7-methyl-2H-benzo-1,5-dioxepin-3(4H)-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.
- L5178Y/MLA Mammalian Cell Gene Mutation Assay (OECD 476, GLP, K, rel. 1): non mutagenic up to cytotoxic concentrations.
- HL/CAT chromosome aberration test (OECD 473, GLP, K, rel. 2, read-across): non clastogenic up to cytotoxic concentrations.

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:

From November 22 to December 13, 2007

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on August 21, 2007/ signed on October 15, 2007)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan gene

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 100, TA1535 and E Coli WP2 sensitive to agents inducing base pair mutation. TA1537 and TA98 sensitive to agents inducing frame-shift mutations.

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 from liver of male Sprague- Dawley rats orally received three consecutive daily doses of phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day prior to S9 preparation on Day 4

Test concentrations with justification for top dose:

Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA 100 and WP2 uvr A strains, with or without S9-mix using the direct plate incorporation method.

Mutation Test (direct plate incorporation method):

Experiment 1 (Range-finding Test)
Salmonella strains and E.coli strain WP2uvrA- (with and without S9): 50, 150, 500, 1500 and 5000 μg/plate, with or without S9-mix. (up to maximum recommended concentration).

Experiment 2 (Main Test)
Salmonella strains and E.coli strain WP2uvrA- (with and without S9): 50, 150, 500, 1500 and 5000 μg/plate, with or without S9-mix. (up to maximum recommended concentration).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test material was insoluble in sterile distilled water at 50 mg/mL , but was fully soluble in DMSO at 50 mg/mL. Therefore, DMSO was selected as vehicle.
- Preparation of test materials: The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 x 10-4 microns.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from the University of California at Berkeley whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Approximately 48 h at 37 °C

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Mutation study: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.

Rationale for test conditions:

Experiment 1 & 2: tested up to 5000 μg/plate (maximum recommended concentration)

Evaluation criteria:

- There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (Kirkland, 1989) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
- A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis recommended by UKEMS (Kirkland, 1989)

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: Not expected
- Water solubility: Not soluble in water
- Precipitation: None
- Other confounding effects: None

PRELIMINARY TOXICITY STUDY:
The test material was non-toxic to the strains TA 100 and WP2uvrA-.

COMPARISON WITH HISTORICAL CONTROL DATA:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the untreated controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate and found to be satisfactory.
- Test material formulation, amino acid supplemented top agar and the S9 mix used in the experiments were shown to be sterile.

Table 7.6.1/2: Preliminary Toxicity Test

S9 mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
- S9	TA 100	114	91	95	110	113	100	102	99	110	84	73
+ S9	TA 100	76	69	82	85	68	76	80	81	73	78	62
- S9	WP2 uvr A	26	26	18	15	35	27	37	18	13	23	18
+ S9	WP2 uvr A	30	36	26	36	36	34	32	38	33	42	15

 

See the attached document for information on tables of results – mutagenicity test

Conclusions:

Under the test conditions, the test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 uvr A were exposed to the test material diluted in DMSO using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations. Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

 

The vehicle 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 material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was tested up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

 

Under the test condition, the test material is not classified as mutagenic with and without metabolic activation to S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 10 to July 23, 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notification No. 1604, 1 November 1999.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on November 20 to 22, 2012/ signed on April 24, 2014)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (tk) locus (TK+/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source: American Type Culture Collection (ATCC), Virginia.
- Type and identity of media: RPMI 1640 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24 h incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196 °C, in heat-inactivated donor horse serum (HiDHS) containing 10 % DMSO. Cultures were used within ten days of recovery from frozen stock.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (5% v/v); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

