2,6-bis(4-ethylphenyl)perhydro-1,3,5,7-tetraoxanaphth-4-ylethane-1,2-diol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following in-vitro studies have been conducted to assess the genetic toxicity of the target substance (EC 406-176-9)

- In-vitro gene mutation in bacteria (Ames) (study on substance itself and supported by results on structurally similar analogue substances.

- In-vitro chromosome aberration in mammalian cells

- In-vitro gene mutation in mammalian cells (Mouse lymphoma assay) - conducted on structurally similar analogue substances.

It is proposed that these results on the structurally similar substances can be used in the assessment of the target substance (EC 406-176-9).

The results of all the above studies were negative.

In-vitro gene mutation in bacteria (Ames):

Study on target substance (EC 406-176-9):

The test material was considered to be non-mutagenic under the conditions of the test.  No indication was observed of the mutagenic potential of the test material with or without metabolic activation in this bacterial reverse mutation assay.

Source Substance; 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-glucitol (EC-413 -110 -2):

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

Source Substance; 1-(2,6-bis(4-tolyl)-1,3-dioxano(5,4-d)-1,3-dioxan-4-yl)ethane-1,2-diol (EC 402-950-5):

It is concluded that, when tested to the limits of solubility in acetone, the test substance was not mutagenic to the bacteria in either the presence or absence of metabolic activation.

In-vitro chromosome aberration in mammalian cells (EC 406-176-9):

The test substance did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system.  The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

In-vitro gene mutation in mammalian cells (Mouse lymphoma assay)

Source Substance; 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-glucitol (EC-413 -110 -2):

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Source Substance; 1-(2,6-bis(4-tolyl)-1,3-dioxano(5,4-d)-1,3-dioxan-4-yl)ethane-1,2-diol (EC 402-950-5):

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

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:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Concentration range in the main test:
-With metabolic activation: 0.5, 0.15, 0.1, 0.05, 0.015, 0.005 mg/plate
-Without metabolic activation: 0.5, 0.15, 0.1, 0.05, 0.015, 0.005 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
5 mg test materiall / ml DMSO

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Concentration of the test substance observed to be toxic to bacteria: no toxicity observed.
Concentration of the test substance resulting in precipitation: No precipitation
Other observations: None

Conclusions:

The test material was considered to be non-mutagenic under the conditions of the test. No indication was observed of the mutagenic potential of the test material with or withour metabolic activation in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-02-18 to 1988-03-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar 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

Target gene:

Histidine

Species / strain / cell type:

bacteria, other: S. typhimurium TA 1535, 1537, 1538, 98, 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix prepared from Aroclor 1254-induced rat liver.

Test concentrations with justification for top dose:

Dose range-finding test: 5000, 500, 50 and 5 µg per plate
Mutation test: 5000, 1500, 500, 150 and 50 µg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone (fine suspension)

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Remarks:

2-Aminoantracene used with S9 mix. ENNG, 9-AC, , 2-NF used without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10 hours
- Exposure duration: 72 hr

NUMBER OF REPLICATIONS:
- 3 replications per concentration per strain per test; test performed twice.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for the reduction of the bacterial background lawn and number of revertant colonies.

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group.

A compound is deemed to provide evidence of mutagenic potential if a statistically significant dose-related increase in the number of revertant colonies of at least twice the concurrent solvent control is obtained in two separate experiments.

Species / strain:

bacteria, other: S. typhimurium TA 1535, 1537, 1538, 98, 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Observed at 5000 μg per plate

RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed in the preliminary dose range-finding study.

MUTATION TEST:
Responses of the positive and negative control groups were within the normal ranges experienced in the laboratory. Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the metabolic activation system.

No significant increases in the revertant colony numbers of any of the five strains were observed following treatment at any dose level, in either the presence or absence of S9 mix.

Conclusions:

It is concluded that, when tested to the limits of solubility in acetone, the test substance was not mutagenic to the bacteria in either the presence or absence of metabolic activation.

