Esterification product of castor oil and tetrahydromethyl-1,3-isobenzofuranedione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

REACH_negative | with and without | S. typhimurium TA98, TA100, TA1535, TA1537, E. coli WP2uvrA | OECD 471 | #key study#

REACH_negative | with and without | human lymphocytes | OECD 473 | #key study#

REACH_negative | with and without | Chinese hamster lung fibroblasts (V79) | OECD 476 | #key study#

Link to relevant study records

Reference

Reference 1

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:

2017-09-07 to 2017-10-18

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 (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

0.01, 0.02, 0.05, 0.10, 0.15, 0.20, 0.25 and 0.5 mg/mL

Vehicle / solvent:

DMSO (1%; v/v)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure)
Expression time (cells in growth medium): 7 - 9 days
Selection time (if incubation with selection agent): approximately 9 - 11 days

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: one experiments with single exposure; cells were seeded in 5 cell culture petri dishes and evaluated
NUMBER OF CELLS SEEDED: 4 x 10^5 cells per petri dish
DETERMINATION OF CYTOTOXICITY: relative survival (RS)

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative control. Additionally, a dose-response realationship was determined in the X² test for trend.

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Precipitation

Precipitation of the test item was noted at concentrations of 0.25 mg/mL and higher without metabolic activation and with metabolic activation at concentrations of 0.2 mg/mL and higher.

Toxicity

A biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in experiment I with and without metabolic activation.

In experiment I, the relative survival was 66% (without metabolic activation) and 8% (with metabolic activation) for the highest tested concentration of 0.50 mg/mL.

Mutagenicity

In the experiment without and with metabolic activation all validity criteria were met. The mutant values of the negative controls fall within the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls are > 50%.

The positive controls, DMBA (1.0 μg/mL) and EMS (300 μg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems.

In the experiment without metabolic activation the mutant values of the negative controls, the solvent controls and all mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 7.8 - 39.7 mutants per 10E6 cells). The positive control EMS induced a distinct increase in mutant frequency with 240.5 mutants/10E6 cells.

The mutant frequencies of the negative controls were 21.5 and 20.1 mutants per 10E6 cells, of the solvent controls 30.4 and 15.6 mutants per 10E6 cells, respectively, and in the range of 9.9 to 31.4 mutants per 10E6 cells with the test item.

The highest mutant frequency was observed at a concentration of 0.25 mg/mL (31.4 mutants per 10E6 cells) with a relative survival of 42%.

The mutant frequencies induced by the test item did not show a biologically relevant increase. None of the observed mutant frequencies were statistically significantly increased over those of the solvent controls.

In the experiment with metabolic activation the mutant values of the negative controls, the solvent controls and all mutant values of the test item concentrations found were within or even lower than the historical control data of the test facility Eurofins Munich (about 9.2 – 38.8 mutants per 10E6 cells). The positive control DMBA induced a distinct increase in mutant frequency with 272.7 mutants/10E6 cells.

The mutant frequencies of the negative controls were 13.4 and 23.1 mutants per 10E6 cells, of the solvent controls 14.6 and 14.0 mutants per 10E6 cells, respectively, and in the range of 4.0 to 21.6 mutants per 10E6 cells with the test item, respectively.

The highest mutant frequency was observed at a concentration of 0.05 mg/mL (21.6 mutants per 10E6 cells) with a relative survival of 72%.

A statistical analysis displayed that one of the mutant frequencies was significantly increased over those of the solvent controls, but there was no evidence for a dose-response relationship. Therefore, this effect was considered as not biologically relevant.

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test item Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.

The selection of the concentrations was based on data from the pre-experiment. With and without metabolic activation were performed as a 4 h short-term exposure assay.

The test item was investigated at the following concentrations:

without metabolic activation: 0.05, 0.10, 0.15, 0.20 and 0.25 mg/mL

with metabolic activation: 0.01, 0.05, 0.10, 0.15, 0.20 and 0.25 mg/mL

Precipitation of the test item was noted at concentrations of 0.2 mg/mL (with metabolic activation) and 0.25 mg/mL (without metabolic activation).