Preliminary toxicity test:
-S9 mix and +S9 mix (3 and 24 h): 3.5, 7.0, 13.9, 27.8, 55.7, 111.4, 222.8, 445.5, 891 and 1782 µg/mL (solubility limit).
Mutation tests:
-S9 mix, 3 h: 50, 100, 150, 200, 250, 300, 350, 400 and 450 µg/mL (limited by cytoxicity),
+S9 mix, 3 h: 50, 100, 200, 400, 600, 800, 1000, 1200, 1400 and 1600 µg/mL (limited by cytoxicity)
+S9 mix, 3 h (additional test): 100, 200, 400, 500, 600, 650, 700, 750, 800, 850, 900 and 1000 µg/mL (limited by cytoxicity)
-S9 mix, 24 h: 50, 100, 125, 150, 175, 200, 225, 250, 275 and 300 µg/mL (limited by cytoxicity)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Preparation of formulation: Test item was found to be soluble at 178.2 mg/mL in dimethyl sulphoxide (DMSO). A solution of 178.2 mg/mL, dosed at 1 % in medium, showed no precipitate in the culture medium. Test item was dissolved and diluted in DMSO, shortly before dosing. The final volume of DMSO added to the cultures was 1 % v/v.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (1 % v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9 mix; 10 µg/mL (3 h exposure) and 5 µg/mL (24 h exposure)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix; 1 µg/mL (3 h exposure)

Details on test system and experimental conditions:

METHOD OF APPLICATION: RPMI 1640 medium
R0: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.
R10p: R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10 % v/v.
R30p: R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30 % v/v.
R10p medium was used for cell culture unless otherwise specified.
R20p medium was used for the cloning efficiency plating. This was prepared by mixing equal volumes of R10p and R30p.

DURATION
- Exposure duration:
Preliminary toxicity test: 3 h in the absence and presence of S9 mix and 24 h in the absence of S9 mix
Main mutation assay: 3 h in the absence and presence of S9 mix and 24 h in the absence of S9 mix
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 7 days for viability plates and approximately 10 to 14 days for mutant plates
- All incubations were performed at 37 °C in a humidified atmosphere of 5 % CO2 in air.

SELECTION AGENT (mutation assays): Selective medium consisted of R10p containing 4 μg/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item and 2 cultures for vehicle control
- Main test: 4 cultures for vehicle control, 2 cultures/dose for test item and positive controls

NUMBER OF CELLS EVALUATED: 1.6 and 2000 cells per well plated for assessing cloning efficiency (CE) and mutant frequency (MF), respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth (RSG) and Relative total growth (RTG)
RSG = (Individual SG x 100) / Mean solvent control SG
Cloning efficiency (CE) = - InP(0) / No. of cells per well
Relative cloning efficiency (RCE) = (Individual CE x 100) / Mean vehicle control CE
RTG = (RSG x Day2 RCE) / 100

OTHER:
Mutant frequency per 10^6 survivors (MF) was calculated as:: CE selective medium / CE non-selective medium

Rationale for test conditions:

There was evidence of toxicity in the preliminary toxicity test, so the maximum concentrations tested in the 3-hour exposure in the absence and presence of S9 mix were 450 and 1600 µg/mL respectively, and in the 24-hour exposure in the absence of S9 mix was 300 µg/mL.

Evaluation criteria:

The following criteria were applied for assessment of individual assay results using data for Mutant Frequency (MF) where the Relative total growth (RTG) normally exceeded 10 %.

The assay was considered valid in accordance with the assay acceptance criteria.

The test agent was regarded as negative if:
- The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
- If the linear trend test was negative, the result was regarded as negative.
- If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.

Statistics:

- The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al., (1989).
- Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend (p<0.05).

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in the pH of the medium of more than 1.0 unit compared with the vehicle control was observed at 1782 μg/mL.
- Effects of osmolality: Osmolality of the test substance in medium was tested at concentrations of 1782 μg/mL; no change in osmolality of the medium of more than 50 mOsm/kg was observed compared with the vehicle control.
- Precipitation: A solution of 178.2 mg/mL, dosed at 1 % in medium, showed no precipitate in the culture medium.

PRELIMINARY TOXICITY TEST:
A 3 h exposure to test item at concentrations from 3.5 to 1782 μg/mL in the absence and presence of S9 mix resulted in relative suspension growth (RSG) values from 98 to 1 % and from 157 to 4 % respectively. A continuous exposure for 24 h to test item at concentrations 3.5 to 1782 μg/mL in the absence of S9 mix resulted in RSG values from 142 to 0 %. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100 % relative total growth (RTG).

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical control data (August 13, 2012 to July 23, 2014)

MAIN TESTS

3 h treatment in the absence of S9 mix:

- Cultures were exposed to test item at concentrations from 50 to 450 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to test item at concentrations from 250 to 450 µg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 100 to 19% were obtained relative to the vehicle control.

