N-(2-hydroxyethyl)dodecanamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2017: Negative

In vitro chromosome aberration test with human lymphocytes (with and without S-9 activation), OECD TG 473, 2017: Negative

In vitro gene mutation to , TK +/-, locus of the L5178Y mouse lymphoma cell line (with and without S-9 activation), OECD TG 490, 2018: Negative

In accordance with the integrated testing strategy under Annexes VII and VIII of REACH, no further testing on genetic toxicity is required. A conclusion of no genetic toxicity can be made for this substance based on the available negative in vitro studies.

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:

27 April 2017 - 16 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

he study was conducted under GLP following the most upto date OECD guideline

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. Room temperature in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Fully miscible in DMSO (dimethyl sulfoxide) and assumed stable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (1.46%) of the test item.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Concentration of stock solution = 50 mg/mL.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Experiment 1 - Plate Incorporation Method : All strains (with and without S9): 1.5, 5, 15, 50, 150, 500, 1500 5000 µg/plate.
Experiment 2 – Pre-Incubation Method:
Salmonella strains TA98 and TA100 and Escherichia coli strain WP2uvrA (with and without S9): 1.5, 5, 15, 50, 150, 500, 1500 5000 µg/plate.
Salmonella strains TA1535 and TA1537 (without S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strains TA1535 and TA1537 (with S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.

Vehicle / solvent:

- Dimethyl sulphoxide was the vehicle used
- Justification: The main vehicle in the guideline

Untreated negative controls:

yes

Remarks:

untreated control

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation and pre-incubation; in suspension (main test)
- Cell density at seeding (if applicable):

DURATION
- Experiment I - Plate incorporation: incubated at 37 ± 3 C for approximately 48 hours
- Experiment II - Preincubation period: 20 mins pre-incubation followed by further incubation at or approximately 48 hours at 37 ± 3 C
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): n/a

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): n/a

STAIN (for cytogenetic assays): n/a

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: n/a

NUMBER OF CELLS EVALUATED: n/a

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n/a

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: presence of bacterial lawn
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:n/a
- Determination of endoreplication: n/a
- Methods, such as kinetochore antibody binding, to characterise whether micronuclei contain whole or fragmented chromosomes (if applicable): n/a

Rationale for test conditions:

The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel (1976 and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out‑of‑historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: not examined
- Precipitation: none
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: In the range-finding test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawn and/or substantial decreases in the revertant colony frequency of Salmonella strains TA1535, TA98 and TA1537 in both the presence and absence of metabolic activation (S9-mix), initially from 500 µg/plate. Consequently, the same maximum dose level (5000 µg/plate) or the toxic limit of the test item was selected as the maximum dose in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: n/a
- Indication whether binucleate or mononucleate where appropriate: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No
- Negative (solvent/vehicle) historical control data: Yes (2009 - 2010 data)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: presence of bacterial lawn
- Other observations when applicable: no

Table 1:       Results for Experiment 1 Plate Incorporation (+/- S9 mix)

Concentration (µg/plate)		Number of revertants (mean) +/-
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
		S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)
Solvent Control (DMSO)		79	96	13	8	32	39	18	21	13	9
1.5 µg		83	81	15	9	24	33	24	16	17	12
5 µg		72	87	15	8	32	36	17	21	8	13
15 µg		65	84	11	8	35	34	19	18	14	9
50 µg		83	84	12	8	38	31	19	17	13	7
150 µg		68	74	13	9	34	39	21	16	8	9
500 µg		70	68	5 S	4	40	41	12	13	8	4
1500 µg		71 P	64 P	3 PS	4 P	37 P	39 P	15 P	11 P	6 PS	3 P
5000 µg		70 P	62 P	0 PV	2 PS	31 P	34 P	12 PS	7 P	2 PS	1 PS
Positive controls	Name	ENNG	2AA	ENNG	2AA	ENNG	2AA	4NQO	BP	9AA	2AA
	Dose Level	3 µg	1 µg	5 µg	2 µg	2 µg	10 µg	0.2 µg	5 µg	80 µg	2 µg
	No. of Revertants	470	859	141	205	624	229	225	206	331	344

