Fatty acids, tall-oil, reaction products with linseed oil, maleic anhydride and methanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic or clastogenic in required in vitro assays

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

guideline study under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver metabolic system

Test concentrations with justification for top dose:

5000, 1580, 500, 158 and 50.0 μg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other:

Details on test system and experimental conditions:

Solutions of the test item, as received, were prepared immediately before use in DMSO. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test item, i.e. the test item was weighed and the necessary volume of solvent was added in order to reach the required concentration.

An initial assay using the plate incorporation method was utilised. The assay was repeated using the pre-incubation method.

Rationale for test conditions:

Solubility of the test item was evaluated in a preliminary trial using DMSO. This solvent was selected since it is compatible with the survival of the
bacteria and the S9 metabolic activity. The test item was found to be soluble at 100 mg/mL.This result permitted a maximum concentration of 5000 μg/plate to be used in the toxicity test.

Evaluation criteria:

Criteria for positive: A two-fold (or more) increase in mean revertant numbers must be observed at two consecutive doses or at the highest practicable dose only. Also, there must be evidence of a dose-response relationship.

Statistics:

Mean plus standard deviation, according to accepted methods.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

A slight decrease in the number of revertant colonies was observed at the

two highest dose levels tested for TA100 tester strain, in the absence and

presence of S9 metabolism. Otherwise, it did not cause reverse mutation.

This does not impact the conclusion that the substance is not mutagenic this assay.

Conclusions:

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium TA 1535, TA 1537, TA 98 or TA 100 or Escherichia coli WP2 uvrA. in the absence or presence of S9 metabolism, under the reported standard experimental conditions of this OECD 471 assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

guideline study under GLP

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

other: mammalian mutagenicity assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79-4, purchased from CLS, Eppelheim, Germany. Cleansed and screened for micoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

S9 enzyme fraction from liver from male rats treated with Arochlor 1254.

Test concentrations with justification for top dose:

0.63 mg/mL, 0.31 mg/mL, 0.16 mg/mL, 0.08 mg/mL, 0.04 mg/mL, 0.02 mg/mL.

Precipitation of the test item was noted at the test item concentrations 2.5 mg/mL and 1.25 mg/mL in the pre-test as well as at the test item concentration of 1.25 mg/mL in the second experiment I.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

The assay was performed in a pre-test and three independent experiments: (experiment I (+S9 and -S9 with an exposure time of 4 h), repetition of experiment I (+S9) and experiment II (-S9 with an exposure time of 24 h)). The pre-test was done to detect a potential cytotoxic effect of the test item.

Start of the experimental phases: first experiment I: 18. Jul. 2016; second experiment I: 08. Aug. 2016; experiment II: 10. Aug. 2016.

The experimental performance in experiment I and II are identical except the treatment period with the test item. In experiment I the test item is incubated for 4 hours (with and without S9) and the cells are afterwards washed two times with PBS Dulbecco (2.5 % HS). In experiment II the incubation time with the test item is 24 hours (without S9) and is not followed by a washing step.

First approximately 106 cells per 10 cm culture dish and approximately 500 cells per 6 cm culture dish were seeded per tested concentration as well as for the solvent and positive controls and incubated for 24 ± 2 hours. The incubation conditions during the whole assay were 37.0° at 1.5 °C in 5 ± 0.5 % CO2.

Afterwards the cells were treated with the test item. Each concentration was prepared in duplicates. After the treatment period (experiment I +S9 and –S9: 4 h; experiment II -S9: 24 h) the cells were washed with PBS Dulbecco (2.5 % HS) twice (not in experiment II –S9). Fresh complete culture medium (5 % HS) was added to the cells before the following incubation. The further implementation of the experiment was divided into the determination of the survival as well as the viability and the mutagenicity.

For the determination of a cytotoxic effect of the test item, the survival of the cells was measured. For this purpose, the cells in the 6 cm dishes were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution after a 7 day-incubation time. The colonies were counted and the cloning efficiency (absolute and relative) was calculated.

For the determination of the second part of the experiment (viability and mutagenicity), the cells in the 10 cm culture dishes were counted and adjusted to 1 * 106 cells per 10 cm cul- ture dish after an incubation time of 68.5 to 72 hours. Subsequently, the cells were incu- bated further for 96 to 98.75 hours. Except in the first experiment I (see deviations from the study plan, page 28), the total expression time was at least 168 h. Afterwards the cells were counted again and seeded into 10 cm culture dishes (5 * 105 cells) for the evaluation of the mutagenicity in selection medium containing 6-TG and into 6 cm culture dishes (500 cells) for the evaluation of the viability in complete culture medium. Both plates were incubated for further 7 days. After this incubation time the cell colonies were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution and counted for the calculation of the cloning efficiency II and the mutation frequency.

