4-(4-isopropoxyphenylsulfonyl)phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of D-8 was tested in three in vitro tests, a bacterial reverse mutation assay (Ames-test) according to OECD TG 471, a mammalian chromosome aberration test according to OECD TG 473 and a mammalian gene mutation test according to OECD TG 476. D-8 showed no signs of genotoxicity in all test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-09-10 to 1987-01-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- => his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98, TA 1538) mutations.

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : rat liver homogenate, Aroclor 1254 induced
- concentration or volume of S9 mix and S9 in the final culture medium: S-9 fraction (10% v/v), MgCl2 (8mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mm), NADH (4 mM)

Test concentrations with justification for top dose:

Test 1: 1500; 500; 150, 50; 15 µg/plate;
Test 2: 5000; 1500; 500, 150; 50; 15; 5 µg/plate;

Vehicle / solvent:

Dimethylsulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene, AA

Remarks:

TA 1535, TA 1537; 2 µg/plate; with S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene, AA

Remarks:

TA 1538, TA 98, TA 100; 0.5 µg/plate; with S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 1538; 2 µg/plate; without S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98; 1 µg/plate; without S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537; 80 µg/plate; without S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

TA 1535; 5 µg/plate; without S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

TA 100; 3 µg/plate; without S-9 mix

Details on test system and experimental conditions:

Preliminary study:
A preliminary study is performed on all tester strains,with and without metabolic activation, in order to evaluate the toxicity of the test article to bacteria.
Increasing concentrations of the test article are used; examples for which are as follows: 5 - 50 - 500 - 5000 mg/plate. The highest dose is 5000 µg/plate if the formulation type allows this dose level to be reached. A minimum of 5 dose levels are used.
Each concentration is tested in duplicate. At the end of the incubation period, the plates are observed for signs of toxicity. The colonies which appears in the presence of the test article are counted and the intensity of the bacterial lawn examined. These observations are compared with those performed in the presence of the vehicle. The signs of toxicity (reduction in bacterial lawn or reduction of the number of colonies) are noted.
Main study:
Two studies are conducted, the second study being performed to confirm or to complement the results of the first one for example by using a closer range near to the top limit dose.
For each study, each concentration of the test article is tested in triplicate with and without metabolic activation on each bacterial strain.
Concentration of the test substance resulting in precipitation: 1500 µg/plate

Evaluation criteria:

The test article will be considered to be clearly mutagenic if:
- The assay is valid
- The number of revertants in presence of the test article is greater than or equal to twice the number of revertants in the negative control at one dose level (the upper dose before toxicity) or more with a dose correlation
- The positive trends/effects are reproducible.

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

The test article D-8 was tested on 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538) with and without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on all tester strains with and without metabolic activation.

Five concentrations (1500, 500, 150, 50 and 15 µg/plate) were tested in triplicate on the strains mentioned above with and without metabolic activation. The results were confirmed in a second independent study, using a closer range near the top of limit dose (5000, 1500, 500, 150, 50, 15, 5 µg/plate). A negative control (vehicle) and a positive control (specific standard mutagen) were included in each study.

Conclusion: Under the experimental conditions employed, it can be concluded that the test article D-8 did not cause an increase in the number of revertants per plate of any of the tester strains used, either in presence or absence of a metabolic activation system in both of the two independent studies performed.

Conclusions:

D-8 was tested for mutagenic activity in the Ames-test with the bacterial strains S. typhimurium TA98, TA100,TA1535, TA1537, TA1538 with and without metabolic activation in two independent studies. No increase of the number of revertants per plate was observed.

Executive summary:

D-8 was tested on 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538) with and without metabolic activation.

Five concentrations (1500, 500, 150, 50 and 15 µg/plate) were tested in triplicate on the strains mentioned above with and without metabolic activation. The results were confirmed in a second independent study, using a closer range near the top of limit dose (5000, 1500, 500, 150, 50, 15 and 5 µg/plate).

A negative control (vehicle) and a positive control (specific standard mutagen) were included in each study.