2AA:          2-Aminoanthracene

9AA:          9-Aminoacridine

BP:             Benzo(a)pyrene

ENN:          N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO:        4-Nitroquinoline-1-oxid

P:              Test Item Precipitate

S:               Sparse bacterial background lawn

V:              Very weak bacterial background lawn

Table 2:              Results for Experiment 2 Pre-Incubation (+/- S9 mix)

Concentration (µg/plate)		Number of revertants (mean) +/-
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
		S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)
Solvent Control (DMSO)		85	110	9	12	36	45	18	20	9	13
0.5 µg		N/T	NT	12	9	N/T	N/T	N/T	N/T	9	12
1.5 µg		85	112	10	11	31	37	18	18	8	12
5 µg		96	117	11	14	40	37	15	21	12	10
15 µg		102	111	11	12	35	43	18	17	11	8
50 µg		97	117	12	13	32	47	17	25	8	9
150 µg		101	119	9 S	12	31	45	15	21	5 S	10
500 µg		72	103	0 T	5 S	41	46	7 S	23	0 T	2 S
1500 µg		70 PS	56 PS	0 PT	0 PT	31 P	43 P	10 PS	10 PS	0 PT	0 PT
5000 µg		44 PS	45 PS	N/T	0 PT	35 P	47 P	7 PS	4 PS	N/T	0 PT
Positive controls	Name	ENNG	2AA	ENNG	2AA	ENNG	2AA	4NQO	BP	9AA	2AA
	Dose Level	3 µg	1 µg	5 µg	2 µg	2 µg	10 µg	0.2 µg	5 µg	80 µg	2 µg
	No. of Revertants	790	1129	165	188	705	191	286	173	329	241

2AA:          2-Aminoanthracene

9AA:          9-Aminoacridine

BP:            Benzo(a)pyrene

ENNG:       N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO:       4-Nitroquinoline-1-oxid

N/T:          Not tested at this dose level

P:              Test Item Precipitate

S:             Sparse bacterial background lawn

Conclusions:

it was concluded that the test itme was non-mutagenic under the conditions of the test.

Executive summary:

OECD 471 (2017) - In a reverse gene mutation assay in bacteria strains: N-(2-hydroxyethyl) dodecanamide potential to induce mutagenic activity in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (using both plate incorporation and pre-Incubation methods) in vehicle dimethyl sulphoxide was evaluated in the presence and absence of metabolic activation.

Up to nine test item concentrations (0.5 and 5000 µg/plate) were used depending on the bacterial strain type and presence or absence of S9-mix i.e  Experiment 1 - Plate Incorporation Method : All strains  (with and without S9): 1.5, 5, 15, 50, 150, 500, 1500 5000 µg/plate.  Experiment 2 – Pre-Incubation Method:  Salmonella strains TA98 and TA100 and Escherichia coli strain WP2uvrA (with and without S9): 1.5, 5, 15, 50, 150, 500, 1500 5000 µg/plate, Salmonella strains TA1535 and TA1537 (without S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate and Salmonella strains TA1535 and TA1537 (with S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.

The test item was tested up to cytotoxic concentrations. 

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background in the test item treated colonies.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 June 2017 - 08 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO at 200 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dissolved in DMSO.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: the test item was accurately weighed, formulated in DMSO and appropriate serial dilutions prepared.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): applied as a liquid

OTHER SPECIFICS: no

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: 18-35 year old non-smoking volunteers (human)
- Suitability of cells: previously screened for suitability

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

4 h without S9 mix: 0, 8, 16, 32, 64, 128, 192 and 256 µg/mL.
4 h with S9 mix: 0, 4, 8, 16, 32, 64, 128 and 256 µg/mL.
24 h without S9 mix: 0, 4, 8, 16, 32, 64, 96 and 128 µg/mL.