Executive summary:

In this in vitro assessment of the mutagenic potential of technical GEL-ALL-MD, histidine-dependent auxotrophic mutants of Salmonella typhirium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) were exposed to test material diluted in acetone which was also used as a negative control.

Two independent mutation tests were performed using agar plates, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary dose range-finding study no toxicity was observed. A top dose level of 5000 micrograms per plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150, 50 µg/plate.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the metabolic activation system.

No evidence of mutagenic activity was seen at any dose level of technical GEL-ALL-MD in either mutation test. Precipitation was observed at the highest dose level tested, 5000 µg/plate.

It is concluded that, when tested to the limits of solubility in acetone, technical GEL-ALL-MD was not mutagenic in either the presence or absence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 26 August 1997 and 11 September 1997.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar 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

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Study: 50, 150, 500, 1500 and 5000 µg/plate
Mutation study (Experiments 1 and 2): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of bacterial strains

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Positive controls (ENNG, 9AA and 4NQO) used without metabolic activation (S9)

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of bacterial strains

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

2AA used with metabolic activation (S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plaing

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in revertant count in one or more strains of bacteria in the presence and/or absence of S9 in both experiments at sub-toxic dose levels.
To be considered negative, the number of revertants at each dose level should be less than twofold that of the vehicle control frequency.

Statistics:

All of the data was analysed using the statistical methods recommended by the UKEMS with Dunnett's method of linear regression used to evaluate the 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Manual counts were performed at and above 1500 µg/plate because of test material precipitation.


PRELIMINARY TOXICITY STUDY:
The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500 and 5000 µg/plate. The test material was non-toxic to the strains of bacterial used (TA100 and WP2uvrA-).

MUTATION STUDY:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

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

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls both with and without metabolic activation are presented in Tables 2 and 3 for Experiment 1 and Tables 4 and 5 for Experiment 2 (see attached background material).

No toxicity was exhibited to any of the strains of bacteria used. A precipitate was observed at and above 1500 µg/plate, but this did not prevent scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the stains of bacteria, 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 and the activity of the S9 fraction was shown to be satisfactory.

Remarks on result:

other: A precipitate was observed at 1500 µg/plate

Preliminary Toxicity Study

The mean number of revertant colonies for the toxicity assay were:

Strain	Dose (µg/plate)
	0	50	150	500	1500	5000
TA100	137	130	139	139	134P	130P
WP2uvrA-	18	18	16	18	21P	19P

P = precipitate.

Conclusions:

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

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with suspensions of the test material using the Ames plate incorporation method 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 was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, 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 and without metabolic activation.

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose of 5000 ug/plate. A precipitate was observed at and above 1500 ug/plate, this 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.

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

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:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical:
1-(2,6-bis(4-tolyl)-1,3-dioxano(5,4-d)-1,3-dioxan-4-yl)ethane-1,2-diol (EC 402-950-5, CAS 87826-41-3)

Taget chemical:
2,6-bis(4-ethylphenyl)perhydro-1,3,5,7-tetraoxanaphth-4-ylethane-1,2-diol (EC 406-176-9, CAS 79072-96-1)

3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see 'Read-across justification to support the REACH registration of EC 406-176-9' document attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

bacteria, other: S. typhimurium TA 1535, 1537, 1538, 98, 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Observed at 5000 μg per plate

RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed in the preliminary dose range-finding study.

MUTATION TEST:
Responses of the positive and negative control groups were within the normal ranges experienced in the laboratory. Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the metabolic activation system.

No significant increases in the revertant colony numbers of any of the five strains were observed following treatment at any dose level, in either the presence or absence of S9 mix.

Conclusions:

It is concluded that, when tested to the limits of solubility in acetone, the test substance was not mutagenic to the bacteria in either the presence or absence of metabolic activation.