- There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum (194 x 10^-6) of the mean concurrent vehicle control mutant frequency (68 x 10^-6) and the Global Evaluation Factor (GEF, 126 x 10^-6), within acceptable levels of toxicity.

 

3 h treatment in the absence of S9 mix:

- Cultures were exposed to test item at concentrations from 50 to 1600 µg/mL. No precipitate was observed by eye at the end of treatment. Relative Suspension Growth values from 98 to 1 % were obtained relative to the vehicle control. As an acceptable range in toxicity was not achieved an additional test was performed.

 

Additional main mutation test – 3 h treatment in the presence of S9 mix:

- Cultures were exposed to test item at concentrations from 100 to 1000 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to test item at concentrations from 200 to

1000 µg/mL were assessed for determination of mutation frequency. RTG values from 92 to 25 % were obtained relative to the vehicle control.

- There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum (238 x 10^-6) of the mean concurrent vehicle control mutant frequency (112 x 10^-6) and the GEF (126 x 10^-6), within acceptable levels of toxicity. All mean mutant frequencies of the test concentrations were within the acceptable vehicle control values and there were no clear increases in the mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05).

 

24 h treatment in the absence of S9 mix:

- Cultures were exposed to test item at concentrations from 50 to 300 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to test item at concentrations from 50 to 250 µg/mL were assessed for determination of mutation frequency. RTG values from 104 to 20 % were obtained relative to the vehicle control.

- There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum (205 x 10^-6) of the mean concurrent vehicle control mutant frequency (79 x 10^-6) and the GEF (126 x 10^-6), within acceptable levels of toxicity.

Table 7.6.1/1: Main mutation test – results:

Groups	Concentration (µg/mL)	3 h Treatment -S9-mix		3 h Treatment +S9-mix		24 h Treatment -S9-mix
		Mean Relative Total Growth (%)	Mean Mutant Freq. (x10^-6)	Mean Relative Total Growth (%)	Mean Mutant Freq. (x10^-6)	Mean Relative Total Growth (%)	Mean Mutant Freq. (x10^-6)
Vehicle control (DMSO)	0	100	68	100	112	100	79
Test item	50	NP	NP	NT	NT	104	82
	150	NP	NP	NT	NT	65	75
	175	NP	NP	NT	NT	45	71
	200	NP	NP	92	101	25	87
	250	100	70	NT	NT	20	76
	300	93	72	NT	NT	NP	NP
	350	59	101	NT	NT	NT	NT
	400	71	78	NP	NP	NT	NT
	450	19	67	NT	NT	NT	NT
	600	NT	NT	74	99	NT	NT
	650	NT	NT	54	119	NT	NT
	900	NT	NT	36	164	NT	NT
	1000	NT	NT	25	141	NT	NT
Methyl methanesulphonate	10	36	1322^a	NT	NT	NT	NT
	5	NT	NT	NT	NT	24	1200^a
Benzo[a]pyrene	1	NT	NT	87	450^a	NT	NT

 

^a Positive control induced an acceptable response

NT Not Tested

NP Not Plated

Conclusions:

Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk^+/-(3.7.2C) mouse lymphoma cells were exposed to test item at the following concentrations:

Preliminary toxicity test:

-S9 mix and +S9 mix (3 and 24 h): 3.5, 7.0, 13.9, 27.8, 55.7, 111.4, 222.8, 445.5, 891 and 1782 µg/mL

Mutation tests:

-S9 mix, 3 h: 50, 100, 150, 200, 250, 300, 350, 400 and 450 µg/mL,

+S9 mix, 3 h: 50, 100, 200, 400, 600, 800, 1000, 1200, 1400 and 1600 µg/mL

+S9 mix, 3 h (additional test): 100, 200, 400, 500, 600, 650, 700, 750, 800, 850, 900 and 1000 µg/mL

-S9 mix, 24 h: 50, 100, 125, 150, 175, 200, 225, 250, 275 and 300 µg/mL 

 

Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 5 % (v/v) S9 mix; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with Aroclor 1254.

 

In the preliminary toxicity test, toxicity was observed following a 3 h exposure to test item in both the absence and presence of S9 mix, and following a 24 h exposure in the absence of S9 mix. At concentrations from 3.5 to 1782 μg/mL, relative suspension growth was reduced from 98 to 1 %, from 157 to 4 % and from 142 to 0 % respectively. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100 % relative total growth (RTG).