The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be lowest precipitating dose level because the onset was prior to the toxicity observed.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Guideline recommendation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO - Batch number 1684307 (>99%)

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 0.75 mL blood

DURATION
- Preincubation period: 48 h
- Exposure duration: 4 h (short-term) or 24 h (continuous)
- Expression time (cells in growth medium): 20 h (short-term) and 0 h (continuous), respectively.
- Selection time (if incubation with a selection agent): Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours and 2.5 hours in the Cell Growth Inhibition Test and Main Test, respectively, before the required harvest time.
- Fixation time (start of exposure up to fixation or harvest of cells): After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 μg/mL)

STAIN (for cytogenetic assays): 5 % Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: When the slides were dry they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

DETERMINATION OF CYTOTOXICITY
- Method: Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
- Any supplementary information relevant to cytotoxicity: no

OTHER EXAMINATIONS:
- Determination of polyploidy: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.
- Determination of endoreplication: If the chromosomes were arranged in closely apposed pairs, i.e. 4 chromatids instead of 2, the cell was scored as endoreduplicated (E).
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): no

- OTHER:

Gaps (g)
Gaps are small areas of the chromosome that are unstained. The chromatids remain aligned as normal and the gap does not extend along the chromatid for a distance greater than the width of a chromatid. If the gap occurs on one chromatid only it is a chromatid gap (g). If a gap appears in both chromatids at the same position it is a chromosome gap (G).

Chromatid Breaks (ctb)
Chromatid breaks (ct) vary in appearance. The chromatid may remain aligned but show a gap which is too large to classify as a gap. Alternatively, the chromatid may be broken so that the broken fragment is displaced. In some cases, the fragment is not seen at all. A chromatid fragment (f) should be scored if the chromosome of origin cannot be identified. Very small fragments are scored as minutes (m).

Chromosome Breaks (csb)
Chromosome breaks (CS) are breaks in both chromatids of the chromosome. A fragment with two chromatids is formed and may be displaced by varying degrees. Breaks are distinguished from gaps by the size of the unstained region. A chromosome break is scored if the fragment is associated with a chromosome from which it was probably derived. However, fragments are often seen in isolation and are then scored as chromosome fragments (F). Very small fragments are scored as minutes (M).

Exchanges (cte and cse)
Exchanges are formed by faulty rejoining of broken chromosomes and may be of the chromosome or chromatid type. Chromatid exchanges (c/c,r) have numerous different forms but are generally not further classified. Where multiple exchanges have occurred each exchange point is counted as one chromatid exchange. Chromosome exchanges generally appear as either a dicentric (D) or a ring (R) form, either of which can be associated with a fragment, which if possible should be scored as part of the exchange.

Multiple Aberrations
If many aberrations are present in one metaphase, the exact details may not be scorable. This is particularly the case when chromosome pulverisation occurs. If the number of aberrations is 10 or more then the cell is classified as X.

Evaluation criteria:

The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range. The level of spontaneous background aberrations was slightly elevated above the normal range and the experiment still considered valid.
• All the positive control chemicals induced a positive response (p ≤ 0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analysed.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Precipitate of the test item observed at and above 125 µg/mL and Hemolysis observed between 125 and 500 µg/mL in the 4(20)-hour and at and above 250 µg/mL in the 24-hour continuous exposure group

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not affected
- Effects of osmolality: not affected
- Evaporation from medium: not affected
- Water solubility: not affected
- Precipitation: precipitation present in prelim test at and above 125 µg/mL. Main test concentrations were tested below this level.
- Definition of acceptable cells for analysis:
- Other confounding effects: hemolysis (cytotoxic response) - see below.


RANGE-FINDING/SCREENING STUDIES:
The dose range for the Cell Growth Inhibition Test was 7.81 to 2000 µg/mL. The maximum dose was the maximum recommended dose level.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 125 µg/mL in the exposure groups in the absence of metabolic activation (S9) and at and above 62.5 µg/mL in the exposure group in the presence of S9.

Hemolysis was observed following exposure to the test item between 125 and 500 µg/mL in the 4(20)-hour exposure groups and at and above 250 µg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 250 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 62.5 µg/mL. The results of the mitotic index of the Cell Growth Inhibition Test are presented in sections 5(2) and 6(2) of Appendix 1. The test item induced evidence of toxicity in all of the exposure groups.

