4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The Ames test and the HPRT test are negative. Hence, the substance is considered to be not mutagenic. However, an in vitro study on chromsome aberration showed positive findings.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-19 to 2017-10-19

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9 (experiment I) non-induced hamster liver (experiment II)

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
Strain WP2 uvrA: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
The remaining strains: 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
Experiment IIa:
Strain TA 100 without S9 mix: 0.3; 1; 3; 10; 33; 100; and 333 µg/plate

No correction for purity was made.

Vehicle / solvent:

Solvent used: DMSO
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 60 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: not soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in experiment I, from 1000 to 5000 µg/plate in experiment II and at 333 µg/plate in experiment IIa. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in experiment I and II and from 100 to 333 µg/plate in experiment IIa. The undissolved particles had no influence on the data recording.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used.

Summary of Experiment I

Study Name: 1862000	Study Code: Envigo 1862000
Experiment: 1862000 VV Plate	Date Plated: 19.09.2017
Assay Conditions:	Date Counted: 22.09.2017

Metabolic Activation	Test Group		TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	DMSO		13 ± 4	10 ± 2	22 ± 3	151 ± 7	31 ± 2
Activation	Untreated		22 ± 8	10 ± 4	20 ± 4	150 ± 6	31 ± 6
	Test item	3 µg	12 ± 4	7 ± 1	26 ± 5	132 ± 15	30 ± 8
		10 µg	13 ± 6	8 ± 2	19 ± 3	135 ± 6	31 ± 6
		33 µg	11 ± 1	9 ± 3	24 ± 4	107 ± 7	31 ± 7
		100 µg	9 ± 2	8 ± 3	19 ± 6	42 ± 9	41 ± 10
		333 µg	7 ± 2^P	8 ± 1^P	13 ± 3^P M	9 ± 2^P M	25 ± 9^P
		1000 µg	5 ± 2^P M	4 ± 1^P M	5 ± 1^P M	4 ± 1^P M	13 ± 2^P M
		2500 µg	4 ± 1^P M	3 ± 1^P M	3 ± 2^P M	2 ± 1^P M	13 ± 1^P M
		5000 µg	1 ± 1^P M	1 ± 1^P M	2 ± 1^P M	1 ± 1^P M	8 ± 2^P M
	NaN3	10 µg	1181 ± 33			2113 ± 150
	4-NOPD	10 µg			293 ± 29
	4-NOPD	50 µg		78 ± 11
	MMS	2.0 µL					994 ± 85
With	DMSO		15 ± 3	13 ± 3	27 ± 6	130 ± 1	38 ± 2
Activation	Untreated		9 ± 2	14 ± 4	35 ± 7	126 ± 13	41 ± 10
	Test item	3 µg	14 ± 3	16 ± 6	28 ± 10	118 ± 10	32 ± 11
		10 µg	11 ± 5	15 ± 5	29 ± 3	123 ± 17	48 ± 6
		33 µg	14 ± 4	11 ± 2	31 ± 10	127 ± 6	34 ± 6
		100 µg	11 ± 1	12 ± 6	27 ± 4	132 ± 8	48 ± 12
		333 µg	8 ± 2^P	18 ± 3^P	23 ± 4^P	42 ± 11^P	30 ± 7^P
		1000 µg	5 ± 1^P M	7 ± 1^P M	12 ± 3^P M	7 ± 2^P M	19 ± 4^P M
		2500 µg	4 ± 1^P M	4 ± 1^P M	5 ± 1^P M	3 ± 1^P M	14 ± 1^P M
		5000 µg	2 ± 1^P M	2 ± 1^P M	3 ± 1^P M	2 ± 1^P M	12 ± 0^P M
	2-AA	2.5 µg	410 ± 30	166 ± 26	3187 ± 195	4245 ± 136
	2-AA	10.0 µg					415 ± 59

Summary of Experiment II

Study Name: 1862000	Study Code: Envigo 1862000
Experiment: 1862000 HV2 Pre	Date Plated: 04.10.2017
Assay Conditions:	Date Counted: 11.10.2017