Executive summary:

In this in vitro assessment of the mutagenic potential of technical GEL-ALL-MD (EC 402-950-5), histidine-dependent auxotrophic mutants of Salmonella typhirium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) were exposed to test material diluted in acetone which was also used as a negative control.

Two independent mutation tests were performed using agar plates, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary dose range-finding study no toxicity was observed. A top dose level of 5000 micrograms per plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150, 50 µg/plate.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the metabolic activation system.

No evidence of mutagenic activity was seen at any dose level of technical GEL-ALL-MD in either mutation test. Precipitation was observed at the highest dose level tested, 5000 µg/plate.

It is concluded that, when tested to the limits of solubility in acetone, technical GEL-ALL-MD was not mutagenic in either the presence or absence of metabolic activation.

It is proposed that this result can be used in the assessment of the target substance (EC 406-176-9).

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical:
1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-glucitol (EC 413-110-2, CAS 135861-56-2)
Taget chemical:
2,6-bis(4-ethylphenyl)perhydro-1,3,5,7-tetraoxanaphth-4-ylethane-1,2-diol (EC 406-176-9, CAS 79072-96-1)

3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see 'Read-across justification to support the REACH registration of EC 406-176-9' document attached in section 13.

Reason / purpose for cross-reference:

read-across source

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Manual counts were performed at and above 1500 µg/plate because of test material precipitation.


PRELIMINARY TOXICITY STUDY:
The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500 and 5000 µg/plate. The test material was non-toxic to the strains of bacterial used (TA100 and WP2uvrA-).

MUTATION STUDY:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. The data are for concurrent untreated control plates performed the same day as the Mutation Study.

No toxicity was exhibited to any of the strains of bacteria used. A precipitate was observed at and above 1500 µg/plate, but this did not prevent scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the stains of bacteria, 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 and the activity of the S9 fraction was shown to be satisfactory.

Remarks on result:

other: A precipitate was observed at 1500 µg/plate

Conclusions:

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

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with suspensions of the test material (EC 413-110-2) using the Ames plate incorporation method 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 was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, 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 and without metabolic activation.

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose of 5000 ug/plate. A precipitate was observed at and above 1500 ug/plate, this 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.

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

It is proposed that this result can be used in the assessment of the target substance (EC 406-176-9).

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 04 February 2020; Experimental completion date: 20 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability.
- Suitability of cells: The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Normal cell cycle time (negative control): Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
- Sex, age and number of blood donors:
Preliminary Toxicity Test: female, aged 25 years
Main Experiment: female, aged 35 years


CELL CULTURE: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Metabolic activation:

with and without

Metabolic activation system:

Induced rat liver homogenate metabolizing system (S9), at a 2% final concentration

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0.49, 0.975, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 to 125 µg/mL.
The maximum dose was limited due to precipitate observed in the solubility test.

Main Experiment
4(20)-hour without S9: 0, 0.5, 1, 2, 4, 8, 16, 32 µg/mL
4(20)-hour with S9 (2%) 0, 1, 2, 4, 8, 16, 32, 64 µg/mL
24-hour without S9 0, 0.5, 1, 2, 4, 8, 16, 32 µg/mL

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited on precipitate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle:
The molecular weight of the test item was given as 414, therefore, the maximum dose level was 2000 µg/mL, the maximum recommended dose level.
The test item was insoluble in Minimal Essential Medium ( MEM) at 20 mg/mL and was insoluble in Dimethyl sulphoxide (DMSO) at 50 mg/mL but was suspendable in Acetone at 100 mg/mL in solubility checks performed in house.
Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 µL aliquots. Consequently, the maximum practical concentration was 500 µg/mL.
Prior to each experiment, the test item was accurately weighed, formulated in acetone and appropriate serial dilutions prepared.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/mL for 4-hour exposure; 0.1 µg/mL for 24-hour exposure

Positive control substance:

mitomycin C

Remarks:

Used in absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

4 µg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

Used in presence of S9-mix

Details on test system and experimental conditions:

CULTURE CONDITIONS:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05-9.10 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.70-0.75 mL heparinized whole blood

4-HOUR EXPOSURE WITH METABOLIC ACTIVATION (S9):
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

4-HOUR EXPOSURE WITHOUT METABOLIC ACTIVATION (S9):
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged and the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

24-HOUR EXPOSURE WITHOUT METABOLIC ACTIVATION (S9):
As the exposure was continuous the cultures were established, at a nominal volume of 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.
The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

PRELIMINARY TOXICITY TEST:
The dose range of test item used was 0.49, 0.975, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 to 125 µg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the Main Experiment.

MAIN EXPERIMENT:
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour recovery period in treatment-free media prior to cell harvest. The dose range of test item used was 0, 0.5, 1, 2, 4, 8, 16 to 32 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour recovery period in treatment-free media prior to cell harvest. The dose range of test item used was 0, 1, 2, 4, 8, 16, 32 to 64 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 0.5, 1, 2, 4, 8, 16 to 32 µg/mL.

CELL HARVEST:
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

PREPARATION OF METAPHASE SPREADS:
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data

STAINING:
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

EVALUATION OF RESPONSE:
QUALITATIVE SLIDE ASSESSMENT:
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

CODING:
The slides were coded using a computerized random number generator.

MITOTIC INDEX:
A total of 1000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

SCORING OF CHROMOSOME DAMAGE:
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Rationale for test conditions:

To assess the potential chromosomal mutagenicity of the test item, EDBS, on the metaphase chromosomes of normal human lymphocytes. Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test.

Evaluation criteria:

Data Evaluation: Following criteria used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.

Criteria for determining study conclusion:
a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations assessed on a case by case basis.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

marginal toxicity in the 4(20) hour exposure in the absence of S9 and the 24 hour continuous exposure group and no toxicity in the 4 (20) hour exposure group in the presence of S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm

PRELIMINARY TOXICITY TEST:
The dose range for the Preliminary Toxicity Test was 0, 0.49, 0.975, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 to 125 µg/mL. The maximum dose was limited due to precipitate observed in the solubility test.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 15.63 µg/mL, in the 4(20)-hour exposure group and the 24 hour continuous exposure group and at and above 31.25 µg/mL in the 4(20) hour exposure group in the presence of S9.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 125 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9) and the continuous exposure group.
The test item induced some evidence of toxicity in the 4(20) hour exposure groups in the absence and presence of S9 and no evidence of toxicity in the 24 hour continuous exposure group.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level and was 32 µg/mL for the 4(20)-hour exposure group in the absence of S9 and was 64 µg/mL for the 4(20) hour exposure group in the presence of S9 and the 24 hour continuous exposure group.

CHROMOSOME ABERRATION TEST - MAIN EXPERIMENT:
The qualitative assessment of the slides determined that precipitate was similar to that observed in the Preliminary Toxicity Test in the 4(20) hour exposure group in the absence of S9 and in the 24 hour continuous exposure group. In the 4(20) hour exposure group in the presence of S9 precipitate was observed at a lower dose level than in the preliminary toxicity test.

There were metaphases suitable for scoring at up to 32 µg/mL in the 4(20) hour exposure group in the absence of S9 and in the 24 hour continuous exposure group. In the 4 (20) hour exposure group in the presence of S9 there were metaphases suitable for scoring at up to 64 µg/mL.

In the 4(20) hour exposure group in the absence of S9 18 and 16% a mitotic inhibition was observed at 16 µg/mL and 32 µg/mL respectively. In the 4(20) hour group in the presence of S9 there was no evidence of toxicity. In the 24 hour continuous exposure group a mitotic inhibition of 32% and 2% was observed at 2 and 4 µg/mL respectively.

The maximum dose level selected for metaphase analysis was 16 µg/mL for all three exposure groups and was the lowest precipitating dose level for each exposure group.

The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.

All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.

The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.

