Benzene-1,3,5-tricarbonyl trichloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2022

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-03-2022 to 02-05-2022

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 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:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine or tryptophan locus

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: Purchased from recognised supplier (dates within full study report) ; S9 Microsomal fraction: Lot No. presented in the full study report.
- method of preparation of S9 mix: Documented in the full study report. S9 was stored at less than or equal to -80ºC; S9-mix was prepared immediately before use and kept refrigerated. 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment or third experiment respectively) ; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution ; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second and third experiments, respectively.
- concentration or volume of S9 mix and S9 in the final culture medium: 5% S9 and/or 10% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively. No issues were reported in the full study report. Furthermore, concurrent positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Test concentrations with justification for top dose:

Experiment 1 (all strains ; direct plate/plate incorporation assay): 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2 (all strains ; direct plate/plate incorporation assay): 0, 5.4, 17, 52, 164, 512 and 1600 µg/plate
Experiment 3 (WP2urvA strain only ; direct plate/plate incorporation assay): 0, 1, 10, 50, 150, 250, 500, 750 and 1000 µg/plate

See 'Additional information on results' section for further information on the justification for the top doses including cytotoxicity and/or precipitation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: Solubility testing was performed based on visual assessment. Due to the limitations of the test item and/or considering specific safety precautions, only dried vehicles could be tested. Dried DMSO and Dried THF were tested within the solubility trial. The test item reacted heavily with dried DMSO and was therefore not suitable as solvent for the OECD TG 471. Subsequently, the test item was found to form a clear colourless solution in dried THF. Test item formulations were used within 3 hours after preparation. Applicant assessment indicates: 'dried DMSO' would possess trace levels of water that could react with the test item, rather than a reaction with DMSO, per se.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 ; 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicate (3)
- Number of independent experiments: At least two for all strains.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Not applicable.
- Test substance added in medium; in agar (plate incorporation). Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (plate incorporation ; Experiment 3 in medium; in agar (plate incorporation). The vehicle was dried tetrahydrofuran as the test item is unstable and reactive with water. For this reason preincubation method was avoided and plate incorporation method was used in this study.
- Other: METHOD OF APPLICATION/EXPERIMENTAL DESIGN: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.05 mL of a dilution of the test item in THF or control solution and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Not applicable.
- Exposure duration/duration of treatment: 37.0 ± 1.0 °C for 48 ± 4 h incubation period.
- Harvest time after the end of treatment (sampling/recovery times): Not applicable.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition (e.g. reduction in bacterial lawn)

METHODS FOR MEASUREMENTS OF GENOTOXICIY: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

- OTHER: In the first mutation experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate in all tester strains. In a follow-up experiment with additional parameters , the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains. A repeat experiment (experiment 3) was performed with tester strain WP2uvrA in the absence and presence of 10% (v/v) S9-mix to further examine: a weak but significant increase of revertants observed in the absence and presence of S9-mix when compared with the concurrent solvent control. The increases observed were up to 2.0- and 2.3-fold. The increases observed in WP2urvA strain in the second experiment were within the historical control data range and were not reproduced in a repeat experiment (Experiment 3). Therefore, the increases were considered not biologically relevant.

Rationale for test conditions:

Selection of an adequate range of doses was based on the first experiment (seven concentrations in triplicate) with all five tester strains, both with and without S9-mix considering cytotoxicity and precipitation. See tables for more information.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 and WP2urvA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 and TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 and WP2urvA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 and TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done. See 'Any other information on materials and methods' and 'evaluation criteria' for details on the acceptability and evaluation criteria of the assay.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Test item was either tested up to cytotoxic or precipitating concentrations or the limit concentration (5000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Test item was either tested up to cytotoxic or precipitating concentrations or the limit concentration (5000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Test item was either tested up to cytotoxic or precipitating concentrations or the limit concentration (5000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Test item was either tested up to cytotoxic or precipitating concentrations or the limit concentration (5000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Test item was either tested up to cytotoxic or precipitating concentrations or the limit concentration (5000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Not applicable. Dried tetrahydrofuran (non-aqueous) vehicle was utilised within the study.
- Data on osmolality: Not reported.
- Possibility of evaporation from medium: Not applicable.
- Water solubility: Not applicable. Dried tetrahydrofuran (non-aqueous) vehicle was utilised within the study.
- Precipitation and time of the determination: Experiment 1: Precipitation of the test item on the plates was observed at concentrations of 512 µg/plate and upwards except for tester strain TA100 in the absence and presence of S9-mix and for tester strains TA98 and WP2uvrA in the presence of S9-mix where precipitate was observed at dose levels of 1600 µg/plate and upwards.
Experiment 2: Precipitation of the test item on the plates was observed at concentrations of 164 µg/plate and upwards except for tester strains TA98, TA1537, TA100 and WP2uvrA in the presence of S9-mix where precipitate was observed at dose levels of 512 µg/plate and upwards.
Experiment 3: Precipitation of the test item on the plates was observed at concentrations of 500 µg/plate and upwards.
- Definition of acceptable cells for analysis: No issues reported.
- Other confounding effects: None reported.

RANGE-FINDING/SCREENING STUDIES (if applicable):
Experiment 1 (direct plate assay) served as a range finding test in all species/strains for subsequent assays.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: In all experiments, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Historical Control Data (HCD) for negative and/or strain-specific positive controls is presented within the full study report.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: See tables.
- Statistical analysis; p-value if any: See tables.
- Any other criteria: Refer to ‘Evaluation Criteria’ section for further information.

Ames test:
- Signs of toxicity: Cytotoxicity was observed. See tables.
- Individual plate counts: See tables.
- Mean number of revertant colonies per plate and standard deviation: See tables.
- Other:
(i) Experiment 1 (plate incorporation method):
All strains (presence of S9-mix): 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
All strains (absence of S9-mix): 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Precipitation of the test item on the plates was observed at concentrations of greater than or equal to 512 µg/plate except for tester strain TA100 in the absence and presence of S9-mix and for tester strains TA98 and WP2uvrA in the presence of S9-mix where precipitate was observed at dose levels of greater than or equal to 1600 µg/plate. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
(ii) Experiment 2 (plate incorporation method): Eight to ten test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item. The dose levels were selected based on the results of Experiment 1.
All strains (presence of S9-mix): 0, 5.4, 17, 52, 164, 512 and 1600 µg/plate
All strains (absence of S9-mix): 0, 5.4, 17, 52, 164, 512 and 1600 µg/plate
Precipitation of the test item on the plates was observed at concentrations of greater than or equal to 164 µg/plate except for tester strains TA98, TA1537, TA100 and WP2uvrA in the presence of S9-mix where precipitate was observed at dose levels of greater than or equal to 512 µg/plate. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies was not observed at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
(iii) Experiment 3 (plate incorporation method):
WP2urvA strain only (presence and absence of S9-mix): 0, 1, 10, 50, 150, 250, 500, 750 and 1000 µg/plate
Precipitation of the test item on the plates was observed at concentrations of 500 µg/plate and upwards. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies was not observed at any of the concentrations tested in the absence and presence of S9-mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: In all experiments, strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Historical Control Data (HCD) strain-specific positive controls is presented within the full study report.
- Negative (solvent/vehicle) historical control data: In all experiments, the negative control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Historical Control Data (HCD) for negative controls is presented within the full study report.

Table 1. Mutagenic response of test item in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay – Experiment 1