The selection of the maximum dose level for main experiment was based on the lowest precipitating dose level because the onset was prior to the toxicity observed.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 125 µg/mL in the exposure groups in the absence of metabolic activation (S9) and at and above 62.5 µg/mL in the exposure group in the presence of S9.

Hemolysis was observed following exposure to the test item between 125 and 500 µg/mL in the 4(20)-hour exposure groups and at and above 250 µg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Table 1:       Short Term Exposure Test – Main Experiment: 4(20)-Hour Without Metabolic Activation (S9)

Concentration (µg/mL)	Replicate	Number and Percentages of Cells Showing Structural Chromosome Aberration (%)							g	Cell Growth Index	Number and Percentages of Cells Showing Numerical Aberration (%)
		Observed	ctb	cte	csb	cse	Others	Total		Mitotic Index (%)	Observed	Polyploids	Others	Total
	A	150	0	0	0	0	0	0	0	4.65	150	0	0	0
	B	150	2	0	0	0	0	2	2	3.95	150	0	0	0
	Total (%)	300 (100)	2 (0.7)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (0.7)	2 (0.7)	(100)	300	0	0	0 (0.0)
32	A	150	1	0	1	0	0	1	0	3.60	150	0	0	0
	B	150	0	0	0	0	0	1	0	5.05	150	0	0	0
	Total (%)	300 (100)	1 (0.3)	0 (0.0)	1 (0.3)	0 (0.0)	0 (0.0)	2 (0.7)	0 (0.0)	(101)	300	0	0	0 (0.0)
64	A	150	0	0	0	0	0	0	0	6.05	150	0	0	0
	B	150	0	0	0	0	0	0	0	4.35	151	1	0	1
	Total (%)	300 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	(121)	301	1	0	1 (0.3)
128P	A	150	1	0	2	0	0	3	2	2.55	150	0	0	0
	B	150	0	0	0	0	0	0	0	3.05	150	0	0	0
	Total (%)	300 (100)	1 (0.3)	0 (0.0)	2 (0.7)	0 (0.0)	0 (0.0)	3 (1.0)	2 (0.7)	(65)	300	0	0	0 (0.0)
Positive Control (MMC) 0.1	A	87^a	9	6	1	0	0	15	0	2.65	87	0	0	0
	B	35^a	10	7	2	0	0	15	2	1.65	35	0	0	0
	Total (%)	122 (100)	19 (15.6)	13 (10.7)	3 (2.5)	0 (0.0)	0 (0.0)	30*** (24.6)	2 (1.6)	(50)	122	0	0	0 (0.0)

MMC       = Mitomycin C

***           = p < 0.001

a              = Slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed

Table 2.       Short Term Exposure Test – Main Experiment: 4(20)-Hour With Metabolic Activation (2%S9).

Concentration (µg/mL)	Replicate	Number and Percentages of Cells Showing Structural Chromosome Aberration (%)							g	Cell Growth Index	Number and Percentages of Cells Showing Numerical Aberration (%)
		Observed	ctb	cte	csb	cse	Others	Total		Mitotic Index (%)	Observed	Polyploids	Others	Total
Negative Control (DSO) 0	A	150	0	0	0	0	0	0	2	5.9	150	0	0	0
	B	150	0	0	0	0	0	0	0	6.05	150	0	0	0
	Total (%)	300 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (0.7)	(100)	300	0	0	0 (0.0)
32	A	150	3	0	0	0	0	3	1	5.45	150	0	0	0
	B	150	1	0	0	0	0	1	1	4.80	150	0	0	0
	Total (%)	300 (100)	4 (1.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	4 (1.3)	2 (0.7)	(86)	300	0	0	0 (0.0)
64	A	150	0	0	0	0	0	0	1	6.40	150	0	0	0
	B	150	1	0	0	0	0	1	1	5.15	150	0	0	0
	Total (%)	300 (100)	1 (0.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.3)	2 (0.7)	(97)	300	0	0	0 (0.0)
128P	A	150	0	0	0	0	0	0	1	5.40	150	0	0	0
	B	150	0	0	0	0	0	0	0	5.00	150	0	0	0
	Total (%)	300 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.3)	(87)	300	0	0	0 (0.0)
Positive Control (CP) 2	A	150	6	1	4	0	0	10	9	2.20	150	0	0	0
	B	150	9	1	1	0	0	10	1	4.20	150	0	0	0
	Total (%)	300 (100)	15 (5.0)	2 (0.7)	5 (1.7)	0 (0.0)	0 (0.0)	20*** (6.7)	10 (3.3)	(54)	300	0	0	0 (0.0)

CP       = Cyclophosphamide

***       = p < 0.001

  a         = Slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed.