Biologically relevant growth inhibition (relative survival < 70%) was observed in the experiment with and without metabolic activation. Without metabolic activation the relative survival was 66% and with metabolic activation 8% for the highest concentration (0.5 mg/mL).

In the experiments no biologically relevant increase of mutants was found after treatment with the test item without and with metabolic activation. All mutant values were within the historical data base of the test facility.

A statistical analysis displayed that one of the mutant frequencies was significantly increased over those of the solvent controls, but there was no evidence for a dose-response relationship.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Conclusion

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-29 to 2018-06-21

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

other: in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction, phenobarbital and β-naphthoflavone induced

Test concentrations with justification for top dose:

Pre-Experiment:
with and without metabolic activation: 2, 5, 10, 20, 40, 100, 250, 700, 2000 and 5000 µg/mL
Experiment I:
without metabolic activation: 10, 30, 50, 70, 100, 150, 200, 250 µg/mL
with metabolic activation: 1, 2, 5, 10, 30, 50, 70, 100, 150, 200 µg/mL
Experiment II without metabolic activation: 5, 10, 30, 50, 70, 100, 150, 200, 500 µg/mL

The following concentrations were evaluated for microscopic analysis:
Experiment I: without metabolic activation: 30, 70 and 150 µg/mL
with metabolic activation: 100, 150 and 200 µg/mL
Experiment II: without metabolic activation: 100, 200 and 500 µg/mL

Vehicle / solvent:

- Justification for choice of solvent/vehicle:
A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 5 mg/mL. Based on the results of the solubility test DMSO was used as solvent (1% DMSO, v/v final concentration). Different test item stock solutions in DMSO were prepared and added to cell culture medium. The test item precipitates at a concentration of 0.5 mg/mL and higher in cell culture medium. The pH was within the physiological range of 7.0±0.4 and for osmolality in the highest evaluated concentration of 500 µg/mL 464 mOsmol/kg was measured (solvent control: 471 mOsmol/kg). The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation [400 and 600 µg/mL]

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation [5.0 µg/mL]

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation)
24 hours (Experiment II without metabolic activation)

FIXATION INTERVAL:
24 hours (Main Experiment)

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 300 cells per concentration (150 cells per culture)
Exceptions:
Experiment I: positive controls (EMS: 130 cells, CPA: 65 cells) due to a clearly positive increase in chromosomal aberrations. In the experiment without metabolic activation, less than 300 cells were evaluated for chromosome aberrations for the concentrations 70 µg/mL (272 cells) as well as 150 µg/mL (183 cells) due to few cells on the slides because of cytotoxic effects. In the experiment with metabolic activation only 221 metaphases were evaluated for the concentration 100 µg/mL.
Experiment II: positive control EMS (100 cells) due to a clearly positive increase in chromosomal aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (MI) and proliferation index (PI)

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the 95% control limits of the historical negative control data.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the 95% control limits of the historical negative control data.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding solvent control. Aberrant cells without gaps were only used for the calculation. Gaps are recorded separately and reported but generally not included in the total aberration frequency calculation according to the guideline.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Precipitation

The test item was suspended in cell culture medium. Precipitate of the test item was noted in experiment I with metabolic activation at a concentration of 200 μg/mL and in experiment II without metabolic activation at a concentration of 500 μg/mL.

Toxicity (Relative Mitotic Index)

In experiment I without metabolic activation, a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted after incubation with the test item at concentrations of 30 μg/mL and higher tested concentrations. In experiment I with metabolic activation, a decrease of the relative mitotic index was noted at concentrations of 70 μg/mL (54% rel. MI) and 100 μg/mL (58% rel. MI). Both concentrations showed strong differences in the rel. mitotic index of each single culture. However, the next higher concentrations did not reveal cytotoxic effects and therefore no dose-dependency was observed. This led to the conclusion that the reduced mitotic indices were not related to cytotoxicity, but rather seemed to be an outlier.

In experiment II without metabolic activation, cytotoxic effects were noted at concentrations of 500 μg/mL (67% rel. MI).

