Tall oil, maleated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

EnvaMul 600 was evaluated for its potential to induce point mutations in Salmonella typhimurium strains, TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA. The experimental design followed the “OECD Guideline for Testing of Chemicals - 471, Bacterial Reverse Mutation Test” (OECD, 1997). EnvaMul 600 was not mutagenic to S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain, WP2 uvrA, under the test conditions.

A gene mutation test in vitro has been performed on mammalian cells ( L5178Y Mouse Lymphoma Cells) according to OECD guideline 490. EnvaMul 600 is not mutagenic in the TK (thymidine kinase) mutation test system.

Furthermore, the substance was investigated in an in vitro chromosome aberration study according to OECD 473. In conclusion, this test showed that EnvaMul 600 is not clastogenic in human lymphocytes under the experimental conditions described in this report. 

Thus, three independent in vitro tests did not show any indications of mutagenicity of the test substance.

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:

Study Initiation:October 8, 2013 Experimental Start:October 9, 2013 Experimental Completion: November 1, 2013 Study Completion:February 24, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other:

Remarks:

histidine auxotrophs

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other:

Remarks:

tryptophan-deficient

Metabolic activation:

with and without

Metabolic activation system:

Rat S9

Test concentrations with justification for top dose:

Based on preliminary testing, the main study doses were designed to reach the test item’s solubility limit of approximately 2 mg/plate in the test system.

For the plate incorporation test, EnvaMulTM 600 was tested at a maximum exposure concentration of 2.0 mg/plate, in the presence and absence of S9.

In the pre-incubation test, EnvaMulTM 600 was tested at a maximum concentration of 2.0 mg/plate for most tester strains with the exception of TA1537 without S9 where the maximum analyzable concentration was 0.25 mg/plate, due to test item toxicity.

Vehicle / solvent:

DMSO
In preliminary testing, EnvaMulTM 600 was determined to be insoluble in water. DMSO was determined to be a suitable solvent as EnvaMulTM 600 was soluble at 20 mg/mL.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

cyclophosphamide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Method of application: plate incorporation experiment and pre-incubation test.

Evaluation criteria:

Negative result (no evidence of genotoxicity) is concluded if there is no substantial increase in the number of colonies per plate; i.e. the results do not exceed the upper 98 percentile limit of the historical solvent/negative control range. A negative result indicates that the test item is non-mutagenic in S. typhimurium or E. coli.
Positive result will be considered positive when there is a significant increase in the number of colonies per plate in comparison to the concurrent negative control and a concentration-related increase over the exposure range tested. A positive result indicates that the test item induces point mutations in S. typhimurium or E. coli.
Equivocal result: If no definite judgment can be made to fit the above criteria, even after repeated experiments, then the result will be described as equivocal. An equivocal result indicates that a definitive result cannot be made by performing the bacterial reverse mutation assay under the conditions described in this study plan.

Statistics:

The numbers of revertant colonies per plate are represented as colony counts in Table 1 for the plate incorporation test and in Table 2 for the pre-incubation test. The tables include the individual plate counts, the means and standard deviations (S.D.). These means and standard deviations were plotted against the dose levels in Figures 1 to 10 for the plate incorporation test and Figures 11 to 20 for the preincubation test. All colony counts were compared to the historical data from this laboratory. The historical ranges in Appendix V are defined by the minimum and maximum mean colony counts. The 95% confidence intervals are also reported.
Statistical analysis was applied to the numbers suspected to be abnormally high or to have a dose-related increase in revertant counts. The colony counts were transformed (square root) to normalize the data prior to using the one-sided Dunnett’s test (Mahon,G.A.T., et al., 1989).

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The pre-incubation test resuts confirmed the plate incorporation test results as negative.

Conclusions:

Tall Oil, Maleated (EnvaMulTM 600) was not mutagenic to S. typhimurium strains, TA98, TA100, TA1535, TA1537, and E. coli strain, WP2 uvrA, under the conditions of the test.

Executive summary:

Tall Oil, Maleated (EnvaMulTM 600) was evaluated for its potential to induce point mutations in Salmonella typhimurium strains, TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA. The experimental design followed the “OECD Guideline for Testing of Chemicals - 471, Bacterial Reverse Mutation Test” (OECD, 1997).