D-8 did not cause an increase in the number of revertants per plate of any of the tester strains used, either in presence or absence of a metabolic activation system in both of the two independent studies performed

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-07-13 to 1988-12-19

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

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung (CHL) cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 from rat liver treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Test without metabolic activation:
24 h treatment : 132, 93, 66, 33 µg/mL; additional: 46.7, 33, 23.3 µg/mL;
48 h treatment : 46, 23, 11.5 µg/mL;
Test with metabolic activation:
6 h treatment: 150, 106, 75, 37.5 µg/mL; additional: 75, 53, 37.5 µg/mL;

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Vehicle and positive controls were established in this test. Duplicate culture were used for each control and each dose level of the test substance.
 Without metabolic activation test:
Three days after culture initiated, growing cells in culture medium were respectively treated with the vehicle control, the positive control and the test substance for two intervals of 22 and 46 hours, in which colcemid was added at 0.2 μg/mL. After 2 hours, the medium was removed and 2 mL of 0.25% Tripsin was added, and the cells were incubated for several minutes. The cells separated from the bottom of the dish were transferred into a test tube to make single cell suspension by the “pipetting”. The tube was centrifuged (800 rpm, 5 min), the supernatant was removed, and the cells were resuspended with 4 mL of 0.07M KCl hypotonic solution at 37°C in water bath. After 10 minutes, the cells were washed 3 times with fixative (methanol : acetic acid, 3:1), dropped onto slide glass and air-dried. Control cultures were also prepared with the same manner.
With metabolic activation:
Three days after culture initiated, cells were respectively treated with the vehicle control, the positive control and the test substance for 6 hours under the condition of the S9mix presence. After the exposure period, the medium was removed and the cells were washed 2 times with fresh medium. The dish was filled with 5 mL of fresh medium, incubated for a further 18 hours and added with colcemid (0.2 μg/mL). After 2 hours, the cells were prepared as described before.

Evaluation criteria:

Data were summarized in tables showing the number of cells scored, the types of aberrations found and the percentages of cells bearing aberrations.
Statistical analysis employed the Fisher's exact test to compare the aberration frequency in treated cells with background data from vehicle controls. The difference is considered significant where p <0.05.

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung (CHL) cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations > 313 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Results Chromosome aberration test

  I - Without metabolic activation test

1) The 24 hours treatment.

Since no mitotic cell was observed at 66 μg/mL and higher concentrations due to the cell lethality, the additional test was performed at 23.3, 33 and 46.7 μg/mL. But, no increase of chromosome aberrations were seen in all concentrations, while the chromosomal aberrations in the vehicle controls were within the normal background range and significantly increased in the positive control of Mitomycin C.

 2) The 48 hours treatment

All concentrations (11.5, 23 and 46 μg/mL) of the test substance had a reduction in mitotic cells.

The frequencies of aberrations in the vehicle control were within the normal background range and that of positive control were increased. As compared with positive control, the frequencies of aberrations in all concentrations of test substance were not increased.

 Based on these results from both tests of 24 and 48 hours treatment, it was concluded that test substance did not induce the chromosome aberration in without metabolic activation test.

 

II - With metabolic activation.

In S9 mix presence dishes, the concentration-related decreases of metaphase cells were found at 37.5 and 75 μg/mL and no mitotic cells were at 106 and 150 μg/ml. In S9 mix absence dishes made as a control for S9 presence dishes. 100 metaphase cells were counted at only 37.5 μg/mL and no cells at 75 μg/mL and higher concentrations. To make up for the complete data, additional aberration test was performed at the concentrations of 37.5, 53 and 75 μg/mL. The frequencies of aberrations in all concentrations of test substance were comparable to that of vehicle control.

Based on this results, it is concluded that the test substance did not induce chromosome aberration in metabolic activation test.

Conclusions:

D-8 was tested for genetic toxicity in a chromosome aberration test in cultured Chinese hamster lung (CHL) cells. The test article did not induce structural chromosome aberrations when tested to the highest practicable concentrations with and without metabolic activation.