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.
	TA1535			TA1537			TA98			TA100			WP2uvrA
Without S9-mix
Positive control	809	± 130		1458	± 279		1557	± 242		644	± 128		1312	± 92
Solvent control	10	± 4		3	± 4		12	± 5		90	± 11		18	± 5
1.7	7	± 1		4	± 2		11	± 4		87	± 2		0	± 0	#a
5.4	9	± 3		2	± 2		11	± 5		78	± 23		12	± 6
17	11	± 1		4	± 1		14	± 4		94	± 12		16	± 5
52	9	± 2		2	± 1		14	± 2		98	± 4		17	± 5
164	5	± 3	NP	4	± 1		8	± 3	NP	76	± 6		13	± 3	NP
512	4	± 1	MP	2	± 1	MP	5	± 3	MP	86	± 2	NP	16	± 5	MP
1600	7	± 1	n MP	1	± 1	n MP	7	± 4	n MP	103	± 15	n SP	8	± 3	n MP
5000			e MP MC			e MP MC			e MP MC			e MP MC			e MP MC
With S9-mix (5%)
Positive control	199	± 36		288	± 82		979	± 264		1611	± 209		523	± 52
Solvent control	11	± 4		3	± 0		15	± 1		76	± 2		34	± 3
1.7	9	± 2		4	± 1		15	± 3		62	± 6		44	± 5
5.4	11	± 4		5	± 1		14	± 6		67	± 17		24	± 4
17	10	± 5		3	± 2		15	± 4		76	± 11		39	± 6
52	10	± 2		4	± 2		11	± 4		68	± 11		40	± 7
164	7	± 3	NP	3	± 1	NP	11	± 1		76	± 6		37	± 6
512	3	± 1	SP	2	± 1	MP	9	± 3	NP	104	± 11		40	± 14	NP
1600	8	± 3	n MP	2	± 2	n MP	7	± 3	n MP	105	± 23	NP	29	± 2	SP
5000			e MP MC			e MP MC			e MP MC			e MP MC	29	± 5	n MP

MC : Microcolonies

NP : No precipitate

SP : Slight Precipitate

MP : Moderate Precipitate

#a : bacterial lawn absent

e : bacterial lawn extremely reduced

n : Normal bacterial background lawn

 

Table 2. Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay – Experiment 2

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.
	TA1535			TA1537			TA98			TA100			WP2uvrA
Without S9-mix
Positive control	776	± 33		1687	± 63		1839	± 45		771	± 16		1857	± 72
Solvent control	11	± 1		8	± 3		20	± 3		131	± 14		22	± 7
5.4	9	± 2		4	± 1		25	± 8		113	± 7		23	± 6
17	8	± 0		6	± 3		24	± 9		108	± 7		34	± 11
52	10	± 4	NP	2	± 1	NP	10	± 1	NP	99	± 14	NP	36	± 4	NP
164	7	± 2	MP	2	± 1	MP	7	± 1	MP	90	± 7	MP	44	± 6	SP
512	7	± 1	MP	2	± 0	MP	9	± 2	MP	79	± 5	MP	29	± 7	MP
1600	5	± 1	n MP	2	± 1	n MP	4	± 3	n MP	73	± 6	n MP	26	± 8	n MP
With S9-mix (10%)
Positive control	88	± 29		108	± 18		356	± 53		947	± 661		466	± 63
Solvent control	6	± 3		6	± 2		22	± 2		65	± 14		28	± 3
5.4	8	± 3		4	± 4		20	± 5		72	± 11		22	± 1
17	7	± 3		2	± 2		15	± 4		71	± 9		52	± 20
52	9	± 3	NP	2	± 1		22	± 2		94	± 12		59	± 7
164	7	± 3	MP	5	± 3	NP	24	± 5	NP	105	± 4	NP	59	± 9	NP
512	10	± 2	MP	3	± 2	MP	17	± 5	SP	113	± 1	SP	65	± 3	SP
1600	7	± 3	n MP	2	± 2	n MP	8	± 3	n MP	81	± 4	n MP	37	± 4	n MP

NP : No precipitate

SP : Slight Precipitate

MP : Moderate Precipitate

n : Normal bacterial background lawn

 

Table 3. Mutagenic response of test substance in the Escherichia coli reverse mutation assay – Experiment 3

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one Escherichia coli strain.
	WP2uvrA
Without S9-mix
Positive control	560	± 49
Solvent control	22	± 7
10	22	± 5
50	15	± 7
150	36	± 6
250	29	± 9	NP
500	17	± 3	n MP
With S9-mix (10%)
Positive control	394	± 33
Solvent control	20	± 4
1	30	± 8
10	24	± 9
50	28	± 14
150	20	± 4
250	32	± 8	NP
500	17	± 1	MP
750	24	± 6	MP
1000	25	± 3	n MP

NP : No precipitate

MP : Moderate Precipitate

n : Normal bacterial background lawn

Conclusions:

Interpretation of results: negative
Under the conditions of this study, the test item was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