Table 3.       Continuous Exposure Test – Main Experiment: 24-Hour Without Metabolic Activation (2%S9).

Concentration (µg/mL)	Replicate	Number and Percentages of Cells Showing Structural Chromosome Aberration (%)							g	Cell Growth Index	Number and Percentages of Cells Showing Numerical Aberration (%)
		Observed	ctb	cte	csb	cse	Others	Total		Mitotic Index (%)	Observed	Polyploids	Others	Total
Negative Control (DSO) 0	A	150	0	0	0	0	0	0	1	4.90	150	0	0	0
	B	150	0	0	0	0	0	0	0	4.35	150	0	0	0
	Total (%)	300 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.3)	(100)	300	0	0	0 (0.0)
16	A	150	0	0	0	0	0	0	0	3.30	150	0	0	0
	B	150	0	0	0	0	0	0	0	4.35	150	0	0	0
	Total (%)	300 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	(83)	300	0	0	0 (0.0)
32	A	150	0	0	0	0	0	0	0	3.35	150	0	0	0
	B	150	0	0	0	0	0	0	0	3.50	150	0	0	0
	Total (%)	300 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	(74)	300	0	0	0 (0.0)
64P	A	150	0	0	0	0	0	0	0	2.65	151	1	0	1
	B	150	3	0	1	0	0	4	0	2.75	150	0	0	0
	Total (%)	300 (100)	3 (1.0)	0 (0.0)	1 (0.3)	0 (0.0)	0 (0.0)	4 (1.3)	0 (0.0)	(58)	301	1	0	1 (0.3)
Positive Control (MMC) 0.2	A	119	7	9	3	0	0	17	6	1.75	119	0	0
	B	58	11	5	4	0	0	16	0	1.40	58	0	0	0
	Total (%)	177 (100)	18 (10.2)	14 (7.9)	7 (4.0)	0 (0.0)	0 (0.0)	33*** (18.6)	6 (3.4)	(34)	177	0	0	0 (0.0)

MMC          = Mitomycin C

*              = p < 0.05

***              = p < 0.001

       a              = Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed

Conclusions:

Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

OECD 473 (2017) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to N-(2-hydroxyethyl) dodecanamide at concentrations of 0, 8, 16, 32, 64, 128, 192 and 256 µg/mL for 4 h without metabolic activation or, 4, 8, 16, 32, 64, 128 and 256 µg/mL for 4 h with metabolic. Additionally, a continuous exposure of 24 h was tested without metabolic activation at test item concentrations of 0, 4, 8, 16, 32, 64, 96 and 128 µg/mL.

N-(2-hydroxyethyl) dodecanamide was tested up to precipitating and cytotoxic concentrations of 120 µg/mL. In the range-finding test, concentrations above 125 μg/mL in all three exposure groups had a reduced pellet which is indicative of toxicity to the cell population present, indicating that maximum exposure has been reached.  Positive controls induced the appropriate response.

 

There was no evidence (or a concentration related positive response) of chromosome aberration induction over background values at the highest tested concentration.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2017 - 25 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO at 200 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dissolved in DMSO.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: the test item was accurately weighed, formulated in DMSO and appropriate serial dilutions prepared.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): applied as a liquid

OTHER SPECIFICS: no

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The test item had a molecular weight greater than 200 therefore the maximum recommended dose level was set at 2000 µg/mL. The purity of the test item was 98.54% which was accounted for in formulation. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Specifically, in the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival.

