2-hydroxy-4-(trifluoromethyl)benzoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Key study: OECD Guideline 471, GLP study. The test item was determined to be non-mutagenic to all of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102 when tested with and without metabolic activation up to 5000 µg/plate.

- Key study: OECD Guideline 490, GLP study. The test item was determined to be non-mutagenic, since no biologically relevant increase in the mutation factor was observed in any treatment with or without metabolic activation.

- Data waiving (in vitro cytogenicity / chromosome aberration study in mammalian cells): an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available.

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:

December 16, 2017 - February 6, 2018

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa mutation (histidine dependence), uvrB mutation (biotin dependence)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Test 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (all strains)
Test 2: 156.25, 512.5, 625, 1250, 2500 and 5000 µg/plate (all strains)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A preliminary solubility and precipitation test was used. The substance was insoluble in distilled water.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Tubes containing 2 mL of molten top agar with 0.5 mM histidine/biotin were maintained at 45 ± 2 °C. A volume of 500 µL of 0.2 M phosphate buffer was added in the absence of metabolic activation system and 500 µL of 5% v/v S9 mix (Test 1) or 10% v/v S9 (Test 2) was added in the presence of metabolic activation system. Volume of 100 µL of the relevant stock solution of test item, DMSO and relevant positive control were used for treatment, as a negative control and as a positive control, respectively. Finally, 100 µL of bacterial culture (1 - 2 x 10E9 bacteria/mL) was added to the tubes and mixed. Two sets were maintained for each concentration of dihydromyrcenyl formate, positive control and negative control. The petriplates were incubated at 37 ± 1 °C for 48 hours and then examined to assess the state of background bacterial lawn inhibition and reduction in number of colonies.

DURATION
- Expression time (cells in growth medium): 48 h

NUMBER OF REPLICATIONS: 2 (Test 1) and 3 (Test 2)

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was detectable as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn.

OTHER EXAMINATIONS:
Cell viability test: Fresh cultures for the test were prepared by inoculating frozen permanent cultures to a flask containing 10 mL of sterile nutrient broth N° 2 (Oxoid). The flasks were then incubated at 37 ± 1 °C in an orbital shaking incubator (120 rpm) for 15 h up to early stationary or late exponential phase. After the incubation period, the culture flasks were removed from the orbital shaking incubator. Aseptically, the cultures were diluted with oxoid nutrient broth (ONB) and the optical density was measured at 660 nm using a Spectrophotometer (Model visiscan 167, manufactured by Systronics). Oxoid nutrient broth was used as the control blank. Cell viability of the tester strains was determined prior to treatment. The optical density of the cultures was found to be in the acceptable range and so they were used for the study.

Genotype confirmation test: The genotype of the tester strain was confirmed for all the strains regularly (once a month). The tester strains of Salmonella typhimurium were tested for histidine dependence, biotin dependence, histidine and biotin dependence, rfa mutation, uvrB mutation through sensitivity to ultraviolet light and the R-factor resistance for ampicillin and tetracycline.

Sterility Check for the Operating System: Top agar, S9 mix, Solvent, Test item, ONB solution, 0.2 M Sodium Phosphate Buffer, MGA Plate.

Rationale for test conditions:

Hence DMSO was selected as the vehicle for treatment. Precipitation was not observed at the tested concentration of 5 µL/plate. Hence 5 µL/plate was selected as the highest concentration to be tested for initial toxicity-mutation test both in the absence and presence (5% v/v S9 mix) of metabolic activation.

Evaluation criteria:

The conditions necessary for determining a positive result were: there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the absence or presence of the metabolic activation system.
Biological relevance of the results was considered first:
Strains TA1535, TA1537:
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.
Strain TA98, TA100 and TA102:
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation test were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing.

Statistics:

Statistical analysis was used as an aid in the evaluation of dose response.

