N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria. Key study. Method according to OECD guideline 471, GLP study. The test item was found to be mutagenic with or without metabolic activation in the Salmonella typhimurium tested strains TA 100, TA 1535 and TA 98.

In vitro micronucleus study in mammalian cells. Key study. Method according to OECD guideline 487, GLP study. The test item was found as non aneugenic and non clastogenic in human lymphocytes up to the concentration of 0.0625 mg/mL both in the presence and absence of metabolic activation under the test conditions.

In vitro gene mutation study in mamalian cells. Data waiving (study scientifically not necessary/other information available): According to the column 1 of REACH Annex VIII, the study does not to be conducted since a positive result was found in the in vitro gene mutation test in bacteria.

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:

22 October 2015 - 20 November 2015

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella thyphimurium

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver

Test concentrations with justification for top dose:

3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.
The test item concentrations to be applied in the main experiment were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (AppliChem Lot No. 410131997).
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

yes

Remarks:

(A. dest.)

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (10 µg/plate for TA 98 and 40 µg/plate for TA 1537 without metabolic activation); 2-Aminoanthracene (2.5 μg/plate for TA1537, TA1535, TA98 and TA100, and 10 µg/plate for TA102 with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 1 x10^9 bacteria/mL

DURATION
- Preincubation period: Samples of each tester strain were grown by culturing for 12 h at 37ºC in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 10e9 cells/mL).
- Exposure duration: 48 h incubation period at 37ºC in the dark.

SELECTION AGENT (mutation assays): All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was characterised by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Rationale for test conditions:

According to OECD TG 471

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high, and if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

(Biologically relevant increases of revertant colony numbers observed at concentrations of 316 µg/plate and higher (-S9) and at concentrations of 2500 µg/plate (+S9))

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

(Biologically relevant increases of revertant colony numbers observed at concentrations of 31.6 µg/plate and higher (±S9))

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

(biologically relevant increases of revertant colony numbers observed at concentrations of 10 µg/plate and higher (-S9) and in all concentrations tested (+S9))

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed at any concentration or tester strain used.
- Other: No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation).


RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with the tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested (3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate ) for toxicity and induction mutations with three plates each. The experimental conditions in this pre-experiment were the same as described for the main experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see tables 4 and 6 in "any other information on results incl. tables"
- Negative (solvent/vehicle) historical control data: see tables 3 and 5 in "any other information on results incl. tables"

Table 3: Historical Laboratory Control Data of the Negative Control (in 2012 - 2014) without S9 (-S9)

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Mean	22.3	95.5	10.9	7.5	230.5
SD	4.8	18.1	5.1	2.4	47.8
Min	13	61	4	2	136
Max	48	182	35	27	415
RSD [%]	21.6	18.9	46.8	31.4	20.8
n	1159	1281	1043	1043	684

Table 4: Historical Laboratory Control Data of the Positive Control (in 2012 - 2014) without S9 (-S9)

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Substance Conc./plate	4-NOPD 10 µg	NaN3 10 µg	NaN3 10 µg	4-NOPD 40 µg	MMS 1 µL
Mean	443.7	704.8	858.3	93.2	1733.5
SD	183.1	272.7	320.2	27.3	408.3
Min	192	132	34	30	162
Max	2213	1498	1472	273	3181
RSD [%]	41.3	38.7	37.3	29.3	23.6
n	1172	1285	1042	1054	688

Table 5: Historical Laboratory Control Data of the Negative Control (in 2012 - 2014) with S9 (+S9)

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Mean	29.9	103.4	9.1	7.6	290.9
SD	6.2	17.4	3.3	2.7	65.3
Min	13	68	4	3	91
Max	61	194	34	31	495
RSD [%]	20.9	16.8	36.1	35.3	22.4
n	1157	1286	1042	1040	683

Table 6: Historical Laboratory Control Data of the Positive Control (in 2012 - 2014) with S9 (+S9)

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Substance Conc./plate	2-AA 2.5 µg	2-AA 2.5 µg	2-AA 2.5 µg	2-AA 2.5 µg	2-AA 10 µg
Mean	2318.0	1839.6	100.0	218.6	663.9
SD	573.6	455.1	60.6	85.2	176.6
Min	128	169	19	28	137
Max	3606	3132	1011	489	2001
RSD [%]	24.7	24.7	60.6	39.0	26.6
n	1156	1284	1041	1039	688