Evaluation criteria:

Assay acceptability
1. the mutant frequency found in the solvent controls is within the laboratory historical control data range
2. the positive control substances shows a significant increase (p < 0.05) in mutant frequency and lies in the range of the historical data.
3. two experimental conditions (+S9 and -S9) are tested.
4. adequate number of cells (spontaneous MF is 5 - 20 * 10-6) and concentrations (minimum of 4) are analysable.
5. the criteria for the selection of top concentration are consistent with the protocol.

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non- mutagenic in this system.

A positive response (classified as mutagenic) is described as follows:
• it reproducibly induces a mutation frequency that is significantly above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
• there is a reproducible concentration-related increase of the mutation frequency.
• if any of the results are outside the historical data of the negative/solvent control.

However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance of a low spontaneous mutation rate within the laboratories historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No increase in mutant frequency was seen with exposure to the test item. Cytotoxicity was not observed in the first experiment in the main test (4 h treatment time, 0.63 mg/ml). As this finding was different from that of the pre-test, an experiment with metabolic activation was repeated and the result did not show actual toxicity but did show a strong cytotoxic tendency, with RS values of 24 -29%. These differences from the pre-test values suggest that the test material may not be fully solubilised in the vehicle. Precipitation of the test item was visible at the next highest dose level. However, the results of experiments 1 and 2 (exposure time of 24 h and without S9) were consistent, and the substance was determined to be non-cytotoxic.

Dose ranges: Experiment 1: 0.02 -0.63 mg/ml.

Repeat Experiment 1: 0.04 -1.25 mg/ml

Experiment 2: 0.02 -0.63 mg/ml

Conclusions:

The test item did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation, in this guideline OECD 476 assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

guideline study under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

human primary lymphocytes

Cytokinesis block (if used):

colchicine

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from liver of rats treated with Arochlor 1254

Test concentrations with justification for top dose:

In experiment one, the concentrations were 40 mg/ml, 30 mg/ml, 20 mg/ml, 10 mg/ml, and 5 mg/ml.
In experiment two, the concentrations were 40 mg/ml, 35 mg/ml, 30 mg/ml, 20 mg/ml, 10 mg/ml, and 5 mg/ml.

According to OECD TG 487, the maximum concentration of the test item should be 2 μl/mL, 2 mg/mL or 10mM, whichever is the lowest. When cytotoxicity occurs, the highest concentration should aim to produce 55 ± 5% cytotoxicity. On the basis of the data found in the pre-experiment, 5 concentrations of the test data were used in experiment 1 and tested with and without metabolic activation.

Vehicle / solvent:

DMSO for test material, 0.9% NaCl for positive controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other:

Details on test system and experimental conditions:

The blood cultures were set up in defined time intervals within 24 h after collection in sterile culture vessels for cell proliferation.
In the pre-experiment and in experiment I with and without metabolic activation, after the initial period of incubation and cell proliferation, the blood cultures were centrifuged (10min, 500*g), the supernatant was discarded and the cells were resuspended in serum free RPMI 1640.Solvent control, positive control or test item was added.
In experiment II without metabolic activation, 48h after seeding, the blood cultures were centrifuged (10min, 500*g). The cell pellet was resuspended in complete culture medium RPMI 1640, cytochalasin B (final concentration 6 μg/mL) and solvent control,test item or positive control was added.

Rationale for test conditions:

As the test item showed complete cytotoxicity(except 0.16 mg/mL with metabolic activation), experiment I was performed with a different range of concentrations.

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
1. the mutant frequency found in the solvent controls falls within the laboratory historical control data range
2. the positive control substances must produce a significant increase (p < 0.05) in mutant frequency and lies in the range of the historical data.
3. two experimental conditions (+S9 and -S9) are tested.
4. adequate number of cells (spontaneous MF is 5 - 20 * 10-6) and concentrations (mini- mum of 4) are analysable.
5. the criteria for the selection of top concentration are consistent with those described in the

The test item is considered to have no genotoxic effects if:
• Neither a statistically significant nor a concentration-related increase of the number of micronucleate cells in the evaluated test concentrations is observed.
• The obtained results lie within the range of the historical laboratory control data for solvent controls.
The test item is considered to have genotoxic effects if:
• At least one test concentration shows a statistically significant increase of micronucleate cells compared to the concurrent solvent control.
• In at least one experimental condition a dose-related increase of micronucleate cells can be observed.
• Any of the results lies outside the range of the historical laboratory control data for solvent controls.

