N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenylpropionamide]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was negative in two Ames studies and a HPRT locus assay. In addition, the substance is predicted to be negative in an Ames test and in a chromosomal aberration study by the QSAR Tool OASIS TIMES. The chemical was within the applicability domain.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1983-03-26)

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvrB-; TA 1537: rfa-, uvrB

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix) prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test: concentrations ranging from 0.08 to 5000 µg/plate.
First test (with and without microsomal activation) 20, 78, 313, 1250, 5000 µg/plate
Second test (with and without microsomal activation): 500, 1000, 2000, 40000, 8000 µg/plate

Vehicle / solvent:

ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: for details see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: triplicates per strain and dose each in 2 independent experiments

POSITIVE CONTROLS:
Without S9 mix:
TA 98: daunorubicin-HCl, 5 and 10 µg/0.1 ml phosphate buffer;
TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 µg/0.1 ml phosphate buffer
TA 1535: sodium azide, 2.5 and 5.0 µg/0.1 ml bidistilled water
TA 1537: 9(5)-aminoacridine hydrochloride monohydrate, 50 and 100 µg/0.1 ml dimethylsulfoxide
With S9 mix:
TA 98, TA 100 and TA 1537: 2-aminoanthracene, 5 µg/0.1 ml dimethylsulfoxide
TA 1535: cyclophosphamide, 250 µg/0.1 ml bidistilled water

Evaluation criteria:

A substance is to be considered as mutagenic when one or both of the following criteria are fulfilled:
1.) doubling of spontaneous mutation rate for strains TA 98, TA 1535 and TA 1537
2.) a mutation rate of at least 1.5 for strain TA 100
a dose response-relationship should be demonstrable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At concentrations of 313 µg/plate and above substance precipitates in soft agar

RANGE-FINDING/SCREENING STUDIES:
- 5000 µg/plate was selected at highest dose from cytotoxicity experiments. Up to 5000 µg/plate no cyctotoxic effect was seen in pretest

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Results of plate incorporation test (test 1) with and without metabolic activation

	Plate Incorporation test 1
Substance	With/ or without S9-mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
			Salmonella typhymurium
			Base-pair substitution type		Frameshift type
			TA 1535	TA 100	TA 1537	TA 98
vehicle only	-	0	16	116	4	31
test substance	-	20	13	99	6	33
test substance	-	78	13	103	4	28
test substance	-	313	11	107	5	29
test substance	-	1250	18	99	5	26
test substance	-	5000	11	85	6	19
Sodium Azide	-	2.5	548
Sodium Azide		5	788
Daunorubicin	-	5				536
Daunorubicin		10				189
4-Nitroquinoline- N-oxid	-	0.125		593
4-Nitroquinoline- N-oxid		0.25		1000
9-Aminoacridine- hydrochloride	-	50			110
9-Aminoacridine- hydrochloride	-	100			977
vehicle only	+	0	25	110	14	42
test substance	+	20	16	115	15	57
test substance	+	78	34	120	15	60
test substance	+	313	25	106	12	44
test substance	+	1250	28	127	12	63
test substance	+	5000	20	119	15	47
Cylophosphamide	+	250	597
2-Aminoanthracene				606	161	1250

Table 2: Results of plate- incorporation test with and without metabolic activation

	Plate Incorporation test 2
Substance	With/ or without S9-mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
			Salmonella typhymurium
			Base-pair substitution type		Frameshift type
			TA 1535	TA 100	TA 1537	TA 98
vehicle	-	0	18	135	9	27
test substance	-	500	14	109	7	29
test substance	-	1000	13	129	5	26
test substance	-	2000	12	132	6	26
test substance	-	4000	22	118	7	24
test substance	-	8000	16	105	9	22
Sodium Azide	-	2.5	461
Sodium Azide	-	5	699
4-Nitroquinoline- N-oxide	-	0.125		683
4-Nitroquinoline- N-oxide	-	0.25		1184
9-Aminoacridine- hydrochloride	-	50			1553
9-Aminoacridine- hydrochloride	-	100			2608
Daunorubicin- HCl	-	5				160
vehicle	+	0	20	145	19	45
test substance	+	500	16	127	17	42
test substance	+	1000	20	113	20	40
test substance	+	2000	18	123	19	34
test substance	+	4000	19	117	20	41
test substance	+	8000	25	96	20	54
2-Aminoanthracene	+	5		586	165	1171
Cyclophosphamid		250	983