The required number of cells and concentrations were analyzed.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in all of the exposure groups.

HISTORICAL CONTROL DATA:
Presented in attached background material.

Conclusions:

EDBS did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction:

This summary describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations.

Methods:

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated;4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected for the Main Experiment were as follows:

Group	Final concentration of test item (µg/mL)
4(20)-hour without S9	0, 0.5, 1, 2, 4, 8, 16, 32
4(20)-hour with S9 (2%)	0, 1, 2, 4, 8, 16, 32, 64
24-hour without S9	0, 0.5, 1, 2, 4, 8, 16, 32

Results

All vehicle (Acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item displayed marginal toxicity in the 4(20) hour exposure in the absence of S9 and the 24 hour continuous exposure group and no toxicity in the 4 (20) hour exposure group in the presence of S9. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

Conclusion:

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

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study conducted between 21st March 2005 and 5th May 2005

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase, TK +/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr J Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr D Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture: Cells were routinely cultured in RPMI 1640 medium
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone beta-naphthaflavone induced rat liver, S9 mix

Test concentrations with justification for top dose:

preliminary study: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/ml
Experiment 1: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 µg/ml
Experiment 2: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as the vehicle because it provided a suspension that was considered to allow accurate dosing of the cultures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

EMS used in the absence of metabolic activation, CP used in the presence of of metabolic activation Migrated to IUCLID6: was used in the absence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration:
Experiment 1: 4 hour exposure with and without metabolic activation.
Experiment 2: Exposure time without metabolic activation increased to 24 hours.
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 14 days


SELECTION AGENT (mutation assays): 5TFT

NUMBER OF REPLICATIONS: duplicate


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

OTHER EXAMINATIONS:
- Other: colony size

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-200 x 10E-6 for the TK+/- locus in L5l78Y cells at this laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10E-6 mutant frequency per survivor are not normally acceptable and will be repeated.

Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the Global Evaluation Factor (GEF) of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:

The UKEMS statistical package was used to review the results.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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: There was no marked change in pH when the test material was dosed into media
- Effects of osmolality: the osmolality did not increase by more than 50 mOsm.
- Precipitation: A precipitate ofthe test material was observed at and above 15.63 µg/ml, and with heavy precipitate aggregation at 1000 µg/ml the maximum exposure was considered to be achieved at 500 µg/ml. In the subsequent mutagenicity experiments the maximum dose was limited to 500 µg/ml by the presence and nature ofthe precipitate.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test: In the 4-hour exposures, both in the absence and presence of metabolic activation (S9) there was an apparent reduction in the Relative Suspension Growth (%RSG) of cells treated with test material when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was also an apparent reduction of %RSG values of cells treated with test material. However, based on the observations in the subsequent mutagenicity test the toxicity was most likely due to cells being washed of with the precipitate rather than true toxicity.
In the subsequent mutagenicity experiments the maximum dose was limited to 500 µg/ml by the presence and nature of the precipitate in the preliminary experiment.

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle (solvent) controls had acceptable mutant frequency values that that were within the normal range for the L5178Y cell line at the TK +I-Iocus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity ofthe metabolising system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the preliminary toxicity test a precipitate oftest material was observed at and above 15.63 µg/ml, increasing with intensity with increase in dose concentration until it aggregated at 1000 µg/ml. It was considered that the aggregation of the precipitate effectively reduced exposure to the cells and, therefore, this dose level was not selected for the mutagenicity tests.

During the course of the study the test material was tested to the limits of toxicity in the presence of metabolic activation, and in the absence of metabolic activation to a dose level where maximum exposure of the cells was achieved.

Conclusions:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction: 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 +/- locus of the L5178Y mouse lymphoma cell line.

Methods: L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls. The entire experiment was repeated to confirm the result of the first experiment. Four-hour exposures were used both with and without activation in Experiment 1. In Experiment 2, the exposure time without activation was increased to 24 hours.

