1,3-bis(hydroxymethyl)urea

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was considered mutagenic in the bacterial reverse mutation assay (reference 7.6.1 -1).

The test substance showed evidence of inducing mutation at the hprt locus in mouse lymphoma cells in the absence of a rat liver metabolic activation system (S9), but did not induce mutation in the same test system when tested in the presence of S9 (reference 7.6.1 -2).

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:

19 September 2016 to 28 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

All Salmonella typhimurium strains contained mutations in the histidine operon.
Escherichia coli WP2 uvrA carried a defect in one of the genes for tryptophan biosynthesis.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver metabolising system (S9 mix)

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system. A maximum concentration of 5000 µg/plate was selected, in order that initial treatments were performed up to the maximum recommended concentration according to current regulatory guidelines (OECD, 1997).

Vehicle / solvent:

- Solvent used: water
- Justification for choice of solvent/vehicle: The selection of the solvent for this assay was undertaken based on the available information from the preliminary solubility test. Ultra pure water showed best performance and was thus used for this experiment at a maximum concentration of 100 µL/plate.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ultra pure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

cumene hydroperoxide

other: 2-Aminoanthracene (2-AA); Daunomycin (DAUN)

Details on test system and experimental conditions:

METHOD OF APPLICATION:plate incorporation

DURATION
- Exposure duration: 2-3 days

NUMBER OF REPLICATIONS: 3 for each test concentration and positive control, 6 for solvent control

DETERMINATION OF CYTOTOXICITY
- Method: reduction of background growth

- OTHER:
Counting of revertant colonies:
Revertant colonies were either scored automatically with the „Sorcerer" or manually with the validated „Ames Study Manager" from Perceptive Instruments, Haverhill, Suffolk, UK. Tables of individual and mean values were generated automatically.
The presence of a background lawn of non-revertant cells was checked for each plate.

Evaluation criteria:

The assessment of test material-induced effects was dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which showed the highest number of colonies.
A test material was to be defined as negative or non-mutagenic in this assay if the assay was to be considered valid, and "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)
For valid data, the test material was considered to be positive or mutagenic if a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2810 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2810 µg/plate

Vehicle controls validity:

valid

Untreated 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:

starting at 2810 µg/plate

Vehicle controls validity:

valid

Untreated 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:

starting at 2810 µg/plate

Vehicle controls validity:

valid

Untreated 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:

starting at 2810 µg/plate

Vehicle controls validity:

valid

Untreated 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test material on the agar plates occurred.

HISTORICAL CONTROL DATA
Observed colony number remained mostly within the historical control values. Values outside those ranges remained without any biological significance.

Table 1: Summary of Series 1

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA 98	TA 100	TA 102	TA1535	TA 1537	WP2 uvrA
	Results without S9
Water	30 ± 5	102 ± 9	221 ± 19	21 ± 4	29 ± 4	36 ± 8
5	35 ± 6	100 ± 22	239 ± 47	22 ± 6	24 ± 6	36 ± 7
15.8	30 ± 4	115 ± 9	251 ± 37	25 ± 5	32 ± 5	34 ± 8
50	30 ± 8	116 ± 5	252 ± 52	25 ± 3	36 ± 5	36 ± 10
158	30 ± 7	102 ± 12	234 ± 44	22 ± 1	30 ± 5	38 ± 8
500	26 ± 6	108 ± 7	245 ± 21	22 ± 2	28 ± 8	32 ± 7
1580	28 ± 4	119 ± 13	352 ± 19	32 ± 9	25 ± 6	44 ± 9
5000	1 ± 1	1 ± 2	3 ± 2	1 ± 0	0 ± 1	42 ± 9
DAUN (1.0)	117 ± 12
NaN3 (2.0)		1446 ± 137		903 ± 15
CUM (200)			757 ± 15
9-AA (50)					1056 ± 286
NQO (2.0)						1509 ± 148
	Results with S9
Water	44 ± 6	125 ± 11	382 ± 33	24 ± 8	29 ± 4	43 ± 6
5	30 ± 8	133 ± 40	397 ± 41	27 ± 6	23 ± 4	37 ± 3
15.8	29 ± 5	126 ± 8	389 ± 27	27 ± 4	31 ± 5	40 ± 2
50	38 ± 2	123 ± 18	420 ± 26	21 ± 7	27 ± 14	46 ± 13
158	34 ± 5	136 ± 10	417 ± 26	25 ± 5	29 ± 2	33 ± 4
500	32 ± 3	116 ± 23	430 ± 18	29 ± 5	32 ± 3	49 ± 7
1580	31 ± 12	127 ± 8	458 ± 6	25 ± 7	26 ± 8	42 ± 40
5000	4 ± 3	9 ± 4	49 ± 10	1 ± 1	8 ± 6	48 ± 4
2-AA (2.0)	763 ± 47	1669 ± 146
2-AA (5.0)				254 ± 17	556 ± 58
2-AA (10)						512 ± 20
B(a)p (10)			1897 ± 213