Executive summary:

A test of bacterial gene mutagenicity for the substance following the OECD guideline 471(1983) was conducted. Following strains were tested: TA 98, TA 100, TA 1535 and TA 1537. Test concentrations ranged from 20 to 8000 μg/ plate. Two experiments were conducted both in absence and presence of S9-mix. For the first experiment, the test concentration ranged from 20 to 5000 µg/plate. For the second experiment, the test concentration was in the range of 500 to 8000 µg/plate. No increase in revertant colonies was noticed, thus the test substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used with or without metabolic activation. Consequently the substance is considered to be nonmutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 24 to August 31, 2020

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:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Rheinland Pfalz, Landesamt für Umwelt; date of inspection: 15.-17. October 2019

Type of assay:

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance preparations. The stability of the test substance under storage conditions was guaranteed until 11 Feb 2024 as indicated by the sponsor and the sponsor holds this responsibility.
- Solubility and stability of the test material in the solvent/vehicle: The stability of the test substance at room temperature in the vehicle DMSO over a period of 4 hours was verified analytically.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The substance was dissolved in DMSO.
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance solutions were prepared immediately before administration.

- Final concentration of a dissolved solid, stock liquid or gel:
concentrations ranging from: first experiment (without S9 mix)
0; 3.3; 5.9; 10.6; 19.1; 34.3; 61.7; 111.1; 200.0 μg/mL; first test (with S9 mix)
0; 1.8; 3.3; 5.9; 10.6; 19.1; 34.3; 61.7; 111.1 μg/mL; second experiment (with S9 mix) 1.8 μg/mL, 3.3 μg/mL, 5.9 μg/mL, 10.6 μg/mL, 19.1 μg/mL, 34.3 μg/mL, 61.7 μg/mL, 111.1 μg/mL

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material (adjustment of pH, osmolality and precipitate) in the culture medium to which the test chemical is added:
In the pre-test the pH value and the osmolality were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.
The pH was measured at least for the top concentrations and for the vehicle controls with and without S9 mix at the beginning of test substance treatment.
Osmolality was measured in at least the top concentrations and the vehicle controls with and without S9 mix at the beginning of test substance treatment.
In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 31.3 μg/mL and above both in the absence and presence of S9 mix. Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.

Target gene:

Hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type of cells: Chinese hamster ovary (CHO) cells
- Source of cells: not specified
- Suitability of cells: suggested in the OECD TG 476
- Normal cell cycle time (negative control): not specified

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: at least 2
- Methods for maintenance in cell culture: For cell cultivation, deep-frozen cell suspensions were thawed at 37°C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5 mL Ham's F12 medium including 10% (v/v) FCS. Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm2 plastic flasks).

Routine passage (preparation of a single cell suspension)
- Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mgfree).
- Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25% [w/v]) to remove the cells from the bottom of the plastic flasks.
- This reaction was stopped by adding 6 mL culture medium incl. 10% (v/v) FCS.
- Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension.
- Cells were counted in a counting chamber or using a cell counter.
- Cell suspensions were diluted with complete culture medium to the desired cell count.
- Cell cycle length, doubling time or proliferation index : doubling time approx.12-16 hours
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: no
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:

All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)

Culture medium
Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).

Treatment medium (without S9 mix)
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) FCS.

Treatment medium (with S9 mix)
Ham's F12 medium containing stable glutamine and hypoxanthine.

Pretreatment medium ("HAT" medium)
Ham's F12 medium supplemented with:
- hypoxanthine (13.6 x 10^3 mg/mL)
- aminopterin (0.18 x 10^3 mg/mL)
- thymidine (3.88 x 10^3 mg/mL)
- 10% (v/v) FCS

Selection medium ("TG" medium)
Ham's F12 medium containing stable glutamine supplemented with:
- 6-thioguanine (10 μg/mL)
- 10% (v/v) FCS