Executive summary:

D-8 was tested in an in vitro cytogenetic assay using Chinese hamster lung (CHL) cells in 2 independent experiments. The test article was dissolved in dimethylsulfoxide (DMSO). Concentrations of 11.5 to 150 µg/mL were tested with and without metabolic activation. All criteria for a valid study were met in both experiments. No significant increase in the number of cells with structural chromosome aberrations was noted in the absence or presence of S9 -mix in both experiments and both sampling times (24 and 48 hours). No abnormal values in the number of numerical aberrations were observed in all cultures in the presence of the test article. D-8 did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its highest practicable concentrations in both the absence or presence of a metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 24, 2019 - October 10, 2019

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 using the Hprt and xprt genes)

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

yes

Remarks:

There is a deviation from the guidelines regarding the confirmation of negative results. Negative results were not confirmed as the confirmation of negative results is not required by the most current Guideline (OECD 476, 29 July 2016).

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

Sub-line (KI)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: ECACC (European Collection of Cell Cultures)
- Suitability of cells: proven
- Absence of Mycoplasma contamination: yes
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Ham's F12 medium containing 10 % fetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 fraction (S9 mix) of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver. The concentration of S9 was 1.5% in medium. The protein concentrations of the S9 batch used in the experiments were 33.8 and 35.5 mg/mL.

Test concentrations with justification for top dose:

5-hour treatment period without S9-mix: 60, 80, 100, 120 and 140 µg/mL
5-hour treatment period with S9-mix: 20, 40, 60, 80 and 100 µg/mL
The concentration levels were chosen mainly based on the cytotoxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium and DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ham's F12 medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x10E6 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 hours
- Harvest time after the end of treatment: 19 hours

- Methods of slide preparation and staining technique used including the stain used: After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.

- Expression time (cells in growth medium between treatment and selection):
During the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x10E6 cells were taken on days 1, 3 and 6 and evaluated on day 8.

- Selection time:
At the end of the expression period, cultures from each dose level were adjusted to 2 x 10E5 cells / dish ( 4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 µg/mL of thioguanine (6-TG).

- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10E6 cells: 2x5 plates at 2 x 10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 10E6 clonable cells.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item 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,
• any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
• the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test item is considered 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 (based 95% control limit).
• The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: The osmolality and the pH values of test item solutions did not show any significant alterations when compared to the concurrent control groups in the Pre-test on Toxicity (Concentration selection) and in the Main Mutation Assay.
- Results from cytotoxicity measurements:
On Day 1, there was very clear evidence of toxicity at the highest tested concentration with the test item in presence and absence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the Pre-test on Toxicity (Concentration selection) assay. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.
- Genotoxicity results: see tables
HISTORICAL CONTROL DATA
The mutation frequency found in the solvent controls was in the range of historical laboratory control data.

Table 1:Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY (5-hour Treatment with and without S9-Mix)

Test group	Dose µg/mL	S9-mix	Treatment/ time/ hour	Number of colonies/200cells/dish			Mean	Relative^a survival in percent
				dish 1	dish 2	dish 3
Untreated Control				205	202	206	204,3	101
Solvent Control (DMSO)	–	–	5	203	202	200	201,7	100
D-8	15.7	–	5	202	204	205	203,7	101
	31.3	–	5	204	202	198	201,3	100
	62.5	–	5	197	193	194	194,7	97
	125	–	5	85	87	84	85,3	42
	250	–	5	0	0	0	0,0	0
	500	–	5	0	0	0	0,0	0
	1000	–	5	0	0	0	0,0	0
	2000		5	0	0	0	0,0	0
Untreated Control				200	200	201	200,3	101
Solvent Control (DMSO)	–	+	5	198	197	200	198,3	100
D-8	15.7	+	5	196	198	196	196,7	99
	31.3	+	5	151	153	157	153,7	77
	62.5	+	5	121	122	120	121,0	61
	125	+	5	0	0	0	0,0	0
	250	+	5	0	0	0	0,0	0
	500	+	5	0	0	0	0,0	0
	1000	+	5	0	0	0	0,0	0
	2000	+	5	0	0	0	0,0	0

^aRelative to Solvent Control

Table 1: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d (5-hour Treatment without S9-Mix)