 

 

Table 2: Summary of Series 2

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA 98	TA 100	TA 102	TA1535	TA 1537	WP2 uvrA
	Results without S9
Water	33 ± 7	117 ± 18	277 ± 16	30 ± 7	30 ± 3	30 ± 7
50	31 ± 3	124 ± 2	300 ± 32	27 ± 4	30 ± 4	36 ± 8
158	29 ± 5	126 ± 11	303 ± 15	18 ± 3	35 ± 3	31 ± 6
500	34 ± 7	117 ± 6	361 ± 39	28 ± 2	29 ± 3	35 ± 11
889	41 ± 11	118 ± 13	368 ± 16	30 ± 2	29 ± 6	34 ± 6
1580	30 ± 7	125 ± 7	476 ± 33	20 ± 2	28 ± 6	30 ± 3
2810	29 ± 6	117 ± 16	316 ± 96	18 ± 8	19 ± 4	42 ± 12
DAUN (1.0)	296 ± 29
NaN3 (2.0)		1709 ± 62		945 ± 27
CUM (200)			894 ± 90
9-AA (50)					1639 ± 183
NQO (2.0)						1608 ± 45
	Results with S9
Water	39 ± 9	132 ± 13	250 ± 28	24 ± 4	32 ± 3	37 ± 7
50	40 ± 8	143 ± 12	282 ± 55	24 ± 4	40 ± 4	39 ± 2
158	37 ± 11	140 ± 13	290 ± 31	25 ± 2	25 ± 8	41 ± 3
500	34 ± 11	139 ± 11	315 ± 37	24 ± 1	36 ± 5	43 ± 3
889	37 ± 2	130 ± 5	297 ± 41	24 ± 3	40 ± 6	38 ± 2
1580	29 ± 7	125 ± 11	414 ± 49	21 ± 8	29 ± 1	42 ± 8
2810	19 ± 6	129 ± 18	571 ± 72	22 ± 3	47 ± 6	48 ± 7
2-AA (2.0)	193 ± 43
2-AA (5.0)		1499 ± 61
2-AA (10)				160 ± 1	341 ± 17	149 ± 12
B(a)p (10)			1176 ± 45

 

Conclusions:

Under the experimental conditions the test substance was considered mutagenic in the bacterial reverse mutation assay.

Executive summary:

A study was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and in the presence of a rat liver metabolising system (S9 mix) according to OECD 471. The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10 % and 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following test item treatments of all the tester strains in the absence and presence of S9 mix, a relevant dose dependent clear increase in revertant numbers was observed in Salmonella typhimurium TA 102 in the presence of S9 mix. It was concluded that the test item was mutagenic under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2017 to 16 August 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)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: In vitro mammalian cell gene mutation test

Target gene:

hprt locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

tk+/- (3.7.2C)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.

MEDIA USED
- RPMI 1640 media supplied containing L-glutamine and HEPES, humidified incubator gassed with 5 ± 1% v/v CO2

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

Cytotoxicity range-finder:
0, 37.56, 75.13, 150.3, 300.5, 601, 1202 µg/mL (with and without metabolic activation)

Experiment 1:
0, 200, 400, 600, 700, 800, 900 µg/mL (without metabolic activation)
0, 200, 400, 600, 700, 800 µg/mL (with metabolic activation)

Experiment 2:
0, 200, 400, 500 µg/mL (without metabolic activation)

top dose applied showing clear cytotoxicity

Vehicle / solvent:

Test article stock solutions were prepared by formulating the test item under subdued lighting in purified water, with the aid of vortex mixing, warming at 37 °C and ultrasonication (where required) to give the maximum required concentration. Subsequent dilutions were made using purified water. The test article solutions were protected from light and used within approximately 2.5 hours of initial formulation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of MolToxTM S-9 were stored frozen in aliquots at <-50°C prior to use (Booth et al, 1980). Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).
The S-9 mix was prepared in the following way:
G6P (180 mg/mL), NADP (25 mg/mL), KCl (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, an aliquot of the mix was added to each cell culture to achieve the required final concentration of test article in a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%.

Cytotoxicity range-finder experiment:
Treatment of cell cultures for the cytotoxicity range-finder experiment was as described below for the mutation experiments. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200Cell concentrations were adjusted to 8 cells/mL and for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v CO2 in air for 7 days. Wells containing viable clones were identified by eye using background illumination and counted.

Treatment mutation assays:
At least 10E7 cells in a volume of 17 mL of RPMI 6 (cells in RPMI 10 diluted with RPMI A [no serum], to give a final concentration of 5% serum in 20 mL) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 2 mL vehicle or test article, or 0.2 mL positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours' incubation at 37±1°C with gentle agitation, cultures were centrifuged (200g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200g) for 5 minutes and finally resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and, where sufficient cells survived, the concentrations adjusted to 2 x 10E5 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies. Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.

Plating for survival:
Following adjustment of the cultures to 2 x 10E5 cells/mL after treatment, samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.