Statistics:

The number of binucleated cells with micronuclei in each treatment group was compared with the solvent control. Statistical significance was tested using Fisher’s exact test at the 5 % level (p ≤ 0.05)

Key result

Species / strain:

lymphocytes: primary human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No increase in micronucleate cells was observed in lymphocytes treated with the test item.
In the pre-experiment, all concentrations caused cytotoxicity except in 0.16 mg/mL. No precipitation was observed, but moderate to strong haemolysis was seen. Therefore, only the positive and solvent controls were scored. Lower concentrations were chosen for the main study.
In experiment 1, only the highest concentration showed complete cytotoxicity with and without metabolic activation. The other concentrations showed little or no cytotoxicity.
In experiment 2, no relevant cytotoxicity was observed, only a slight decrease in growth at the highest concentration.

Results of Experiment 1:

Treatment	Aver- age CBPI	Cyto- toxicity (%)	Total No. of BINC examined	Total No. of MBNC	% MBNC
exposure period 4 h without S9 mix
Solvent control DMSO 0.5% v/v	1.858	--	2020	7	0.35
Solvent control 0.9% NaCl 0.5% v/v	1.772	--	2011	5	0.25
Positive control MMC 0.3 µg/mL	1.686	4.8	2010	50	2.49**
Test item 0.15 mg/mL	1.549	16.6	2002	11	0.55
Test item 0.10 mg/mL	1.594	14.2	2014	10	0.50
Test item 0.05 mg/mL	1.777	4.4	2006	7	0.35
exposure period 4 h with S9 mix
Solvent control DMSO 0.5% v/v	1.640	--	2009	5	0.25
Solvent control 0.9% NaCl 0.5% v/v	1.680	--	2006	5	0.25
Positive control CPA 30 µg/mL	1.169	30.4	2004	56	2.79**
Test item 0.20 mg/mL	1.389	15.3	2008	4	0.20
Test item 0.15 mg/ml	1.479	9.8	2014	4	0.20
Test item 0.10 mg/mL	1.398	14.8	2007	8	0.40

BINC binucleated cells

MBNC binuceated cells with micronuclei

In experiment 1 without and with metabolic activation, complete cytotoxicity was observed at the highest chosen concentration (without metabolic activation, at 0.20 mg/mL; with metabolic activation, at 0.30 mg/mL) of the test item, the remaining concentrations showed only a small or no (without S9, at 0.05 mg/mL) cytotoxic effect.

No relevant increase of the number of binucleated cells with micronuclei was detected at the evaluated concentrations and a 2nd experiment (experiment 2 without metabolic activation, extended exposure) was performed.

Results of Experiment 2:

Treatment	Aver- age CBPI	Cyto- toxicity (%)	Total No. of BINC examined	Total No. of MBNC	% MBNC
exposure period 23 h without S9 mix
Solvent control DMSO 0.5% v/v	1.749	--	2018	14	0.69
Solvent control 0.9% NaCl 0.5% v/v	1.722	--	2018	7	0.35
Positive control MMC 0.3 µg/mL	1.368	20.5	2002	76	3.80**
Positive control Colchicine 0.03 µg/mL	1.074	37.6	1399	101	7.22**
Test item 0.20 mg/mL	1.425	18.6	2001	7	0.35
Test item 0.175 mg/mL	1.561	10.8	2000	10	0.50
Test item 0.15 mg/mL	1.635	6.6	2001	7	0.35

BINC binucleated cells

MBNC binuceated cells with micronuclei

In experiment 2, no relevant cytotoxicity was observed, only a slight decrease of growth at the highest concentration (0.20 mg/mL). No relevant increase of the number of binucleated cells with micronuclei was detected at the evaluated concentrations.

Conclusions:

The test item is considered nongenotoxic under the conditions of this in vitro micronucleus assay (OECD 487) in primary human lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

All in vitro tests for mutagenicity and genotoxicity are negative (non-genotoxic).

Justification for classification or non-classification

Experimental data on the test material on mutagenesis and genotoxicity do not meet the criteria for classification in Regulation EC No. 1272/2008.