Study code:				813-476-5054
Test item:				D-8 (without S9-mix)
Test date of Main Mutation Assay:				September 24, 2019 - October 10, 2019				Expression period:						8 days
Solvent:				DMSO				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control a	202.3	±	1.53		100	100	1		0	2	1	3	7		102	6.86
Pos. control (EMS 1.0µL/mL) a	53.7	±	2.89		27	64	198		200	196	204	190	988		65	1520.00**
TEST ITEM
60 µg/mL a	196.7	±	2.52		97	101	1		1	1	1	1	5		103	4.85
80 µg/mL a	182.7	±	2.08		90	100	2		2	1	1	1	7		101	6.93
100 µg/mL a	162.7	±	1.53		80	99	0		2	0	2	2	6		101	5.94
120 µg/mL a	87.0	±	1.00		43	97	0		0	2	2	2	6		99	6.06
140 µg/mL a	25.0	±	1.00		12	98	1		1	1	3	0	6		100	6.00

a = parallel of first culture, abs.C.E. = Absolute Cloning Efficiency, EMS=Ethyl methanesulfonate, ** = p < 0.01 to the concurrent negative control and to the historical control

Table 2: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d (5-hour Treatment without S9-Mix) continued

Study code:				813-476-5054
Test item:				D-8 (without S9-mix)
Test date of Main Mutation Assay:				September 24, 2019 - October 10, 2019				Expression period:						8 days
Solvent:				DMSO				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control b	203.7	±	1.15		100	100	2		2	0	2	0	6		101	5.94
Pos. control (EMS 1.0µL/mL) b	54.3	±	1.53		27	63	191		194	204	200	199	988		64	1543.75**
TEST ITEM
60 µg/mL b	197.7	±	2.52		97	100	2		0	1	0	2	5		101	4.95
80 µg/mL b	181.3	±	1.53		89	100	0		2	1	2	1	6		101	5.94
100 µg/mL b	161.7	±	0.58		79	99	1		1	1	0	2	5		100	5.00
120 µg/mL b	87.0	±	1.00		43	98	1		1	2	0	2	6		100	6.00
140 µg/mL b	24.3	±	1.15		12	98	2		1	2	0	1	6		100	6.00

b= parallel of first culture, abs.C.E. = Absolute Cloning Efficiency, EMS=Ethyl methanesulfonate, ** = p < 0.01 to the concurrent negative control and to the historical control

Table 3: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d(5-hour Treatment without S9-Mix) continued

Study code:				813-476-5054
Test item:				D-8 (without S9-mix)
Test date of Main Mutation Assay:				September 24, 2019 - October 10, 2019				Expression period:						8 days
Solvent:				DMSO				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control c	203.7	±	2.31		100	100	1		1	2	2	0	6		101	5.94
Pos. control (EMS 1.0 µL/mL) c	52.7	±	2.08		26	67	192		198	207	202	209	1008		67	1504.48**
TEST ITEM
60 µg/mL c	199.0	±	2.65		98	100	1		2	1	2	0	6		101	5.94
80 µg/mL c	185.7	±	2.08		91	101	0		0	2	3	1	6		102	5.88
100 µg/mL c	168.0	±	1.73		82	98	1		0	1	2	2	6		99	6.06
120 µg/mL c	88.7	±	1.53		44	98	0		1	0	3	1	5		98	5.10
140 µg/mL c	23.7	±	1.15		12	98	2		1	1	2	1	7		98	7.14

c= parallel of first culture, abs.C.E. = Absolute Cloning Efficiency, EMS=Ethyl methanesulfonate, ** = p < 0.01 to the concurrent negative control and to the historical control

Table 4: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d(5-hour Treatment without S9-Mix) continued