Expression period:
Cultures were maintained in flasks for a period of 7 days during which the hprt mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask. From observations on recovery and growth of the cultures during the expression period, appropriate cultures were selected to be plated for viability and 6TG resistance.

Plating for viability:
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10E5 cells/mL in readiness for plating for 6TG resistance. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (9 to 10 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for 6TG resistance:
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10E5 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 pg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (11 to 12 days) and wells containing clones were identified as above and counted.

Analysis of results:
From the zero term of the Poisson distribution the probable number of clones/well (P) on microtitre plates in which there are EW empty wells (without clones) out of a total of TW wells is given by: P = -ln (EW/TW).
Cloning Efficiency (CE) in any given culture is therefore: CE = P/No of cells plated per well
and as an average of 1.6 cells/well were plated on all survival and viability plates: CE = P/1.6.
Percentage Relative Survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures:
% RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted as follows:
Adj. % RS = RS % x (post-treatment cell concentration treated / post-treatment cell concentration vehicle control)
All %RS values were adjusted as described above.
Mutant Frequency (MF) is usually expressed as "mutants per 10E6 viable cells". In order to calculate this, the cloning efficiencies of both mutant and viable cells in the same culture were calculated: MF = [CE (mutant)/CE (viable)] x 10E6.
From the formulae given and with the knowledge that 2 x 10E4 cells were plated/well for mutation to 6TG resistance:
CE (mutant) = P (mutant)/2 x 104
CE (viable) = P (viable)/1.6
where, in each case, P = -ln (EW/TW).
Therefore, MF = [P (mutant)/2 x 10E4] x [1.6/P (viable)] x 10E6 = {-ln [EW/TW (mutant)]/-ln [EW/TW (viable)]} x 80.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p<0.05).
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p<0.05).
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF +/-2 standard deviations).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity:
In the cytotoxicity range-finder experiment, six concentrations were tested in the absence and presence of S-9 ranging from 37.56 to 1202 µg/mL (equivalent to 10 mM). No precipitate was observed. The highest concentration to give >10% RS was 601 µg/mL, which gave 48% and 43% RS in the absence and presence of S-9, respectively. No marked changes in osmolality or pH were observed in the range-finder at the highest concentrations tested (1202 µg/mL), compared to the concurrent vehicle controls.
In experiment 1 ten concentrations, ranging from 100 to 1202 µg/mL, were tested in the absence and presence of S-9. No precipitate was observed. Following the treatment incubation period, cultures tested at 100 µg/mL in the absence and presence of S-9 were not plated for survival as there were sufficient concentrations to determine an appropriate toxicity profile and were discarded. Seven days after treatment, the highest three concentrations in the absence of S-9 (1000 to 1202 µg/mL) and the highest four concentrations in the presence of S-9 (900 to 1202 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 900 µg/mL in the absence of S-9 and 800 pg/mL in the presence of S-9, which gave 10% and 19% RS, respectively.
In experiment 2 eleven concentrations, ranging from 200.0 to 1202 µg/mL, were tested in the absence of S-9. No precipitate was observed. Seven days after treatment, the highest eight concentrations (600 to 1202 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected. The highest concentration analysed was 500 µg/mL, which gave 12% RS.

Mutation frequency:
The acceptance criteria were met with the exception that in experiment 2, only three concentrations (up to a maximum of 500 µg/mL) gave >10% RS and were
acceptable for analysis. However, these concentrations, ranging from 200 to 500 µg/mL, covered a wide range of toxicity from non-toxic to toxic (80% to
12% RS), therefore the data were considered acceptable and the study was accepted as valid.
In the absence of S-9 when tested up to toxic concentrations in experiment 1, a statistically significant increase in MF was observed at the highest concentration analysed (900 µg/mL) and there was a statistically significant linear trend (p<0.01). The individual MF values in both replicate cultures at 900 µg/mL exceeded the historical mean vehicle control MF at the time of experiment 2 (5.04: range 1.45 to 8.63) and some individual MF values at lower test article concentrations also exceeded the historical control range, which was indicative of a positive result.
In the absence of S-9 when tested up to toxic concentrations in experiment 2, a statistically significant increase in MF was observed at the highest concentration analysed (500 µg/mL) and there was a statistically significant linear trend (p<0.01). The individual MF values in both replicate cultures at 500 µg/mL exceeded the historical mean vehicle control MF, which was again indicative of a positive result.
In the presence of S-9 when tested up to toxic concentrations in experiment 1, no statistically significant increases in mean MF, compared to the vehicle control MF value, were observed at any concentration analysed and there was no statistically significant linear trend.