S9: metabolic activation  

Conc.: concentration

Mean: mean of revertants/plate

Min.: minimum of revertants/plate

Max.: maximum of revertants/plate

SD: Standard Deviation

RSD: Relative Standard Deviation

n: Number of control values

Conclusions:

Under the experimental conditions reported, the test substance caused gene mutations in the tester strains TA 100, TA 1535 and TA 98. Therefore, the test substance is considered to be mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study). The experiments were carried out using Salmonella typhimurium strains TA1537, TA1535, TA98, TA100 and TA102 in the presence and absence of metabolic activation (S9 mix prepared from the livers of rats). The test item was dissolved in DMSO and diluted prior to treatment. In the initial toxicity-mutation test the bacterial cultures were exposed to test item at 8 concentrations (three plates/concentration) between 3.16 and 5000 µg/plate. No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation). In the main test, the cultures were exposed to test item at 8 concentrations (three plates/concentration) between 3.16 and 5000 µg/plate both in the absence and presence (S9 mix) of metabolic activation. All the values for the negative control were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies. All validity criteria were met. Under the experimental conditions reported, the test substance caused gene mutations by base pair changes in the genome of the tester strains TA 100 and TA 1535 and frameshifts in the genome of the tester strain TA 98. Therefore, the test substance is considered to be mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 January 2020 - 06 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test method according OECD Guideline 487. GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: peripheral blood lymphocytes from human donors.
- Suitability of cells: healthy, young, non-smoking donors with no known recent exposure to genotoxic chemicals or radiation were used. Blood from individual was collected in sodium heparin vacutainer and analyzed using Advia 2120. All the parameters were within the acceptable range.
- Normal cell cycle time (negative control): 12 to 15 hours.

For lymphocytes:
- Sex, age and number of blood donors: One male of 28 and one female of 24 years old were used, one for the initial cytotoxicity and the other for the micronucleus test.
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: No
- Mitogen used for lymphocytes: Phytohemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics (1% Penicillin-Streptomycin), 5±1% CO2, 37±1ºC.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B (CytoB)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver of male Wistar rat induced with sodium phenobarbtone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively.
- method of preparation of S9 mix: 1 mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution containing 4 mM Nicotinamide Adenine Dinucleotide Phosphate (NADP), 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) at pH 7.3.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL (10% v/v) of S9 mix and 0.05 mL (1% v/v) of S9 homogenate.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Batch of S9 homogenate was assessed for sterility, protein content and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 tester strain.

Test concentrations with justification for top dose:

0.015625, 0.03125 and 0.0625 mg/mL.
Justification for top dose: Based on the results of solubility, precipitation, osmolality and pH tests, the initial cytotoxicity test was conducted at concentrations of 0.0625, 0.125, 0.25 and 0.5 mg/mL. The average cytotoxicity (CBPI) ranged from 74.58% to 100% at 0.125 mg/mL to 0.5 mg/mL and from 51.67% to 52.54% at 0.0625 mg/mL. As the only dose in the range of 55 ± 5% cytotoxicity was 0.0625 mg/mL, this was selected as the highest concentration for the micronucleus test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: N, N-dimethyl formamide.
- Justification for choice of solvent/vehicle: A solubility test was conducted to determine the maximum concentration or workable solution/suspension of the test item in the vehicle compatible with the test system at 200 mg/mL using distilled water, dimethyl Sulphoxide, acetone and N, N-dimethyl formamide. The test item was found soluble in N, N-dimethyl formamide at 200 mg/mL. Precipitation, osmolality and pH test was conducted at 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post incubation, heavy precipitation was observed at 1 and 2 mg/mL and mild and moderate precipitation and no change in pH were observed at 0.25 and 0.5 mg/mL. No precipitation was observed in any other concentrations tested. No change in pH and osmolality was observed in any of the concentrations tested. Untreated controls were inlcuded in the test in order to demonstrate that no deleterious or chromosomal effects were induced by this solvent.
- Justification for percentage of solvent in the final culture medium: 50 µL of test item solutions were used for each concentration and the final volume in the test tubes was made up to 5 mL with culture media. Thus, percentage of solvent in the final culture medium was 1% (v/v), that is, not higher than 1% (v/v) as recommended by OECD TG 487 for organic solvents. In any case, untreated controls were inlcuded in the test in order to assure that this solvent had no adverse effect.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

N, N-dimethyl formamide

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

Remarks:

3 h treatment, without metabolic activation (0.03 µg/mL)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

N, N-dimethyl formamide

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

24 h treatment, without metabolic activation (0.075 µg/mL)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

N, N-dimethyl formamide

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

3 h treatment, with metabolic activation (10 µg/mL)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 44 to 48 hours, with phytohaemagglutinin (PHA) to induce cell division prior to exposure.
- Exposure duration/duration of treatment: 3 h (short term treatment, +S9 and -S9); 24 h (long term treatment, -S9).
- Harvest time after the end of treatment (sampling/recovery times): 20 to 24 hours (1.5- 2 cell cycle length).