In the plate incorporation experiment, Tall Oil, Maleated (EnvaMulTM 600) denoted as EnvaMulTM 600, was tested at a highest concentration of 2.0 mg/plate in the presence and absence of S9. At this level, slight precipitate was visible with a microscope. Precipitate was not observed for any lower concentrations. A clear pattern of toxicity was not observed for all conditions when compared to the concurrent negative controls. For all conditions, a normal background lawn was observed but for some conditions, a greater than 30% reduction in colony counts from the concurrent negative controls was observed. Thus, the test item was evaluated at the limit of solubility in the test system (OECD, 1997). Finally, for all strains and conditions, all 5 concentrations were analyzable for mutagenicity (OECD, 1997). For all conditions, the test item did not produce any statistically significant increases (p>0.01) in colony counts over the concurrent negative controls and a dose-response was not observed. A more sensitive pre-incubation test was designed to confirm the negative results of the plate incorporation test.

In the pre-incubation test, EnvaMulTM 600 was tested at a maximum concentration of 2.0 mg/plate for all tester strains; however, due to extreme toxicity at 0.5, 1.0, and 2.0 mg/plate, for TA1537 without S9, the maximum concentration evaluated for this strain was 0.25 mg/plate. Slight and moderate precipitate were observed under the microscope at 1.0 and 2.0 mg/plate without S9, respectively. Slight precipitate was observed with a microscope at 2.0 mg/plate with S9. Test item precipitate was not observed for all lower concentrations. For some conditions, a reduction of the background lawn and/or the number of colony counts was observed for at least the highest concentration. Only for TA1537 without S9 was toxicity clearly the limiting factor in the selection of concentrations tested. In this case, test item toxicity reduced the maximum analyzable concentration from 2.0 mg/plate to 0.25 mg/plate. Therefore, EnvaMulTM 600 was tested at the limit of solubility in the test system for most conditions with the exception of TA1537 without S9 where the test item was tested at the limit of toxicity (OECD, 1997). For all strains and conditions, at least 5 concentrations were analyzable for mutagenicity (OECD, 1997). For all conditions, the test item did not produce any statistically significant increases (p>0.01) in colony counts over the concurrent negative control and a dose-response was not observed. Therefore, the pre-incubation test was concluded as negative and confirmed the results of the plate incorporation test.

Thus, it was concluded that Tall Oil, Maleated (EnvaMulTM 600) was not mutagenic to S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain, WP2 uvrA, under the test conditions.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 27 Jun 2017, experimental completion date: 29 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/--3.7.2C mouse lymphoma cells

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 31.3 to 300 µg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test:
Without and with S9-mix: 20, 30, 40, 50, 75, 100 and 125 μg/mL exposure medium.
In the absence and presence of S9-mix, the dose levels of 150 to 250 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without and with S9-mix: 20, 30, 40, 50, 75, 100 and 125 μg/mL exposure medium.
To obtain more information about the possible mutagenicity of EnvaMul 600, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period.
Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 1.88, 3.75, 7.5, 15, 30, 40, 50, 60, 70, 80, 90 and 100 µg/mL exposure medium.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

Remarks:

dimethyl sulfoxide (DMSO), the vehicle of the test item

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Test System L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale Recommended test system in international guidelines (e.g. OECD, EC).
Source American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in liquid nitrogen (-196 °C). The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 10E6 cells/mL.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

valid

Positive controls validity:

valid

Conclusions:

In conclusion, EnvaMul 600 is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of EnvaMul 600 by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.  

The study procedures described in this report were based on the most recent OECD guideline. 

Batch HD0379UD11 of EnvaMul 600 was a viscous brown liquid. The vehicle of the test item was dimethyl sulfoxide.

In the first experiment, EnvaMul 600 was tested up to concentrations of 125 µg/mL in the absence and presence of S9-mix. The incubation time was 3 hours. Relative total growth (RTG) was 8 and 4% in the absence and presence of S9-mix, respectively. EnvaMul 600 did not precipitate in the culture medium at this dose level.

In the second experiment, EnvaMul 600 was tested up to concentrations of 90 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was 18%. EnvaMul 600 did not precipitate in the culture medium at this dose level.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. 

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, EnvaMul 600 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, EnvaMul 600 did not induce a significant increase in the mutation frequency.

In conclusion, EnvaMul 600 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Batch HD0379UD11

Target gene:

n/a

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines ( e.g. OECD).
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to
35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016) are presented below:
Dose-range finding study: age 31, AGT = 13.2 h
First cytogenetic assay: age 35, AGT = 15.2 h
Second cytogenetic assay: age 22, AGT = 13.7 h

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

colchicine

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate

Test concentrations with justification for top dose:

Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay:
Without and with S9-mix: 39, 156, 250 and 313 µg/mL culture medium (3 h exposure time, 24 h fixation time).