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	DMSO		9 ± 1	12 ± 3	30 ± 7	155 ± 9	32 ± 5
Activation	Untreated		9 ± 3	13 ± 2	32 ± 6	162 ± 1	39 ± 8
	Test item	3 µg	7 ± 2	15 ± 3	23 ± 5	139 ± 5	36 ± 10
		10 µg	5 ± 2	15 ± 6	29 ± 5	130 ± 7	34 ± 11
		33 µg	6 ± 1	12 ± 4	20 ± 3	61 ± 5	32 ± 6
		100 µg	6 ± 1	10 ± 3	12 ± 2	36 ± 11	25 ± 2
		333 µg	6 ± 1^P	9 ± 3^P M	2 ± 1^P M	2 ± 1^P M	18 ± 4^P
		1000 µg	3 ± 2^P M	4 ± 1^P M	0 ± 1^P M	0 ± 1^P M	18 ± 4^P
		2500 µg	2 ± 1^P M	3 ± 1^P M	0 ± 1^P M	0 ± 0^P M	10 ± 2^P M
		5000 µg					6 ± 2^P M
	NaN3	10 µg	1213 ± 71			1961 ± 62
	4-NOPD	10 µg			314 ± 6
	4-NOPD	50 µg		89 ± 9
	MMS	2.0 µL					858 ± 90
With	DMSO		14 ± 5	17 ± 3	33 ± 3	140 ± 13	45 ± 7
Activation	Untreated		11 ± 5	17 ± 7	40 ± 8	193 ± 35	54 ± 12
	Test item	3 µg	15 ± 4	16 ± 8	36 ± 10	144 ± 17	47 ± 2
		10 µg	14 ± 3	20 ± 2	35 ± 7	145 ± 14	39 ± 5
		33 µg	18 ± 4	14 ± 6	39 ± 4	127 ± 12	44 ± 8
		100 µg	13 ± 4	15 ± 6	41 ± 8	100 ± 9	42 ± 6
		333 µg	10 ± 4^P	15 ± 5^P	29 ± 8^P	8 ± 3^P M	26 ± 9^P
		1000 µg	9 ± 3^P	6 ± 2^P M	7 ± 2^P M	2 ± 1^P M	26 ± 5^P
		2500 µg	7 ± 3^P M	6 ± 1^P M	1 ± 1^P M	0 ± 1^P M	15 ± 4^P M
		5000 µg					13 ± 1^P M
	2-AA	2.5 µg	378 ± 11	104 ± 17	4405 ± 188	3595 ± 36
	2-AA	10.0 µg					532 ± 25

Summary of Experiment IIa

Study Name: 1862000	Study Code: Envigo 1862000
Experiment: 1862000 HV2a Pre	Date Plated: 19.10.2017
Assay Conditions:	Date Counted: 23.10.2017

Metabolic Activation	Test Group	Dose Level (per plate)	TA 100 Revertant Colony Counts (Mean ±SD)
Without	DMSO		145 ± 17
Activation	Untreated		192 ± 14
	Test item	0.3 µg	137 ± 7
		1 µg	145 ± 12
		3 µg	152 ± 6
		10 µg	130 ± 9
		33 µg	71 ± 9
		100 µg	1 ± 1^P M
		333 µg	0 ± 0^P M
	NaN3	10 µg	2092 ± 34

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) usingSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in three independent experiments with and without liver microsomal activation (S9 mix). Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:          3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

Strain WP2uvrA:                              3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The remaining strains:                       3; 10; 33; 100; 333; 1000; and 2500 µg/plate

Experiment IIa:

Strain TA 100 without S9 mix:         0.3; 1;3; 10; 33; 100; and 333 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in experiment I, from 1000 to 5000 µg/plate in experiment II and at 333 µg/plate in experiment IIa. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in experiment I and II and from 100 to 333 µg/plate in experiment IIa. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I without S9 mix	Experiment I with S9 mix	Experiment II without S9 mix	Experiment II with S9 mix	Experiment IIa without S9 mix
TA 1535	1000 – 5000	1000 – 5000	1000 – 2500	/	-
TA 1537	1000 – 5000	1000 – 5000	1000 – 2500	1000 – 2500	-
TA 98	1000 – 5000	2500 – 5000	100 – 2500	1000 – 2500	-
TA 100	100 – 5000	333 – 5000	33 – 2500	333 – 2500	100 – 333
WP2uvrA	1000 – 5000	2500 – 5000	2500 – 5000	2500 – 5000	-

/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)

- = not performed

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-11 to 2017-10-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 7.6, 13.3, 23.2, 40.6, 71.1, 124, 218, 381, 667, 2000 µg/mL

Without metabolic activation:
Experiment I: 7.6, 13.3, 23.2, 40.6, 71.1, 124, 218, 381, 667, 2000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (0.5%)
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