 

In main tests, following 3 h treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 h treatment in the absence and presence of S9 mix were 450 and 1000 µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 19 and 25 % respectively. In the 24 h treatment, the maximum concentration assessed for mutant frequency was 250 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The RTG was reduced to 20 %. In all tests the concurrent vehicle and positive control were within acceptable ranges.

 

Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical and genotoxic properties because of their structural similarity. It has to be highlighted that source and target substances behave similarly under neutral and acid pH, they are all stable at those pHs and no hydrolysis products are formed.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are both mono-constituent substances.

3. ANALOGUE APPROACH JUSTIFICATION
- In vitro gene mutation study in bacteria - Ames test
No (reverse) gene mutations were detected in the Ames test performed on both the source and the target substances.
- In vitro gene mutation study in mammalian cells
In the hprt test performed on the target substance, none of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases.
- In vitro cytogenicity study in mammalian cells
In the CAT performed on the source substance, no significant increases in the frequency of cells with aberrations was induced. The CAT test on the target substance provided in this dossier was performed according to OECD TG 473. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the CAT conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.3.

4. DATA MATRIX
[Further information is included as attachment to Iuclid section 13]

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

lymphocytes: humans

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Effects of pH: No significant change in pH when the test material was added into media.
Effects of osmolality: Osmolality did not increase by more than 50 mOsm.

RANGE-FINDING/SCREENING STUDIES:
Test material showed some evidence of toxicity in all exposure groups. A red colouration was observed in the parallel blood-free cultures at the end of the exposure period, at and above 128.88 μg/mL, in the 4(20)-hour pulse exposure group without metabolic activation and at and above 1031 μg/mL, in the 4(20)-hour pulse exposure group with metabolic activation. In the continuous exposure group a cloudy precipitate was seen at 2062 μg/mL. Haemolysis was observed at and above 515.5 μg/mL in the 4(20)-hour exposure groups with and without S9, and at and above 1031 μg/mL in the 24-hour exposure group. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 515.5 μg/mL in the 4(20)-hour treatment in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 257.75 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: All vehicle and solvent controls were in the range of historical laboratory control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
EXPERIMENT 1: The results of the mitotic indices (MI) from the cultures after their respective treatments showed 32% and 76% growth inhibition respectively at 257.75 and 515.5 μg/mL in the absence of S9. In the presence of S9 the data show 26% and 67% growth inhibition respectively was achieved at 257.75 and 515.5 μg/mL. However due to excessive toxicity the dose level 515.5 μg/mL was not suitable for metaphase analysis in either exposure group. A precipitate of the test material was not seen at the end of the treatment period in either exposure group; however haemolysis was seen in both exposure groups at and above 515.5 μg/mL, at the end of the treatment period. The maximum dose level selected for metaphase analysis was based on toxicity and was the highest dose level with acceptable levels of toxicity (257.75 μg/mL).
EXPERIMENT 2: The results of the mitotic indices (MI) from the cultures after their respective treatments showed 31% growth inhibition at 257.75 μg/mL in the absence of S9 and 24% and 70% growth inhibition respectively was achieved at 386.65 and 515.5 μg/mL in the presence of S9. A precipitate of the test material was not seen at the end of the treatment period in either exposure group; however haemolysis was seen in both exposure groups, in the continuous exposure group at and above 64.44 μg/mL and at and above 257.75 μg/mL in the presence of S9. The maximum dose level selected for metaphase analysis was 257.75 μg/mL for the 24-hour exposure group, and 515.5 μg/mL for the 4(20)-hour exposure group in the presence of S9.

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation. The test item did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

None

Conclusions:

The source material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The source and target substances are therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, human primary lymphocytes were exposed to test item diluted in DMSO at the following concentrations:

Preliminary Toxicity Test (Cell Growth Inhibition Test):

0, 8.05, 16.11, 32.22, 64.44, 128.88, 257.75, 515.5, 1031 and 2062 μg/mL; 4 h exposure time with and without metabolic activation followed by a 20 h recovery period, and a continuous exposure of 24 hours without metabolic activation

Experiment 1: 4 hour treatment followed by 24 hour harvest after the start of treatment

without and with S9 (2 % S9): 0, 64.44, 128.88, 257.75, 515.5, 773.33 and 1031 μg/mL

Experiment 2:

without S9: 0, 16.11, 32.22, 64.44, 128.88, 257.75 and 515.5 μg/mL; 24 hour treatment followed by harvest at the end of treatment

with S9 (1 % S9): 0, 64.44, 128.88, 257.75, 386.65, 515.5 and 773.33 μg/mL; 4 hour treatment followed by 24 hour harvest after the start of treatment

Mitotic activity was arrested by addition of colcemid at 0.1 µg/mL for each culture, two hours before the harvest. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Vehicle and positive controls were also included in this test.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition.