Key result

Species / strain:

S. typhimurium, other: TA1537, TA1535, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 5000 µg/plate)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No increase in the number of revertant colonies was observed both in the absence and presence of metabolic activation at any of the tested concentration in all the tester strains.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No observed.
- Definition of acceptable cells for analysis:
- Other confounding effects: Cell viability and genotype confirmatory tests were performed (see above). The cells were determined to be suitable for the test.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Revertants per plate (mean ± SD)
Without S9 mix
TA1537: 274.27 ± 94.89
TA 1535: 376.70 ± 113.61
TA98: 542.14 ± 148.27
TA100: 880.92 ± 175.36
TA102: 1058.59 ± 152.48

With S9 mix
TA1537: 300.52 ± 98.11
TA 1535: 419.02 ± 122.05
TA98: 671.47 ± 171.10
TA100: 968.94 ± 158.04
TA102: 1088.70 ± 154.86

Positive controls in the absence of metabolic activation:
TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate).
Positive controls in the presence of metabolic activation
2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)

- Negative (DMSO) historical control data: Revertants per plate (mean ± SD)
Without S9 mix
TA1537: 8.40 ± 2.73
TA 1535: 18.37 ± 4.44
TA98: 22.79 ± 4.92
TA100: 131.37 ± 10.65
TA102: 228.89 ± 12.48

With S9 mix
TA1537: 8.96 ± 3.02
TA 1535: 20.03 ± 4.97
TA98: 24.24 ± 5.43
TA100: 134.50 ± 10.53
TA102: 232.55 ± 13.12

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Normal growth in bacterial background lawn pattern was observed up to the tested concentration of 5000 µg/plate. Cytotoxicity was characterised by the reduction in the number of revertant colonies:
Test 1: Significant reduction in number of revertant colonies was observed (i.e. 92.2% to 100%) at the tested concentration of 5000 µg/plate both in the absence and presence of the metabolic activation (5% v/v S9 mix) in all the tester strains.
Test 2: Significant reduction in number of revertant colonies was observed (i.e. 84.43% to 100%) at the tested concentration of 5000 µg/plate both in the absence and presence of the metabolic activation (10% v/v S9 mix) in all the tester strains. Reduction in number of revertant colonies was observed (i.e. 36.61% to 45.88%) in both presence and absence of metabolic activation in all tester strains up to the tested concentration of 2500 µg/plate.
- Other observations when applicable: No.

Test 1: Without metabolic activation (-S9)

Concentration (µg/plate)	His^+Revertant Colonies/Plate (Absence of Metabolic Activation)
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	6.00	1.41	14.50	0.71	16.00	4.24	141.00	5.66	237.50	6.36
1.5	7.00	2.83	14.00	5.66	20.50	3.54	134.00	5.66	244.50	9.19
5	4.50	0.71	14.00	1.41	19.50	7.78	125.00	5.66	230.50	3.54
15	5.50	0.71	16.50	3.54	18.50	2.12	138.00	14.14	230.50	2.12
50	6.00	0.00	16.00	1.41	18.00	7.07	136.50	3.54	231.50	13.44
150	6.00	4.24	14.50	6.36	17.00	4.24	138.00	8.49	232.50	6.36
500	2.50	0.71	15.00	4.24	17.00	5.66	140.50	7.78	237.00	22.63
1500	5.00	2.83	17.00	2.83	16.00	4.24	131.50	12.02	231.50	16.26
5000	0.00	0.00	0.00	0.00	0.00	0.00	10.50	4.95	17.50	4.95
PC	239.50	37.48	324.00	39.60	495.00	87.68	961.00	8.49	1106.00	289.91
2Aa	-	-	-	-	-	-	140.50	12.02	-	-

Test 1: With metabolic activation (+S9)