Table 2: Range-Finder Experiment - 3 Hour Treatment in the Absence and Presence of S-9

Concentration [µg/mL]	3 hour treatment -S-9	3 hour treatment +S-9
	% RS	% RS
0	100	100
37.56	100	100
75.13	143	119
150.3	120	97
300.5	101	71
601	48	43
1202	2	1

% RS Percent Relative Survival

Table 3: Experiment 1-3 Hour treatment in the Absence and Presence of S-9

3 Hour Treatment -S-9			3 Hour Treatment +S-9
Concentration [µg/mL]	% RS	MF§	Concentration [µg/mL]	% RS	MF§
0	100	6.59	0	100	7.48
200	100	3.90 NS	200	73	7.56 NS
400	82	7.90 NS	400	74	6.14 NS
600	42	10.53 NS	600	55	6.42 NS
700	17	7.06 NS	700	34	8.05 NS
800	20	8.36 NS	800	19	6.11 NS
900	10	15.00*
NQO 0.15	69	22.36	B[a]P 2	69	18.02
NQO 0.20	61	25.74	B[a]P 3	41	56.84

Linear trend test on mutant frequency -S-9: p≤0.01

Linear trend test on mutant frequency +S-9: NS

§         6-TG resistant mutants/106 viable cells 7 days after treatment

%RS   Percent relative survival adjusted by post treatment cell counts

NS      Not significant

*          Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level

*, **, ***           Test for linear trend:χ2 (one-sided), significant at 5%, 1% and 0.1% level respectively

Table 4: Experiment 2 - 3 Hour Treatment -S-9

Concentration [µg/mL]	% RS	MF§
0	100	5.26
200	80	7.72 NS
400	29	7.52 NS
500	12	12.72*
NQO 0.150	50	32.74
NQO 0.200	22	83.39

Linear trend test on mutant frequency -S-9: P≤0.01

§         6-TG resistant mutants/106 viable cells 7 days after treatment

%RS   Percent relative survival adjusted by post treatment cell counts

NS      Not significant

*          Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level

*, **, ***           Test for linear trend: χ2 (one-sided), significant at 5%, 1% and 0.1% level respectively

 

Conclusions:

It is concluded that the test item showed evidence of inducing mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence of a rat liver metabolic activation system (S-9) in two independent experiments, but did not induce mutation in the same test system when tested up to toxic concentrations for 3 hours in the presence of S-9 under the experimental conditions described.

Executive summary:

The test item was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finder experiment followed by a mutation experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). A second mutation experiment was subsequently performed in the absence of S-9 to confirm a weak positive result obtained for the first experiment under this treatment condition. The test article was formulated in purified water.

A 3 hour treatment incubation period was used for all experiments. In the cytotoxicity range-finder experiment, six concentrations were tested in the absence and presence of S-9 ranging from 37.56 to 1202 μg/mL (equivalent to 10 mM). The highest concentration to give ≥10% relative survival (RS) was 601 μg/mL, which gave 48% and 43% RS in the absence and presence of S-9, respectively.

In experiment 1 ten concentrations, ranging from 100.0 to 1202 μg/mL, were tested in the absence and presence of S-9. The highest concentrations analysed to determine viability and 6TG resistance were 900 μg/mL in the absence of S-9 and 800 μg/mL in the presence of S-9, which gave 10% and 19% RS, respectively.

In experiment 2 eleven concentrations, ranging from 200.0 to 1202 μg/mL, were tested in the absence of S-9. The highest concentration analysed to determine viability and 6TG resistance was 500 μg/mL, which gave 12% RS.

Vehicle and positive control treatments were included in the mutation experiments in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

In the absence of S-9 when tested up to toxic concentrations in experiment 1, a statistically significant increase in MF was observed at the highest concentration analysed (900 μg/mL) and there was a statistically significant linear trend (p≤0.01).

The individual MF values in both replicate cultures at 900 μg/mL exceeded the historical mean vehicle control MF at the time of experiment 2 (5.04: range 1.45 to 8.63) and some individual MF values at lower test article concentrations also exceeded the historical control range, which was indicative of a positive result.

In the absence of S-9 in experiment 2 there was a shift in toxicity and only three concentrations (up to a maximum of 500 μg/mL) gave ≥10% RS. However, these concentrations, ranging from 200 to 500 μg/mL, covered a wide range of toxicity from 80% to 12% RS and the data were considered acceptable. When tested up to toxic concentrations in the absence of S-9 in experiment 2, a statistically significant increase in MF was observed at the highest concentration analysed (500 μg/mL) and there was a statistically significant linear trend (p≤0.01). The individual MF values in both replicate cultures at 500 μg/mL exceeded the historical mean vehicle control MF.

In the presence of S-9 when tested up to toxic concentrations in experiment 1, no statistically significant increases in mean MF, compared to the vehicle control MF value, were observed at any concentration analysed and there was no statistically significant linear trend.

It is concluded that the test item showed evidence of inducing mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence of a rat liver metabolic activation system (S-9) in two independent experiments, but did not induce mutation in the same test system when tested up to toxic concentrations for 3 hours in the presence of S-9 under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Under the experimental conditions reported the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse (reference 7.6.2-1).