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Cytokinesis was blocked using Cytochalasin B at 6 µg/mL. For the short treatment, cytoB was added at the end of treatment and incubated for 20-24 h. For the long treatment, cytoB was added at the beggining of the treatment.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After treatment, cell suspension was mixed with 3 mL of freshly prepared warm 0.56% Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 3 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was incubated for 10 minutes at room temperature and later suspension was centrifuged at 2000 rpm for 10 minutes. The procedure was repeated twice by adding 4 mL of cold acetic acid: methanol fixative (1:3). Clean slides were stored in a beaker with distilled water and kept in the refrigerator for at least 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension were aspirated and dropped onto the chilled slide pre labeled with study number, with (+S9) or without metabolic activation (-S9), treatment/group and slide number. The slides were air dried. Minimum of 3 slides were prepared for each treatment replicate. Smear was stained using Acridine Orange stain by allowing the stain to retain for 5 minutes.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Measurement of the relative frequencies of mononucleate, binucleate and multi-nucleate cells in the culture was made to determine cell proliferation and the cytostatic activity of the treatment to ensure that only cells that divided during or after treatment were scored. Slides were evaluated for the presence of micronuclei in at least 2000 binucleates per concentration (at least 1000 per culture).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity: CBPI was determined from 500 cells per culture.

Evaluation criteria:

Positive result if, in any of the experimental conditions examined:
a. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
b. The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
c. Any of the results are outside the distribution of the historical negative control data.

Negative result if, in all experimental conditions examined:
a. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
b. There is no concentration-related increase when evaluated with an appropriate trend test.
c. All results are inside the distribution of the historical negative control data.

Statistics:

Data was analyzed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p<0.05).

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the initial cytotoxicity test, the test item induced cytotoxicity at 0.0625 mg/mL (51.67 to 52.54%) when compared to vehicle control and thus this was selected as the top concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In an initial cytotoxicity test, the test item was tested at the concentrations of 0.0625, 0.125, 0.25 and 0.5 mg/mL. The average % cytotoxicity ranged from 74.58 to 100 at 0.125 mg/mL to 0.5 mg/mL and the average cytotoxicity ranged from 51.67 to 52.54 at 0.0625 mg/mL. As the % cytotoxicity of test item was in the range of 55 ± 5% at 0.0625 mg/mL, this was selected as the highest concentration for the micronucleus test. The other concentrations selected were 0.015625 and 0.03125 mg/mL as low and mid concentrations respectively.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
Vehicle control: average percentage of micronucleus of 0.24% (short treatment +S9 and -S9, and long treatment).
Negative control: average percentage of micronucleus of 0.25% (short treatment +S9), 0.19% (short treatment -S9 and long treatment)
Positive controls: average percentage of micronucleus of 1.06% (short treatment +S9), 1.03% (short treatment -S9 and 1.15% (long treatment)

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o CBPI and distribution of mono-, bi- and multi-nucleated cells: see table XXX below
- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture from binucleated cells: see table XXX below

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see table XXX below. Values obtained with concurrent positive controls were within the 95% control limits of the distribution of the laboratory’s historical positive controls database.
- Negative (solvent/vehicle) historical control data: see table XXX below. The concurrent vehicle control values were within the 95% control limits of the distribution of the laboratory’s historical vehicle control database.