One experiment was performed. The exposure period was 4 hours with and without metabolic activation. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 150 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 150 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 2 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

moderate toxicity in the absence of S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item, dissolved in DMSO, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Only one experiment was performed, since the test item was considered to be mutagenic after the first experiment. The exposure period was 4 hours with and without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 150 metaphases per culture were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 2000 µg/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
Precipitation of the test item in the culture medium was observed at 124 µg/mL and above in the absence and presence of S9 mix at the end of treatment.
No relevant influence on osmolarity or pH was observed.
In the absence of S9 mix, moderate cytotoxicity was observed up to the highest evaluable concentration. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration. Concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.
In the absence of S9 mix, statistically significant increases were observed in all evaluated concentrations (3.7 up to 9.3 % aberrant cells, excluding gaps). These values were clearly out of the range of the laboratory historical solvent control data (0.1 – 2.8 % aberrant cells, excluding gaps). Dose dependency, tested by a trend test, which was marginally not statistically significant (p-value: 0.064), did not occurr. In the presence of S9 mix, statistically significant increases were observed in the two highest evaluated concentrations (5.0 and 5.7 % aberrant cells, excluding gaps). All three evaluated concentrations were clearly out of the range of the laboratory historical solvent control data (0.4 – 3.2 % aberrant cells, excluding gaps). Dose dependency, tested by a trend test, which was statistically significant, occurred.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
Either EMS (825 µg/mL) or CPA (10.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Summary of results of the chromosomal aberration study with the test item

Exposure period 4 hrs without S9 mix

Exp.	Preparationinterval	Test item concentration (µg/mL)	Mitotic indices (%of control)	Aberrant cells(%)incl. gaps*	Aberrant cells(%)excl. gaps*	Aberrant cells(%) carrying exchanges
I	22 hrs	Solvent control^1	100.0	1.0	0.7	0.0
		Positive control^2	47.6	9.3	9.0^S	1.7
		23.2	82.1	4.7	4.7^S	0.0
		40.6	63.8	4.0	3.7^S	0.3
		71.1	54.1	9.7	9.3^S	0.3

*      Including cells carrying exchanges

^S       Aberration frequency statistically significant higher than corresponding control values

¹       DMSO        0.5% (v/v)

²         EMS        825 µg/mL

³       CPA       10.0 µg/mL

 

Exposure period 4 hrs with S9 mix

Exp.	Preparationinterval	Test item concentration (µg/mL)	Mitotic indices (%of control)	Aberrant cells(%) incl. gaps*	Aberrant cells(%) excl. gaps*	Aberrant cells(%) carrying exchanges
I	22 hrs	Solvent control^1	100.0	2.3	2.0	0.0
		Positive control^3	43.7	11.0	11.0^S	1.3
		23.2	91.0	4.0	4.0	0.0
		40.6	90.3	5.3	5.0^S	0.0
		71.1	83.5	6.0	5.7^S	0.0

*      Including cells carrying exchanges

^S       Aberration frequency statistically significant higher than corresponding control values

¹       DMSO        0.5% (v/v)

²         EMS        825 µg/mL

³       CPA       10.0 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, the test item is considered to be clastogenic in this chromosome aberration test, when tested up to the highest evaluable concentration.

Executive summary:

The test item dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in one experiment. The following study design was performed:

	With and Without S9 mix
	Exp. I
Exposure period	4 hrs
Recovery	18 hrs
Preparation interval	22 hrs

In each experimental group two parallel cultures were analyzed. Per culture 150 metaphases were evaluated for structural chromosomal aberrations.

The highest applied concentration in this study (2000 µg/mL of the test item) was chosen with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473.

In the absence of S9 mix, moderate cytotoxicity was observed up to the highest evaluable concentration. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration. Concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

In the absence of S9 mix, statistically significant increases were observed in all evaluated concentrations (3.7 up to 9.3 % aberrant cells, excluding gaps). These values were clearly out of the range of the laboratory historical solvent control data (0.1 – 2.8 % aberrant cells, excluding gaps). Dose dependency, tested by a trend test, which was marginally not statistically significant (p-value: 0.064), did not occurr. In the presence of S9 mix, statistically significant increases were observed in the two highest evaluated concentrations (5.0 and 5.7 % aberrant cells, excluding gaps). All three evaluated concentrations were clearly out of the range of the laboratory historical solvent control data (0.4 – 3.2 % aberrant cells, excluding gaps). Dose dependency, tested by a trend test, which was statistically significant, occurred.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 2017 until 17 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