Concentration (µg/plate)	His^+Revertant Colonies/Plate [Presence of Metabolic Activation (5% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	6.50	0.71	15.50	0.71	19.00	5.66	135.00	4.24	243.50	10.61
1.5	6.50	3.54	17.50	0.71	19.00	7.07	136.00	19.80	242.00	4.24
5	8.50	0.71	15.50	4.95	21.00	2.83	134.50	12.02	240.50	13.44
15	5.50	0.71	17.00	4.24	20.00	2.83	134.50	13.44	236.50	21.92
50	6.50	4.95	16.50	3.54	21.50	2.12	132.50	0.71	245.00	2.83
150	6.00	1.41	18.50	4.95	17.50	2.12	134.00	15.56	232.50	0.71
500	5.50	2.12	15.50	2.12	20.00	4.24	135.00	5.66	249.00	15.56
1500	6.50	2.12	16.50	3.54	19.50	2.12	131.50	6.36	247.00	14.14
5000	0.00	0.00	0.00	0.00	0.00	0.00	7.50	2.12	19.00	12.73
PC-2Aa	295.00	84.85	322.00	16.97	720.00	46.67	904.00	19.80	910.50	36.06

Test 2: Without metabolic activation (-S9)

Concentration (µg/plate)	His^+Revertant Colonies/Plate [Absence of Metabolic Activation]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	6.00	2.00	14.00	3.00	23.67	6.51	133.00	6.56	222.00	11.53
156.25	8.00	2.65	15.33	3.51	20.67	2.52	133.33	8.62	230.00	15.39
312.5	6.33	0.58	12.33	1.53	22.00	5.57	132.00	8.19	232.00	19.31
625	7.00	3.00	14.00	5.29	21.33	3.06	132.00	5.29	229.00	3.00
1250	7.00	3.61	13.33	2.31	21.00	5.57	132.00	8.00	232.00	15.52
2500	3.33	0.58	7.67	2.08	14.00	2.65	78.33	13.65	134.00	26.51
5000	0.00	0.00	0.00	0.00	0.00	0.00	14.67	3.51	30.67	14.57
PC	209.67	27.30	360.00	56.31	437.33	113.07	878.67	130.37	951.67	134.24
2Aa	-	-	-	-	-	-	132.00	8.19	-	-

Test 2: With metabolic activation (+S9)

Concentration (µg/plate)	His^+Revertant Colonies/Plate [Presence of Metabolic Activation (10% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	8.00	3.61	15.67	2.52	23.33	7.77	133.33	7.37	244.00	8.54
156.25	8.00	2.00	16.33	4.51	23.00	4.58	130.00	2.65	236.00	16.64
312.5	9.33	1.53	15.33	2.52	19.00	4.58	133.67	8.08	237.67	16.04
625	8.33	0.58	14.33	4.04	22.67	7.02	134.00	10.82	234.00	9.85
1250	8.33	1.53	13.67	6.43	23.33	7.09	130.33	10.02	233.67	14.01
2500	4.33	1.53	9.33	2.08	14.00	2.65	82.33	11.50	154.67	27.68
5000	0.00	0.00	0.00	0.00	0.00	0.00	10.00	3.61	38.00	19.67
PC-2Aa	223.67	34.49	322.67	28.01	642.00	170.71	969.33	147.77	972.33	44.97

Conclusions:

The test item was determined to be non-mutagenic to all of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102 when tested with and without metabolic activation up to 5000 µg/plate.

Executive summary:

A bacterial reverse mutation assay was performed according to OECD Guideline 471 (GLP study) using five histidine deficient mutant tester strains of Salmonella typhimurium (i.e., TA1537, TA1535, TA98, TA100, and TA102). Two independent experiments, in the absence and presence of metabolic activation, were performed. In the initial test, bacterial cultures were exposed to the test item at 8 concentrations (two plates/concentration) between 1.5 and 5000 µg/plate. No increase in the number of revertant colonies (no mutagenic effect) were observed both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in all the tester strains. To confirm the negative results obtained in the initial toxicity mutation test, confirmatory mutation test was conducted with an increased S9 concentration i.e., 10% v/v S9 mix and modified concentration spacing. In the confirmatory test, bacterial cultures were exposed to the test item at 6 concentrations (three plates/concentration) between 156.25 and 5000 µg/plate both in the absence and presence (10 % v/v S9 mix) of metabolic activation system. After 48 hours of incubation at 37 ± 1°C, the revertant colonies were scored. Test item did not induce any significant increase in the number of revertants, in both trials, with and without S9 mix, in any of the five tester strains. All the values for the negative control were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system. All criteria for a valid study were met. From the results of this study, under the specified experimental conditions, test item was concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 11, 2019 - October 22, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection (ATCC, Manassas,Virginia, USA)
- Suitability of cells: L5178Y TK+/-, -3.7.2 C cells are selected on the basis of growth ability in culture, stability of karyotype, chromosome number, sensitivity to chemical mutagens, a high cloning efficiency and stable spontaneous mutant frequency. Furthermore, it is the test system recommended by the OECD guidelines.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: DMEM supplemented with antibiotics and with 10% fetal bovine serum (FBS) is used for growth and multiplication of L5178Y cells, for washing and dilution of cells, and as treatment medium for prolongued exposure of the cells in the absence of metabolic activation. DMEM supplemented with antibiotics and 20% FBS is used for the determination of cell viability (Cloning medium, CM). CM supplemented with the selective agent trifluorothymidine (TFT) at 4 µg/mL is used for the selection of mutants. All laboratory cultures will be maintained at 37 ± 1ºC and 5 ± 1% CO2.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 (sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate)

Test concentrations with justification for top dose:

Initial cytotoxicity test: 0.0625, 0.125, 0.25, 0.5, 1 and 2.0 mg/mL (based on pH, solubility and precipitation tests).
Main experiment: 0.25, 0.5, 1.0 and 2.0 mg/mL. The selection of the concentrations used in the main experiment was based on the initial cytotoxicity test results.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. Based on the results of the solubility test the test item was dissolved in DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1 x 10^7 cells/tube

DURATION
- Exposure duration: 3-4 hours (short-term treatment) or 20-24 hours (long-term treatment)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 days
- Incubation time for cloning efficiency (RCE) determination: 13/14 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT).

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: all cells were scored

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, and relative total growth

Rationale for test conditions:

In accordance with OECD 490 (17) "Media and culture conditions".

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined, the increase in mutant frequency (MF) above the concurrent background exceeds the Global Evaluation Factor (GEF) and the increase is concentration related (e.g., using a trend test). Subsequently, a test item 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.

Statistics:

Significance of difference between vehicle control values over different dose groups and the positive control were carried out using a method of analysis based on χ2. All analysis and significance tests were evaluated at 5 % level of significance (p ≤ 0.05).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No statistically significant increases in the frequency of mutation frequency were observed over the concentration range tested (0.25, 0.5, 1 and 2 mg/mL).

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No. In the pre-tests, no change in pH was observed 24 hours of incubation at the highest dose tested.
- Water solubility: The test item was soluble in DMSO at 200 mg/mL.
- Precipitation: Slight precipitation was observed at 2 mg/L (top dose) in the pre-test, but not at lower doses.
- Other confounding effects: None reported.

RANGE-FINDING/SCREENING STUDIES: The relative total growth (RTG) values obtained for test item treated at 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mLwere 93.98%, 85.81%, 85.87%, 77.78%, 86.13% and 81.29%, respectively, in the presence of metabolic activation and 99.94%, 97.43%, 91.67%, 93.00%, 82.11% and 83.32%, respectively, in the absence of metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: cyclophosphamide (short treatment with S9): range = 412.06 - 524.05, mean = 468.06, CL 95%; methylmethanesulphonate (short treatment without S9): range = 479.37 - 603.23, mean = 541.30, CL95%; methylmethanesulphonate (long treatment without S9): range = 479.37- 603.23, mean = 541.30, CL 95%.
- Negative (solvent/vehicle) historical control data: short treatment with S9: range = 54.32 - 68.81, mean = 61.57, CL 95%; short term treatment without S9: range = 53.49 - 7072, mean = 62.10, CL 95%; long treatment without S9: range = 53.49 - 70.72, mean = 62.10, CL95%.
- Results obtained for positive and negative controls were within historical control ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cell survival was assessed by determining the Relative Cloning Efficiency (RCE), Relative Suspension Growth (RSG) and Relative Total Growth (RTG) in the test.