Study code:				813-476-5054
Test item:				D-8 (without S9-mix)
Test date of Main Mutation Assay:				September 24, 2019 - October 10, 2019				Expression period:						8 days
Solvent:				DMSO				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control d	203.0	±	1.00		100	100	0		1	3	2	1	7		101	6.93
Pos. control (EMS 1.0µL/mL) d	54.0	±	0.00		27	66	192		198	197	208	210	1005		66	1522.73**
TEST ITEM
60 µg/mL d	199.0	±	1.73		98	99	1		2	1	1	2	7		100	7.00
80 µg/mL d	183.7	±	0.58		90	100	2		1	0	1	1	5		101	4.95
100 µg/mL d	163.3	±	2.08		80	99	2		2	1	1	1	7		99	7.07
120 µg/mL d	89.3	±	1.53		44	98	2		1	3	0	0	6		99	6.06
140 µg/mL d	23.3	±	2.08		11	99	0		2	1	2	1	6		99	6.06

d= parallel of first culture, abs.C.E. = Absolute Cloning Efficiency, EMS=Ethyl methanesulfonate, ** = p < 0.01 to the concurrent negative control and to the historical control

Table 5: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d (5-hour Treatment with S9-Mix)

Study code:				813-476-5054
Test item:				D-8 (with S9-mix)
Test date of Main Mutation Assay:				September 24, 2019 - October 10, 2019				Expression period:						8 days
Solvent:				DMSO				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control a	201.0	±	1.00		100	100	2		1	1	2	0	6		101	5.94
Pos. control (DMBA 20 µg/mL) a	119.3	±	2.31		59	74	112		108	109	119	106	554		75	738.67**
TEST ITEM
20 µg/mL a	189.0	±	2.65		94	100	1		1	1	1	2	6		101	5.94
40 µg/mL a	159.3	±	1.15		79	98	2		0	1	1	2	6		100	6.00
60 µg/mL a	126.7	±	1.53		63	98	2		2	1	1	1	7		99	7.07
80 µg/mL a	112.0	±	2.00		56	97	1		2	0	1	2	6		98	6.12
100 µg/mL a	41.0	±	1.00		20	98	1		1	1	2	1	6		99	6.06

a = parallel of first culture , abs.C.E. = Absolute Cloning Efficiency, DMBA= 7,12-Dimethyl benzanthracene, ** = p < 0.01 to the concurrent negative control and to the historical control

Table 5:CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d (5-hour Treatment with S9-Mix) continued

Study code:				813-476-5054
Test item:				D-8 (with S9-mix)
Test date of Main Mutation Assay:				September 24, 2019 - October 10, 2019				Expression period:						8 days
Solvent:				DMSO				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control b	202.3	±	2.08		100	100	1		1	0	2	2	6		101	5.94
Pos. control (DMBA 20 µg/mL) b	118.3	±	2.31		58	74	102		108	111	106	104	531		75	708.00**
TEST ITEM
20 µg/mL b	191.7	±	1.53		95	99	2		1	1	0	1	5		100	5.00
40 µg/mL b	159.3	±	0.58		79	99	2		1	2	2	0	7		100	7.00
60 µg/mL b	124.7	±	1.15		62	98	0		2	1	2	1	6		100	6.00
80 µg/mL b	112.3	±	1.15		56	98	0		1	2	3	0	6		99	6.06
100 µg/mL b	39.7	±	0.58		20	99	2		3	0	0	1	6		100	6.00

b = parallel of first culture , abs.C.E. = Absolute Cloning Efficiency, DMBA= 7,12-Dimethyl benzanthracene,** = p < 0.01 to the concurrent negative control and to the historical control

Table 6: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d(5-hour Treatment with S9-Mix) continued

Study code:				813-476-5054
Test item:				D-8 (with S9-mix)
Test date of Main Mutation Assay:				September 24, 2019 - October 10, 2019				Expression period:						8 days
Solvent:				DMSO				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control c	200.7	±	1.15		100	100	0		2	2	2	1	7		101	6.93
Pos. control (DMBA 20 µg/mL) c	117.7	±	2.52		59	74	113		108	104	103	115	543		75	724.00**
TEST ITEM
20 µg/mL c	189.7	±	2.08		95	99	2		2	2	1	0	7		100	7.00
40 µg/mL c	160.0	±	1.00		80	100	0		3	3	1	0	7		101	6.93
60 µg/mL c	129.0	±	2.00		64	98	0		1	1	2	2	6		99	6.06
80 µg/mL c	112.3	±	1.15		56	98	2		1	0	1	1	5		99	5.05
100 µg/mL c	38.7	±	0.58		19	98	1		1	2	1	2	7		99	7.07

c = parallel of first culture , abs.C.E. = Absolute Cloning Efficiency, DMBA= 7,12-Dimethyl benzanthracene,** = p < 0.01 to the concurrent negative control and to the historical control