Under the conditions of an OECD 489 compliant assay (Comet assay), the test item was concluded to be negative for the induction of DNA damage in liver.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2012 to 09 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

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:

other: Mammalian Erythrocyte Micronucleus Test

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: 8 - 11 weeks
- Weight at study initiation: mean value 34.8 g (SD ± 1.7 g)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: single, Makrolon Type II/III, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: ad libitum, pelleted standard diet (Harlan Laboratories B.V.; Postbus 6174; 5960 AD Horst; The Netherlands)
- Water: ad libitum, tap water
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): 35 - 65
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: sterile water
- Justification for choice of solvent/vehicle: relative non-toxicity for the animals
- Amount of vehicle: All animals received a single standard volume orally. A correction factor of 1.26 was applied based on preliminary data of the sponsor.
- Lot/batch no.: 113318061

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was dissolved in sterile water.

Duration of treatment / exposure:

24 h for all dose groups and in addition 48 h for additional animals of vehicle control and 2000 mg/kg bw group

Frequency of treatment:

1 application

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

2 groups of animals were treated with this dose for collection after 24 and 48 h.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 in test groups, 5 in control groups

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 40 mg/kg bw, Volume of 10 ml/kg bw (in sterile water)
- Lot/batch no.: A0302605

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 48 hours. Three adequately spaced dose levels spaced by a factor of 2 were administered.

TREATMENT AND SAMPLING TIMES: Sampling of the bone marrow was done 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald /Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

A test item was classified as mutagenic if it induced either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.

A test item that failed to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes was considered non-mutagenic in this system.

Statistics:

nonparametric Mann-Whitney test

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose: The animals treated with 2000 mg/kg bw showed no clinical signs.
- Clinical signs of toxicity in test animals: no
- Other: No sex specific differences were observed with regard to clinical signs. In accordance with the test guidelines the main study was performed using males only.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In comparison to the corresponding vehicle controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. In fact the mean values of micronuclei observed after treatment with the test substance were below the value of the vehicle control group.
- Statistical evaluation: Statistical analysis was performed only for the positive control (p = 0.004).
- Clinical signs of toxicity in test animals: Ruffled fur was detected in some animals of the 2000 mg/kg bw dose groups. The treated animals in other dose groups did not express any clinical signs.

Table 1: Summray of the results

Test group	Dose [mg/kg bw]	Sampling time [h]	PCEs with micronuclei [%]	Range	PCE per 2000 erythrocytes
Vehicle	0	24	0.15	1-6	1441
Test item	500	24	0.143	1-7	1502
Test item	1000	24	0.136	1-4	1377
Test item	2000	24	0.186	0-5	1335
Positive control	40	24	1.58	22-56	1437
Vehicle	0	48	0.19	1-7	1455
Test item	2000	48	0.143	2-5	1387

Conclusions:

Under the experimental conditions reported the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

A study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in sterile water, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h (vehicle and 2000 mg/kg bw only) after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The dose levels of the test item were investigated based on results of a pre-experiment. Administered doses were 500, 1000 and 2000 mg/kg bw. A correction factor of 1.26 was applied based on test item purity and data provided by the sponsor. After treatment with the test item the mean number of PCEs per 2000 erythrocytes in the high dose group after 24 and 48 hour treatment was slightly lower as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control. However, only few individual values in the high dose group fell minimally below the lowest individual vehicle control value thus indicating that the test item exerts only a minimal cytotoxic effect in the bone marrow, if at all. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that under the experimental conditions reported the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 August 2016 - 30 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Hsd:SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS, Inc.
- Age at study initiation: 6 - 8 weeks
- Weight at randomization: males 150 - 350 g, females 120 - 250 g
- Assigned to test groups randomly: yes, under following basis: randomization function within Microsoft Excel
- Fasting period before study: not specified
- Housing: up to 5 animals/sex per cage
- Diet: certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet) ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20%
- Air changes: not specified
- Photoperiod: 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: water
- Justification for choice of solvent/vehicle: solubility properties of the test item, non-toxicity to animals
- Concentration of test material in vehicle: 50, 100 and 200 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: not applicable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared at least once on each day of use, each concentration was prepared by mixing an appropriate amount of the test substance with the appropriate volume of the vehicle, the formulation was vortexed, sonicated, homogenized, and/or stirred in order to achieve workable or soluble formulations, if needed

Duration of treatment / exposure:

24 hours

Frequency of treatment:

2 treatments, second treatment approx. 21 hours after first dosing

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 animals in the dose groups, 3 animals in the positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate
- Justification for choice of positive control: appropriate reference control based on historical laboratory control data
- Route of administration: oral (gavage)
- Dose: 200 mg/kg bw

Tissues and cell types examined:

liver, stomach and duodenum

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: determination of MTD in dose range finding experiment

TREATMENT AND SAMPLING TIMES: approx. 3-4 hours after last treatment tissues were prepared in mincing solution
- Liver was minced with fine scissors and the cell suspension was strained
- Glandular stomach and duodenum was scraped with plastic spatula and the cell suspension was strained

