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Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the study OECD (476), Method B17 of Commission Regulation (EC) No. 44012008 of 30 May 2008 conducted to assess the potential mutagenicity of the test material on the thymidine kinase, TK +I-, locus of the L5178Y mouse lymphoma cell line. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

In the study OECD 473 and Method B10 of Commission Regulation (EC) 440/2008.The in vitro study for the detection of strucutural chromosomal aberrations in cultured mammalian cells. The test material was considered to be non-clastogenic to human lymphocytesin vitro.

In the study OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines. The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. The test material was considered to be non-mutagenic under the conditions of this test.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

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

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

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect.
- Effects of osmolality: no effects.
- Evaporation from medium: no effects.
- Water solubility: no effects.
- Precipitation: no effects.


RANGE-FINDING/SCREENING STUDIES: yes.

COMPARISON WITH HISTORICAL CONTROL DATA: see attachment Appendix 3.

ADDITIONAL INFORMATION ON CYTOTOXICITY: mp cutptpxocoty observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

1. Preliminary Toxicity Test:

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

 The numbers of revertant colonies for the toxicity assay were:

 

With (+) or without (-) S9­mix	Strain	Dose (mg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	122	111	107	109	108	126	151	128	102	84	92P
+	TA100	99	98	110	102	112	117	101	101	101	88	93P
-	WP2uvrA"	30	19	20	14	20	26	25	22	22	24	25P
+	WP2uvrA"	37	32	42	47	30	32	44	23	36	35	44P

 

2. Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

A history profile of vehicle and positive control values is presented in Appendix 3.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. An oily precipitate was observed at and above 1500 μg/plate and at 5000 μg/plate, without and with S9-mix, respectively. An associated oily film was also observed at 5000 μg/plate in all strains without S9 only. These observations did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. An oily precipitate was observed at and above 1500 μg/plate and at 5000 μg/plate, without and with S9-mix, respectively. An associated oily film was also observed at 5000 μg/plate in all strains without S9 only. These observations did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

primary culture, other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Scorable metaphase cells were obtained, and 51%~85% mitotic inhibition was achieved in the main test.

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: NDA
- Effects of osmolality: NDA
- Evaporation from medium: NDA
- Water solubility: insoluble.
- Precipitation: at and above 320 ug/ml in exposure groups in the absence of S9, and at and above 640 ug/ml in the presence of S9.
- Other confounding effects: NDA

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was conducted, and the dose range was 19.53 to 5000 ug/ml, the maximum dose was based on the maximum recommended dose level. Metaphase cells were present up to 625 ug/ml in the 4(20)-hour exposure group in the absence of S9, and up to 1250 ug/ml in the presence of metabolic activation (S9). The test material induced clear evidence of toxicity in all three of the exposure groups and, therefore, the selection of the maximum dose level for the main test was based on toxicity for all three exposure groups. The maximum dose levels were 320 ug/ml in the 4(20)-hour group in the absence of S9, 640 μg/ml in the 4(20)-hour group in the presence of S9 and 160 μg/ml in the 24-hour continuous exposure group.

COMPARISON WITH HISTORICAL CONTROL DATA:
The data collected for the vehicle and untreated control cultures were compared to historical aberration ranges, and were satisfactory.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 58% mitotic inhibition was achieved at 320 ug/ml in the 4(20)-hour exposure group in the absence of S9. In the presence of S9, 51% and 85% mitotic inhibition was achieved at 640 and 960 ug/ml respectively, the response observed at 960 ug/ml precluded this dose level from being assessed for the presence of chromosome aberrations. In the 24-hour continuous exposure cultures 55% and 83% mitotic inhibition was achieved at 160 and 320 ug/ml respectively. The response seen at 320 ug/ml also precluded this dose level from being assessed for chromosome aberrations.

Remarks on result:

other: strain/cell type: Human lymphocytes

Remarks:

Migrated from field 'Test system'.

The vehicle (solvent) controls had frequencies of cells with aberrations within the expected range. The positive control materials induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolizing system.The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either of the 4(20)-hour exposure groups. However, a statistically significant increase was observed at the maximum dose level scored, 160 ug/ml, in the 24-hour continuous exposure group. The response was very modest, only just above the upper limit of the historical range and did not include any exchange-type aberrations. The majority of the aberrations were present in one of the duplicate cultures (B) which exhibited the greatest level of toxicity (60%) and was not part of a true dose related response. It was considered that the response was most likely due to a cytotoxic mechanism and not a genotoxic mechanism and, therefore, did not have any toxicological significance or biological relevance.