Table 7: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d(5-hour Treatment with S9-Mix) continued

Study code:				813-476-5054
Test item:				D-8 (with S9-mix)
Test date of Main Mutation Assay:				September 24, 2019 - October 10, 2019				Expression period:						8 days
Solvent:				DMSO				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control d	201.3	±	0.58		100	100	3		1	1	2	0	7		101	6.93
Pos. control (DMBA 20 µg/mL) d	117.3	±	1.53		58	74	107		115	114	104	112	552		75	736.00**
TEST ITEM
20 µg/mL d	194.0	±	4.58		96	99	0		0	2	2	1	5		100	5.00
40 µg/mL d	159.7	±	2.52		79	100	0		1	3	3	0	7		101	6.93
60 µg/mL d	128.3	±	1.53		64	99	1		2	2	1	0	6		99	6.06
80 µg/mL d	111.0	±	1.00		55	99	2		1	1	1	1	6		99	6.06
100 µg/mL d	38.0	±	1.00		19	99	2		1	1	0	2	6		99	6.06

d = parallel of first culture , abs.C.E. = Absolute Cloning Efficiency, DMBA= 7,12-Dimethyl benzanthracene,** = p < 0.01 to the concurrent negative control and to the historical control

Conclusions:

D-8 tested up to cytotoxic concentrations with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency.

Executive summary:

The test item dissolved in DMSO was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD TG 476. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.

5-hour treatment period without S9-mix:

60, 80, 100, 120 and 140 µg/mL

5-hour treatment period with S9-mix:

20, 40, 60, 80 and 100 µg/mL

In the absence and presence of metabolic activation clear cytotoxicity (survival between 11-20 %) of the test item was observed at the highest concentration applied (140 µg/mL in the absence and 100 µg/mL in the presence of S9 mix).

The osmolality and the pH values of test item solutions did not show any significant alterations when compared to the concurrent control groups in the Pre-test on Toxicity (Concentration selection) and in the Main Mutation Assay.

Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95% control limit).

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µL/mL) and 7, 12-Dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

D-8 tested up to cytotoxic concentrations with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo mutagenicity assay was performed with D-8 in the mouse micronucleus test according to EU Method B.12/OECD Guideline 474. D-8 showed no signs of mutagenic activity in this test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-02-24 to 2009-07-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi Coop Ltd., 1103 Budapest, Cserkesz u. 90;
- Age at study initiation: young adult mice, 7 - 8 weeks;
- Weight at study initiation: Preliminary test: Males: 30.3 - 35.1 g; Females: 26.5 - 28.5 g; Main test: Males: 31.6 - 35.4 g;
- Assigned to test groups randomly: All animals were sorted according to body weight by computer and divided to weight ranges.
- Fasting period before study: No
- Housing: Group caging (5 animals/cage, except of highest dose, where 7 animals/cage (incl. 2 reserve mice))
- Cage type: II type polypropylene/polycarbonate
- Diet: Animals received ssniff® SM R/M-Z+H “Autoclavable complete feed for rats and mice” produced by ssniff Spezialdiäten GmbH (D-59494 Soest/ Germany) ad libitum;
- Water: tap water from the municipal supply from 500 mL bottle, ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C;
- Humidity: 30 - 70 % R.H.;
- Air changes: 15 - 20 air changes/h by central air-condition system;
- Photoperiod: 1h hours light from 6:00 a.m. to 6:00 p.m.

