5,7-Di-t-butyl-3-[3,5-dimethyl-4-[(1,3,7,9-tetra-t-butyl-5-methyl-5H-benzo[d][1,3,2]benzodioxaphosphocin-11-yl)oxy]phenyl]-3H-benzofuran-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the abscence and presence of metabolic activation.

The test item is also not mutagenic in the HPRT assay in the presence and absence of metabolic activation.

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:

2016-09-16 to 2016-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature; under light exclusion ; no direct sunlight

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver S9 mix

Test concentrations with justification for top dose:

1st experiment and 2nd experiment:
0; 33; 100; 333; 1000; 2500; 5000 µg/plate
Top dose of 5 mg/plate was selected in agreement with the current guidelines.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: DMSO has been demonstrated to be suitable for bacterial reverse mutation tests and historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 4-nitro-o-phenylenediamine (NOPD) without S9, strain: TA 98; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) without S9, strains: TA 1535 and TA 1537; 2-aminoanthracene (2-AA) with S9 all strains and without S9, strains: TA1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

SELECTION AGENT: -his and -trp deficient medium

NUMBER OF REPLICATIONS: 3 plates per dose

DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants (factor <= 0.6)
- clearing or diminution of the background lawn

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: DMSO was used as vehicle
- Precipitation: was found from 33 µg/plate onward with and without S9 mix

HISTORICAL CONTROL DATA
- Positive historical control data: please refer to "Any other information on results" table 4
- Negative (solvent/vehicle) historical control data: please refer to "Any other information on results" table 4

CYTOTOXICITY
A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 1000 μg/plate onward. In the preincubation assay weak bacteriotoxicity (slight decrease in the number of his+ revertants) was occasionally observed depending on the strain and test conditions from about 33 μg/plate onward. Please also refer to "Any other information on results" table 3.

Table 1. Experiment 1 standard plate incorporation test

Dose (µg/plate)	Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli
	TA 100	TA 98	TA 1535	TA 1537	WP2 uvrA
	Results without S9
DMSO	103.0 ± 14.0	21.7 ± 4.2	8.3 ± 1.5	8.0 ± 1.0	19.7 ± 1.2
33	107.0 ± 7.0	28.3 ± 6.4	12.7 ± 2.1	6.0 ± 1.0	13.0 ± 4.4
100	107.3 ± 15.0	25.0 ± 9.5	9.0 ± 3.5	9.0 ± 2.0	13.3 ± 3.2
333	103.3 ± 6.0	28.0 ± 11.3	9.0 ± 2.6	7.0 ± 1.0	15.0 ± 1.0
1000	94.7 ± 8.6	24.0 ± 1.0	7.3 ± 2.3	3.3 ± 1.5	12.7 ±4.5
2500	82.7 ± 15.0	10.0 ± 4.0	6.0 ± 3.6	3.7 ± 2.1	15.7 ± 1.5
5000	85.0 ± 5.6	8.3 ± 2.5	7.7 ± 1.5	4.3 ± 2.5	17.7 ± 6.1
MNNG (5)	4149.7 ± 147.0	-	4984.7 ± 175.6	-	-
NOPD (10)	-	909.7 ± 30.1	-	-	-
AAC (100)	-	-	-	1997.7 ± 133.0	-
4-NQO (5)	-	-	-	-	1130.3 ± 48.2
	Results with S9
DMSO	112.0 ± 16.0	25.7 ± 5.5	9.0 ± 1.7	9.7 ± 3.5	26.3 ± 8.5
33	101.7 ± 7.5	28.3 ± 9.1	7.7 ± 2.3	12.0 ± 4.0	23.0 ± 9.6
100	95.3 ± 4.7	29.3 ± 6.7	8.0 ± 3.6	11.0 ± 2.6	25.0 ± 4.6
333	92.0 ± 27.6	28.7 ± 1.5	10.3 ± 2.1	7.7 ± 4.0	29.7 ± 8.1
1000	78.7 ± 7.5	22.7 ± 4.5	8.7 ± 2.1	6.7 ± 2.1	25.0 ± 1.7
2500	68.3 ± 8.4	12.7 ± 2.1	5.3 ± 0.6	6.0 ± 2.6	17.0 ± 3.6
5000	109.0 ± 14.2	21.7 ± 7.2	8.3 ± 2.1	8.3 ± 4.2	18.7 ± 3.5
2- AA (2.5)	1467.3 ± 116.7	1234.3 ± 41.5	149.3 ± 12.4	118.7 ± 3.8	93.3 ± 21.2