DETAILS OF SLIDE PREPARATION:
At least four slides/wells per animal were prepared per organ/tissue. An aliquot of 2.5-7.5 μL of each cell suspension per slide was mixed with 0.5% low melting agarose. The cell/agarose suspension was applied to microscopic slides coated with 1% normal melting agarose. The slides were placed at 2-8 ºC for at least 15 minutes, to allow the gels to solidify. Three slides/wells were used for scoring.
Each slide was submerged in a lysis solution at least overnight at 2-8 ºC. The lysis solution was composed of 100 mM EDTA (disodium), 2.5 M sodium chloride, 10 mM tris hydroxymethyl aminomethane in purified water; pH 10; 1% triton-X100 and 10% DMSO was added on the day of use.
After cell lysis, slides were washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH ~ 7.5) and placed in an electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer (300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water, pH > 13) for ~ 20 minutes at 2-10 ºC, protected from light. Electrophoresis was conducted for 30 minutes at 0.7 volts/cm.
The slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides were dehydrated with 200-proof ethanol for at least 5 minutes, then air-dried and stored at room temperature with desiccant. Slides were stained with SybrgoldTM prior to scoring.

METHOD OF ANALYSIS: Three slides/wells per organ/animal were used. Fifty randomly selected cells per slide/well were scored (total of 150 cells per animal). The following endpoints of DNA damage were assessed and measured:
- Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail.
- % Tail DNA (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
- Tail Moment (also known as Olive Tail Moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/100 ].
Each slide/well was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was determined by scanning 150 cells per animal (percentage of “clouds” is calculated by adding the total number of clouds for all slides scored, dividing by the total number of cells scored and multiplying by 100). The “clouds”, also known as “hedgehogs”, are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis or apoptosis. A “cloud” is produced when almost the entire cell DNA is in the tail of the comet and the head is reduced in size, almost nonexistent. “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis.

OTHER: tissue samples were fixed with formalin for possible histopathology

Evaluation criteria:

A test substance is considered to have induced a positive response if
a) at least one of the group mean for the % tail DNA of the test substance doses exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
b) when multiple doses are examined at a particular sampling time, the increase is dose-related (p ≤ 0.01) and
c) results of the group mean or of the individual animals of at least one group are outside the distribution of the historical negative control database for that tissue.
A test substance is considered to have induced a clear negative response if none of the criteria for a positive response were met and there is direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue has been demonstrated.
If the response is neither clearly positive nor clearly negative, or in order to assist in establishing the biological relevance of a result, the data are evaluated by expert judgment and/or further investigations.

Statistics:

The median %tail DNA for the Comets scored on each slide is determined and the mean of the median values is calculated for each animal. The mean of the individual animal is then used to calculate a group mean.
The group variances for % tail DNA generated for the vehicle and test substance groups is compared using Levene’s test (significance level of p ≤ 0.05). If the differences and variations between groups are found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test is performed (significance level of p ≤ 0.05). If Levene’s test indicates heterogeneous group variances (significance level of p ≤ 0.05), the suitability of a transformation of the original data is evaluated (e.g. using logarithm transformed values of the original data) in an attempt to meet the normality criteria. Afterwards, statistical analysis is performed using the parametric tests described above. If parametric tests are not acceptable, non-parametric statistical methods (Kruskal Wallis and/or Mann Whitney test) are used in evaluation of data.
A linear regression analysis is conducted to assess dose responsiveness in the test substance treated groups (significance level of p ≤ 0.01).
A pair-wise comparison (Student’s T-test, significance level of p ≤ 0.05) is used to compare the positive control group to the concurrent vehicle control group. If parametric tests are not acceptable, non-parametric statistical methods (Kruskal Wallis and/or Mann Whitney test) are used in evaluation of data.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000, 2000 mg/kg bw/d
- Solubility: The test item was well soluble in deionized water
- Clinical signs of toxicity in test animals: No mortality occurred at any dose level during the course of the dose range finding assay. No considerable reductions in mean group body weights were seen in the test substance treated groups during the course of the study. All rats appeared normal throughout the observation period.
- Evidence of cytotoxicity in tissue analyzed: no
- Rationale for exposure: according to guideline


RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: according to guideline and previous DRF

Table 1. % Tail DNA in Liver Cells Following Administrations of test item (Samples collected 3 to 4 hours post-last dose)

Treatment (10 mL/kg/treatment)	Number of Animals	Group Mean % of Clouds	Tail DNA (%)^A Mean ± S. D.
Vehicle Control: Deionized Water	6	0.0	0.19± 0.25
Test Item 500 mg/kg bw/d	6	0.8	0.10± 0.14
Test Item 1000 mg/kg bw/d	6	0.5	0.13± 0.11
Test Item 2000 mg/kg bw/d	6	0.5	0.35± 0.61
Positive Control EMS 200 mg/kg^B	3	1.0	16.60± 2.96*