I. Preliminary toxicity test: 0 (control), 0.98, 1.95, 3.91, 7.81, 15.63 31.25, 62.5, 125 and 250 μg/mL
Within three exposure groups:
i) 4-hours exposure to the test item without S9-mix
ii) 4-hours exposure to the test item with S9-mix (2%)
iii) 24-hour exposure to the test item without S9-mix

There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups. The toxicity curve was very steep in all three exposure groups. Precipitate of the test item was observed at and above 62.5 µg/mL in both 4-hour exposure groups. Precipitate of the test item was observed and above 125 µg/mL in the 24-hour exposure group.

II. Main Test:
4-hour without S9: 0 (control), 4, 8, 16, 32, 40, 48, 56, 64 μg/ml and EMS 400 μg/ml
4-hour with S9: 0 (control), 4, 8, 16, 32, 40, 48, 56, 64 μg/ml and CP 1.5 μg/ml
24-hour without S9: 0 (control), 4, 8, 16, 32, 40, 48, 56, 64 μg/mL and EMS 150 μg/ml
where:
EMS = Ethylmethanesulphonate
CP = Cyclophosphamide

The cultures were then incubated for 24 hours, and sub-cultured subject to acceptable limits of mean cell count. Following a further 24 hours the cultures were counted and discarded. Giving total of 48 hours for expression period.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was soluble in DMSO at 2000 mg/mL in solubility checks performed. The maximum dose level (determined prior to the test based on molecular weight) was 2000 µg/mL, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 2000 μg/mL range (full results recorded in the full study report). The test item was formulated within two hours of it being applied to the test system.
Applicant assessment indicates: The test item had been demonstrated to be insoluble in Minimal Essential Medium (MEM) in previous mammalian cell line testing. DMSO is a guideline accepted vehicle with an available laboratory historic control data set

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: incubation with test item.
- Cell density at seeding (if applicable): Preliminary Test: 4-hour exposure: 5 x10^5 cells/mL ; 24-hour exposure: 1.5 x10^5 cells/mL ; Definitive Test: 4-hour exposure: 5 x10^5 cells/mL; 24-hour exposure: 1.5 x10^5 cells/mL

DURATION
- Preincubation period: Preliminary Test: 4-hour exposure: 45  minutes; 24-hour exposure: 45 minutes ; Definitive Test: 4-hour exposure: 55 minutes ; 24-hour exposure: 55 minutes
- Exposure duration: Treatment was for 4 hours at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air, post exposure (total time 12 to 14 days from exposure start). To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells.

SELECTION AGENT (mutation assays): 5-Trifluorothymidine at 4 μg/mL

SPINDLE INHIBITOR (cytogenetic assays): Not applicable.

STAIN (for cytogenetic assays): Not applicable.

NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air, post exposure. Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air, post exposure with two hours incubation.

NUMBER OF CELLS EVALUATED: 2000 cells/well were seeded with selection agent. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) analysis. Scoring of plates daily for %RSG and %V to obtain RTG. Mutation scoring of plates was ultimately performed for the presence of mutant colonies; large and small colonies analyses was additionally conducted.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):  Not applicable.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Not applicable.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG) and cloning efficiency via viability (%)
- Any supplementary information relevant to cytotoxicity: percentage relative suspension growth (%RSG) (post exposure toxicity during the expression period); viability (%) from non-selective medium cultures.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable.
- Determination of endoreplication: Not applicable.
- Methods, such as kinetochore antibody binding, to characterise whether micronuclei contain whole or fragmented chromosomes (if applicable): Not applicable.

Rationale for test conditions:

The rationale for selection of concentrations was based on preliminary toxicity testing and number of cultures was in accordance with the guideline (duplicate). Optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved.

Evaluation criteria:

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered
unable to induce mutations in this test system.

Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.