Based on the results obtained, it is concluded that the test item is non mutagenic and does not induce forward mutations at the thymidine kinase (TK) locus of L5178Y TK+/- mouse lymphoma cells at and up to 2 mg/mL both in short term and long term treatment, both in presence and absence of metabolic activation as it showed no evidence of increase in the induction of mutation frequency under the test conditions.

Conclusions:

The test item was determined to be not mutagenic with or without metabolic activation under the experimental conditions reported.

Executive summary:

The genetic toxicity of the test item was evaluated by an In vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay), according to OECD TG 490, under GLP conditions. The potential to induce mutations at the mouse lymphoma thymidine kinase locus was studied using the cell line L5178Y. The selection of the test item concentrations used in the main experiment was based on the results of preliminary solubility/precipitation, pH and cytotoxicity assays. Mouse lymphoma cells at an initial concentration of 1E07 cells/mL were exposed to 0.25, 0.5, 1 and 2 mg/mL test item for 3 and/or 24 hours with and without metabolic activation by S9-mix (sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate prepared from male Wistar rats). Concurrent untreated/solvent control and two positive controls were performed.

In the main experiment, no biologically relevant increase in the mutation factor was observed in any treatment with or without metabolic activation in the concentration range tested. All the acceptance criteria were met. In conclusion, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Key study. Cytogenicity. Read-across from analogue substance. An acute micronucleus study in bone marrow of CFLP mice (method similar to OECD 474, no GLP). For the analogue, no significant increase in the number of micronucleated PCE was observed at any dose tested, including the highest dose,which induced mortality. Based on the available data for the read-across approach, the target substance is not mutagenic under test conditions.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline available

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

bone marrow sampling time 6h after last dose instead of 18h; highest dose tested caused deaths; verification of bone marrow exposure to the test substance not specified.

Principles of method if other than guideline:

REFERENCES: [1] Boller, K., Schmid W. (1970) Humangenetik 11, 35; [2] Heddle J.A. (1973) Mutation Res. 18, 187; [3] Matter B., Schmid W. (1971) Mutation Res. 12, 417; [4] Schmid W. (1973) Agents and Actions, 3, 77; [5] Schmid W. (1975) Mutation Res. 31, 9; [6] Von Ledebur M., Schmid W. (1973) Mutation Res. 19, 109.

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CF-1

Remarks:

(CFLP, SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Anglia Laboratory Animals (Alconbury, Huntingdon, UK)
- Age at study initiation: not specified
- Weight at study initiation: 19 - 23 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, overnight
- Housing: each group of 5 mice was kept in a plastic disposable cage and maintained in a controlled environment.
- Diet: 'Grain Harvester' Anglia Laboratory Animal Diet, ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 50 ± 5% RH

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% MC (methyl cellulose)
- Concentration of test material in vehicle: see experimental design in 'Any other information on materials and methods'.
- Dosing volume: 0.1 ml / 100 g bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was prepared as a suspension in 1% methylcellulose, by direct addition, using a high speed mixer.