^AMean of 3 or 6 animals means of medians ^BEthyl methanesulfonate (EMS administered only once at 3 to 4 hours prior to organ collection day 2

S.D. = Standard Deviation *p ≤ 0.05 (Student’s t-test); Statistically significant increase relative to the vehicle control

 

Table 2. % Tail DNA in Glandular Stomach Cells Following Administrations of test item (Samples collected 3 to 4 hours post-last dose)

 

Treatment (10 mL/kg/treatment)	Number of Animals	Group Mean % of Clouds	Tail DNA (%)^A Mean ± S. D.
Vehicle Control: Deionized Water	6	12.5	12.06±2.89
Test Item 500 mg/kg bw/d	6	17.0	7.73± 1.97@
Test Item 1000 mg/kg bw/d	6	7.7	1.71± 1.27@
Test Item 2000 mg/kg bw/d	6	10.5	0.24± 0.18@
Positive Control EMS 200 mg/kg^B	3	33.7	32.97± 4.10*

^AMean of 3 or 6 animals means of medians ^BEthyl methanesulfonate (EMS administered only once at 3 to 4 hours prior to organ collection day 2

S.D. = Standard Deviation *p ≤ 0.05 (Student’s t-test); Statistically significant increase relative to the vehicle control

@p ≤ 0.01 (regression analysis): Statistically significant relative to the vehicle control.

 

 Table 3. % Tail DNA in Duodenal Cells Following Administrations of test item (Samples collected 3 to 4 hours post-last dose)

Treatment (10 mL/kg/treatment)	Number of Animals	Group Mean % of Clouds	Tail DNA (%)^A Mean ± S. D.
Vehicle Control: Deionized Water	6	22.7	1.89± 0.91
Test Item 500 mg/kg bw/d	6	46.3	3.33± 1.67
Test Item 1000 mg/kg bw/d	6	16.0	2.05± 0.99
Test Item 2000 mg/kg bw/d	6	18.2	1.94±0.61
Positive Control EMS 200 mg/kg^B	3	27.0	16.51±1.51*

^AMean of 3 or 6 animals means of medians ^BEthyl methanesulfonate (EMS administered only once at 3 to 4 hours prior to organ collection day 2

S.D. = Standard Deviation *p ≤ 0.05 (Student’s t-test); Statistically significant increase relative to the vehicle control

 

Conclusions:

Under the conditions of this in vivo comet assay, the test item was concluded to be negative for the induction of DNA damage in liver.

Executive summary:

The test item was evaluated for genotoxicity using the Comet assay to evaluate its potential to induce DNA damage in liver, stomach and duodenum cells of male rats. Deionized water was selected as the vehicle. Test substance and/or control formulations were administered at a dose volume of 10 mL/kg/day by oral gavage. In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/dose. The additional dose levels tested were 500 and 1000 mg/kg/day in three animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/day, which is the highest guideline recommended dose for this assay.

The definitive assay dose levels tested were 500, 1000, and 2000 mg/kg/day with males only. The definitive assay was repeated due to technical issues. The data from the initial definitive assay is included in the raw data workbook. Only the data from the repeat definitive assay is included in this report.

The test substance gave a negative (non-DNA damaging) response in this assay in liver, stomach and duodenum for males in % tail DNA. None of the test substance treated animal multi-well slides had statistically significant increases in the % tail DNA compared to the respective vehicle controls. However, the test substance treated multi-well slides for stomach did have a statistically significant dose response but the response was decreasing. The vehicle control % tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, stomach and duodenum.

Under the conditions of this study, the administration of the test item at doses up to and including a dose of 2000 mg/kg/day did not cause a significant increase in DNA damage in liver, stomach and duodenum relative to the concurrent vehicle control. Therefore, the test item was concluded to be negative in the in vivo Comet Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity

in vitro

A study (reference 7.6.1 -1) was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and in the presence of a rat liver metabolising system (S9 mix) according to OECD 471. The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10 % and 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following test item treatments of all the tester strains in the absence and presence of S9 mix, a relevant dose dependent clear increase in revertant numbers was observed in Salmonella typhimurium TA 102 in the presence of S9 mix. It was concluded that the test item was mutagenic under the experimental conditions described.

A bacterial reverse gene mutation assay with and without metabolic activation (S9 mix) using the test material was conducted (reference 7.6.1 -3). The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium strains TA 98, TA 100 and TA102. The preincubation method was performed with test item concentrations of 0.21, 0.42, 0.83, 2.08, 4.16, 8.33 µmol/plate. Vehicle and positive control treatments were included for all strains. Cytotoxicity was detected for TA 98 and TA 100 without metabolic activation. A dose dependent increase in revertant numbers was detected in all test strains with and without metabolic activation. Therefore the test item was determined to be mutagenic in bacterial reverse mutation assay under the described conditions.

