Fatty acids, tall-oil, reaction products with acrylic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (Ames test equivalent or similar to OECD471): negative with and without metabolic activation Cytogenicity in mammalian cells (Chromosome Aberration Assay according to OECD473): negative with and without metabolic activation Gene mutation in mammalian cells (Mouse Lymphoma Assay according to OECD476): negative with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

not relevant

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12 nutrient medium supplemented with 10% heat-inactivated fetal bovine serum (HIFBS) and 2 mM L-glutamine (stock medium) and medium containing 10% HIFBS, 2 mM L-glutamine, 50 units/mL of penicillin and 50 ug/mL of streptomycin (test medium).
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction and "CORE" (1:4)."CORE" = 24 mg NADP and 45 mg DL-isocitric acid per mL in deionized, distilled water

Test concentrations with justification for top dose:

Without metabolic activation: 150, 100, 75, 50, 25 and 13 ug/mL
With metabolic activation: 100, 75, 50, 25, 13 and 6.3 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: easily miscible

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Without activation: 16 hr
With activation: 2 hr
- Fixation time (start of exposure up to fixation or harvest of cells): 15.25 hours until harvested

SPINDLE INHIBITOR (cytogenetic assays): Colcmid (0.1 µg/mL), last 2 hr of incubation
STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: 4 replicate cultures (2 for cytoxicity determination in the Parallel Toxicity Test, and 2 for the evaluation of induced chromosome aberration)

NUMBER OF CELLS EVALUATED: 100 metaphases per dose

DETERMINATION OF CYTOTOXICITY
- Method: A range-finding test at the maximum concentration of 5000 µg/mL and nine lower concentrations was performed to determine the doses for the main test. Cytotoxicity was evaluated on the basis of Relative Cell Growth (RCG). Furthermore, the Average Generation Time (AGT) was determined to fix the harvest time for the main test.

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: yes

OTHER: Validity: the assay was considered valid based on:
1 - Negative control: the number of cells with aberrations (excl. gaps and endoreplications) should not exceed 6%
2 - At least 25% of scored cells in the positive control should show one or more chomosome aberrations
3 - At least one of the scored test doses should show more than 25% reduction in the RCG

Evaluation criteria:

A result was considered a positive response if:
1) The test article showed a positive dose-response trend and a statistically significant increase over that of the solvent controls in the number of cells with aberrations at one or more dose levels.
2) In the event there is no positive dose-response trend, at least two consecutive test doses should show a statistically significant increase in the number of cells with chromosome aberrations.

The test substance was considered to have caused an equivocal response if:
1) One of the test doses shows a statistically significant increase in the number of cells with aberrations without an accompanying positive dose-response trend.
2) The test article shows a statistically significant dose-response trend, but none of dose levels shows a significant increase in the number of cells with aberrations.

The test article was considered to have caused a negative reponse if no indication of a positive dose response was observed and none of the test doses showed a statistically significant increase in the percentage aberrant cells.

Statistics:

A statistical analysis was not performed for the test doses since the percentage of cells with aberrations in the test dose was lower than in the solvent control both with and without metabolic activation.

The data for the positive controls were analyzed using a Chi-square test (P ≤ 0.05 was considered significant).

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Cytotoxic from a dose of 500 ug/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: Test doses showed an osmolality value below 427
- Water solubility: formed a turbid suspension with water
- Precipitation: Precepitation was only observed at concentrations of 5000, 1000 and 500 ug/mL, not in the test doses.
- Other confounding effects: not relevant

RANGE-FINDING/SCREENING STUDIES: Calls did not survive at concentrations >500 µg/mL both with and without metabolic activation. The highest dose at which cells did not survive was 100 µg/mL, in which RCG was reduced to 54% without metabolic activation and 13% with metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: no information available

ADDITIONAL INFORMATION ON CYTOTOXICITY: no information available

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Percentage of cells with Aberrations without metabolic activation:

	Solvent control	Positive control	Negative control	Tested concentrations
				100µg/mL	75µg/mL	50µg/mL	25µg/ml
% of cells with aberrations	4.0	58	4.0	3.0	3.0	4.0	1.0
P-value	-	0.000	-	-	-	-	-

 

Percentage of cells with Aberrations with metabolic activation:

	Solvent control	Positive control	Negative control	Tested concentrations
				75µg/mL	50µg/mL	25µg/mL	13µg/ml
% of cells with aberrations	3.0	29	3.0	2.0	1.0	2.0	1.0
P-value	-	0.000	-	-	-	-	-

 

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

In an in vitro Chromosome Aberration assay, Diacid 1550 did not cause a significant increase in the number of cells with aberrations or a positive dose response trend for concentrations of 6.3 to 100 ug/mL in Chinese Hamster Ovary Cells with and without metabolic activitation under the conditions of this test.

Executive summary:

In this study the ability of Diacid 1550 to induce chromosome aberrations in Chinese Hamster Ovary Cells was investigated. The study procedures used in this study were according to OECD Guideline 473 and the study was performed under GLP conditions..