The dose range of test material, plated for expression of mutant colonies, was selected based on the results and observations of a preliminary toxicity test and was 3.91 to 500 µg/ml in the absence of metabolic activation, and 3.91 to 62.5 µg/ml in the presence of metabolic activation for the first experiment. For the second experiment the dose range was 3.91 to 500 µg/ml both with and without activation.

Results: The maximum dose level used was limited by the presence and nature of the precipitate observed. In the preliminary toxicity test a precipitate of test material was observed at and above 15.63 µg/ml, increasing with intensity with increase in dose concentration until it aggregated at 1000 µg/ml. It was considered that the aggregation of the precipitate effectively reduced exposure to the cells and, therefore, this dose level was not selected for the mutagenicity tests. The vehicle (solvent) controls had acceptable mutant frequency values that that were within the normal range for the L5178Y cell line at the TK +/- 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 a statistically significant or dose-related increase in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

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

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical:
1-(2,6-bis(4-tolyl)-1,3-dioxano(5,4-d)-1,3-dioxan-4-yl)ethane-1,2-diol (EC 402-950-5, CAS 87826-41-3)

Taget chemical:
2,6-bis(4-ethylphenyl)perhydro-1,3,5,7-tetraoxanaphth-4-ylethane-1,2-diol (EC 406-176-9, CAS 79072-96-1)

3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see 'Read-across justification to support the REACH registration of EC 406-176-9' document attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Methods:L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material (source substance EC 402-950-5) at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls. The entire experiment was repeated to confirm the result of the first experiment. Four-hour exposures were used both with and without activation in Experiment 1. In Experiment 2, the exposure time without activation was increased to 24 hours.

The dose range of test material, plated for expression of mutant colonies, was selected based on the results and observations of a preliminary toxicity test and was 3.91 to 500 µg/ml in the absence of metabolic activation, and 3.91 to 62.5 µg/ml in the presence of metabolic activation for the first experiment. For the second experiment the dose range was 3.91 to 500 µg/ml both with and without activation.

Results:The maximum dose level used was limited by the presence and nature of the precipitate observed. In the preliminary toxicity test a precipitate of test material was observed at and above 15.63 µg/ml, increasing with intensity with increase in dose concentration until it aggregated at 1000 µg/ml. It was considered that the aggregation of the precipitate effectively reduced exposure to the cells and, therefore, this dose level was not selected for the mutagenicity tests. The vehicle (solvent) controls had acceptable mutant frequency values that that were within the normal range for the L5178Y cell line at the TK +/- 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 a statistically significant or dose-related increase in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

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

It is proposed that this result can be used in the assessment of the target substance (EC 406-176-9).

Reference 9

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 10 June 2013 and 20 August 2013

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell culture:
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO 2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (master stocks of cells were tested and found to be free of mycoplasma).

- Periodically "cleansed" against high spontaneous background: yes
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/ml), Hypoxanthine (15 μg/ml), Methotrexate (0.3 μg/ml) and Glycine (22.5 μg/ml). For the following 24 hours the cells were cultured in THG medium
(i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthoflavone induced rat liver (S9-mix)

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 4.05 to 1036.25 µg/ml
Mutagenicity Test:
Experiment 1 and 2: 64.77, 129.53, 259.06, 518.13, 777.19, 1036.25 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle:
Following solubility checks performed in-house, the test item was accurately weighed and formulated in dimethyl sulfoxide (DMSO) prior to serial dilutions being prepared.
The molecular weight of the test item was 414.49 and the purity was> 99%. Due to formulation problems the maximum achievable dose was 1036.25 µg/ml. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate (EMS) was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) was used as the positive control in the presence of metabolic activation.