4 hours treatment without S9 mix: 1.56; 3.13; 6.25; 12.5; 25.0 µg/mL
4 hours treatment with S9 mix: 3.9; 7.8; 15.63; 31.25; 62.5 µg/mL

p = precipitation visible to the unaided eye at the end of treatment

Vehicle / solvent:

DMSO (0.5% v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours with and without metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: approx. 1000000 cells


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

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

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.36 in the solvent control versus pH 7.38 at 2000.0 µg/mL)
- Effects of osmolality: No increase (396 mOsm in the solvent control versus 372.mOsm at 2000 µg/mL)
- Evaporation from medium: Not examined
- Precipitation: No precipitation in the evaluated concentrations noted
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 4.6 µg/mL and 2000 µg/mL were used. The highest concentration was chosen with respect to the current OECD Guideline 476.
In the pre-experiment relevant toxic effects were observed after 4 hours treatment at 41.7 µg/mL and above without metabolic activation, and at 125.0 µg/mL and above with metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Precipitation occurred 125.0 µg/mL and above with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
The concentrations used in the main experiment were selected based on the cytotoxicity data of the pre-experiment. The individual concentrations were generally spaced by a factor of 2. Narrower spacing was used at higher concentrations to cover the cytotoxic range more closely.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by an adjusted cloning efficiency I below 50% in both cultures occurred at 25.0 µg/mL without metabolic activation.

Summary Table

Culture I

				relative	relative	rel. adjusted	mutant	0.95%
	conc.	P	S9	cloning	cell	cloning	colonies/	confidence
	µg/mL		mix	efficiency I	density	efficiency I	10 exp. 6	interval
				%	%	%	cells
Solvent control with DMSO		-	-	100.0	100.0	100.0	18.4	1.7 - 30.2
Positive control (EMS)	300.00	-	-	80.4	78.5	63.1	304.4	1.7 - 30.2
Test item	1.56	-	-	91.4	74.0	67.6	26.5	1.7 - 30.2
Test item	3.13	-	-	82.5	86.8	71.6	21.6	1.7 - 30.2
Test item	6.25	-	-	76.5	77.9	59.6	17.5	1.7 - 30.2
Test item	12.50	-	-	68.6	71.5	49.1	18.9	1.7 - 30.2
Test item	25.00	-	-	20.4	49.3	10.1	22.1	1.7 - 30.2
Test item	37.50	-	-	5.1	8.2	0.4	#	#
Test item	50.00	P	-	#	4.9	#	#	#
Test item	100.00	P	-	#	2.7	#	#	#
Solvent control with DMSO		-	+	100.0	100.0	100.0	14.2	2.0 - 29.4
Positive control (DMBA)	2.30	-	+	101.5	73.8	74.9	132.7	2.0 - 29.4
Test item	3.90	-	+	101.0	108.9	110.0	13.0	2.0 - 29.4
Test item	7.80	-	+	104.5	72.5	75.8	22.0	2.0 - 29.4
Test item	15.63	-	+	105.4	78.0	82.2	18.4	2.0 - 29.4
Test item	31.25	-	+	103.7	68.7	71.2	21.6	2.0 - 29.4
Test item	62.50	-	+	107.6	71.2	76.6	21.8	2.0 - 29.4
Test item	78.13	P	+	3.3	74.7	2.5	#	#
Test item	93.75	P	+	8.2	10.6	0.9	#	#
Test item	125.00	P	+	#	4.4	#	#	#