Prior to the Chromosome Aberration assay, a miscibility test, osmolality test and a range finding test were performed in order to determine suitable doses for the main test.

In the Chromosome Aberration assay, concentrations of 150, 100, 75, 50 25, and 13 ug/mL were used without metabolic activation, whereas concentrations of 100, 75, 50, 25, 13 and 6.3 ug/mL were used with metabolic activation. Cells were exposed to the test substance for 16 hours without metabolic activation and for 2 hours with metabolic activation. After harvesting, cells were air dried and stained in Giemsa stain. A total of 100 metaphases were scored per dose for chromosome aberrations.

Diacid 1550 did not cause a significant increase in the number of cells with aberrations or a positive dose response trend for concentrations of 6.3 to 100 ug/mL in Chinese Hamster Ovary Cells with and without metabolic activitation under the conditions of this test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April 1991 to 28 May 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Ames et al. (1975)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

His-gene (HisG 46, HisC 3076, HisD 3052)

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver S9

Test concentrations with justification for top dose:

2.5 and 8 µL of a 10 mg/L solution
2.5, 8, and 25 µL of a 100 mg/L

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: 2-anthramine, N-methyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 ± 4 hours

SELECTION AGENT (mutation assays): Agar containing Histidine

NUMBER OF REPLICATIONS: experiments performed in triplicate

NUMBER OF CELLS EVALUATED: no data available

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (number of revertant colonies per plate) in TA100

Evaluation criteria:

The average number of revertant colonies (± the standard deviation) per plate is tabulated. A positive reponse is indicated if a sample substance produces a consistent bacterial response which is two times that of the solvent or spontaneous reversions for TA 98 and TA 100. For the strains TA 15335, TA 1537 and TA 1538, a sample producing a consistent bacterial reponse three times that of the solvent or spontaneous control is indicated as positive.

Statistics:

No information available

Species / strain:

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

Remarks:

100 mg/mL induced cytotoxicity in TA 100

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: sample was not soluble above 100 mg/mL in dimethylsulfoxide

RANGE-FINDING/SCREENING STUDIES: Sample was diluted in dimethylsulfoxide and tested for cytoxicity in TA 100; 100 mg/mL induced cytotoxicity

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Average test results per strain ± standard deviation with activation:

Tester Strain	Solvent control	Positive control	10 mg/mL		100 mg/mL
			2.5µL	8µL	2.5µL	8µl	25µl
TA 98	28±2	3842±935	28±5	23±6	28±6	30±8	28±6
TA 100	88±3	2894±176	82±12	75±15	56±9	61±16	53±13
TA 1535	13±1	589±200	14±2	8±3	8±2	11±6	11±3
TA 1537	15±2	135±32	14±4	19±0	31±5	14±6	2±1
TA 1538	28±3	461±254	24±3	16±5	24±3	20±5	41±25

 

Average test results per strain ± standard deviation without activation:

Tester Strain	Solvent control	Positive control	10 mg/mL		100 mg/mL
			2.5µL	8µL	2.5µL	8µl	25µl
TA 98	14±5	1443±93	10±3	16±3	11±4	11±4	11±5
TA 100	43±2	2764±79	33±7	29±4	32±10	32±10	27±6
TA 1535	32±4	1386±33	26±6	20±3	4±3	5±2	8±5
TA 1537	38±16	1973±606	42±7	19±6	10±3	4±2	3±2
TA 1538	28±20	1024±213	6±1	4±2	4±2	2±2	2±1

 

Conclusions:

Interpretation of results (migrated information):
negative no mutagenicity with and without activation

In this Ames test, Diacid 1550 did not show any mutagenic activity in the Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 with and without activitation under the conditions of this test.

Executive summary:

In this study the ability of Diacid 1550 to induce mutations in the Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 was investigated. The study procedures used in this study were based on the Ames Test as described by Ames et al. (1975).

The samples were diluted in dimethylsulfoxide and were incubated on agar plates for 48 ± 4 hours at 37°C (plate incorporation assay). After incubation, the agar plates were examined for the average number of revertant colonies. Prior to the Ames test, a cytoxicity test was performed in TA100 restulting in a cytoxic dose of 100 mg/mL.

Diacid 1550 dit not show any mutagenic activity up to limit concentrations in the Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 with and without activitation under the conditions of this test.

Reference 3

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:

The experimental phases of the study were performed between 03 April 2012 and 27 June 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Remarks:

Date GLP of inspection: 19 - 21 July 2011 Date of signature on GLP form: 31 August 2011

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
RPMI 1640

- Properly maintained:
yes

- Periodically checked for Mycoplasma contamination:
yes

- Periodically checked for karyotype stability:
no

- Periodically "cleansed" against high spontaneous background:
yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch BCBG1395V 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 A0302605 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
Solvent (DMSO) treatment groups were used as the vehicle controls.

- Justification for choice of solvent/vehicle:
Suitable for dosing at the required concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

This study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al, 1980; Jotz and Mitchell, 1981).