Table 2. Experiment 2 Pre-incubation test

Dose (µg/plate)	Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli
	TA 100	TA 98	TA 1535	TA 1537	WP2 uvrA
	Results without S9
DMSO	94.7 ± 6.1	20.3 ± 6.4	15.3 ± 3.8	9.7 ± 3.1	20.0 ± 7.5
33	84.0 ± 3.5	19.0 ± 4.4	7.7 ± 1.2	9.0 ± 2.0	14.3 ± 2.5
100	92.0 ± 3.6	15.0 ± 3.6	7.7 ± 2.9	7.7 ± 2.1	13.7 ± 3.1
333	90.7 ± 8.1	18.0 ± 2.6	10.3 ± 6.5	11.0 ± 3.0	14.0 ± 2.6
1000	84.7 ± 12.9	13.0 ± 5.3	8.7 ± 2.1	11.7 ± 4.0	15.0 ± 5.6
2500	90.3 ± 14.6	17.7 ± 1.2	7.0 ± 2.6	11.0 ±4.4	13.0 ± 6.1
5000	76.0 ±8.7	13.0 ± 4.0	8.7 ± 1.5	6.3 ± 2.5	13.3 ± 3.1
MNNG (5)	2189.0 ± 191.8	-	2491.7 ± 96.4	-	-
NOPD (10)	-	811.0 ±41.4	-	-	-
AAC (100)	-	-	-	1201.7 ± 148.5	-
4-NQO (5)	-	-	-	-	322.0 ± 18.1
	Results with S9
DMSO	124.0 ±16.7	22.3 ± 3.6	12.0 ± 3.5	11.0 ± 5.3	22.0 ± 3.6
33	71.3 ± 1.2	16.7 ± 3.8	8.7 ± 1.2	6.7 ± 3.8	16.7± 3.8
100	76.0 ± 8.0	16.0 ± 1.0	9.0 ± 4.4	9.0 ± 1.7	16.0 ± 1.0
333	67.0 ± 2.6	17.0 ± 2.6	8.0 ±4.6	10.3 ± 3.1	17.0 ± 2.6
1000	73.3 ± 18.5	14.7 ± 4.2	9.0 ± 4.6	9.0 ± 0.0	14.7 ± 4.2
2500	73.3 ± 8.3	16.7 ± 3.1	8.3 ± 2.3	9.0 ± 1.0	16.7 ± 3.1
5000	82.0 ± 11.3	14.7 ± 4.6	4.3 ± 0.6	5.3 ± 1.5	14.7 ± 4.6
2- AA (2.5)	1187.7 ± 57.6	111.7 ± 6.4	167.7 ± 10.7	156.7 ±17.6	111.7 ± 6.4

Table 3. Decreased revertant numbers (cytotoxicity) observed at following concentrations (µg/plate)

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E. coli
1st - SPT	Without	-	-	1000 - 5000	2500 - 5000	-
	With	2500
2nd - PIT	Without	1000 - 5000	-	-	5000	-
	With	5000	33 - 2500	5000	-	-

Table 4. Historical control data

		Bacterial strains
Historical control data of DMSO control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	18	95	10	7	22
		SD	3.1	9.9	2.2	1.5	4.0
		Minimum	12	69	6	5	12
		Maximum	30	140	17	12	33
	+S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	24	100	10	8	23
		SD	4.7	12.3	2.1	2.2	4.0
		Minimum	13	76	6	5	13
		Maximum	35	150	18	16	35
Historical control data of positive control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	486	3331	4063	975	884
		SD	183.3	1021.5	1308.8	339.9	442.4
		Minimum	271	1125	1174	253	133
		Maximum	431	5557	6612	1858	1552
	+S9 (2-AA		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	1427	1792	217	130	98
		SD	361.9	448.9	50.0	33.6	26.6
		Minimum	431	361	108	56	54
		Maximum	2427	2819	367	241	169

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains in a reverse mutation assay. Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were used as tester strains with a dose range of 33 μg - 5000 μg/plate in both standard plate test and preincubation test with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect was observed depending on the strain and test conditions from about 33 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance GSID 1212253 is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-15 to

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)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro gene mutation test in CHO cells (HPRT locus assay)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch number GSID 1212253-1 OP3
- Expiration date of the lot/batch: 2017-12-31
- Purity test date: not reported (stated in analytical report 16L00238, BASF SE)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (under light exclusion)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test susbtance is soluble and stable in DMSO., the stability over a period of 4 hours was verified analytically.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- doubling time of about 12 - 16 hours
- Methods for maintenance in cell culture: Stocks of the CHO cell line (1 mL portions) are maintained at -196 °C in liquid nitrogen using 7 % (v/v) DMSO in culture medium as a cryoprotectant.

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix, phenobarbital (i.p.) and β-naphthoflavone (orally) - induced

Test concentrations with justification for top dose:

Test concentrations were chosen based on the results of a pretest for dose selecetion and taking into accout the current guidelines.
Test concentrations of the 1st experiment:
without and with S9 mix [µg/mL]: 3.13; 6.25; 12.5; 25; 50; 100

Test concentrations of the 2nd experiment:
without and with S9 mix [µg/mL]: 4.69; 9.38; 18.75; 37.50; 75; 100

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to insolubility of the test substance in water, dimethyl sulfoxide (DMSO) was selected as appropriate vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7-9 days
- Selection time: 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): cells were fixed with methanol directly after the selection period.