Culture II

				relative	relative	rel. adjusted	mutant	0.95%
	conc.	P	S9	cloning	cell	cloning	colonies/	confidence
	µg/mL		mix	efficiency I	density	efficiency I	10 exp. 6	interval
				%	%	%	cells
Solvent control with DMSO		-	-	100.0	100.0	100.0	22.4	1.7 - 30.2
Positive control (EMS)	300.00	-	-	87.6	108.2	94.8	252.4	1.7 - 30.2
Test item	1.56	-	-	88.6	108.2	95.9	14.3	1.7 - 30.2
Test item	3.13	-	-	83.9	113.6	95.4	21.9	1.7 - 30.2
Test item	6.25	-	-	78.6	119.8	94.2	17.5	1.7 - 30.2
Test item	12.50	-	-	78.6	112.1	88.2	16.4	1.7 - 30.2
Test item	25.00	-	-	36.8	96.6	35.6	15.5	1.7 - 30.2
Test item	37.50	-	-	12.7	12.7	1.6	#	#
Test item	50.00	P	-	#	3.0	#	#	#
Test item	100.00	P	-	#	3.4	#	#	#
Solvent control with DMSO		-	+	100.0	100.0	100.0	13.1	2.0 - 29.4
Positive control (DMBA)	2.30	-	+	107.6	84.5	91.0	131.2	2.0 - 29.4
Test item	3.90	-	+	106.1	84.4	89.6	26.2	2.0 - 29.4
Test item	7.80	-	+	109.0	87.5	95.4	26.0	2.0 - 29.4
Test item	15.63	-	+	112.2	77.8	87.3	31.6	2.0 - 29.4
Test item	31.25	-	+	109.8	73.8	81.0	22.1	2.0 - 29.4
Test item	62.50	-	+	112.4	67.0	75.3	12.8	2.0 - 29.4
Test item	78.13	P	+	11.0	40.4	4.5	#	#
Test item	93.75	P	+	3.3	10.3	0.3	#	#
Test item	125.00	P	+	#	2.9	#	#	#

P = precipitation visible at the end of treatment

#    culture was not continued due to exceedingly severe cytotoxic effects

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test tiem is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of the test test to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (2000 µg/mL) was chosen with the respect to the current OECD guideline. The highest concentration in the main experiment was limited by cytotoxicity observed in the pre-experiment.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiment up to the maximum concentration.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The OECD 474 assay was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. After expulsion of the main nucleus, PCE are released into peripheral blood. Peripheral blood was sampled and analysed for presence of micronucleated PCE. The test item prepared in 0.25% aqueous hydroxypropyl methyl cellulose(Methocel^®). The volume administered orally was 10 mL/kg bw for each application. Peripheral blood samples were collected for micronuclei analysis44h after the final application of the test item. Basedon the outcome of the dose range finding study, a dose of 250 mg/kg bw/day was selected as maximum tolerated dose (MTD. Inthe main experiment three dose levels were used covering a range from the maximum tolerated dose to little or no toxicity.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. Therefore, the test itme is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus 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:

02/2022 - 09/2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 474.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 474 “Mammalian Erythrocyte Micronucleus Test“, adopted July 29, 2016, recommends using mice. The mouse is an animal which has been used for many years as a suitable experimental animal in cytogenetic investigations. There is an abundance of available data, which may aid the interpretation of the results of the in vivo micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments may also be useful for the design and performance of the micronucleus test

Source: Charles River, 97633 Sulzfeld, Germany
Number of animals: 5 males per dose group (7 males for 1 MTD dose group)
Initial age at start of
acclimatisation: 6 - 12 weeks
Age at start of treatment: Minimum 7 weeks

Sex:

male

Details on test animals or test system and environmental conditions:

Housing: 5 animals of identical sex per cage
Cage type: IVC cage (Polysulphone), Type II L
Bedding: Altromin saw fiber bedding (Batch: 0131)
Feed: Free access to Altromin 1324 (Batch: 0939) maintenance diet for rats and mice
Air change: At least 10 x per hour
Water: Free access to tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
Environment: Temperature 22 ± 3 °C
Relative humidity 55 ± 10%
Artificial light 6:00 - 18:00

Route of administration:

oral: gavage

Vehicle:

0.25% aqueous hydroxypropyl methylcellulose (Methocel®) as vehicle of the test item was used as negative control. All control animals were handled in an identical manner to the test group subjects.
The sampling time for the negative control was 44 h after final treatment.
Name: 0.25% aqueous hydroxypropyl methylcellulose
Route and frequency of administration: oral, twice
Volume administered: 10 mL/kg bw

Name: Aqua ad iniectabilia
Supplier: Deltamedica
Batch No.: 2006003
Physical State/Colour:liquid/colorless
Storage: ambient, at room temperature
Expiry date: May 2023
Name: hydroxypropyl methylcellulose (Methocel®)
Supplier: Colorcon
Batch No.: D180I7S002
Storage: ambient, at room temperature
Expiry date: 27 July 2023

Details on exposure:

The animals received the test item twice at a 24 h interval by the oral route. Four hours before treatment all animals were under fasting condition.

Duration of treatment / exposure:

administrations on 2 consecutive days by oral gavage.