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system
- source of S9: liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation)
- method of preparation of S9 mix: S9 fraction was prepared according to Ames et al. in an AAALAC-approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive and was prepared freshly prior to each experiment. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am – 6 pm and darkness from 6 pm – 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at + 4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
- the concentrations of the cofactors in the S9 mix: MgCl2 8 mM, KCl 33 mM, glucose-6-phosphate 5 mM, NADP 4 mM, phosphate buffer (pH 7.4) 15 mM
- concentration or volume of S9 mix and S9 in the final culture medium: 2% S9 fraction and 20% of S9 mix in the final culture medium
- Content of protein in S9 fraction: 24.3 g/L
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Sterility controls of S9 mix were performed; enzymatic activity was characterized with benzo[a]pyrene

Test concentrations with justification for top dose:

Based on the data and the observations from the pre-test and taking into account the current
guidelines, the following doses were selected in this study:

first experiment (without S9 mix):
0; 3.3; 5.9; 10.6; 19.1; 34.3; 61.7; 111.1; 200.0 μg/mL,

first test (with S9 mix):
0; 1.8; 3.3; 5.9; 10.6; 19.1; 34.3; 61.7; 111.1 μg/mL;

second experiment (with S9 mix):
1.8 μg/mL, 3.3 μg/mL, 5.9 μg/mL, 10.6 μg/mL, 19.1 μg/mL, 34.3 μg/mL, 61.7 μg/mL, 111.1 μg/mL;

In the pre-test the pH value and the osmolality were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. No precipitation of the test substance in the vehicle dimethyl sulfoxide (DMSO) was observed in the stock solution (200 mg/mL). In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 31.3 μg/mL and above both in the absence and presence of S9 mix. After 4 hours treatment in the absence and presence of S9 mix, cytotoxicity was not observed as indicated by a reduced RS of about or below 20% of control up to 31.3 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: insolubility of the test substance in the aqueous culture medium, DMSO has been demonstrated to be suitable in the CHO/HPRT assay. Furthermore, historical control data were available.
- Percentage of solvent in the final culture medium: 1% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

positive controls: 7,12-dimethylbenz[a]anthracene (DMBA) was tested with S9 Mix, ethyl methanesulfonate (EMS) without S9 Mix; historical positive control data available

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20x10^6 cells in 40 mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Attachment period of the cells: 20 - 24 hours
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time: 7-9 days
- Selection time: 6-7 days
- Selection medium: Ham's F12 medium containing stable glutamine supplemented with 6-thioguanine (10 μg/mL) and 10% (v/v) FCS

METHODS FOR MEASUREMENT OF CYTOTOXICITY

The cloning efficiency (CE, %) was calculated for each test group as follows:

total number of colonies in the test group
CEabsolute = ————————————————————— x 100
total number of seeded cells in the test group

CEabsolute of the test group
CErelative = —————————————— x 100
CEabsolute of the vehicle control


The number of colonies in every petri dish was counted and recorded. Using the formula above the values of absolute cloning efficiencies (CEabsolute, CE1 absolute and/or CE2 absolute) were calculated. Based on these values the relative cloning efficiencies (CErelative, CE1 relative and/or CE2 relative) of the test groups were calculated and reported as a percentage of the respective CEabsolute value of the corresponding vehicle control (vehicle control = 100%).
In addition, with regard to cell loss while exposure period, relative survival (RS) was calculated based on CE of cells plated immediatedly after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in vehicle controls.

number of cells at the end of treatment
Adjusted CE = —————————————————— x CE1
number of seeded cells

adjusted CE of the test group
RS = ———————————————————— x 100
adjusted CE of the vehicle control


METHODS FOR MEASUREMENTS OF GENOTOXICIY

The number of colonies in each flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized per every 10^6 cells seeded. The uncorrected mutant frequency (MFuncorr.) per 10^6 cells was calculated for each test group as follows:

total number of mutant colonies
MFuncorr. = —————————————–— x 10^6
number of seeded cells

The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.):

MFuncorr.
MFcorr. = —–——–— x 100
CE2 absolute

OTHER:
Each test group were fixed with methanol, stained with Giemsa and counted.

Evaluation criteria:

The test substance was considered to be clearly positive if all following criteria were met:
• A statistically significant increase in mutant frequencies was obtained.
• A dose-related increase in mutant frequencies was observed.
• The corrected mutation frequencies exceeded both the concurrent vehicle control value and the range of the laboratory’s historical negative control data (95% control limit). Isolated increases of mutant frequencies above the historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but were not regarded as sufficient evidence of mutagenicity.