Toxicity (Proliferation Index)

The BrdU-technique was used for determining the proliferation index to detect a possible effect on the proliferation rate after treatment with the test item and thus indicating cell cycle delay. In the experiment I, the values of the proliferation index of the negative and solvent controls were 1.34 and 1.33 (without metabolic activation) as well as 1.36 and 1.44 (with metabolic activation), respectively. The proliferation index of the highest dose groups evaluated were 1.19 (150 μg/mL, without metabolic activation) and 1.13 (200 μg/mL, with metabolic activation). In the experiment II, the values of the proliferation index of the negative and solvent controls were 1.34 and 1.50 (without metabolic activation). The proliferation index of the highest dose group evaluated without metabolic activation (500 μg/mL) was 1.54. A biologically relevant decrease of the proliferation index was indicated in experiment I with metabolic activation in the highest evaluated concentration compared to the solvent control.

Clastogenicity

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations for at least one of the dose groups, which is higher than the laboratory negative control range.

In experiment I without metabolic activation, the aberration rate of the negative control (2.0%), the solvent control (3.3%) and all evaluated concentrations were within the historical control data of the testing facility (-0.09 to 3.47% aberrant cells exclusive gaps).

In experiment I with metabolic activation, the aberration rates of the negative control (2.0%), the solvent control (2.7%) and the highest test item concentrations 150 μg/mL (1.7%) and 200 μg/mL (2.7%) were within the historical control data of the testing facility (-0.41 to 3.42% aberrant cells exclusive gaps). The lowest evaluated concentration of the test item was marginally above the historical control range with a mean value of 3.2% aberrant cells. As no statistical significance or dose-dependency was observed and the value was only slightly above the laboratory control range, this effect was not regarded as biologically relevant.

In experiment II without metabolic activation, the aberration rate of the negative control (2.3%), the solvent control (1.3%) and all dose groups treated with the test item (100 μg/mL: 1.3%, 200 μg/mL: 1.3% and 500 μg/mL: 2.7%) were within the historical control data of the testing facility (-0.57 to 3.16% aberrant cells exclusive gaps).

The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding solvent control in experiment I and II.

EMS (400 and 600 μg/mL) and CPA (5 μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the dose groups of the test item evaluated in experiment I and II.

The χ² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation.

Polyploid Cells

No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

	Dose group	Concen-tration [µg/mL]	Relative Mitotic Index [%]	Proliferation Index	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipi-tation (+/-)^a	Statistical Signifi-cance^b
					incl. Gaps	excl. Gaps
without 4 h treatment, 24 h preparation interval	C	0	107	1.34	5.0	2.0	-0.09 % - 3.47 % aberrant cells excl. gaps	-	-
	S	0	100	1.33	4.3	3.3		-	/
	2	30	52	/	3.7	3.3		-	-
	4	70	62	/	2.2	1.8		-	-
	6	150	47	1.19	3.8	2.2		-	-
	EMS	600	50	/	13.8	13.8		-	+
with 4 h treatment, 24 h preparation interval	C	0	128	1.36	3.0	2.0	-0.41 % - 3.42% aberrant cells excl. gaps	-	-
	S	0	100	1.44	3.7	2.7		-	/
	8	100	58	/	4.5	3.2		-	-
	9	150	74	/	2.3	1.7		-	-
	10	200	129	1.13	5.7	2.7		+	-
	CPA	5	42	/	16.9	16.9		-	+

	Dose group	Concen-tration [µg/mL]	Relative Mitotic Index [%]	Proliferation Index	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipi-tation (+/-)^a	Statistical Signifi-cance^b
					incl. Gaps	excl. Gaps
without 24 h treatment, 24 h preparation interval	C	0	134	1.34	3.3	2.3	-0.57 % - 3.16 % aberrant cells excl. gaps	-	-
	S	0	100	1.50	2.3	1.3		-	/
	6	100	104	/	2.0	1.3		-	-
	8	200	76	/	2.7	1.3		-	-
	9	500	67	1.54	3.3	2.7		+	-
	EMS	400	20	/	33.0	30.0		-	+

The mitotic index was determined in 1000 cells per culture of each test group. The relative values of the mitotic index are related to the solvent controls. 