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

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay.

The study was conducted on an analogue substance. The supporting substance is considered adequate for read-across purpose (see IUCLID section 13 for additional justification).

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 07 to October 08, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on August 19, 2008/ signed on March 4, 2009)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: humans

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum, at 37ºC with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at approximately 90 μg/ml final concentration.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

2 % S9 (final concentration); S9 fraction was obtained from the liver homogenates of male Sprague Dawley rats treated with phenobarbitone and β-naphthoflavone

Test concentrations with justification for top dose:

Preliminary Toxicity Test (Cell Growth Inhibition Test): 0, 8.05, 16.11, 32.22, 64.44, 128.88, 257.75, 515.5, 1031 and 2062 μg/mL
Experiment 1: without and with S9 (2 % S9): 0, 64.44, 128.88, 257.75, 515.5, 773.33 and 1031 μg/mL (limited by cytotoxicity)
Experiment 2:
without S9: 0, 16.11, 32.22, 64.44, 128.88, 257.75 and 515.5 μg/mL. (limited by cytotoxicity)
with S9 (1 % S9): 0, 64.44, 128.88, 257.75, 386.65, 515.5 and 773.33 μg/mL. (limited by cytotoxicity)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Formulation preparation: The test material was melted prior to testing; this allowed the test material to be accurately weighed. The test material was dissolved in Dimethyl sulphoxide (DMSO) and serial dilutions prepared. The molecular weight of the test material was given as 206.2, therefore the maximum dose level was 2062 μg/mL, which was equivalent to 10 mM.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9 mix; 0.4 and 0.2 μg/mL for 4(20)-hour and 24-hour cultures respectively

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9 mix; 5 μg/mL

Details on test system and experimental conditions:

TEST SYSTEM: Cultures prepared from the blood of a human volunteer who had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.

CELL CULTURE: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 15 % foetal calf serum, at 37 ºC with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at approximately 90 μg/mL final concentration.

DURATION
- Exposure duration: 4 hours (± S9) in Experiment 1, 4 hours (+S9) and 24 hours (-S9) in Experiment 2
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (± S9) in Experiment 1 and 2

SPINDLE INHIBITOR (cytogenetic assays): Mitotic activity was arrested by addition of demecolcine (Colcemid 0.1 μg/mL), two hours before the harvest time.
STAIN (for cytogenetic assays): 5 % Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures/dose

NUMBER OF CELLS EVALUATED:
- Total of 2000 lymphocyte cell nuclei was counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
- Where possible the first 100 consecutive well-spread metaphases from each culture were counted. Where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the International System for Chromosome Nomenclature (1985) as described by Scott et al and compatible and equitable to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: If greater than 44 chromosomes are scored and the number is a multiple of the haploid count then the cell is classified as a polyploid cell.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated (E).

Rationale for test conditions:

Experiment 1 & 2: Mitotic index data was used to estimate test material toxicity and for selection of the dose levels for the main test.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes: humans

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Effects of pH: No significant change in pH when the test material was added into media.
Effects of osmolality: Osmolality did not increase by more than 50 mOsm.

