Phosphoric acid, butyl ester, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in the bacterial reverse mutation assay.

The read across-substance (CAS 107-66-4) was not clastogenic in the chromosome aberration study and it was not mutagenic in vitro in mammalian cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1979

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only one experiment was performed

GLP compliance:

not specified

Remarks:

study conducted before GLP was introduced

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- test material: Da7934/3

Target gene:

histidine operon

Species / strain / cell type:

other: Salmonella typhimurium TA1535, TA1537; TA1538; TA100 and TA98

Additional strain / cell type characteristics:

other: histidine and biotine auxotrophic, rfa and uvrB character

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix, Aroclor 1254 induced

Test concentrations with justification for top dose:

0, 10, 25, 100, 250, 1000, 2500 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

methylmethanesulfonate

other: 2 -aminofluorene (with S9 mix, all strains), ethylmethansulfonate (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertant colonies

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A reversion rate at least two-fold higher than the spontaneous reversion rate of the respective strain is considered as a positive mutation test result.

Statistics:

not necessary

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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:

no 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:

no 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A study was performed to assess the cytotoxic property of the test item at 2500 µg/plate or lower. No cytotoxic property was revealed at the tested concentrations.

Table 1: Number of revertants per plate (mean of three plates),

	strain TA 1535		strain TA 1537		Strain TA 1538		strain TA 100		strain TA 98
conc. [µg] per plate	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA
2500	10	8	5	3	13	6	76	68	12	13
1000	6	6	6	6	12	10	74	66	10	14
250	9	7	4	3	12	8	77	68	16	15
100	9	11	7	5	12	6	80	75	15	12
25	8	9	5	4	13	8	76	77	15	10
10	7	10	4	5	12	7	79	65	12	13
0	8	8	6	4	13	7	79	71	13	13
2-aminofluorene 10 µg	14		35		>1000		>1000		>1000
methylmethansulfonate 1000 µg		13		6		15		>1000		25
ethylmethansulfonate 5000 µg		>1000		5		22		>1000		25
2-Aminoacridine  10 µg		13		291		14		140		18

 

Conclusions:

The test item was not mutagenic in the bacterial reverse mutation assay.

Executive summary:

An Ames test was conducted with the test item in five strains of Salmonella typhimurium: TA98, TA100, TA1535, TA1537, TA1538. The substance was tested at the following concentrations: 0, 10, 25, 100, 250, 1000, 2500 µg/plate, with and without metabolic activation (Aroclor 1254 induced rat liver S9-mix). DMSO served as vehicle. The positive control substances 2-aminofluorene, methylmethanesulfonate, ethylmethanesulfonate and 9-aminoacridine led to revertant colony counts clearly exceeding a two-fold increase over the spontaneous mutation rate of each strain and therefore a positive mutation result. The revertant colony counts of the test item treated plates were below the fold increase of the spontaneous mutation rate. Therefore, the substance was considered to be not mutagenic in this assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1979

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

yes

Remarks:

only one experiment was performed, only one bacterial strain tested

GLP compliance:

not specified

Remarks:

study conducted before GLP was introduced

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- test material: Da7934/3

Target gene:

trp operon

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan auxotrophic, uvrA character

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix, Aroclor 1254 induced

Test concentrations with justification for top dose:

0, 10, 25, 100, 250, 1000, 2500 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: dimethylnitrosamine (with S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertant colonies

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A reversion rate at least two-fold higher compared to the spontaneous reversion rate is considered as a positive mutation test result.

Statistics:

not necessary

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A study was performed to assess the cytotoxic property of the test item at 2500 µg/plate or lower. No cytotoxic property was revealed at the tested concentrations.

Table 1: Number of revertants per plate (mean of three plates),

	E. coli WP2 uvrA
conc. [µg] per plate	+MA	-MA
2500	24	20
1000	26	25
250	25	25
100	27	22
25	22	22
10	31	18
0	26	25
methylmethansulfonate 1000 µg		212
dimethylnitrosamine 5000 µg	397	11

Conclusions:

The test item was not mutagenic in the bacterial reverse mutation assay in the E. coli strain WP2 uvrA..