Statistics:

Statistical guidelines recommended by the UKEMS (Robinson W. D. et al., Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp 102-140). The statistical package used indicates the presence of statistically significant increases and linear-trend events.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Precipitate of the test item was observed at and above 62.5 µg/mL in both 4-hour exposure groups. Precipitate of the test item was observed and above 125 µg/mL in the 24-hour exposure group

Table 1:       Results of preliminary cytotoxicity test

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
0.98	110	116	81
1.95	99	93	122
3.91	107	93	137
7.81	110	92	114
15.63	89	88	110
31.25	91	103	72
62.5	1 p	98 p	1
125	0 p	1 p	0 p
250	0 p	0 p	0 p

P= precipitate

Table 2:       Definitive Test - summary of results: 4-hour exposure - with and without S9  and 24-h without S9

Treatment (µg/ml)	4-hours-S-9			Treatment (µg/ml)	4-hours+S-9			Treatment (µg/ml)	24-hours-S-9
	%RSG	RTG	MF§		%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1.00	130.62	0	100	1.00	134.37	0	100	1.00	140.54
4	113	1.04	127.15	4Ø	106			4	115	1.21	116.65
8	108	1.07	124.24	8Ø	91			8	99	1.06	1118.37
16	105	1.09	132.58	16	100	1.03	132.28	16	74	0.88	126.82
32	93	1.01	114.28	32	93	1.16	107.12	32	35	0.71	112.47
40	90	1.17	100.38	40	96	1.00	131.48	40	29	0.68	103.30
48	8	0.10	83.19	48	101	1.16	113.91	48	11	0.25	119.94
56 Ø	1			56	91	0.96	133.05	56 Ø	1
64P Ø	1			64P	85	0.87	144.28	64P Ø	0
MF threshold for a positive response=256.62				MF threshold for a positive response=260.37				MF threshold for a positive response=266.54
EMS 400	76	0.63	1293.51	CP 1.5	77	0.57	872.82	EMS 150	44	0.42	1147.39

$               =       Cell counts (x105 cells/ml).  Set up on previous day to 2 x 105 cells/ml unless otherwise stated in parenthesis.

%RSG       =       Relative Suspension Growth

RTG        =       Relative Total Growth

%V        =       Viability Day 2

§ or #        =       Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis

CP        =       Cyclophosphamide

EMS        =       Ethylmethanesulphonate

MF§        =       5-TFT resistant mutants/106 viable cells 2 days after exposure

Ø               =       Not plated surplus to requirements

P               =       Precipitate present at the end of the exposure period

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

The study was performed to the requirements of OECD TG 490 and EU Method B.17, US EPA OPPTS 870.5300 and Japan guidelines for screening mutagenicity testing of chemicals under GLP conditions to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary cytotoxicity test at up to 250 μg/mL concentration, a definitive test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at ten dose levels in duplicate, together with vehicle (DMSO), and positive controls. Exposures were conducted using a 4-hour exposure with and without metabolic activation (2% S9), and a 24-hour exposure without metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item exhibited dose-related toxicity to the cells in each of the three exposure groups of the preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were: 4-hour and 24-hour without S9 (2%): 4, 8,16, 32, 40, 48 μg/mL and 4- with S9 (2%): 16, 32, 40, 48, 56, 64 μg/mL. Additional concentrations were excluded, as applicable. The maximum dose level used in the definite test was limited by test item-induced toxicity. The concentrations of 56 and 64 µg/mL in the 4-hour and 24-hour exposures in the absence of metabolic activation were not plated out for 5-TFT resistance and viability due to excessive toxicity.  Precipitate of the test item was observed at 64 µg/mL in all three exposure groups.  The presence of precipitate in the 4-hour exposure in the presence of metabolic activation at 64 µg/mL satisfies the requirements of  the OECD guideline. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. Vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolism activation system.  It is concluded that the test item did not induce any increases in mutant frequency above the Global Evaluation Factor (GEF). Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable, no adverse effects were observed in the available studies.