The test substance was considered to be clearly negative if the following criteria were met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies was observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups was close to the concurrent vehicle control value and within the range of the laboratory’s historical negative control data (95% control limit).

Statistics:

A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency and the independent variable was the dose. The calculation was performed using EXCEL function RGP. A pair-wise comparison of each test group with the control group was carried out using
Fisher's exact test with Bonferroni-Holm correction. Both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: Osmolality and pH values were not influenced by test substance treatment.

- Precipitation and time of the determination: In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 31.3 μg/mL and above both in the absence and presence of S9 mix. In the 1st Experiment, in the absence of S9 mix, test substance precipitation was observed in culture medium at the end of treatment at 19.1 μg/mL and above. In the presence of S9 mix precipitation was observed at 19.1 μg/mL and above in the 1st and 2nd Experiment. All other higher concentrations used were not further considered.

RANGE-FINDING/SCREENING STUDIES:
An initial range-finding cytotoxicity test for the determination of the experimental doses was conducted.

STUDY RESULTS
The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

For all test methods and criteria for data analysis and interpretation:
- At least four concentrations were evaluated to describe a possible dose response relationship.

- Genotoxicity results:
please see table 1 in section "Any other information on results incl. tables"

HISTORICAL CONTROL DATA
please see table 2 and 3 in section "Any other information on results incl. tables"

Table 1: Summary of results 

 

Exp.	Exposure period (h)	Test groups [µg/mL]	S9 mix	Prec.	Genotoxicity**	Cytotoxicity***
					MFcorr. [per 10^6 cells]	RS	CE2
						[%]	[%]
1	4	Vehicle control^1	-	n.d.	2.96	100.0	100.0
		3.3	-	-	3.78	117.2	84.7
		5.9	-	-	3.70	129.3	99.8
		10.6	-	-	1.74	112.2	85.0
		19.1	-	+	1.20	114.0	82.3
		34.3	-	+	n.c.1	n.c.1	n.c.1
		61.7	-	+	n.c.1	n.c.1	n.c.1
		111.1	-	+	n.c.1	n.c.1	n.c.1
		200.0	-	+	n.c.1	n.c.1	n.c.1
		Positive control^2	-	n.d	126.61s	106.2	80.5
1	4	Vehicle control^1	+	n.d.	3.51	100.0	100.0
		1.8	+	-	n.c.	126.5	n.c.
		3.3	+	-	2.13	113.3	82.5
		5.9	+	-	2.04	97.0	114.9
		10.6	+	-	8.65s	101.0	101.5
		19.1	+	+	0.96	82.1	91.2
		34.3	+	+	n.c.1	n.c.1	n.c.1
		61.7	+	+	n.c.1	n.c.1	n.c.1
		111.1	+	+	n.c.1	n.c.1	n.c.1
		Positive control^3	+	n.d.	61.09s	73.4	85.7
2	1	Vehicle control^1	+	n.d.	1.93	100.0	100.0
		1.8	+	-	2.46	82.0	78.5
		3.3	+	-	0.00	64.2	94.2
		5.9	+	-	1.66	60.7	97.1
		10.6	+	-	1.63	51.6	79.1
		19.1	+	+	0.76	37.8	84.6
		34.3	+	+	n.c.1	n.c.1	n.c.1
		61.7	+	+	n.c.1	n.c.1	n.c.1
		111.1	+	+	n.c.1	n.c.1	n.c.1
		Positive control^3	+	n.d.	110.83s	30.3	77.2

 

+ Microscopically or macroscopically visible precipitation in culture medium at the end of exposure period
** Mutant frequency MFcorr.: mutant colonies per 10^6 cells corrected with the CE2 value
*** Cloning efficiency related to the respective vehicle/negative control
s Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)
n.c. Culture was not continued since a minimum of only four analysable concentrations is required
n.c.1 Culture was not continued since only one concentration beyond the solubility limit is required
n.d. Not determined
1 DMSO 1% (v/v); 2 EMS 400 μg/mL;  3 DMBA 1.25 μg/mL