Executive summary:

An Ames test was conducted with the test item in the E.coli strain WP2 uvrA. The substance was tested at the following concentrations: 0, 10, 25, 100, 250, 1000, 2500 µg/plate, with and without metabolic activation (Aroclor 1254 induced rat liver S9-mix). DMSO served as vehicle. The positive control substances methylmethanesulfonate and dimethylnitrosamine led to revertant colony counts clearly exceeding a two-fold increase over the spontaneous mutation rate and therefore a positive mutation result. The revertant colony counts of the test item treated plates were below the fold increase of the spontaneous mutation rate. Therefore, the substance was considered to be not mutagenic in this assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2008-06-20 to 2008-09-09

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: cHA0044776
- Expiration date of the lot/batch: April 07, 2009

Target gene:

histidine locus

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: besides being histidine auxotrophic, the strains of S. typhimurium carry an additional rfa mutation and are deficient in DNA excision repair. Strain WP2 uvrA is defective in tryptophan biosynthesis and deficient in DNA excision repair.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/p-Naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

ln the pre-experiment the concentration range of the test item was 3 - 5000 pµg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. The following concentrations were tested in experiment ll: 33; 100; 333; 1000; 2500; and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (without metabolic activation, strains TA 1537, TA 98); 2-aminoanthracene (all strains, with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I: in agar (plate incorporation); Experiment II: preincubation

DURATION
- Preincubation period: in the preincubation assay (Experiment II): 60 min
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h (+ 1 h if preincubated)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

according to Guideline

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Minor toxic effects were observed in experiment ll without metabolic activation at 5000 µg/plate.

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

Remarks:

Minor toxic effects were observed in experiment ll without metabolic activation at 5000 µg/plate.

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:

no 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

Remarks:

Minor toxic effects were observed in experiment ll without metabolic activation at 5000 µg/plate.

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:

no 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: no effects observed
- Effects of osmolality: no effects observed
- Precipitation: no precipitation observed

 

Table 1: Number of revertants per plate (mean of three plates), Experiment I

	strain TA 1535		strain TA 1537		Strain TA 98		strain TA 100		strain WP2 uvrA
conc. [µg] per plate	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
water	12	12	14	12	39	44	167	162	60	81
untreated	10	13	15	12	33	42	158	143	62	65
Test item 3 µg	13	14	15	15	36	51	144	162	56	65
Test item 10 µg	12	12	16	14	39	41	165	161	56	77
Test item 33 µg	12	12	15	14	38	50	144	152	75	73
Test item 100 µg	13	14	15	14	35	41	144	164	68	75
Test item 333 µg	12	13	14	15	33	49	160	153	69	71
Test item 1000 µg	10	11	13	16	42	44	136	148	71	68
Test item 2500 µg	9	9	11	15	29	36	124	157	64	75
Test item 5000 µg	7	10	7	12	26	42	82	132	67	71
Sodium azide 10 µg	1795						1975
4-NOPD 10 µg					475
4-NOPD 50 µg			132
MMS 3 µg									1336
2-Aminoanthracene 2.5 µg		488		261		1840		1828
2-Aminoanthracene 10 µg										358

 

 

Table 2: Number of revertants per plate (mean of three plates), Experiment II

	strain TA 1535		strain TA 1537		Strain TA 98		strain TA 100		strain WP2 uvrA
conc. [µg] per plate	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
water	23	23	9	18	34	43	144	173	43	52
untreated	19	22	10	18	36	46	160	166	41	52
Test item 33 µg	23	24	12	19	34	50	135	164	42	63
Test item 100 µg	19	30	13	20	34	44	116	173	45	61
Test item 333 µg	23	20	13	16	39	46	132	170	44	60
Test item 1000 µg	21	23	12	18	32	43	107	159	45	57
Test item 2500 µg	11	19	9	13	34	40	71	154	40	51
Test item 5000 µg	6	15	2	10	26	34	39	109	44	48
Sodium azide 10 µg	1797						1987
4-NOPD 10 µg					666
4-NOPD 50 µg			128
MMS 3 µg									288
2-Aminoanthracene 2.5 µg		190		113		1108		1459
2-Aminoanthracene 10 µg										354

Conclusions:

The test item was not mutagenic in the bacterial reverse mutation assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment l) and the pre-incubation test (experiment ll) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment l: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment ll: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in experiment I. Reduced background growth was observed in experiment ll without metabolic activation at 5000 µg/plate in strains TA 1535, TA 1537, and TA 100. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment I with and without metabolic activation. Minor toxic effects were observed in experiment ll without metabolic activation in strains TA 1535, TA 1537, and TA 100 at 5000 µg/plate. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Please refer to the attached read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

inhibition was observed at 156.2 µg/plate

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

Remarks:

inhibition was observed at 156.2 µg/plate

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

Remarks:

inhibition was observed at 156.2 µg/plate

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

Remarks:

inhibition was observed at 156.2 µg/plate

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

Remarks:

inhibition was observed at 156.2 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The test substance was not mutagenic in the bacterial reverse mutation assay.