To obtain more information about the possible clastogenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to EnvaMul 600 in the absence of S9-mix for 24 or 48 hours. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 50, 125, 150, 175, 200, 225 and 250 µg/mL culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time).

Vehicle / solvent:

dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Evaluation criteria:

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations.

Statistics:

The positive control data have been analyzed by the Fisher’s exact test (one-sided, p <0.05).
All results have been calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
STATISTICAL ANALYSIS
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p <0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p <0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

lymphocytes:

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

Remarks on result:

other: Both in the absence and presence of S9-mix EnvaMul 600 did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

Mitotic Index of Human Lymphocyte Cultures Treated with EnvaMul 600 in the First Cytogenetic Assay

EnvaMul 600                                                Number of metaphases^a)

concentration (µg/ml)                                    Absolute               Number              Percentage
                                                                                              of cells scored     of control

Without metabolic activation (-S9-mix)

3 h exposure time, 24 h fixation time

Control^b)		45	-	61		1010	-	1011		100
39		41	-	57		1017	-	1005		92
156		40	-	46		1003	-	1015		81
250	^c)	26	-	30		1012	-	1008		53
313	^c)	13	-	19		1017	-	1007		30
MMC-C; 0.5 µg/ml		23	-	24		1000	-	1009		44
MMC-C; 0.75 µg/ml		19	-	17		1002	-	1005		34

 

With metabolic activation (+S9-mix)

3 h exposure time, 24 h fixation time

Control^b)		48	-	56		1003	-	1006		100
39		43	-	56		1008	-	1002		95
156		43	-	53		1004	-	1014		92
250	^c)	27	-	31		1000	-	1011		56
313	^c)	18	-	15		1013	-	1005		32
CP; 10 µg/ml		19	-	24		1009	-	1004		41

^a)   Duplicate cultures.

^b)    Dimethyl sulfoxide.

^c)   EnvaMul 600 precipitated in the culture medium.

Mitotic Index of Human Lymphocyte Cultures Treated with EnvaMul 600 in the Second Cytogenetic Assay

EnvaMul 600                                               Number of metaphases^a)

concentration (µg/ml)                                    Absolute             Number              Percentage
                                                                                                  of cells scored    of control

Without metabolic activation (-S9-mix)

24 h exposure time, 24 h fixation time

Control^b)		82	-	77		1000	-	1003		100
50		66	-	61		1000	-	1001		80
125		51	-	55		1007	-	1014		67
150		46	-	49		1007	-	1000		60
175		31	-	37		1003	-	1000		43
200		17	-	22		1000	-	1000		25
225	^c)	10	-	13		1002	-	1000		14
250	^c)	6	-	3		1001	-	1001		6
MMC-C; 0.2 µg/ml		25	-	26		1000	-	1000		32
MMC-C; 0.3 µg/ml		16	-	17		1000	-	1000		21

 

48 h exposure time, 48 h fixation time

Control^b)		66	-	67		1000	-	1000		100
50		52	-	45		1000	-	1000		73
125		41	-	43		1000	-	1000		63
150		34	-	32		1000	-	1003		50
175		25	-	23		1000	-	1003		36
200	^c)	15	-	12		1000	-	1000		20
225	^c)	6	-	8		1000	-	1000		11
250	^c)	4	-	2		1000	-	1000		5
MMC-C; 0.1 µg/ml		45	-	41		1002	-	1001		65
MMC-C; 0.15 µg/ml		37	-	40		1000	-	1003		58

^a)   Duplicate cultures.

^b)    Dimethyl sulfoxide

^c)   EnvaMul 600 precipitated in the culture medium.

Conclusions:

EnvaMul 600 is not clastogenic in human lymphocytes.

Executive summary:

The objective of this study was to evaluate EnvaMul 600 for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

The possible clastogenicity of EnvaMul 600 was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

Batch HD0379UD11 of the test item was a viscous brown liquid. The vehicle of the test item was dimethyl sulfoxide. 

In the first cytogenetic assay, EnvaMul 600 was tested up to 250 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. The test item precipitated in the culture medium at this dose level.

In the second cytogenetic assay, EnvaMul 600 was tested up to 175 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 150 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

EnvaMul 600 did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No biologically relevant effects of EnvaMul 600 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that EnvaMul 600 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. 

In conclusion, this test is valid and EnvaMul 600 is not clastogenic in human lymphocytes under the experimental conditions described in this report. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was tested in three independent in vitro mutagenicity studies and was found consistently negative for mutagenicity. Therefore, the substance does not require classification for mutagenicity according to CLP (Regulation EC No 1272/2008).