 

Table 2: HISTORICAL POSITIVE CONTROL DATA

 

Without S9 mix 400 µg/mL ethyl methanesulfonate (EMS)
	Corrected Mutant Frequency*
Exposure period	4 hrs
Mean	160.94
Minimum	42.47
Maximum	438.29
Standard Deviation	73.52
95% Lower Control Limit	14.83
95% Upper Control Limit	307.05
No. of Experiments	124
With S9 mix 1.25 µg/mL 7,12-Dimethylbenz[a]anthracene (DMBA)
	Corrected Mutant Frequency*
Exposure period	4 hrs
Mean	126.62
Minimum	21.52
Maximum	270.48
Standard Deviation	54.78
95% Lower Control Limit	17.81
95% Upper Control Limit	235.44
No. of Experiments	129

 

* = mutant frequency (per 1 million cells) corrected with the cloning
efficiency at the end of the expression period (CE2)

 

Table 3: HISTORICAL NEGATIVE CONTROL DATA

 

	Without S9 mix - all vehicles*	With S9 mix - all vehicles*
Corrected Mutant Frequency**
Exposure period	4 hrs	4 hrs
Mean	2.76	2.93
Minimum	0.00	0.00
Maximum	8.00	9.93
Standard Deviation	1.73	2.09
95% Lower Control Limit	0.00	0.00
95% Upper Control Limit	6.21	7.08
No. of Experiments	130	132

 

* = culture medium, water 10% (v/v), DMSO 1% (v/v), acetone 1% (v/v)
** = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in an in vitro mammalian cell gene mutation test (HPRT-locus) in absence and in the presence of metabolic activation.

Executive summary:

The test substance was evaluated for genotoxic potential in a HPRT locus assay using CHO cells according to OECD TG 476 (GLP compliant). Two independent experiments in a dose range from 1.8-111.1 µg/ml were carried out, both with and without (1st experiment only) the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The test item was considered to be non-mutagenetic under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OASIS TIMES v.2.30.1.11

2. MODEL (incl. version number)
Ames mutagenicity S9 activated Model version: v.17.17

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC(C)(C)c1cc(CCC(=O)NCCCCCCNC(=O)CCc2cc(C(C)(C)C)c(O)c(C(C)(C)C)c2)cc(C(C)(C)C)c1O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
see attached QMRF

5. APPLICABILITY DOMAIN
see attached QMRF

6. ADEQUACY OF THE RESULT
The chemical falls within the applicability domain and the result is threfore adequate and reliable

Qualifier:

no guideline required

Principles of method if other than guideline:

see attached QMRF

GLP compliance:

no

Type of assay:

other: QSAR prediction of an Ames test

Species / strain:

other: QSAR model

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Conclusions:

The QSAR predition is negative and the chemical falls within the applicability domain of the model.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OASIS TIMES v.2.30.1.11

2. MODEL (incl. version number)
Chromosomal Aberrations S9 activated v.17.17

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC(C)(C)c1cc(CCC(=O)NCCCCCCNC(=O)CCc2cc(C(C)(C)C)c(O)c(C(C)(C)C)c2)cc(C(C)(C)C)c1O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
see attached QMRF

5. APPLICABILITY DOMAIN
see attached QMRF

6. ADEQUACY OF THE RESULT
The chemical falls within the applicability domain and the result is threfore adequate and reliable

Qualifier:

no guideline required

Principles of method if other than guideline:

see attached QMRF

GLP compliance:

no

Type of assay:

other: QSAR prediction of a chromosomal aberration assay in vitro

Species / strain:

other: QSAR model

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Conclusions:

The QSAR predition is negative and the chemical falls within the applicability domain of the model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test article was tested negative in a micronucleus Test, Sister Chromatid Exchange assay and in a Dominant Lethal Assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5915 (In Vivo Sister Chromatid Exchange Assay)

Version / remarks:

(1998)

Deviations:

yes

Remarks:

(4 animals per sex and dose)

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5915 (In Vivo Sister Chromatid Exchange Assay)

Deviations:

yes

Remarks:

(4 animals per sex and dose)

Principles of method if other than guideline:

Procedure according to Perry et al. (1974), Goto et al. (1978), Allen et al. (1977), Perry et al. (1975), Marquardt et al. (1978)
PERRY, P. and S. WOLFF Nature 251 , 156-158 (1974).
GOTO, K., S. MAEDA, Y. KANO and T. SUGIYAMA Chromosoma (Berl.) 66, 351-359 (1978).
ALLEN, J.W., C F . SHULER, R.W. MENDES and S.A. LATT
Cytogenet. Cell Genet. 18, 231-237 (1977).
PERRY, P. and H.J. EVANS Nature 258, 121-125 (1975).
MARQUARDT, H. and U. BAYER Mutation Res. 56, 169-176 (1978).