Executive summary:

The mutagenicity of the test substance was investigated by means of a reverse mutation study using bacteria. The bacteria used wereSalmonella typhimuriumTA100, TA1535, TA98 and TA1537, andEscherichia coliWP2uvrA. The following concentrations were used ( with and without metabolic activation, phenobarbital (PB) and 5,6-benzoflavone (BF) induced rat liver S9-mix):0, 4.882, 9.765, 19.53, 39.06, 78.12, 156.2 µg/plate. In the assay, 3 negative control plates, 3 positive control plates and 3 plates per dose were used. The assay was undertaken twice, and no increase in the number of revertant colonies was observed at any dose, for any of the 5 types of bacteria used. In the positive control group, an increase in the number of revertant colonies was observed for all bacteria used, and in the negative control group the total number of revertant colonies was within historical control values. The test item was deemed to have no mutagenic potential in the test systems used.

Reference 5

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:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Please refer to the attached read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Key result

Species / strain:

other: Chinese hamster CHL

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50 % growth inhibition was observed in Experiment I at 0.24 mg/mL, and at 0.54 mg/mL (with S9 mix) and 0.41 mg/mL (without S9 mix) in Experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The read across-substance was found to have no clastogenic potential in this assay.

Executive summary:

The clastogenic potential of the test item was investigated using cultured Chinese hamster (CHL) cells. In the direct method (Experiment I), a concentration of 0.24 mg/mL afforded approximately 50 % growth inhibition. In the metabolic activation method, concentrations of 0.54 mg/mL and 0.41 mg/mL afforded approximately 50 % growth inhibition, in the presence of S9 mix and without S9 mix, respectively. In the chromosome aberration test, therefore, the high concentrations were 0.24 mg/mL for the direct method, 0.54 mg/mL for the metabolic activation method in the presence of S9 mix, and 0.41 mg/mL for the metabolic activation method without S9 mix; the middle concentrations were half, and the low concentrations one quarter, the respective high concentrations. CHL cells were treated for 24 hours and 48 hours in the direct method, and no chromosome structure aberration or polyploidy inducing effects were observed in any of the treated groups. In the metabolic activation method, at the maximum treatment concentration (0.41 mg/mL) without S9 mix there was mitotic inhibition, and so chromosome analysis was not possible. No chromosome structure aberration or polyploidy inducing effects were observed in the other concentration groups, or in any of the groups treated in the presence of S9 mix. The test item did not induce chromosome structure aberration or polyploidy in CHL cells in any of the treatment groups on treatment for 24 hours (0.06-0.24 mg/mL) or on treatment for 48 hours (0.06-0.24 mg/mL) by the direct method. Furthermore, no chromosome structure aberration or polyploidy inducing effects were observed in the metabolic activation method, either in the presence of S9 mix (0.14-0.54 mg/mL) or without S9 mix (0.10-0.21 mg/mL). It was therefore concluded that the test item does not induce chromosome aberration under the test conditions described above. Using positive controls, cells with structure aberrations such as chromatid exchange and chromatic break were observed at high frequency in the mitomycin C treated group in the direct method, and in the cyclophosphamide treated group in the presence of S9 mix.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Please refer to the attached read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Relevant cytotoxic effects were only noted at the maximum concentration of the first experiment without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment l) and the pre-incubation test (experiment ll) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment l: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment ll: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in experiment I. Reduced background growth was observed in experiment ll without metabolic activation at 5000 µg/plate in strains TA 1535, TA 1537, and TA 100. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment I with and without metabolic activation. Minor toxic effects were observed in experiment ll without metabolic activation in strains TA 1535, TA 1537, and TA 100 at 5000 µg/plate. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

A further Ames test was conducted with the test item in five strains of Salmonella typhimurium: TA98, TA100, TA1535, TA1537, TA1538. The substance was tested at the following concentrations: 0, 10, 25, 100, 250, 1000, 2500 µg/plate, with and without metabolic activation (Aroclor 1254 induced rat liver S9-mix). DMSO served as vehicle. The positive control substances 2-aminofluorene, methylmethanesulfonate, ethylmethanesulfonate and 9-aminoacridine led to revertant colony counts clearly exceeding a two-fold increase over the spontaneous mutation rate of each strain and therefore a positive mutation result. The revertant colony counts of the test item treated plates were below the fold increase of the spontaneous mutation rate. Therefore, the substance was considered to be not mutagenic in this assay.