Duration of treatment / exposure:

30h

Frequency of treatment:

2 equal doses separated by a 24-h interval

Post exposure period:

6h

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C;
- Route of administration: intraperitoneal injection
- Doses / concentrations: 14 mg/kg bw

Tissues and cell types examined:

Polychromatic erythrocytes (PCE) from bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: preliminary studies, based on information provided, indicated that the LD50 of the test item is approximtely 400 mg/kg bw and that a top dosage of 1000 mg/kg bw would cause 1-2 deaths.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Treatment: rats were administered the test item by oral gavage at total doses of 250, 500, and 1000 mg/kg bw, as two equal doses separated by 24h (both administered after overnight fasting) of 10 ml/kg-bw dose volume.

DETAILS OF SLIDE PREPARATION:
- Following the last dose, the animals were observed for a further 6h before killing and all mortalities and signs of malreaction during the experiment were recorded. The animals were killed by cervical dislocation, the femurs dissected out and bone marrow smears prepared. The smears were fixed in methanol, deffated in xylene and stained with Giemsa.

METHOD OF ANALYSIS:
- The stained smears were examined by light microscopy. 2000 polychromatic erythrocites (PCEs) per mouse were scored for the frequency of micronucleated cells.

Evaluation criteria:

In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 400 - 1400 mg/kg bw.
- Clinical signs of toxicity in test animals: No toxic reactions were observed up to 600 mg/kg bw. At doses of 800 - 1400 mg/kg bw, hypopnoea was observed. Mortality data is summarized in table 1.
- High dose with and without activation: a dose of 1000 mg/kg bw was chosen as the highest dose that would be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: No reactions were observed up to 500 mg/kg bw. At 1000 mg/kg bw, hypopnoea was observed, and there were three mortalities between 24 and 30 hours after administration of the second dose.
- Induction of micronuclei (for Micronucleus assay): No significant increase observed at any dose tested. The negative control group gave a mean count of 2.6 micronucleated cells, within the laboratory historical value range. The mean micronucleated cell counts for groups treated at 250 and 500 mg/kg bw were comparable with the concurrent control value. The mean micronucleated cell count for the group treated at 1000 mg/kg bw was 2.7, slightly higher than the concurrent control. However, individual counts were within the historical range for negative controls. Therefore, the value was not considered statistically significant. The positive control gave a mean count of 92.7 micronucleated cells.
- Ratio of PCE/NCE (for Micronucleus assay): not examined.

Table 1. Preliminary toxicity study: mortality data.

Phase	Group	Material	Total dosage over 24 h (mg/kg bw)	Mortality ratio (no. deaths/no. dosed)			Period during which deaths occurred (h)
				♂	♀	Combined	0 – 8	8 – 24	24 – 30
I	1a	1% MC	-	0/2	0/2	0/4
	2	UR1501	400	0/2	0/2	0/4
	3		600	0/2	0/2	0/4
	4		800	0/2	0/2	0/4
	5		1000	0/2	0/2	0/4
	6		1200	1/2	1/2	2/4		1	1
	7		1400	1/2	1/2	2/4			2
II	1b	1% MC	-	0/5	0/5	0/10
	8	UR1501	1000	1/5	2/5	3/10			3
	9		1100	0/5	4/5	4/10	2		2
	10		1200	5/5	3/5	8/10	6		2
	11		1300	3/5	4/5	7/10	2		5
	12		1400	5/5	3/5	8/10	5		3

Table 2. Main study: Group mean number of micronucleated cells per 2000 polychromatic erythrocytes per animal.

Group	Material	Total dosage over 24 h (mg/kg bw)	Period during which deaths occurred (h)
			Mean	Range
1	1% MC (vehicle control)	-	2.6	1 – 6
2	UR1501	250	2.2	1 – 5
3		500	2.1	0 – 6
4		1000	2.7	1 – 4
5	Mitomycin C (positive c.)	14	92.7	71 – 126

Table. Number of micronucleated cells (mn) per 2000 polychromatic erythrocytes – individual values.