GLP compliance:

no

Type of assay:

sister chromatid exchange assay

Species:

hamster, Chinese

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 20-28 g (females), 25-30 g (males)
- Diet (e.g. ad libitum): NAFAG No. 924
- Water (e.g. ad libitum): tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22- 24
- Humidity (%): 54-62
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.7% aqueous solution of CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

Two hrs before application of test or control substance each animal is applied with 5 -bromodeoxyuridine (BUdR) (45 mg tab as subcutaneous implantation in the neck (Fa. Heinrich Mack, D-Illertissen, Germany)). Twenty-four hours after administration of test substance and 2hrs after intraperitoneal injection with colcemide animals were sacrificed.

Duration of treatment / exposure:

24 hrs

Frequency of treatment:

single dose

Post exposure period:

24 hrs (test substance)

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

Dose / conc.:

6 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

4 (only chromatides of 2 animals per group and sex examined)

Control animals:

yes, concurrent vehicle

Positive control(s):

7,12-dimethylbenzanthracene
- Route of administration: oral, gavage
- Doses / concentrations: 100 mg/kg bw in 0.7% aqueous solution of sodium-carboxymethylcellulose (CMC)

Tissues and cell types examined:

Bone marrow, 25 cells per animal in metaphase

Details of tissue and slide preparation:

Bone marrow from the shafts of both femurs was suspended in balanced salt solution and diluted to hypotonicity with distilled water, kept in a waterbath at 4 to 6°C for 23 min and then centrifuged for 10 min at 200 x g. The pellets were then fixed in methanol-acetic acid 3:1 for a period of 30 min, resuspended, centrifuged for 5 min at 150 x g, and stored in renewed fixative overnight at 4°C. Finally the pellets again were centrifuged for 5 min at 150 x g, and resuspended in some 0.5 mL fixative.
These specimens were pipetted onto wet slides and air-dried. The air-dried slides then were treated with a solution of bisbenzimide for 15 min, rinsed in McIlvaine-buffer pH 8.0 and irradiated in this buffer at 50°C with UV-light of 350 nm. Following the development of the fluorochrome-UV-light reaction in 60°C 2 x SSC (standard sodium citrate) for 90 min, the slides were stained in 40% Giemsa for 20-40 min, well rinsed, cleared in Xylol and mounted in Eukitt.
The slides of two female and two male animals each of the treatment groups and of the control groups were examined. Twenty-five differently stained metaphases of the second cell-cycle with 5-bromodeoxyuridine (BUdR)-substitution were analysed.

Evaluation criteria:

Analyze of Sister Chromatide Exchanges (SCE's) according to Marquardt et al. (1978)
MARQUARDT, H. and BAYER, U. Mutation Res. 56, 169-176 (1978).

Statistics:

Significance of differences between treatment and control group was assessed by the t-test (p < 0.01)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

When comparing the number of SCE’s between negative control and the test groups (1500, 3000 and 6000 mg/kg bw test substance) no statistical difference between treated groups and control could be seen. In contrast, the positive control group showed a highly significant increase of SCE's per cell (12.8) in comparison with the negative control (5.28 SCE's/cell).

