treatment. The pH of the stock solutions were adjusted to physiological values using small amounts of 2 N NaOH.
In this study, in the absence and the presence of S9 mix no precipitation of the test substance in culture medium was observed up to the highest applied test substance concentration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

2-ethanoic acid was negative in a micronucleus test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attached file for justification of read across

Reason / purpose for cross-reference:

read-across source

Additional information on results:

Negative results in micronucleus test.

Executive summary:

2-ethanoic acid was negative in a micronucleus test,.

Reference 2

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:

1993-11-11 till 1994-02-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant; guideline study; available as unpublished report; ne restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female CD-1 mice were supplied by Harlan Olac Limited, Shaw's Farm, Blackthorn, Bicester, Oxfordshire. On arrival from the supplier, all mice were examined for signs of disease or ill health. Any animal judged unsuitable for testing was automatically excluded from the study. The mice were housed individually in non-SPF conditions in a room with a 12 h light -dark cycle. Animals were kept individually in polypropylene and stainless steel cages measuring 48 cm x 15 cm x 13 cm . White wood shavings provided the bedding in the cages. Benches were washed and floors were swept and disinfected with a mop impregnated with 0.5% Tego 2000 (Th Goldschmidt & Company Limited, Middlesex, England), an ampholytic detergent, at least once each day during the experiments . Walls were washed with Tego and cage racks and water bottles changed on a weekl y basis during the animal housing period .

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Immediately prior to dosing, the test compound was dissolved in corn oil to give the required test concentration . The dose volume used for both the control and test compound treated animals was a constant 10 ml.kg-1 body weight .
Food and water were freely available to the mice at all times except for a brief 2-3 h period prior to dosing when food was withheld . The diet used was SDS Rat and Mouse Maintenance Diet No. 1 obtained from Special Diet Services Limited, Witham, Essex , England.

Frequency of treatment:

Dosing 0 h + 24 h

Post exposure period:

DRF: 6 day
main test: 2 day
micronucleus test: 0 h

Remarks:

Doses / Concentrations:
400 mg.kg-1.day-1
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
800 mg.kg-1.day-1
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
1600 mg.kg-1.day-1
Basis:
nominal in diet

No. of animals per sex per dose:

DRF:
6 groups of 1 male and 1 female; 50 - 125 - 350 - 800 - 2000 - 5000 mg.kg-1.day-1
Main test:
3 groups of 3 male and 3 female; 2000 - 3000 - 4000 mg.kg-1.day-1
Micronucleus test:
400 mg.kg-1.day-1: 5 male and 5 female
800 mg.kg-1.day-1: 5 male and 5 female
1600 mg.kg-1.day-1: 8 male and 8 female

Control animals:

yes, concurrent vehicle

Positive control(s):

One control group of male and female CD-1 mice received the positive control agent, 50 mg cyclophosphamide.kg-1.day-1

Tissues and cell types examined:

polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

Mice were killed by cervical dislocation . The iemora were quickly dissected out and freed of adherent tissue . A small hole was made in the neck of onefemur and the marrow flushed, using a 1 ml syringe fitted with a gauge 25 needle, into a centrifuge tube containing 3 ml of a 1:1 mixture of foetal calserum and 0.8% trisodium citrate in Sorensen's buffer, pH 6 .8 (Sorensen's buffer, pH 6 .8 = 2 .84 g Na2HPO4.L-1 plus 2 .72 g KH2P04.L-1 distilled water). This mildly hypotonic treatment served to make the micronuclei clearly visible and to distinguish them from surrounding artefacts. Following completion of the sampling procedure the contents of the tubes were briefly agitated on a vortex mixer to allow separation of the cells. The tubes were centrifuged for 5 min at 1000 r.p.m. to pellet the cells . All but a few drops of supernatant fluid were discarded. The cells were then resuspended on a vortex mixer in this residual amount of supernatant liquid. Clean slides were assigned numbers corresponding to the tube numbers. A drop of the suspension was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube and animal. The smear was left to air dry, fixed in methanol for ca 5 min and immersed for 30 min in modified Wright stain (0.3 % w/v buffered at pH 6.9 in methanol) to give optimum erythrocyte discrimination. The stained smears were rinsed in 2-3 changes of distilled water and air dried. Permanent slide preparations were made by sealing glass coverslips onto the microscopic slides using DPX mounting medium.

Evaluation criteria:

1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micronucleated cells (MN-PCE) determined .As a control against inclusion of artefacts, or action of a mutagen on the G2 and/or mitotic phase of the cell cycle, the number of micronucleated normochromatic erythrocytes (MN-NCE) in mature red blood corpuscles were also recorded.a mean frequency of MN-PCE of 0.128% per mouse and 0.122% in CD-1 mice for both sexes combined.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

However, in the high dose group males a slight increase in MN-PCE to 0.28% was noted. This micronucleus frequency was marginally outside the established control range of 0.00-0.25% for a random group of 8 mice . The response was therefore classified as equivocal in accordance with protocol defined criteria. To clarify whether the obtained increase was due to a biological chance event, it was recommended to the Sponsor that a repeat analysis of the high dose males should be performed using a greater sample size of cells.The additional assessment was performed on re-randomised slides from the high, vehicle and positive control group. Both analyses were performed by the same assessor and a total of 2000 polychromatic erythrocytes per mouse examined for micronuclei. The observed frequency of MN-PCE (0. 10%) in the high dose from this additional assessment was well within the normal negative response range. The vehicle (0.09%) and positive control (1.17 % ) groups showed MN-PCE frequencies which were similar to
the first assessment. No bone marrow toxicity was detected in the 2-ethylhexanoic acid exposed mice, as the PCE/NCE ratios were of a normal order for all 3 test material groups. It was concluded that 2-ethvlhexanoic acid did not induce micronuclei in bone marrow when given as maximum tolerated oral doses to male and female mice.

Conclusions:

Interpretation of results (migrated information): negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There is no reason to believe that the negative results would not be relevant to humans.

Justification for classification or non-classification

According to the study results on crude tall oil and 2 -ethylhexanoic acid, the substance requires no clasification, because the results are not sufficient for classification.