Table 1. Summary of Cytokinesis Block Proliferation Index and cytotoxicity

Treatment	Metabolic Activation	Concentration (mg/mL)	Duration (hours)	Replicates No.	Total Number of Cells	CBPI	Mean CBPI	% of Cytotoxicity
Vehicle Control	With S9	0	3 to 6	1	536	1.61	1.61	NA
				2	534	1.61
Negative Control		0		1	536	1.57	1.60	1.64
				2	525	1.63
POLY-U		0.0625		1	508	1.29	1.29	52.46
				2	517	1.28
		0.125		1	556	1.15	1.15	75.41
				2	552	1.15
		0.25		1	522	1.05	1.05	91.80
				2	526	1.04
		0.50		1	526	1.00	1.01	98.36
				2	519	1.01
Vehicle Control	Without S9	0	3 to 6	1	553	1.58	1.59	NA
				2	588	1.60
Negative Control		0		1	537	1.59	1.58	1.69
				2	547	1.57
POLY-U		0.0625		1	519	1.28	1.28	52.54
				2	527	1.28
		0.125		1	554	1.14	1.15	74.58
				2	569	1.16
		0.25		1	536	1.04	1.04	93.22
				2	538	1.04
		0.50		1	513	1.00	1.00	100.00
				2	526	1.01
Vehicle Control	Without S9	0	20 to 24	1	573	1.59	1.60	NA
				2	573	1.60
Negative Control		0		1	571	1.59	1.59	1.67
				2	567	1.58
POLY-U		0.0625		1	528	1.29	1.29	51.67
				2	515	1.30
		0.125		1	550	1.15	1.15	75.00
				2	536	1.16
		0.25		1	544	1.05	1.04	93.33
				2	524	1.04
		0.50		1	516	1.01	1.01	98.33
				2	523	1.01

Table 2. Summary of micronuclei incidence with metabolic activation for 3 to 6 hours

Treatment	Concentration (mg/mL)	Replicate No.	Average CBPI	Average % of Cytotoxicity	Total No. of Binucleates	Total No. of Micronucleus	Average Percentage of Micronucleus
Vehicle Control	0	1	1.61	1.61	2062	5	0.24
		2
Negative Control	0	1	1.62	0.00	2011	5	0.25
		2
POLY-U	0.015625	1	1.46	24.59	2141	5	0.24
		2
	0.03125	1	1.39	36.07	2021	4	0.20
		2
	0.0625	1	1.30	50.82	2026	5	0.25
		2
Cyclophosphamide Monohydrate	10 µg/mL	1	1.55	9.84	2079	22	1.06*
		2

* Statistically significant.

Table 3. Summary of micronuclei incidence without metabolic activation for 3 to 6 hoursx

Treatment	Concentration      (mg/mL)	Replicate No.	Average CBPI	Average % of Cytotoxicity	Total No. of Binucleates	Total No. of Micronucleus	Average Percentage of Micronucleus
Vehicle Control	0	1	1.59	1.67	2080	5	0.24
		2
Negative Control	0	1	1.60	0.00	2078	4	0.19
		2
POLY-U	0.015625	1	1.44	25.42	2103	3	0.14
		2
	0.03125	1	1.38	35.59	2034	5	0.25
		2
	0.0625	1	1.29	50.85	2036	4	0.20
		2
Colchicine	0.03 µg/mL	1	1.54	8.47	2048	21	1.03*
		2

* Statistically significant.

Table 4. Summary of micronuclei incidence without metabolic activation for 20 to 24 hours

Treatment	Concentration      (mg/mL)	Replicate No.	Average CBPI	Average % of Cytotoxicity	Total No. of Binucleates	Total No. of Micronucleus	Average Percentage of Micronucleus
Vehicle Control	0	1	1.60	3.23	2089	5	0.24
		2
Negative Control	0	1	1.62	0.00	2090	4	0.19
		2
POLY-U	0.015625	1	1.44	26.67	2085	5	0.24
		2
	0.03125	1	1.39	35.00	2006	5	0.25
		2
	0.0625	1	1.29	51.67	2030	4	0.20
		2
Mitomycin C	0.075 µg/mL	1	1.53	11.67	2007	23	1.15*
		2

* Statistically significant.

Table 5. Individual data of Cytokinesis Block Proliferation Index and cytotoxicity