Route of administration:

oral: gavage

Vehicle:

Polyethylene glycol 400 (PEG 400)

Details on exposure:

Justification of the dose selection: Groups of two male and female mice were treated on one occasion by oral gavage at dose levels of 500, 1000, 1500 and 2000 mg/kg bw/day. The treatment volume was 10 mL/kg bw/day. Animals were examined regularly for toxic signs and mortalities. On the basis of results of preliminary toxicity test, doses for the Mouse Micronucleus Test were: 500, 1000 and 2000 mg/kg bw/day. The preliminary study indicates no sex difference in toxicity, then one sex (male) was used in the main study.
The test/vehicle control items were administered by oral gavage on one occasion. In the low and mid dose groups the sampling was made once at 24 hours after treatment. In the high dose and vehicle control groups, mice were treated at 24 and 48 hours before sampling. Five male animals per dose group were used for sampling on each occasion. Cyclophosphamide (positive control) was administered intraperitoneally one occasion at a treatment volume of 10 mL/kg bw/day. Sampling was performed 24 hours after the treatment and five male animals were used for sampling. The mice were examined regularly for visible signs of reactions to treatment, immediately after dosing, and at intervals until sacrifice.

Duration of treatment / exposure:

One occasion

Frequency of treatment:

Once

Post exposure period:

24 hours (24 and 48 hours for negative control and high-dose test item)

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

Basis: actual ingested

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Basis: actual ingested

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Basis: actual ingested

No. of animals per sex per dose:

5 males per dose (additional 5 males in negative control and high-dose test item group)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide: 60 mg/kg bw/day

Tissues and cell types examined:

The animals from each group were weighed and bone marrow was obtained from two exposed femurs of the mice from every dose- time point immediately after sacrificing. The bone marrow was flushed with foetal bovine serum (5 mL). After vortex mixing, the cell suspension was concentrated by centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides. Slides were then dried at room temperature.

Details of tissue and slide preparation:

Subsequently the slides were stained as follow:
1. Fixed for a minimum of 5 minutes in methanol and allowed to air-dry.
2. Stained with Giemsa solution (99 mg/mL distilled water) for 25 minutes.
3. Rinsing in distilled water.
4. Drying at room temperature (at least 12 hours).
5. Coating with EZ-mounting.

Evaluation criteria:

The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative control group.
The test item would have been considered to have shown genotoxic activity in this study if the following criteria had been met:
- increases in the frequency of micronucleated polychromatic erythrocytes were observed in treated animals compared to the corresponding negative controls.
- the increases were dose-related
- the increases were statistically significant.

Statistics:

Statistically analysis was performed using Kruskall Wallis Non Parametric ANOVA test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Clinical signs and Mortality

Preliminary Toxicity Test (Pretest)

A preliminary toxicity test was performed to identify the appropriate maximum dose level for the main test. Groups of two male and female mice were treated on one occasion by oral gavage at dose levels of 500, 1000, 1500 and 2000 mg/kg bw/day. The treatment volume was 10 mL/kg bw/day. Animals were examined regularly for toxic signs and mortalities. The animals treated with dose levels of D-8 expressed mortality as shown in the table:

D-8 (mg/kg bw./day)	Number of Animals	Mortality
500	2 males and 2 females	0 males and 0 females
1000	2 males and 2 females	0 males and 0 females
1500	2 males and 2 females	0 males and 0 females
2000	2 males and 2 females	0 males and 0 females

No adverse reactions to treatment were observed in the mice dosed 500 and 1000 mg/kg body weight/day. One male animal dosed 1500 mg/kg body weight showed moderate decrease in activity, narrow palpebra 3, 4 hours after the treatment and a slight decrease in activity between 4 and 48-hour after the treatment. The other male and two female were symptom free in this dose group. One male animal dosed 2000 mg/kg body weight showed a moderate and a slight decrease in activity.The symptom was observed between one and four hours after the treatment. The other male and two female were symptom free in this dose group.

Micronucleus Test :

Based on the results of the preliminary toxicity test, the doses of the MNT were following: 500, 1000 and 2000 mg/kg bw/day.

The mice were examined for visible signs of reactions to treatment, immediately after dosing and at intervals until sacrifice.

No animals died during the study.

No adverse reactions to treatment were observed in the mice of the vehicle and positive control groups. The mice dosed at the 500, 1000 mg/kg body weight/day dose level were symptom-free during the study.