The study was performed to the requirements of OECD TG 471 and EU Method B.13/14 guidelines in accordance with GLP, to evaluate the mutagenic activity of the test item in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (by the the plate incorporation method) in both the presence and absence of S-9 metabolic activation system. The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in at least two independent experiments, both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). The vehicle of the test item was dried tetrahydrofuran as the test item is unstable and reactive with water. For this reason preincubation method was avoided and plate incorporation method was utilised within the study. In Experiment 1, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 512 μg/plate and upwards except for tester strain TA100 in the absence and presence of S9-mix and for tester strains TA98 and WP2uvrA in the presence of S9-mix where precipitate was observed at dose levels of 1600 µg/plate and upwards. No significant increase in the number of revertants were observed. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains at dose levels of 5000 µg/plate in the absence and presence of S9-mix. In Experiment 2, the test item was tested at a concentration range of 5.4 to 1600 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 164 μg/plate and upwards except for tester strains TA98, TA1537, TA100 and WP2uvrA in the presence of S9-mix where precipitate was observed at dose levels of 512 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested. No significant increase in the number of revertants was observed in all Salmonella typhimurium strains. In tester strain WP2uvrA in the absence of S-9 a 2.0-fold increase was observed at a single dose (164 µg/plate), In the presence of S9-mix, the test item induced a 2.1, 2.1 and 2.3 -fold increases in the number of revertant colonies at: 52, 164 and 512 µg/plate, respectively when compared to the solvent control. These weak increases were within the historical control data range however required further clarification within a further experiment. In Experiment 3, using WP2uvrA strain only in the presence of 10% (v/v) S9-mix at a concentration range of 1 to 1000 µg/plate and in the absence of S9-mix at 10 to 500 µg/plate, it was observed that: the test item precipitated at dose levels of 500 μg/plate and upwards and the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant increase in the number of revertants was observed. It was considered this indicated the weak increases observed in the second experiment were incidental and not biologically relevant. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate, and that the metabolic activation system functioned properly. Under the conditions of this study, it is concluded that that the test item was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key study : OECD TG 471, 2022 : The study was performed to the requirements of OECD TG 471 and EU Method B.13/14 guidelines in accordance with GLP, to evaluate the mutagenic activity of the test item in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (by the the plate incorporation method) in both the presence and absence of S-9 metabolic activation system. The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in at least two independent experiments, both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). The vehicle of the test item was dried tetrahydrofuran as the test item is unstable and reactive with water. For this reason preincubation method was avoided and plate incorporation method was utilised within the study. In Experiment 1, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 512 μg/plate and upwards except for tester strain TA100 in the absence and presence of S9-mix and for tester strains TA98 and WP2uvrA in the presence of S9-mix where precipitate was observed at dose levels of 1600 µg/plate and upwards. No significant increase in the number of revertants were observed. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains at dose levels of 5000 µg/plate in the absence and presence of S9-mix. In Experiment 2, the test item was tested at a concentration range of 5.4 to 1600 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 164 μg/plate and upwards except for tester strains TA98, TA1537, TA100 and WP2uvrA in the presence of S9-mix where precipitate was observed at dose levels of 512 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested. No significant increase in the number of revertants was observed in all Salmonella typhimurium strains. In tester strain WP2uvrA in the absence of S-9 a 2.0-fold increase was observed at a single dose (164 µg/plate), In the presence of S9-mix, the test item induced a 2.1, 2.1 and 2.3 -fold increases in the number of revertant colonies at: 52, 164 and 512 µg/plate, respectively when compared to the solvent control. These weak increases were within the historical control data range however required further clarification within a further experiment. In Experiment 3, using WP2uvrA strain only in the presence of 10% (v/v) S9-mix at a concentration range of 1 to 1000 µg/plate and in the absence of S9-mix at 10 to 500 µg/plate, it was observed that: the test item precipitated at dose levels of 500 μg/plate and upwards and the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant increase in the number of revertants was observed. It was considered this indicated the weak increases observed in the second experiment were incidental and not biologically relevant. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate, and that the metabolic activation system functioned properly. Under the conditions of this study, it is concluded that that the test item was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

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