C:       Negative Control (Culture Medium)

S:        Solvent Control (1% DMSO; v/v)

EMS: Ethylmethanesulfonate

CPA:  Cyclophosphamide

a:        - without precipitation; + with precipitation

b:        statistical significant increase compared to solvent controls (Fisher’s exact test, p< 0.05),
+ significant; - not significant

Conclusions:

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters did not induce structural chromosomal aberrations in human lymphocyte cells.
Therefore, Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

A chromosome aberration assay was carried out in order to investigate a possible potential of Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters to induce structural chromosome aberrations in human lymphocytes.

Precipitate of the test item was noted in experiment I with metabolic activation at a concentration of 200 μg/mL and in experiment II without metabolic activation at a concentration of 500 μg/mL.

In experiment I without metabolic activation, a biologically relevant decrease of the relative mitotic index was noted after incubation with the test item at concentrations of 30 μg/mL and higher.

In experiment I with metabolic activation, a decrease of the relative mitotic index was noted at concentrations of 70 μg/mL and 100 μg/mL. However, the next higher concentrations did not reveal cytotoxic effects. Therefore the reduced mitotic indices did not seem to be related to cytotoxicity.

In experiment II without metabolic activation, cytotoxic effects were noted at a concentration of 500 μg/mL.

A biologically relevant decrease of the proliferation index was indicated in experiment I with metabolic activation in the highest evaluated concentration compared to the solvent control.

In both experiments, neither a biologically relevant increase of the aberration rates nor an increase in the frequencies of polyploid cells was noted after treatment with the test item without and with metabolic activation.

The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the dose groups of the test item evaluated in experiment I and II.

The χ² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation.

EMS (400 and 600 μg/mL) and CPA (5 μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for In vitro Mammalian Chromosome Aberration Test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

hisC3076 (frameshift): S. typhimurium TA1537
hisD3052/R-factor (frameshift): S. typhimurium TA98
hisG46 (base-pair substitutions): S. typhimurium TA1535
hisG46/R-factor (base-pair substitutions): S. typhimurium TA100
WP2uvrA (base-pair substitutions): E.coli

R-factor: plasmid pKM101 (increases error-prone DNA repair)

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537, Escherichia coli WP2uvrA

Additional strain / cell type characteristics:

other: each Salmonella strain, additional mutations: rfa, gal, chl, bio, uvrB

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix (prepared from adult male Wistar rats, liver induced by phenobarbital and ß-naphthoflavone)

Test concentrations with justification for top dose:

1st experiment (dose range finding): 100, 333, 1000, 3330 and 5000 μg/plate; for TA100 and WP2uvrA additionally 3, 10, 33 μg/plate
2nd experiment: 100, 333, 1000, 3330 and 5000 μg/plate

Vehicle / solvent:

The test substance was dissolved in dimethyl sulfoxide. The stock solutions were treated with ultrasonic waves until the test substance had completely dissolved. Test substance concentrations were used within 15 minutes to 2 hours after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 4-nitroquinoline N-oxide (-S9); 2-aminoanthracene (+S9)

Details on test system and experimental conditions:

DOSE RANGE FINDING TEST
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of [Substance] used in the subsequent mutation assay was 5 mg/plate.

MUTATION ASSAY
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10E9cells/mlL of one of the tester strains, 0.1 mL of a dilution of the test substance in dimethyl sulfoxide and eilher 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 h. After this period revertant colonies (histidine independent (His +) for Salmonella typhimurium bacteria and tryptophan independent (Trp +) for Escherichia co/i were counted.

Evaluation criteria:

Acceptability of the assay
The assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range lor each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535. TA1537, TA98 or WP2uvrA is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537, Escherichia coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Precipitation on the plates was observed at the start of the incubation period at concentrations of 1000 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period.

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within the laboratory historical control date ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurlum reverse mutation assay and in the Escherlchla coll reverse mutation assay.

Conclusions:

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The study procedures described in this report were based on the most recent OECD and EEC guidelines.

The test substance was dissolved in dimethyl sulfoxide. In the dose range finding test, the substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA.

Precipitation was observed on the plates at the dose level of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first experiment of the mutation assay. Based on the results of the dose range finding test, the substance was tested in the first mutation assay at a concentration range of 100 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, the test item was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Precipitation was observed on the plates at the top dose of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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 test substance is predicted to be non-mutagenic in bacterial cells, and did not produce mutagenicity in mammalian or human cell culture with and with metabolic activation. The substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.