Table 1: The effect of test substance on bone marrow cells of chinese hamster

Group	Dose [mg/kg]	overall no of animals  (male+ female)	group mean no.  of SCE's	standard- deviation	t-value
Control (0.7% CMC in water)		2m +2f	5.28	2.95
Control (DMBA)	100	2m +2f	12.79	6.25	10.88*
Test substance	1500	2m +2f	6.02	3.16	1.72
Test substance	3000	2m +2f	4.81	2.37	1.24
Test substance	6000	2m +2f	4.88	3.91	0.82
* p≤ 0.01 DMBA= 7,12 Dimethylbenzanthracene

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1983-03-26)

Principles of method if other than guideline:

The test was conducted according to the procedures of Boller et al. (1970), Matter et al. (1970) and Müller et al. (1972).
Boller, K. and W. Schmid (1970) Humangenetik 11, 35-54.
Matter, B. and W. Schmid (1971) Mutation Res. 12, 417-425.
Müller, D., M. Langauer, R. Rathenberg, F.F. Strasser and R. Hess (1972) Verh. Dtsch. Ges. Path. 56, 381-384.

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

hamster, Chinese

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: male: 22-30 g, female: 20-26 g
- Diet (e.g. ad libitum): NAFAG No. 924
- Water (e.g. ad libitum): tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24°C
- Humidity (%): 51 - 59%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.7% aqueous solution of CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

24 hrs after second application animals were sacrificed

Duration of treatment / exposure:

48 hrs

Frequency of treatment:

2 doses per animal; once daily

Post exposure period:

24 hrs

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

Dose / conc.:

6 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 128 mg/kg bw in 20mL/kg in 0.7% aqueous solution CMC

Tissues and cell types examined:

blood cells of bone marrow

Details of tissue and slide preparation:

Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 μL rat serum the bone marrow was drawn up. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it on behind a cover glass, and the preparations were air-dried. After 3 hrs, the slides were stained in undiluted May-Gruenwald solution for 2 min, then in May-Gruenwald solution/water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol 55% for 5-8 s and washed off twice in water, they were left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylol and mounted in Eukitt.

Evaluation criteria:

Slides of 3 female and 3 male animals each of the negative and positive control group and from the different test groups were examined.

1000 bone marrow cells each were scored per animal and the following anomalies were registered: a) Single Jolly
bodies, b) fragments of nuclei in erythrocytes, c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells,
e) polyploid cells.

Statistics:

Significance of differences between doses were assessed by chi-square test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In all tested doses (1500, 3000 and 6000 mg/kg bw) the percentage of cells with anomalies was not significantly different from the negative control.

In the positive control group (Cyclophosphamide 128 mg/kg bw) a significant increase (p < 0.05) of anomalies was found (percentage of anomalies: 8.5 vs. 0.12 in negative control)

Table 1: The effect of test substance and Cyclophosphamide on bone marrow cells in chinese hamster

Test group	dose  [mg/kg bw]	average number of cell anomalies per animal
negative control (carboxymethyl cellulose)	0	0.12
positive control (Cyclophosphamide)	128	8.5
test substance	1500	0.08
test substance	3000	0.03
test substance	6000	0.07

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

only 2 doses applied, highest dose should be 5000 mg/kg bw

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Version / remarks:

(1984-04-04)

Principles of method if other than guideline:

study conducted according to Machemer et al. (1971)
Machemer, L., and Hess, R. (1971), Comparative Dominant Lethal Studies with Phenylbutazone, Thio-TEPA and MMS in the Mouse. Experientia 27, 1050-1052

GLP compliance:

no

Remarks:

(GLP was not mandatory when study was done)

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

Tif:MAGf

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 2.5 - 6 months (males), 2 months (females)
- Diet (e.g. ad libitum): NAFAG No. 890
- Water (e.g. ad libitum): tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Humidity (%): 60 ± 5
- Photoperiod (hrs dark / hrs light): 14/10

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 2% aqueous solution of CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

Only males were treated.
Immediately after treatment each male was placed in a cage with 2 untreated females. After the end of the week, the females were removed and replaced by another group of 2 females. The procedure was continued for six consecutive weeks. The females were daily examined for successful mating
indicated by the occurrence of a vaginal plug. The day that the vaginal plug was observed was designated as "day 0" of gestation. Overall 6 mating periods were included.

Duration of treatment / exposure:

6 weeks

Frequency of treatment:

single dosing

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 males per dose
12 females per male (Two females are placed in a cage with one treated male, after one week females are replaced; all females are untreated)

Control animals:

yes, concurrent vehicle

Positive control(s):

no positive control

Tissues and cell types examined:

male germinal cells, zygotes (before implantation), embryo

Evaluation criteria:

number of implantations and embryonic deaths were compared in the different groups. The data were further compared with the cumulative of untreated controls (historical controls)

Statistics:

total number of implantations (inclusive pre-implantation losses) in the control and test groups were compared by student's t-test or Mann-Whitney U-test.
Number of mated and pregnant dams or embryonic deaths were compared with Fisher's exact test or chi-square test.