Conclusions:

The test material was considered to be non-clastogenic to human lymphocytesin vitro.

Executive summary:

This report describes the results of an in vitro study for the detection of strucutural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al, 1990). The method used follow OECD 473 and Method B10 of Commission Regulation (EC) 440/2008.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, ie. 4 hours exposure in the presence of 2% induced S9 system, with cell harvest after a 20-house expression period and a 24 hours continuous exposure in the absence of metabolic activation.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test material was toxic and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that induced a dose level that achieved or exceeded approximately 50% mitotic inhibition.

The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

01 July 2009 - 28 July 200

Reason / purpose for cross-reference:

read-across source

Species / strain:

other: The L5178Y TK+/-3.7.2c mouse lymphoma cell line

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: The L5178Y TK+/-3.7.2c mouse lymphoma cell line

Preliminary Toxicity Test. The dose range of the test material used in the preliminary toxicity test was 3.91 to 1000 ug/ml. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (ug/ml)	% R8G (-S9) 4-Hour Exposure	% R8G (+S9) 4-Hour Exposure	% R8G (-S9) 24-Hour Exposure
0	100	100	100
19.53	78	60	77
39.06	56	56	45
78.13	29	51	32
156.25	2	39	5
312.5	0	33	0
625	0	13	0
1250	0	0	0
2500	0	0	0
5000	0	0	0

In all three of the exposure groups there was a marked reduction in the relative suspension growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The toxicity curve was steep in all three of the exposure groups. A precipitate of the test material was observed at and above 312.5 ug/ml. In the subsequent mutagenicity experiments the maximum dose was limited by test material-induced toxicity.

Mutagenicity Test

The maximum dose level used limited by test material-induced toxicity. A precipitate of test material was observed at and above 78.13 ug/ml in the 4-hour exposure group in the absence of metabolic activation, and at and above 156.25 ug/ml in the 4-hour exposure group in the presence of metabolic activation, in the main test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK^+I-locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups.

A summary of the results from the test is presented in Table 1 (attached)

Key to Tables 1

% RSG =Relative Suspension Growth

RTG = Relative Total Growth

CP =   Cyclophosphamide

EMS= Ethylmethanesulphonate

MF§ = 5-TFT resistant mutants/10⁶viable cells 2 days after treatment

NP = Not plated due to toxicity or surplus to requirements

Ø= Not plated for viability or TFT resistance

NS = Not significant

Conclusions:

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +I-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 44012008 of 30 May 2008.

One main experiment was performed. In this main experiment, L5178Y TK +I- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls. The exposure groups used were as follows: 4-hour exposures both with and without metabolic activation, and 24 hours without metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 4.88 to 156.25 ug/ml for the 4-hour exposure group in the absence of metabolic activation, 9.77 to 625 ug/ml for the 4-hour exposure group in the presence of metabolic activation, and 5 to 160 ug/ml for the 24-hour exposure group in the absence of metabolic activation.

The maximum dose level used limited by test material-induced toxicity. A precipitate of test material was observed at and above 78.13 ug/ml l in the 4-hour exposure group in the absence of metabolic activation, and at and above 156.25 ug/ml in the 4-hour exposure group in the presence of metabolic activation, in the main test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK^+I-locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups.

Conclusion.The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic and germ cell study: gene mutation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

None of the in-vitro tests revealed a mutagenic potential of the test substance. It can therefore be concluded that the substance is non mutagenic in-vitro, and possible in vivo.

Short description of key information:
In-vitro: negative in Ames Test with Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA (with and without metabolic activation)
in-vitro: negative in Chromosome-Aberration in Chinese Hamster Lung (CHL) cell line (CHL/IU)
in-vitro: negative in Mouse Lymphoma Assay

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

There are conclusive but not sufficient data to classify the substance with regard to mutagenicity. The test substance is not classified for this endpoint in accordance with the CLP Regulation (EC) No 1272/2008.