Metabolic Activation and Duration	Treatment	Concentration (mg/mL)	Replicate No.	No. ofMononucleates	No. ofBinucleates	No. ofMultinucleates
With S9 and 3 to 6 hours	Vehicle Control	0	1	255	235	46
			2	258	228	48
	Negative Control	0	1	275	216	45
			2	245	230	50
	POLY-U	0.0625	1	380	108	20
			2	392	106	19
		0.125	1	487	53	16
			2	485	52	15
		0.25	1	500	20	2
			2	506	19	1
		0.5	1	524	2	0
			2	516	3	0
Without S9 and 3 to 6 hours	Vehicle Control	0	1	276	232	45
			2	285	256	47
	Negative Control	0	1	265	226	46
			2	276	231	40
	POLY-U	0.0625	1	395	101	23
			2	399	106	22
		0.125	1	488	52	14
			2	496	56	17
		0.25	1	516	18	2
			2	520	16	2
		0.5	1	513	0	0
			2	522	2	1
Without S9 and 20 to 24 hours	Vehicle Control	0	1	284	240	49
			2	275	252	46
	Negative Control	0	1	279	246	46
			2	286	234	47
	POLY-U	0.0625	1	398	105	25
			2	385	108	22
		0.125	1	485	50	15
			2	466	56	14
		0.25	1	522	19	3
			2	506	17	1
		0.5	1	514	0	2
			2	520	2	1

Table 6. Individual data of micronuclei incidence with metabolic activation (+S9, 3  to 6 hours)

Treatment	Concentration(mg/mL)	Replicate No.	No. of Mononucleates	No. of Binucleates	No. of Multinucleates	No. of Binucleates (MN Scoring)	CBPI	No. of Micronucleus
Vehicle Control	0	1	250	231	44	1032	1.61	2
		2	255	230	46	1030	1.61	3
Negative Control	0	1	245	240	45	1009	1.62	3
		2	250	244	47	1002	1.62	2
POLY-U	0.015625	1	310	160	35	1064	1.46	3
		2	310	165	32	1077	1.45	2
	0.03125	1	340	145	25	1012	1.38	2
		2	335	140	27	1009	1.39	2
	0.0625	1	386	115	20	1016	1.30	3
		2	380	110	22	1010	1.29	2
Cyclophosphamide Monohydrate	10 µg/mL	1	265	230	28	1034	1.55	10
		2	270	224	28	1045	1.54	12

Table 7. Individual data of micronuclei incidence without metabolic activation (-S9, 3 to 6 hours)

Treatment	Concentration(mg/mL)	Replicate No.	No. of Mononucleates	No. of Binucleates	No. of Multinucleates	No. of Binucleates (MN Scoring)	CBPI	No. of Micronucleus
Vehicle Control	0	1	276	228	42	1034	1.57	3
		2	270	258	45	1046	1.61	2
Negative Control	0	1	270	230	45	1036	1.59	2
		2	265	254	41	1042	1.60	2
POLY-U	0.015625	1	328	159	35	1054	1.44	1
		2	320	164	33	1049	1.44	2
	0.03125	1	350	135	28	1020	1.37	2
		2	348	145	30	1014	1.39	3
	0.0625	1	408	105	25	1017	1.29	2
		2	400	105	25	1019	1.29	2
Colchicine	0.03 µg/mL	1	275	210	32	1011	1.53	10
		2	280	225	33	1037	1.54	11

Table 8. Individual data of micronuclei incidence without metabolic activation (-S9, 20 to 24 hours)

Treatment	Concentration(mg/mL)	Replicate No.	No. of Mononucleates	No. of Binucleates	No. of Multinucleates	No. of Binucleates (MN Scoring)	CBPI	No. of Micronucleus
Vehicle Control	0	1	286	244	50	1030	1.59	3
		2	274	253	47	1059	1.60	2
Negative Control	0	1	270	250	49	1036	1.61	2
		2	265	248	50	1054	1.62	2
POLY-U	0.015625	1	325	155	35	1029	1.44	2
		2	330	159	36	1056	1.44	3
	0.03125	1	350	130	31	1005	1.38	3
		2	356	145	30	1001	1.39	2
	0.0625	1	400	110	23	1015	1.29	2
		2	386	105	23	1015	1.29	2
Mitomycin C	0.075 µg/mL	1	270	201	30	1006	1.52	11
		2	268	201	31	1001	1.53	12

Table 9. Historical data

Positive control

Frequency of Micronucleated Binucleates (%)
95% Confidence level
	With S9 (3 to 6 hours) 10 µg/mL of Cyclophosphamide	Without S9 (3 to 6 hours)		Without S9 (20 to 24 hours) 0.075 µg/mL of Mitomycin C
		0.075 µg/mL of Mitomycin C	0.03 µg/mL of Colchicine
Average	0.91	0.88	0.95	0.98
Standard Deviation	0.31	0.26	0.32	0.35
Sample Size	19	16	20	20
Margin of Error	0.14	0.13	0.14	0.15
Upper Bound	1.05	1.01	1.09	1.14
Lower Bound	0.77	0.76	0.81	0.83
95% Confidence Level	1.96	1.96	1.96	1.96
Max	1.52	1.40	1.47	1.73
Min	0.44	0.56	0.45	0.49