In addition to the above mentioned Ames test, a test was conducted with the test item in the E.coli strain WP2 uvrA. The substance was tested at the following concentrations: 0, 10, 25, 100, 250, 1000, 2500 µg/plate, with and without metabolic activation (Aroclor 1254 induced rat liver S9-mix). DMSO served as vehicle. The positive control substances methylmethanesulfonate anddimethylnitrosamine led to revertant colony counts clearly exceeding a two-fold increase over the spontaneous mutation rate and therefore a positive mutation result. The revertant colony counts of the test item treated plates were below the fold increase of the spontaneous mutation rate. Therefore, the substance was considered to be not mutagenic in this assay.

Read across study (Ames)

The mutagenicity of the read across substance was investigated by means of a reverse mutation study using bacteria. The bacteria used wereSalmonella typhimuriumTA100, TA1535, TA98 and TA1537, andEscherichia coliWP2uvrA. The following concentrations were used ( with and without metabolic activation, phenobarbital (PB) and 5,6-benzoflavone (BF) induced rat liver S9-mix):0, 4.882, 9.765, 19.53, 39.06, 78.12, 156.2 µg/plate. In the assay, 3 negative control plates, 3 positive control plates and 3 plates per dose were used. The assay was undertaken twice, and no increase in the number of revertant colonies was observed at any dose, for any of the 5 types of bacteria used. In the positive control group, an increase in the number of revertant colonies was observed for all bacteria used, and in the negative control group the total number of revertant colonies was within historical control values. The read across-substance was deemed to have no mutagenic potential in the test systems used.

Read across (Chromosome aberration)

The clastogenic potential of the read across-substance was investigated using cultured Chinese hamster (CHL) cells. In the direct method (Experiment I), a concentration of 0.24 mg/mL afforded approximately 50 % growth inhibition. In the metabolic activation method, concentrations of 0.54 mg/mL and 0.41 mg/mL afforded approximately 50 % growth inhibition, in the presence of S9 mix and without S9 mix, respectively. In the chromosome aberration test, therefore, the high concentrations were 0.24 mg/mL for the direct method, 0.54 mg/mL for the metabolic activation method in the presence of S9 mix, and 0.41 mg/mL for the metabolic activation method without S9 mix; the middle concentrations were half, and the low concentrations one quarter, the respective high concentrations. CHL cells were treated for 24 hours and 48 hours in the direct method, and no chromosome structure aberration or polyploidy inducing effects were observed in any of the treated groups. In the metabolic activation method, at the maximum treatment concentration (0.41 mg/mL) without S9 mix there was mitotic inhibition, and so chromosome analysis was not possible. No chromosome structure aberration or polyploidy inducing effects were observed in the other concentration groups, or in any of the groups treated in the presence of S9 mix. The read across-substance did not induce chromosome structure aberration or polyploidy in CHL cells in any of the treatment groups on treatment for 24 hours (0.06-0.24 mg/mL) or on treatment for 48 hours (0.06-0.24 mg/mL) by the direct method. Furthermore, no chromosome structure aberration or polyploidy inducing effects were observed in the metabolic activation method, either in the presence of S9 mix (0.14-0.54 mg/mL) or without S9 mix (0.10-0.21 mg/mL). It was therefore concluded that the read across-substance does not induce chromosome aberration under the test conditions described above. Using positive controls, cells with structure aberrations such as chromatid exchange and chromatic break were observed at high frequency in the mitomycin C treated group in the direct method, and in the cyclophosphamide treated group in the presence of S9 mix.

Read across (HPRT):

The study was performed to investigate the potential of the read across substance (CAS: 107 -66 -41) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD guideline 476. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 2200 µg/mL was equal to approximately 10 mM. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item was negative in this HPRT assay.

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. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the twelth time in Regulation (EU) No 2019/521.