Frequency of treatment:

daily

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Top dose selection is based on results of the dose range finding experiment performend as pre test using 1000, 500, and 250 mg/kg bw.

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 M

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide
CAS No.: 50-18-0
Supplier: Sigma
Catalogue No.: C0768
Batch No.: MKCL2547
Dissolved in: physiological saline
Dosing: 40 mg/kg bw
Route and frequency of
administration: ip, single
Volume administered: 10 mL/kg bw
Expiry date: October 2022

Tissues and cell types examined:

Clinical observation, Blood cell analysis

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

A preceding study on toxicity was performed with the same strain and under identical conditions as in the main study. The maximum dose that was applied was 1500 mg/kg bw for females and 1000 mg/kg bw for males based on data concerning toxicity of the test item provided by the sponsor and animal welfare aspects. The maximum volume which was administered was 10 mL/kg bw. The animals received the test item once.
The use of the maximum tolerated dose is generally recommended. In cases where the test item cannot be dissolved or evenly suspended in any of the well-known vehicles, the highest dose which can be formulated and administered reproducibly is used. The volume to be administered is compatible with physiological space available.
The maximum tolerated dose is defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period (for example, by inducing body weight depression or hematopoietic system cytotoxicity, but not death or evidence of pain, suffering or distress necessitating humane euthanasia).
Animals treated with the highest dose (1 MTD) showed toxic effects with reduction of spontaneous activity, hunched posture, prone position, ataxia, constricted abdomen, piloerection and half eyelid closure up to 4 h after each application. 24 h after the final application all animals had fully recovered. Mice treated with 125 mg/kg bw/day (0.5 MTD) showed the same signs of toxicity as displayed for the 1 MTD dose group animals except for ataxia. These signs of toxicity were in total less intensely developed than in the 1 MTD group animals. 24 h after the final application all animals had fully recovered. The animals treated with 50 mg/kg bw/day (0.2 MTD) showed no signs of toxicity after the treatment with the test item.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling of the peripheral blood was carried out on animals 44 h after final treatment.

METHOD OF ANALYSIS:
Cell suspensions flow cytometry

OTHER:

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits), and
- bone marrow exposure to the test item occurred.

Statistics:

Poisson-based 95% control limits

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

reduction of spontaneous activity, hunched posture, prone position, ataxia, constricted abdomen, piloerection and half eyelid closure up to 4 h after each application. 24 h after the final application all animals had fully recovered.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose Formulation Analysis Results
The quantification of the test item formulations showed that measured concentrations lay below the expected nominal concentrations and below the acceptance criterion. This could be due to the preparation method of the test item that resulted in a suspension of the test item with the presence of small particles. Albeit all formulations were shown to be homogenous, the presence of particles and the use of relatively small volumes for formulation analysis could be the cause for the discrepancy.
The test item formulations for the pre-experiment to determine the maximum tolerated dose and for the main experiment were prepared following the same procedure and were applied to the animals as suspensions under continuous stirring.
Additionally, the clinical signs of systemic toxicity of the animals of the highest dose group (1 MTD, 250 mg/kg) in the main experiment were consistent with the observations of the pre-experiment indicating that the doses applied in both experiments were consistent. The application of a higher dose would have led to a level of systemic toxicity contradicting animal welfare.
Thus in both experiments of this study the maximum tolerated dose of the test item was applied according to OECD 474 and the criteria for selection of the maximum dose were fulfilled and the study is considered acceptable and valid.

Bioanalysis
For analytical purposes blood samples of additional three male animals were obtained two hours after the final application of the maximum tolerated dose of the test item.
The analysis of the blood plasma showed that the test material could be detected in all samples demonstrating systemic bioavailability after oral administration which is considered as evidence for exposure of bone marrow to the test item.


For further details on results incl. historical control please refer to study report attached as a confidential attachment.

Dose Group	Concentration [mg/kg bw/day]	Sampling time [h]	Rel. PCE	Proportion rel. PCE to Control [%]	MN [%]
NC	0	44	2.48	100	0.25
CPA	40	44	0.47	19.0	1.95
0.2 MTD	50	44	1.85	74.6	0.20
0.5 MTD	125	44	1.59	64.1	0.19
1 MTD	250	44	1.90	76.6	0.24

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, Art. 277455 (PY-2-B(OH)2) is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The provided information is sufficient for non classification for this endpoint according to the EU Regulation (EC) No 1272/2008 on Classification,Labelling and Packaging of Substances and Mixtures.