SELECTION AGENT: 6-thioguanine

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF CELLS EVALUATED: The number of all colonies in each flask was counted and recorded for determination of mutant frequency and cloning efficiency.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95 % control limit)

Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95 % control limit)

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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: pH values were not influenced by test substance treatment.
- Effects of osmolality: Osmolality was not influenced by test substance treatment.
- Precipitation: Precipitation was observed macroscopically In the 1st Experiment in the absence and the presence of S9 mix at the end of treatment at 50 μg/mL and above, and in the 2nd Experiment in the absence of S9 mix at 37.50 μg/mL and above and in the presence of S9 mix at 18.75 μg/mL and above.

Summary of results

Table 1: Experiment 1, 4 hour exposure period

Test group [µg/mL]	S9 mix	Cloning efficiency 1 (survival)		Cloning efficiency 2 (viability)		Mutant Frequency (per 10^6 cells)
		Abs [%]	Rel [%]*	Abs [%]	Rel [%]	uncorrected	corrected**
Vehicle control (DMSO	-	170.0	100	83.5	100	3.25	3.89
	+	99.8	100	84.3	100	1.75	2.08
3.13	-	119.3	114.0	94.5	113.2	5.75	6.08
	+	106.5	115.6	86.0	102.1	1.75	2.03
6.25	-	153.0	149.8	100.0	119.8	3.25	3.25
	+	87.0	104.8	100.5	119.3	3.0	2.99
12.5	-	111.0	114.7	91.0	109.0	3.0	3.30
	+	96.3	107.6	102.3	121.4	1.75	1.71
25.0	-	114.3	119.1	98.0	117.4	2.25	2.30
	+	95.3	98.9	87.3	103.6	1.25	1.43
50.0	-	111.5	104.3	99.3	118.9	3.0	3.02
	+	79.8	98.6	87.3	103.6	1.0	1.15
100.0	-	96.5	97.4	93.8	112.3	3.5	3.73
	+	87.5	100.2	88.0	104.5	0.75	0.85
EMS 400.0	-	95.0	75.1	66.8	79.9	74.00	110.86
DMBA 1.25	+	70.5	77.2	77.5	92.0	74.0	95.48

* with regard to cell loss while exposure period, relative survival (RS) is calculated based on CE of cells plated immediatedly after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in vehicle/negative controls.

**correction on the basis of the absolute cloning efficiency 2 at the end of the expression period

 

Table 2: Experiment 2, 4 hour exposure period

Test group [µg/mL]	S9 mix	Cloning efficiency 1 (survival)		Cloning efficiency 2 (viability)		Mutant Frequency (per 10^6 cells)
		Abs [%]	Rel [%]*	Abs [%]	Rel [%]	uncorrected	corrected**
Vehicle control (DMSO	-	87.5	100.0	91.8	100.0	4.0	4.36
	+	75.3	100.0	82.8	100.0	3.5	4.23
4.69	-	87.3	97.9	85.0	92.6	3.75	4.41
	+	76.8	123.2	75.8	91.5	1.25	1.65
9.38	-	87.0	103.0	98.5	107.4	5.0	5.08
	+	77.5	127.3	84.7	102.4	1.75	2.06
18.75	-	78.3	93.4	82.0	89.4	2.75	3.35
	+	72.8	118.2	106.0	128.1	2.25	2.12
37.5	-	69.5	87.6	72.3	78.7	1.75	2.42
	+	71.3	115.6	76.8	92.7	3.75	4.89
75.0	-	72.0	82.5	96.8	105.4	2.75	2.84
	+	67.5	116.1	75.3	90.9	1.75	2.33
100.0	-	67.8	81.3	95.3	103.8	4.0	4.20
	+	62.0	104.8	71.8	86.7	6.5	9.06
EMS 400.0	-	66.8	74.5	81.5	88.8	120.25	147.55
DMBA 1.25	+	51.3	88.5	64.0	77.3	74.0	115.63

* with regard to cell loss while exposure period, relative survival (RS) is calculated based on CE of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in vehicle/negative controls.

 **correction on the basis of the absolute cloning efficiency 2 at the end of the expression period

 

Conclusions:

The test substance is not mutagenic in the HPRT locus assay using CHO cells with and without metabolic activation under the experimental conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay 

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. 

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5000 μg/plate (SPT); 33 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: Precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was observed depending on the strain and test conditions from about 33 μg/plate onward.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor≥2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

 

CONCLUSION: Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

 

 

 

HPRT assay

The substance was assessed for its potency to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- andβ-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested and evaluated for gene mutations:

1st Experiment

without S9 mix 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00μg/mL

with S9 mix 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00μg/mL

2nd Experiment

without S9 mix 0; 4.69; 9.38; 18.75; 37.50; 75.00; 100.00μg/mL

with S9 mix 0; 4.69; 9.38; 18.75; 37.50; 75.00; 100.00μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, dose selection was based on the solubility properties of the test substance. The highest applied concentrations in both main experiments showed clear test substance precipitates in culture medium macroscopically at the end of exposure period. In both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations tested for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant or statistically significant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The test item was found to be not mutagenic in a bacterial reverse mutation assay and in a HPRT assay. As a result the substance is not considered to be classified as genotoxic under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.