Additional information

OECD TG 471, 2017 - The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using both the Ames plate incorporation and pre incubation methods at nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 g/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations.  The dose range was amended following the results of Experiment 1 and ranged between 0.5 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth of the bacterial lawn in all strains up to 5000 µg/plate. A test item precipitate (greasy and particulate in appearance) was noted at and above 1500 g/plate, this observation did not prevent the scoring of revertant colonies. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9‑mix) in Experiment 1 (plate incorporation method). weakened bacterial background lawns were noted to all the Salmonella tester strains in the absence of S9‑mix from 150 µg/plate (TA1535 and TA1537), 500 µg/plate (TA98) and 1500 µg/plate (TA100).  In the presence of S9-mix, weakened bacterial lawns were noted from 500 µg/plate (TA1535 and TA1537) and 1500 µg/plate (TA100 and TA98). No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence of S9. It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

 

OECD TG 473, 2017 - The study was performed to the requirements of OECD TG 473 and EU Method B.10 under GLP conditions to assess the potential chromosomal mutagenicity of the test substance, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The exposure conditions were divided between experiment 1 which was 4(20)-hour with and without S9 activation, and experiment 2 which was 4(20)-hour with S9 activation and 24-hour exposure without S9 activation. The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test (Cell Growth Inhibition Test) where the results indicated that the maximum concentration should be limited to the lowest precipitating dose level with toxicity also being taken into account in the dose selection. The dose levels selected for the Main Test were as follows, 4(20)-hour with and without S9-Mix (2%): 0, 8, 16, 32, 64, 128, 192, 256 μg/mL. 4(20)-hour with S9 mix (2%): 0, 4, 8, 16, 32, 64, 128, 256 μg/mL. In the 24-hour continuous exposure without S9 mix activation the doses levels were: 0, 4, 8, 16, 32, 64, 96, 128 μg/mL. All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. In the main experiment, A precipitate of the test item was observed in the parallel blood-free cultures at 128 µg/mL levels tested in the 4(20)-hour exposure groups and at and above 64 µg/mL in the 24-hour continuous exposure group. Hemolysis was observed at and above 128 µg/mL in the 4(20)-hour exposure groups in the presence and absence of S9 and at and above 96 µg/mL in the 24-hour continuous exposure group. Moderate dose-related inhibition of mitotic index was observed in the exposure groups in the absence of S9 only. In the 4(20)-hour exposure group in the absence of S9, 35% mitotic inhibition was achieved at 128 µg/mL.  This dose level coincided with the onset of precipitate so the maximum dose level selected for metaphase analysis was 128 µg/mL. In the 24-hour continuous exposure group, 26% and 42% inhibition of mitotic index was observed at 32 and 64 µg/mL, respectively.  Above this dose level, there were too few metaphases available to score.  However, 64 µg/mL coincided with the onset of precipitate so this was the maximum dose level selected for metaphase analysis.  In the presence of S9, no inhibition of mitotic index of was noted, but the onset of precipitate was 128 µg/mL so this was the maximum dose level selected for metaphase analysis. The test item did not induce a statistically significant increase in the frequency of cells with aberrations either in the absence or presence of metabolic activation. No statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups was observed. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

 

The study was performed to the requirements of OECD TG 490 and EU Method B.17, US EPA OPPTS 870.5300 and Japan guidelines for screening mutagenicity testing of chemicals under GLP conditions to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary cytotoxicity test at up to 250 μg/mL concentration, a definitive test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at ten dose levels in duplicate, together with vehicle (DMSO), and positive controls. Exposures were conducted using a 4-hour exposure with and without metabolic activation (2% S9), and a 24-hour exposure without metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item exhibited dose-related toxicity to the cells in each of the three exposure groups of the preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were: 4-hour and 24-hour without S9 (2%): 4, 8,16, 32, 40, 48 μg/mL and 4- with S9 (2%): 16, 32, 40, 48, 56, 64 μg/mL. Additional concentrations were excluded, as applicable. The maximum dose level used in the definite test was limited by test item-induced toxicity. The concentrations of 56 and 64 µg/mL in the 4-hour and 24-hour exposures in the absence of metabolic activation were not plated out for 5-TFT resistance and viability due to excessive toxicity.  Precipitate of the test item was observed at 64 µg/mL in all three exposure groups.  The presence of precipitate in the 4-hour exposure in the presence of metabolic activation at 64 µg/mL satisfies the requirements of the OECD guideline. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. Vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolism activation system.  It is concluded that the test item did not induce any increases in mutant frequency above the Global Evaluation Factor (GEF). Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity.