Benzyl propionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material did not induce the frequency of revertant colonies in bacteria with and without metabolic activation. Furthermore,The test item did not induce gene mutations at the HPRT locus in V79 cells under the conditions chosen. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2015 and March 2016

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)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian gene mutation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Doubling time: 12 – 16 h
- Modal number of chromosomes: 22


MEDIA USED
- Type and identity of media including CO2 concentration: Minimum Essential Medium (MEM) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1%)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Concentrations applied: 12.5, 25.0, 50.0, 100.0, 200.0 and 300.0 µg/mL
The maximum test item concentration of the pre-experiment (1642 μg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiment was limited by phase separation of the test item.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

- Cell density at seeding: 0.7 – 1.2 x 10E+7 cells

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time: 8 days

SELECTION AGENT: 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: two independent experiments with each 5 replicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The colonies were stained with 10% methylene blue in 0.01% KOH solution.
NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted.

DETERMINATION OF CYTOTOXICITY
- Method: adjusted cloning efficiency

- OTHER: In a pre-experiment the medium was checked for precipitation as well as pH and osmolarity changes.

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Other confounding effects: Phase separation occurred at 300 µg/mL at the beginning and at the end of treatment with and without S9 mix.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 12.8 μg/mL and 1642.0 μg/mL (equal to a molar concentration of approximately 10 mM) were used. In the pre-experiment a relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 410.5 μg/mL and above in the absence of metabolic activation. In the presence of metabolic activation no relevant cytotoxic effect was determined up to the highest concentration.
The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Phase separation occurred at 205.3 μg/mL and above after 4 hours treatment without and 102.6 μg/mL and above with metabolic activation. The dose range of the main experiment was set based on the main experiment. The individual concentrations were spaced by a factor of 2.0. Narrower spacing was used at the two highest concentrations to cover possible toxic effects more closely.

HISTORICAL CONTROL DATA
- Positive historical control data (in mutant colonies per 10E+6 cells):
EMS: range = 53.9 – 889.0, mean = 153.0, SD = 88.5
DMBA: range = 59.6 – 2042.6, mean = 424.6, SD = 291.4

- Negative (solvent/vehicle) historical control data (in mutant colonies per 10E+6 cells):
+ S9: Solvent control (water, DMSO, medium): range = 1.6 – 42.8, mean = 15.0, SD = 7.4, 95% CL = 0.2 – 29.7
- S9: Solvent control (water, DMSO, medium): range = 2.4 – 44.2, mean = 14.6, SD = 7.0, 95% CL = 0.6 – 28.7

Summary of results

	Conc. μg/mL	PS	S9 mix	Relative cloning efficiency I	Relative cell density	Relative adjusted CE I	Mutant colonies / 10^6cells	95% confidence interval	Relative cloning efficiency I	Relative cell density	Relative adjusted CE I	Mutant colonies / 10^6cells		95 % confidence interval
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12		13
Experiment I /4h treatment				Culture I					Culture II
Solvent control with DMSO			-	100.0	100.0	100.0	17.0	0.2 – 29.7	100.0	100.0	100.0		6.4	0.2 – 29.7
Positive control (EMS)	300.0		-	123.9	67.9	84.1	300.3	0.2 – 29.7	81.9	95.3	78.1		211.7	0.2 – 29.7
Test item	12.5		-	124.3	71.2	88.5	#		94.7	95.0	89.9		#
Test item	25.0		-	113.4	111.8	126.8	19.1	0.2 – 29.7	116.8	80.5	94.1		9.2	0.2 – 29.7
Test item	50.0		-	118.6	88.0	104.4	19.2	0.2 – 29.7	99.9	72.3	72.3		15.2	0.2 – 29.7
Test item	100.0		-	115.7	74.9	86.7	24.5	0.2 – 29.7	98.3	88.4	86.9		8.6	0.2 – 29.7
Test item	200.0		-	101.0	96.7	97.6	12.9	0.2 – 29.7	49.8	83.2	41.5		16.3	0.2 – 29.7
Test item	300.0	PS	-	86.4	80.7	69.7	24.2	0.2 – 29.7	54.6	65.9	36.0		17.9	0.2 – 29.7
Solvent control with DMSO			+	100.0	100.0	100.0	6.4	0.6 – 28-7	100.0	100.0	100.0		16.6	0.6 – 28-7
Positive control (DMBA)	2.2		+	75.6	102.1	77.3	95.6	0.6 – 28-7	94.0	128.3	120.6		133.6	0.6 – 28-7
Test item	12.5		+	76.6	118.9	91.1	#		98.4	112.9	111.1		#
Test item	25.0		+	69.5	101.1	70.3	8.7	0.6 – 28-7	102.5	121.2	124.3		17.9	0.6 – 28-7
Test item	50.0		+	86.8	101.6	88.2	12.3	0.6 – 28-7	102.8	86.9	89.3		15.0	0.6 – 28-7
Test item	100.0		+	79.8	95.0	75.8	12.3	0.6 – 28-7	102.3	103.5	105.9		24.1	0.6 – 28-7
Test item	200.0		+	66.1	106.8	70.6	11.5	0.6 – 28-7	93.5	100.4	93.8		10.5	0.6 – 28-7
Test item	300.0	PS	+	65.9	107.5	70.8	18.6	0.6 – 28-7	89.1	97.5	86.9		6.7	0.6 – 28-7

PS = Phase separation at the beginning and at the end of treatment

# = culture was not continued as a minimum of only four analysable concentrations is required

Conclusions:

The test item did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Thus, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

A GLP conform study according to OECD TG 476 was performed to assess the potential of the test item to induce gene mutations at the HPRT locus in V79 cells. An appropriate cell number was treated with test item concentrations of 12.5, 25.0, 50.0, 100.0, 200.0 and 300.0 µg/mL for 4 hours in the absence and presence of S9 mix.

Phase separation was observed at the highest concentration of 300 μg/mL in the presence and absence of metabolic activation.

No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation. Moderate cytotoxic effects limited to just one of the parallel cultures were noted at 200 and 300 μg/mL without metabolic activation.

The positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Thus, the test item did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Thus, the test item is considered to be non-mutagenic in this HPRT assay.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2015 and March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 September 2014

Deviations:

yes

Remarks:

To get distinct responses of statistical significance when using the specified positive controls the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified (non-GLP validation experiments).

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Healthy non-smoking female donors not receiving medication.
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Sex, age and number of blood donors: one female (35 years old) for experiment I and one female (27 years old) for experiment II
- Whether whole blood or separated lymphocytes were used: whole blood were used
- Methods for maintenance in cell culture: Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours.
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection.

MEDIA USED
- Type and identity of media including CO2 concentration: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Concentrations applied: 10.7, 18.7, 32.7, 57.2, 100.0, 175.1, 306.4, 536.2, 938.3, 1642.0 µg/mL (experiment I); 100.0, 175.1, 306.4, 536.2, 938.3, 1642.0 µg/mL (experiment II)
With regard to the molecular weight of the test item, 1642.0 μg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (pulse exposure) and 20 hours (continuous exposure)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were fixed with an ice-cold mixture of methanol and glacial acetic acid. The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The micronuclei were counted in cells showing a clearly visible cytoplasm area.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity: CBPI was determined in 500 cells per culture.

OTHER: The osmolarity and pH were determined in the solvent control and the maximum concentration without metabolic activation.

Evaluation criteria:

The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

Negative result:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data

Positive result:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data

Statistics:

Statistical significance was confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Definition of acceptable cells for analysis: The micronuclei were counted in cells showing a clearly visible cytoplasm area.
- Other confounding effects: In Experiment I, phase separation of the test item in the culture medium was observed at 536.2 μg/mL and above in the absence of S9 mix and at 938.3 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment II in the absence of S9 mix at 536.2 μg/mL and above at the end of treatment.

RANGE-FINDING/SCREENING STUDIES: Test item concentrations ranging from 10.7 to 1642.0 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Using a reduced Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity, no cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 1642.0 μg/mL was chosen as top treatment concentration for Experiment II.

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells: comparable to solvent control

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: 1000 binucleate cells per culture (in total 2000 binucleated cells per concentration and controls)

HISTORICAL CONTROL DATA
- Positive historical control data:
-S9 mix (MMC; pulse treatment): mean = 11.66, range = 4.15 - 24.00, 95% CL = 1.48 – 21.85, SD = 5.09
-S9 mix (Demecolcin, continuous treatment): mean = 3.55, range = 2.10 – 6.40, 95% CL = 1.69 – 5.41, SD = 0.93
+S9 mix (CPA; pulse treatment): mean = 4.80, range = 2.25 - 11.30, 95% CL = 0.88 – 8.73, SD = 1.96

- Negative (solvent/vehicle) historical control data:
-S9 mix (pulse treatment): mean = 0.61, range = 0.15 - 1.25, 95% CL = 0.02 – 1.15, SD = 0.27
-S9 mix (continuous treatment): mean = 0.55, range = 0.05 - 1.43, 95% CL = 0.05 – 1.05, SD = 0.25
+S9 mix (pulse treatment): mean = 0.64, range = 0.15 - 1.35, 95% CL = 0.08 – 1.20, SD = 0.28

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Summary of results

Exp.	Preparation interval	Test item concentration in μg/mL	Proliferation index CBPI	Cytostasis in %*	Micronucleated cells in %**
Exposure period 4 hrs without S9 mix
I	40 hrs	Solvent control^1	1.93		0.95
		Positive control^2	1.68	27.3	12.70^S
		175.1	1.96	n.c.	0.80
		306.4	1.88	6.0	0.90
		536.2^PS	1.90	3.3	0.95
Exposure period 20 hrs without S9 mix
II	40 hrs	Solvent control^1	1.87		0.85
		Positive control^3	1.69	20.8	3.05^S
		175.1	1.92	n.c.	0.40
		306.4	1.90	n.c.	0.60
		536.2^PS	1.88	n.c.	0.75
Exposure period 4 hrs with S9 mix
I	40 hrs	Solvent control^1	1.95		0.55
		Positive control^4	1.49	48.3	7.15^S
		306.4	1.99	n.c.	0.40
		536.2	1.91	4.2	0.45
		938.3^PS	1.97	n.c.	0.30

*     For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^PS    Phase separation occurred at the end of treatment

^s     The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1    DMSO          0.5 % (v/v)

2    MMC             1.0 μg/mL

3    Demecolcin   75.0 ng/ml

4    CPA              15.0 μg/mL

Conclusions:

The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic (non-clastogenic and non-aneugenic) in this test, when tested up to phase separating concentrations.

Executive summary:

To assess the potential of the test item to induce micronuclei in human lymphocytes, an in vitro micronucleus assay according to OECD 487 under GLP conditions was performed. Two independent experiments were performed. In experiment I, the exposure period was 4 hours with and without S9 mix. In experiment II, the exposure period was 20 hours without S9 mix. In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. Based on the results of the pre-experiment, cells were treated with test item concentrations ranging from 10.7 to 1642.0 µg/mL (experiment I) and 100.0 to 1642.0 µg/mL (experiment II). To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. No precipitation occurred and no osmolality and pH changes were observed. In Experiment I, phase separation of the test item in the culture medium was observed at 536.2 μg/mL and above in the absence of S9 mix and at 938.3 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment II in the absence of S9 mix at 536.2 μg/mL and above at the end of treatment. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. The positive controls induced statistically significant increases in cells with micronuclei.

In conclusion, the test item did not induce micronuclei in human lymphocytes under the test conditions chosen. Thus, the test item is considered to be non-mutagenic (non-clastogenic and non-aneugenic), when test up to the phase separating concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2001-08-03 and 2001-08-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

only one strain was tested

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate (S9) from Aroclor 1254 pretreated rats

Test concentrations with justification for top dose:

1000, 1500, 2250, 3000 and 4000 µg/plate with and without S9 mix

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide; +S9: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 replicates (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of revertant colonie and/or a diminution of the background lawn was taken as an indication of bacteriotoxicity.

Statistics:

X2-test

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: done

ADDITIONAL INFORMATION ON CYTOTOXICITY: Both in the absence and presence of S9 mix the test item was not bacteriotoxic towards the strain TA1535.

Induction of revertants per plate (mean of three plates) in S. typhimurium TA1535 in the absence and presence of S9 mix

Substance concentration (µg/plate)	- S9 mix		+ S9 mix
	I	II	I	II
control	33	22	26	14
solvent control	29	27	27	15
1000	38	31	21	13
1500	41	38	21	11
2250	33	38	24	15
3000	34	32	23	19
4000	30	28	22	13
Sodium azide (0.7)	400	383	-	-
2-aminoanthracene (0.9)	-	-	205	157

Conclusions:

The results indicated that the test item was not mutagenic to Salmonella typhimurium strain TA1535 in the absence and presence of a metabolizing system.

Executive summary:

The reason for this study was to reinvestigate the mutagenicity of the test substance after its purification by distillation in the mutant strain TA1535 of Salmonella typhimurium. The study was conducted with and without S9 mix induced with Aroclor 1254 in male rats. The test item was dissolved in DMSO and tested in concentrations of 1000 to 4000 µg per plate in the presence and absence of S9. Both in the absence and presence of S9 mix the test item was not bacteriotoxic towards the strain TA1535. Precipitation of the test compound on the plates was not observed. Sodium azide, and 2 -aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strain as well as the efficacy of the metabolizing system. The test substance induced a very weak, dose related increase in the mutation frequency of the tester strain TA1535 in the absence of a metabolic activation system. However, significant values were not reached. The presence of the metabolizing system suppressed the effect completely. Under the experimental conditions, the test item was not mutagenic to Salmonella typhimurium strain TA 1535 in the absence and presence of a metabolizing system.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2000-08-31 and 2001-02-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

no pre-incubation test was performed

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Supernatant of liver homogenates from Sprague-Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

15, 50, 150, 500, 1500, 5000 µg/plate with and without S9 mix

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (TA100, TA1535), 2-nitrofluorene (TA98), 9-aminoacridine (TA1537), Mitomycin C (TA 102); + S9: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 replicates (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies and/or diminution of the background lawn

Statistics:

X2-test

Key result

Species / strain:

S. typhimurium, other: TA1537, TA98, TA100, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: done

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the absence of S9-mix the test item was bacteriotoxic towards the strain TA102 at 1500 µg/plate and towards the strains TA98 and TA100 at 5000 μg/plate. In the presence of S9-mix the test item was bacteriotoxic towards the strain TA102 at 5000 µg/plate.

Table 1: Revertants/plate (mean of three plates) in the absence of metabolizing system

Dose µg/plate I/II	TA 98		TA 100		TA102		TA1535		TA1537
	I	II	I	II	I	II	I	II	I	II
control	41	21	97	92	275	308	13	21	13	12
Solv. Contr	36	21	95	85	257	287	17	21	16	13
15	-	-	-	-	-	316	-	-	15	-
50	34	21	88	79	225	327	20	-	15	11
150	35	31	100	93	262	305	21	-	13	9
500	30	32	90	89	253	264	35	25	14	8
1000	-	-	-	-	-	-	-	27	-	-
1500	26	20	100	77	180 T	199 T	47 *	33	13	7
3000	-	-	-	-	-	-	-	41 *	-	-
5000	15 T	25	72 T	85	30T	-	32	37	-	8
Pos. contr.
Sodium azide (0.7)			409	296			714	965
2-nitrofluorene (2.5)	581	587
9-aminoacridine (50)									305	529
Mitomycin C					685	753

Solvent control: DMSO 50 µL/plate; T: bacteriotoxic; * significantly different from control (p < 0.01)

Table 2: Revertants/ plate (mean of three plates) in the presence of metabolizing system

Dose µg/plate I/II	TA 98		TA 100		TA102		TA1535		TA1537
	I	II	I	II	I	II	I	II	I	II
control	17	31	124	91	295	348	12	10	18	13
Solv. Contr	14	28	98	85	271	305	10	9	15	14
15	-	-	-	-	-	-	-	-	12	-
50	15	31	114	95	272	295	13	10	15	12
150	14	28	98	75	284	332	11	10	17	14
500	12	33	102	78	269	329	8	9	15	18
1500	9	27	96	79	253	296	12	9	16	17
5000	13	28	104	90	180 T	243 T	11	11	-	17
Pos. contr.
2-aminoanthracene (0.8)	211	845	429	352	434	633	197	134
2-aminoanthracene (1.7)									143	188

Solvent control: DMSO 50 µL/plate; T: bacteriotoxic

Conclusions:

The results indicate that the test substance under the experimental conditions was not mutagenic in TA1537, TA100, TA98 and TA102 in the absence and presence of a metabolizing system as well as in TA1535 with a metabolizing system. The results in the strain TA1535 (without S9 mix) can be interpreted as ambiguous.

Executive summary:

The mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102) according to OECD 471. The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test item was dissolved in DMSO and tested in concentrations of 15 to 5000 µg per plate in the presence and absence of S9. In the absence of S9 mix the test item was bacteriotoxic towards the strain TA102 at 1500 µg/plate and towards the strains TA98 and TA100 at 5000 µg/plate. In the presence of S9 mix the test item was bacteriotoxic towards the strain TA102 at 5000 µg/plate. Precipitation of the test compound on the plates was not observed. Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system. The test item induced a dose related increase in the mutation frequency of the tester strain TA 1535 in the absence of metabolic activation system which was significant at 1500 µg test item per plate. However, the number of reversions was not three times higher than the reversion control of the vehicle control.In addition, the increase of mutation frequency induced by the positive control was above the historical control data range. In conclusion, the increase in the mutation frequency observed in strain TA1535 (without S9 mix) may have been overstated and can be interpreted as ambiguous. The results indicated that the test substance under the experimental conditions was not mutagenic in TA1537, TA100, TA98 and TA102 in the absence and presence of a metabolizing system as well as in TA1535 with a metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

WoE Approach - Ames test

The mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102) according to OECD 471 in a weight of evidence approach. The test item was dissolved in DMSO and tested in concentrations of 15 to 5000 µg per plate. In the first experiment, the test substance under the experimental conditions was not mutagenic in TA1537, TA100, TA98 and TA102 in the absence and presence of a metabolizing system as well as in TA1535 with a metabolizing system. The test item induced a dose related increase in the mutation frequency of the tester strain TA 1535 in the absence of metabolic activation system which was significant at 1500 µg test item per plate. However, the number of reversions was not three times higher than the reversion control of the vehicle control. In addition, the increase of mutation frequency induced by the positive control was above the historical control data range. Thus, the increase in the mutation frequency observed in strain TA1535 (without S9 mix) may have been overstated and can be interpreted as ambiguous. The second Ames test reinvestigate the mutagenicity of the test substance in the mutant strain TA1535 of Salmonella typhimurium. The results indicated, that the test item was not mutagenic to Salmonella typhimurium strain TA1535 in the presence and absence of a metabolizing system. Based on the results of the weight of evidence approach, the test substance was not mutagenic to Salmonella typhimurium strain TA1537, TA100, TA98, TA102 and TA1535 in the absence and presence of a metabolizing system.

Key study - HPRT test

A GLP conform study according to OECD TG 476 was performed to assess the potential of the test item to induce gene mutations at the HPRT locus in V79 cells. An appropriate cell number was treated with test item concentrations of 12.5, 25.0, 50.0, 100.0, 200.0 and 300.0 µg/mL for 4 hours in the absence and presence of S9 mix.

Phase separation was observed at the highest concentration of 300 μg/mL in the presence and absence of metabolic activation.

No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation. Moderate cytotoxic effects limited to just one of the parallel cultures were noted at 200 and 300 μg/mL without metabolic activation.

The positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Thus, the test item did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Thus, the test item is considered to be non-mutagenic in this HPRT assay.

Key study - MNT in vitro

To assess the potential of the test item to induce micronuclei in human lymphocytes, an in vitro micronucleus assay according to OECD 487 under GLP conditions was performed. Two independent experiments were performed. In experiment I, the exposure period was 4 hours with and without S9 mix. In experiment II, the exposure period was 20 hours without S9 mix. In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. Based on the results of the pre-experiment, cells were treated with test item concentrations ranging from 10.7 to 1642.0 µg/mL (experiment I) and 100.0 to 1642.0 µg/mL (experiment II). To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. No precipitation occurred and no osmolality and pH changes were observed. In Experiment I, phase separation of the test item in the culture medium was observed at 536.2 μg/mL and above in the absence of S9 mix and at 938.3 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment II in the absence of S9 mix at 536.2 μg/mL and above at the end of treatment. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. The positive controls induced statistically significant increases in cells with micronuclei.

In conclusion, the test item did not induce micronuclei in human lymphocytes under the test conditions chosen. Thus, the test item is considered to be non-mutagenic (non-clastogenic and non-aneugenic), when test up to the phase separating concentrations.

Overall Conclusion

Based on the results of the Weight of Evidence approach of the Ames test, the test item did not increased the frequency of revertant colonies in Salmonella typhimurium strains TA100, TA1535, TA102, TA1537 and TA98 with and without S9 mix. Additionally, the test item did not induce gene mutations at the HPRT locus in V79 cells and did not induce micronuclei in human lymphocytes. Therefore, the test item ist considered to be non-mutagenic in bacteria and mammalian cells. Furthermore, the test item is non-clastogenic and non-aneugenic in human lymphocytes.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The results indicate that the substance is non-mutagenic, non-clastogenic and non-aneugenic. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP),

as amended for the tenth time in Regulation (EC) No 2017/776.