Vehicle control

Frequency of Micronucleated Binucleates (%)
95% Confidence level
	With S9  (3 to 6 hours)	Without S9 (3 to 6 hours)	Without S9   (20 to 24 hours)
Average	0.16	0.19	0.18
Standard Deviation	0.05	0.09	0.06
Sample Size	14	14	14
Margin of Error	0.03	0.05	0.03
Upper Bound	0.19	0.24	0.21
Lower Bound	0.13	0.14	0.15
95% Confidence Level	1.96	1.96	1.96
Max	0.24	0.33	0.30
Min	0.09	0.00	0.10

Conclusions:

In an in vitro micronucleus test, the test item was found as non aneugenic and non clastogenic in human lymphocytes up to the concentration of 0.0625 mg/mL both in the presence and absence of metabolic activation under the test conditions.

Executive summary:

The test item was evaluated for formation of micronuclei in human lymphocytes according to OECD Guideline 487, following the Principles of GLP. Based on the results of the solubility and precipitation tests, the test item was formulated in N, N-dimethyl formamide as vehicle and 0.5 mg/mL was selected as the highest concentration in the Initial Cytotoxicity Test. This test was conducted at concentrations of 0.0625, 0.125, 0.25 and 0.5 mg/mL. The average cytotoxicity ranged from 74.58% to 100% at 0.125 mg/mL to 0.5 mg/mL and from 51.67% to 52.54% at 0.0625 mg/mL. As the cytotoxicity of the test item was in the range of 55 ± 5% at 0.0625 mg/mL, this value was selected as the highest concentration for the main test. Cytochalasin B (cytoB) was used as cytokinesis blocker. Cytotoxicity was assessed by Cytokinesis-Block Proliferation Index (CBPI). Cultured human peripheral blood lymphocytes previously incubated for 44 -48 h with PHA in RPMI medium were treated with test item in N, N-dimethyl formamide at the concentrations of 0.015625, 0.03125 and 0.0625 mg/mL. The treatment was carried out in duplicates for the short term period (3 h) both in the presence and absence of metabolic activation (S9 mix) and for the long term period (24 h) in the absence of metabolic activation. The presence of micronuclei was evaluated in at least 2000 binucleate cells per concentration (at least 1000 per culture). Cyclophosphamide Monohydrate (+S9 for short term) at 10 µg/mL, Colchicine (-S9 for short term) at 0.03 µg/mL and Mytomycin C at 0.075 µg/mL (-S9 long term) were used as positive controls. In the micronucleus test the test item induced cytotoxicity at 0.0625 mg/mL (50.82 to 51.67%) when compared to vehicle control. There was no statistically significant increase in the percentage of micronuclei in binucleated cells observed in any of the tested concentrations when compared to the vehicle control. Values obtained with concurrent vehicle and positive controls were within the 95% control limits of the distribution of their respective laboratory’s historical control databases. All acceptability criteria were met. Based on these results, it is concluded that the test item is non clastogenic and non aneugenic in cultured human lymphocytes at and up to 0.0625 mg/mL both in short term and long term treatments (presence and absence of metabolic activation).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because a positive result was found in in vitro gene mutation study in bacteria

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to the column 1 of REACH Annex VIII, the study does not to be conducted since a positive result was found in the in vitro gene mutation test in bacteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Data waiving (study scientifically not necessary): It is possible to make a conclusive hazard assessment in accordance with Annex I of REACH without additional testing on the basis of structure-activity relationship with a known mutagen.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Based on the document "Guidance on information requirements and chemical safety assessment Chapter R.7a: Endpoint specific guidance" (Version 6.0-July 2017), page 566, since the substance shares structural characteristics with known mutagens (harmonised CLP classification of analogue CAS 52234-82-9: Mutagen Category 2) it is possible to make a conclusive hazard assessment in accordance with Annex I of REACH without additional testing on the basis of structure-activity relationship alone. Furthermore, a justification is provided below on the basis of point 1.5 of Annex XI for adaptation of the standard testing regime in order to demonstrate that the information used is adequate for the purpose of classification and labelling.

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information, the substance is classified as Mutagen Category 2 in accordance with CLP Regulation (EC) no 1272/2008.