RANGE-FINDING/SCREENING STUDIES:
Test material showed some evidence of toxicity in all exposure groups. A red colouration was observed in the parallel blood-free cultures at the end of the exposure period, at and above 128.88 μg/mL, in the 4(20)-hour pulse exposure group without metabolic activation and at and above 1031 μg/mL, in the 4(20)-hour pulse exposure group with metabolic activation. In the continuous exposure group a cloudy precipitate was seen at 2062 μg/mL. Haemolysis was observed at and above 515.5 μg/mL in the 4(20)-hour exposure groups with and without S9, and at and above 1031 μg/mL in the 24-hour exposure group. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 515.5 μg/mL in the 4(20)-hour treatment in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 257.75 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: All vehicle and solvent controls were in the range of historical laboratory control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
EXPERIMENT 1: The results of the mitotic indices (MI) from the cultures after their respective treatments showed 32% and 76% growth inhibition respectively at 257.75 and 515.5 μg/mL in the absence of S9. In the presence of S9 the data show 26% and 67% growth inhibition respectively was achieved at 257.75 and 515.5 μg/mL. However due to excessive toxicity the dose level 515.5 μg/mL was not suitable for metaphase analysis in either exposure group. A precipitate of the test material was not seen at the end of the treatment period in either exposure group; however haemolysis was seen in both exposure groups at and above 515.5 μg/mL, at the end of the treatment period. The maximum dose level selected for metaphase analysis was based on toxicity and was the highest dose level with acceptable levels of toxicity (257.75 μg/mL).
EXPERIMENT 2: The results of the mitotic indices (MI) from the cultures after their respective treatments showed 31% growth inhibition at 257.75 μg/mL in the absence of S9 and 24% and 70% growth inhibition respectively was achieved at 386.65 and 515.5 μg/mL in the presence of S9. A precipitate of the test material was not seen at the end of the treatment period in either exposure group; however haemolysis was seen in both exposure groups, in the continuous exposure group at and above 64.44 μg/mL and at and above 257.75 μg/mL in the presence of S9. The maximum dose level selected for metaphase analysis was 257.75 μg/mL for the 24-hour exposure group, and 515.5 μg/mL for the 4(20)-hour exposure group in the presence of S9.

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation. The test item did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

None

Conclusions:

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, human primary lymphocytes were exposed to test item diluted in DMSO at the following concentrations:

Preliminary Toxicity Test (Cell Growth Inhibition Test):

0, 8.05, 16.11, 32.22, 64.44, 128.88, 257.75, 515.5, 1031 and 2062 μg/mL; 4 h exposure time with and without metabolic activation followed by a 20 h recovery period, and a continuous exposure of 24 hours without metabolic activation

Experiment 1: 4 hour treatment followed by 24 hour harvest after the start of treatment

without and with S9 (2 % S9): 0, 64.44, 128.88, 257.75, 515.5, 773.33 and 1031 μg/mL

Experiment 2:

without S9: 0, 16.11, 32.22, 64.44, 128.88, 257.75 and 515.5 μg/mL; 24 hour treatment followed by harvest at the end of treatment

with S9 (1 % S9): 0, 64.44, 128.88, 257.75, 386.65, 515.5 and 773.33 μg/mL; 4 hour treatment followed by 24 hour harvest after the start of treatment

Mitotic activity was arrested by addition of colcemid at 0.1 µg/mL for each culture, two hours before the harvest. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Vehicle and positive controls were also included in this test.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition.

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

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Safepharm, 2008	Ames Test (OECD 471) K, rel.1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100	-S9 +S9	Up to recommended maximum level	-S9 : non mutagenic +S9 : non mutagenic
2    HLS, 2014	L5178YTK+/- MLA test (OECD 476) K, rel. 1	Gene mutation	L5178Y tk+/-(3.7.2C) mouse lymphoma cells	-S9 +S9	Up to cytotoxic concentration	-S9 : non mutagenic +S9 : non mutagenic
3 (RA)   Harlan, 2009	HL/CAT (OECD 473) K, rel. 1	Chromosomal aberration	Human Lymphocytes	-S9 +S9	Up to cytotoxic concentration	-S9 : non clastogenic +S9 : non clastogenic

Gene mutation Assays (Tests n° 1 and 2):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies.The substance is therefore considered as non-mutagenic according to the Ames test.

- An inability to produce gene mutation was observed in mammalian cells using an in vitro gene mutation assay in L5178Y tk+/-(3.7.2C) mouse lymphoma cells (L5178Y TK+/- MLA test) (Test n°2). None of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore the substance is considered as negative for inducing gene mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay.

Therefore this result confirms the results of the Ames test and it demonstrates the non-mutagenic effect of the substance.

  

Chromosomal aberration (Test n°3)

The clastogenic potential of the substance was determined with a supporting substance (see IUCLID section 13 for additional justification) using an in vitro chromosome aberration test in human lymphocytes (OECD guideline No. 473), which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of three experiments. The substance does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.The substance and the supporting substance are therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.