	Material
	1% MC		UR1501						Mitomicyn C
Group	1		2		3		4
Dose (mg/kg)	-		250		500		1000		14
Sex	Animal	mn	Animal	mn	Animal	mn	Animal	mn	Animal	mn
♂	1	1	6	3	11	2	16	D	21	81
	2	6	7	1	12	4	17	2	22	77
	3	2	8	1	13	1	18	2	23	126
	4	3	9	5	14	2	19	4	24	93
	5	2	10	2	15	1	20	1	25	118
♀	26	2	31	3	36	2	41	4	46	77
	27	2	32	2	37	2	42	D	47	71
	28	4	33	2	38	6	43	D	48	94
	29	1	34	1	38	1	44	4	49	102
	30	3	35	2	40	0	45	2	50	88

D: animal died.

Table. Laboratory standard (historical) values in the period Feb 1972 – Feb 1978.

Negative control No. of experiments	Total no. of animals examined	Mean no. of micronucleated cells counted per 2000 PCE	Range of individual counts of micronucleated cells per 2000 PCE per animal
81	783	1.65	0 – 6

 

Conclusions:

The test item was not mutagenic under test conditions.

Executive summary:

An acute micronucleus study in bone marrow of CF-1 (CFLP, SPF) mice was performed for the test item, by a method similar to OECD 474 (non-GLP). Based on the results of a preliminary toxicity study were the LD50 was found to be around 400 mg/kg bw, five animals per sex per dose group were exposed to 250, 500 or 1000 mg/kg bw of test item in 1% methylcellulose by oral gavage, administered in two equal doses separated by a 24-hour resting period. Negative (vehicle, 1% aqueous methylcellulose) and positive (14 mg/kg bw Mitomicyn C by i.p. injection) controls were run in parallel. 6 hours after the last injection, the animals were killed by cervical dislocation, the femurs dissected out and and bone marrow spreads were prepared, air-dried, fixed using absolute methanol, defatted in xylene and stained with Giemsa. At termination, bone marrow analysis included assessment of the frequency of MN-PCE among 2,000 PCE per animal. All control values obtained were within the historical control range. No significant increase in the number of micronucleated PCE was observed at any dose tested, including the highest dose, which induced mortality. Since the results were clearly negative at all doses tested, the test item was not mutagenic under test conditions.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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 (see "Attached justification")

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance is a breakdown product of the analogue substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Triflusal.
- Target: TFMSA (see "Test material" for further information).

3. ANALOGUE APPROACH JUSTIFICATION
Please find Reporting Format in "Attached justification".

4. DATA MATRIX
Please find data Matrix included in "Attached justification".

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Based on the available data for the read-across approach, the target substance is not mutagenic under test conditions.

Executive summary:

Read-across from supporting substance (structural analogue or surrogate): An acute micronucleus study in bone marrow of CF-1 (CFLP, SPF) mice was performed for the analogue substance triflusal, by a method similar to OECD 474 (non-GLP). Based on the results of a preliminary toxicity study were the LD50 was found to be around 400 mg/kg bw, five animals per sex per dose group were exposed to 250, 500 or 1000 mg/kg bw of test item in 1% methylcellulose by oral gavage, administered in two equal doses separated by a 24-hour resting period. Negative (vehicle, 1% aqueous methylcellulose) and positive (14 mg/kg bw Mitomicyn C by i.p. injection) controls were run in parallel. 6 hours after the last injection, the animals were killed by cervical dislocation, the femurs dissected out and and bone marrow spreads were prepared, air-dried, fixed using absolute methanol, defatted in xylene and stained with Giemsa.At termination, bone marrow analysis included assessment of the frequency of MN-PCE among 2,000 PCE per animal. All control values obtained were within the historical control range. No significant increase in the number of micronucleated PCE was observed at any dose tested, including the highest dose,which induced mortality. Since the results were clearly negative at all doses tested, the test item was not mutagenic under test conditions. Based on the available data for the read-across approach, the target substance is not mutagenic under test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, the substance is not classified for mutagenicity according to the CLP Regulation (EC) no. 1272/2008.