The method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances. The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).

Evaluation criteria:

Please see "Any other information on materials and methods incl. tables" section.

Statistics:

Please see "Any other information on materials and methods incl. tables" section.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test

The dose range of the test item used in the preliminary toxicity test was 19.53 to 5000 µg/ml. In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult. A greasy / oily precipitate of the test item was observed at and above 625 µg/ml in all three of the exposure groups. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

There was once again evidence of marked dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (Tables 3 and 6). The levels of toxicity observed were very similar to those of the preliminary toxicity test. There was evidence of a marked reduction in viability (%V) in the absence of metabolic activation, therefore indicating that residual toxicity had occurred. However, it should be noted that the reduction was only observed at a dose level that had been excluded from the statistical analysis due to toxicity. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in both the absence and presence of metabolic activation. In both the absence and presence of metabolic activation, a dose level that exceeded the usual upper limit of acceptable toxicity was plated for viability and 5 TFT resistance as sufficient cells were available at the time of plating. However, whilst excluded from the statistical analysis due to the toxicity exceeding the upper limit of 90%, there was no evidence of any increase in mutant frequency at these, or any of the other dose levels. The excessive toxicity observed at 90 µg/ml in both the absence and presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Table 3 and Table 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation. Precipitate of the test item was not observed at any of the dose levels.

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

As was seen previously, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (Tables 9 and 12). There was no evidence of any reductions in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at and above 70 µg/ml in the absence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item. It should be noted that the lowering of the S9 concentration to 1% in this second experiment resulted in lower levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2% metabolic activation in the Preliminary Toxicity Test and Experiment 1.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation. Precipitate of the test item was not observed at any of the dose levels.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Remarks on result:

other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Remarks:

Migrated from field 'Test system'.

Please see Attached "Tables 1 to 13"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Conclusions:

Interpretation of results (migrated information):
other: Non-mutagenic

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

Introduction. 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Thethod was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In VitroMammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

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 item at 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 item at up to ten 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 item was selected following the results of a preliminary toxicity test and was 7.5 to 90 µg/ml in both the absence and presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 1.25 to 80 µg/ml in the absence of metabolic activation, and 5 to 80 µg/ml in the presence of metabolic activation.

Results. 

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of test item 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 items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion. 

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

Three in vitro gene mutation and cytogenicity tests are available.

In the Ames test, which was performed equivalent or similar to OECD471 (according to Ames et al., 1975), the ability of Diacid 1550 to induce mutations in Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 was investigated. The samples were diluted in dimethylsulfoxide and were incubated on agar plates for 48 ± 4 hours at 37°C (plate incorporation assay). After incubation, the agar plates were examined for the average number of revertant colonies. Prior to the Ames test, a cytoxicity test was performed in TA100 restulting in a cytoxic dose of 100 mg/mL.

Diacid 1550 dit not show any mutagenic activity up to limit concentrations in the Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 with and without activitation under the conditions of this test.

In the Chromosome Aberration assy in Chinese Hamster Ovary Cells (according to OECD473 and under GLP-conditions), the ability of Diacid 1550 to induce chromosome aberrations in mammalian cells was investigated. Prior to the Chromosome Aberration assay, a miscibility test, osmolality test and a range finding test were performed in order to determine suitable doses for the main test.

In the Chromosome Aberration assay, concentrations of 150, 100, 75, 50 25, and 13 ug/mL were used without metabolic activation, whereas concentrations of 100, 75, 50, 25, 13 and 6.3 ug/mL were used with metabolic activation. Cells were exposed to the test substance for 16 hours without metabolic activation and for 2 hours with metabolic activation. After harvesting, cells were air dried and stained in Giemsa stain. A total of 100 metaphases were scored per dose for chromosome aberrations.

Diacid 1550 did not cause a significant increase in the number of cells with aberrations or a positive dose response trend for concentrations of 6.3 to 100 ug/mL in Chinese Hamster Ovary Cells with and without metabolic activitation under the conditions of this test.

In the Mouse Lymphoma Assay (MLA) dose ranges of 7.5 - 90 ug/mL (experiment 1) and 1.25 -80 ug/mL (experiment 2) were tested in the presence and absence of metabolic activation. The maximum dose levels used in the assay were limited by test-item induced toxicity. Precipitation of test-item was not observed at any of the dose levels. Mutation frequency values of controls were in the normal range and positive control substances showed marked increases in mutation frequencies, indicating that the test system worked properly. Phosphonic acid did not induce any toxicologically significant dose-related increases in mutation frequency at any dose level, either with or without metabolic acitivation, in either the first or the second experiment.

It is concluded that phosphonic acid is not mutagenic to L5178Y cells under the conditions of the test.

Justification for selection of genetic toxicity endpoint
No mutagenic effects were observed in three in vitro tests.

Justification for classification or non-classification

Based on the available information, Diacid 1550 does not need to classified as mutagenic in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2002/EC.