A micronucleus test in vitro was conducted with the test substance using V79 cells. The test substance was applied in concentrations from 3-333 µg/L with and without metabolic activation (S9). For each concentration 2 independent slides were prepared and each 500 cells were evaluated. Relative density of cells was used to analyze cytotoxic effects. The test substance was determined to induce micronuclei at 100 and 333 µmol/L and to be cytotoxic at 333 µmol/L in the absence of metabolic activation. With metabolic activation no cytotoxic and no mutagenic effects of the test substance were observed (reference 7.6.1 -4).

The test item was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol (reference 7.6.1 -2). A 3 hour treatment incubation period was used for all experiments.

In experiment 1, ten concentrations, ranging from 100.0 to 1202 μg/mL, were tested in the absence and presence of S-9. The highest concentrations analysed to determine viability and 6TG resistance were 900 μg/mL in the absence of S-9 and 800 μg/mL in the presence of S-9, which gave 10% and 19% RS, respectively.

In experiment 2 eleven concentrations, ranging from 200.0 to 1202 μg/mL, were tested in the absence of S-9. The highest concentration analysed to determine viability and 6TG resistance was 500 μg/mL, which gave 12% RS.

Vehicle and positive control treatments were included in the mutation experiments in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

In the absence of S-9 when tested up to toxic concentrations in experiment 1, a statistically significant increase in MF was observed at the highest concentration analysed (900 μg/mL) and there was a statistically significant linear trend (p≤0.01).

The individual MF values in both replicate cultures at 900 μg/mL exceeded the historical mean vehicle control MF at the time of experiment 2 (5.04: range 1.45 to 8.63) and some individual MF values at lower test article concentrations also exceeded the historical control range, which was indicative of a positive result.

In the absence of S-9 in experiment 2 there was a shift in toxicity and only three concentrations (up to a maximum of 500 μg/mL) gave ≥10% RS. However, these concentrations, ranging from 200 to 500 μg/mL, covered a wide range of toxicity from 80% to 12% RS and the data were considered acceptable. When tested up to toxic concentrations in the absence of S-9 in experiment 2, a statistically significant increase in MF was observed at the highest concentration analysed (500 μg/mL) and there was a statistically significant linear trend (p≤0.01). The individual MF values in both replicate cultures at 500 μg/mL exceeded the historical mean vehicle control MF.

In the presence of S-9 when tested up to toxic concentrations in experiment 1, no statistically significant increases in mean MF, compared to the vehicle control MF value, were observed at any concentration analysed and there was no statistically significant linear trend.

It is concluded that the test item showed evidence of inducing mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence of a rat liver metabolic activation system (S-9) in two independent experiments, but did not induce mutation in the same test system when tested up to toxic concentrations for 3 hours in the presence of S-9 under the experimental conditions described.

in vivo

A study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (reference 7.6.2-1). The test item was dissolved in sterile water, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h (vehicle and 2000 mg/kg bw only) after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The dose levels of the test item were investigated based on results of a preexperiment. Administered doses were 500, 1000 and 2000 mg/kg bw. A correction factor of 1.26 was applied based on test item purity and data provided by the sponsor. After treatment with the test item the mean number of PCEs per 2000 erythrocytes in the high dose group after 24 and 48 hour treatment was slightly lower as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control. However, only few individual values in the high dose group fell minimally below the lowest individual vehicle control value thus indicating that the test item exerts only a minimal cytotoxic effect in the bone marrow, if at all. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that under the experimental conditions reported the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.

The test item was evaluated for genotoxicity using the Comet assay to evaluate its potential to induce DNA damage in liver, stomach and duodenum cells of male rats (reference 7.6.2 -2). Deionized water was selected as the vehicle. Test substance and/or control formulations were administered at a dose volume of 10 mL/kg/day by oral gavage. In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/dose. The additional dose levels tested were 500 and 1000 mg/kg/day in three animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/day, which is the highest guideline recommended dose for this assay.

The definitive assay dose levels tested were 500, 1000, and 2000 mg/kg/day with males only. The definitive assay was repeated due to technical issues. The data from the initial definitive assay is included in the raw data workbook. Only the data from the repeat definitive assay is included in this report.

The test substance gave a negative (non-DNA damaging) response in this assay in liver, stomach and duodenum for males in % tail DNA. None of the test substance treated animal multi-well slides had statistically significant increases in the % tail DNA compared to the respective vehicle controls. However, the test substance treated multi-well slides for stomach did have a statistically significant dose response but the response was decreasing. The vehicle control % tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, stomach and duodenum.

Under the conditions of this study, the administration of the test item at doses up to and including a dose of 2000 mg/kg/day did not cause a significant increase in DNA damage in liver, stomach and duodenum relative to the concurrent vehicle control. Therefore, the test item was concluded to be negative in the in vivo Comet Assay.

Conclusion

Based on negative findings in two in vivo studies covering both, mutagenicity as well as cytogenicity endpoints, the positive finding in the bacterial reverse mutation assay was not confirmed. According to ECHA Guidance R7a (2017) results obtained in vivo are of greater relevance than results of in vitro tests when assessing genotoxicity potential of a substance.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No 2017/776.