Sex:

male

Genotoxicity:

negative

Remarks:

no significant difference between test and control groups in mating ratio, number of implantations and embryonic deaths

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not applicable

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic Toxicity in vitro

 

Ames-Test:

In a reverse gene mutation assay in bacteria following OECD guideline 471, strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium were exposed to test substance in ethanol at concentrations of 20, 78, 313, 1250 and 5000 µg/plate (first experiment) and 500, 1000, 2000, 4000 and 8000 µg/plate (second experiment) in the presence and absence of mammalian metabolic activation.  A test was to be considered as positive when a dose-dependency for mutagenic effects can be derived, the number of revertants for strain TA98, TA 1535 and TA 1537 is at least twice as high and/ or the number of revertants of strain TA 100 is at least factor of 1.5 or higher. The positive controls induced the appropriate responses in the corresponding systems. There was no evidence of induced mutant colonies over background. This study is classified as acceptable.

In an older gene mutation assay in bacteria strains (TA 98, TA 100, TA 1535, TA 1537) of S. typhimurium were exposed to test substance in DMSO at concentrations of 25, 75, 225, 675 and 2025 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding systems. A test was to be considered as positive when a dose-dependency for mutagenic effects can be derived and the number of revertants for all strains is at least twice as high. For strain TA 100 in experiments without metabolic activation an increase in the number of revertants was seen. Nevertheless, the increase was smaller than a factor of 2 and no clear dose-response was seen. For the other tester strains no increase was seen in experiments with and without metabolic activation. There was no evidence of induced mutant colonies over background. This study is classified as acceptable.

OASIS TIMES predicted the chemical to be negative in an Ames test. This prediction was valid, and the chemical was within the applicability domain of the model. This model also includes the missing E. coli strain.

 

HPRT-Test:

The test substance was evaluated for genotoxic potential in a HPRT locus assay using CHO cells according to OECD TG 476 (GLP compliant). Two independent experiments in a dose range from 1.8-111.1 µg/ml were carried out, both with and without (1st experiment only) the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The test item was considered to be non-mutagenetic under the conditions of the test.

Chromosomal aberration in vitro:

OASIS TIMES predicted the chemical to be negative in an in vitro chromosome aberration study. This prediction was valid, and the chemical was within the applicability domain of the model.

Genetic Toxicity in vivo

 

Micronucleus-Test

In a Chinese hamster bone marrow micronucleus assay, (6 animals/sex/dose) were treated orally with test substance at doses of 0, 1500, 3000, 6000 mg/kg bw. The vehicle was 0.7 % aqueous carboxymethylcellulose. There were no signs of toxicity during the study. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data with some restrictions (no GLP study).

 

Sister chromatid exchange study (SCE)

In a Chinese hamster sister chromatid exchange study (SCE), (6 animals/sex/dose) were treated orally with test substance at doses of 0, 1500, 3000, 6000 mg/kg bw. The vehicle was 0.7% aqueous Carboxy-methylcellulose (CMC) as single dose by gavage. There were no signs of toxicity during the study.  The positive control (7,12 Dimethylbenzanthracene) induced the appropriate response. There was not a significant increase in the frequency of SCE’s after any treatment time. This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5915 for in vivo cytogenetic mutagenicity data with some deficiencies (no GLP study, substance purity not reported).

 

Dominant lethal assay

A test of mutagenic effects on male germinal cells was done for the test substance by a single oral application. The test was conducted on 20 male albino mice with doses at 0, 1000 and 3000 mg/kg bw. There were no compound related effects in implantation, embryonic death and lethality for the progenies. The study is classified as acceptable. The study satisfies the requirement for test guideline OECD 478 with some restrictions (no GLP study, only 2 doses applied, highest dose lower than 5000 mg/kg bw, substance purity not reported).

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse findings on genotoxicity were observed in in vitro or in vivo studies. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.