One male animal dosed 2000 mg/kg body weight showed slight piloerection, slight narrow palpebra and slight decrease in activity on the day of treatment. The other male in this dose group showed slight narrow palpebra and slight, moderate decrease in activity on the day of treatment. In this animal slight and moderate piloerection were observed during the study. No adverse reactions to treatment were observed in the other mice dosed at the 2000 mg/kg body weight/day.

No notable effect of treatment on body weights was observed.

Conclusions:

The frequencies of micronucleated polychromatic erythrocytes (MPCEs) for the negative and positive control mice were within acceptable ranges and compatible with the historical control data. No biologically significant increases in the frequency of MPCEs were seen in the groups of mice treated with D-8 compared to the vehicle control group. D-8 did not show any genotoxic activity in this Mouse Micronucleus Test.

Executive summary:

Potential mutagenic activity of D-8 was examined in bone marrow of male CRL: NMRI BR mice according to EU Method B.12. The doses of the test item for the Micronucleus Test were determined according to a preliminary oral toxicity test performed within this study, the doses selected were 500, 1000 and 2000 mg/kg body weight/day of D-8. The single oral administration of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight of D-8 did not induce any biologically relevant increase in the frequency of MPCEs in male mice at either 24 or 48 hours after the treatment compared to the vehicle control. No biologically significant increases in the frequency of MPCEs were seen in the groups of mice treated with D-8 compared to the vehicle control group. D-8 did not show any genotoxic activity in this mouse micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

in vitro

D-8 was tested on 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538) with and without metabolic activation in a study according to OECD TG 471. Five concentrations (1500, 500, 150, 50 and 15 µg/plate) were tested in triplicate on the strains mentioned above with and without metabolic activation. The results were confirmed in a second independent study, using a closer range near the top of limit dose (5000, 1500, 500, 150, 50, 15 and 5 µg/plate). A negative control (vehicle) and a positive control (specific standard mutagen) were included in each study. D-8 did not cause an increase in the number of revertants per plate of any of the tester strains used, either in presence or absence of a metabolic activation system in both of the two independent studies performed

D-8 was tested in an in vitro cytogenetic assay according OECD TG 473 using Chinese hamster lung (CHL) cells in 2 independent experiments. The test article was dissolved in dimethylsulfoxide (DMSO). Concentrations of 11.5 to 150 µg/mL were tested with and without metabolic activation. All criteria for a valid study were met in both experiments. No significant increase in the number of cells with structural chromosome aberrations was noted in the absence or presence of S9 -mix in both experiments and both sampling times (24 and 48 hours). No abnormal values in the number of numerical aberrations were observed in all cultures in the presence of the test article. D-8 did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its highest practicable concentrations in both the absence or presence of a metabolic activation system.

The test item dissolved in DMSO was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD TG 476. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.

5-hour treatment period without S9-mix:

60, 80, 100, 120 and 140 µg/mL

5-hour treatment period with S9-mix:

20, 40, 60, 80 and 100 µg/mL

In the absence and presence of metabolic activation clear cytotoxicity (survival between 11-20 %) of the test item was observed at the highest concentration applied (140 µg/mL in the absence and 100 µg/mL in the presence of S9 mix).

The osmolality and the pH values of test item solutions did not show any significant alterations when compared to the concurrent control groups in the Pre-test on Toxicity (Concentration selection) and in the Main Mutation Assay.

Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95% control limit).

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µL/mL) and 7, 12-Dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

D-8 tested up to cytotoxic concentrations with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency.

in vivo

Potential mutagenic activity of D-8 was examined in bone marrow of male CRL: NMRI BR mice according to EU Method B.12. The doses of the test item for the Micronucleus Test were determined according to a preliminary oral toxicity test performed within this study, the doses selected were 500, 1000 and 2000 mg/kg body weight/day of D-8. The single oral administration of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight of D-8 did not induce any biologically relevant increase in the frequency of MPCEs in male mice at either 24 or 48 hours after the treatment compared to the vehicle control. No biologically significant increases in the frequency of MPCEs were seen in the groups of mice treated with D-8 compared to the vehicle control group. D-8 did not show any genotoxic activity in this mouse micronucleus test.

Justification for classification or non-classification

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

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.