Details on test system and experimental conditions:

DURATION
- Exposure duration:
Experiment 1: 4 hours with and without metabolic activation
Experiment 2: 4 hours with metabolic activation and 24 hours without metabolic activation
- Expression time: 2 days

NUMBER OF REPLICATIONS: Duplicate
NUMBER OF CELLS EVALUATED: 2000 cells/well

PRELIMINARY TOXICITY TEST:
A preliminary toxicity test was performed on cell cultures at 5 x 10E5 cells/ml, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E5 cells/ml using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 4.05 to 1036.25 μg/ml for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10E5 cells/ml.

The cultures were incubated at 37 °C with 5% CO 2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10E5 cells/ml. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/ml or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required).


MUTAGENICITY TEST:
EXPERIMENT 1:
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10E6 cells/ml in 10 ml aliquots in R10 medium in unvented 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentratio) at six dose levels of the test item (64.77 to 1036.25 μg/ml in both the absence and presence of metabolic activation), vehicle and positive controls. To each flask was added 2 ml of S9-mix if required, 0.2 ml of the treatment dilutions (0.2 ml for the positive control), and sufficient R0 medium to bring the total volume to 20 ml.

The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

EXPERIMENT 2:
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10E6 cells/ml in 10 ml cultures in R10 medium in unvented 25 cm2 tissue culture flasks for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3 x 10E6 cells/ml in 10 ml cultures were established in unvented 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (1% S9 final concentration) at six dose levels of the test item (64.77 to 1036.25 μg/ml in both the absence and presence of metabolic activation), vehicle and positive controls. To each flask was added 2 ml of S9-mix if required, 0.2 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to give a final volue of 20 ml (R10 is used for the 24-hour exposure group).

The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.


MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY:
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10E5 cells/ml. The cultures were incubated at 37°C with 5% CO 2 in air and subcultured every 24 hours for the expression period of two days, by counting and diluting to 2 x 10E5 cells/ml, unless the mean cell count was less than 3 x 10E5 cells/ml in which case all the cells were maintained.

On Day 2 of the experiment, the cells were counted, diluted to 10E4 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

PLATE SCORING:
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °C with 5% CO 2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two to three hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Evaluation criteria:

For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely,
when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:

Small significant increases designated by the UKEMS statistical package will be reviewed using the above evaluation criteria, and may be disregarded at the Study Director's discretion.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

optimum levels of toxicity were achieved in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Preliminary toxicity test: Precipitate observed at and above 16.19 µg/ml.
Mutagenicity Tests: Precipitate observed at and above 64.77 µg/ml.


COMPARISON WITH HISTORICAL CONTROL DATA:
None of the vehicle control mutatant frequency values were outside the acceptable range of 50 to 170 x 10-6 viable cells.
Positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

Preliminary Toxicity Test:

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose Level (µg/ml)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
4.05	70	94	99
8.1	58	97	101
16.19	64	94	133
32.88	85	105	94
64.77	87	75	82
129.53	89	93	92
259.06	77	98	81
518.13	67	99	74
1036.25	36	96	74

In the 4-hour and 24 hour exposure groups in the absence of metabolic activation there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 4 hour exposure in the presence of metabolic activation there was no evidence of marked test item induced toxicity. Overall, precipitate of the test item was observed at and above 16.19 µg/ml. Based on the %RSG values observed, the maximum dose level in the subsequent Mutagenicity Test would be set at 1036.25 µg/ml.

Mutagenicity Test

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

Experiment 1

The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7 (attached background material).

There was evidence of marked dose-related toxicity following exposure to the test item in both the absence of metabolic activation, as indicated by the RTG and %RSG values (Tables 3 and 6). There was no evidence of a reduction in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Based on the RTG and %RSG values, it was considered that optimum levels of toxicity were achieved in the absence of metabolic activation. Whilst optimum levels of toxicity were not achieved in the presence of metabolic activation, the test item was exposed to the cultures at the maximum achievable concentration due to formulation issues above this concentration. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels (Tables 3 and 6). It should also be noted that the GEF was not exceeded at any of the dose levels. Precipitate of the test item was observed at and above 64.77 µg/ml in both exposure groups.

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13 (attached background material).

There was evidence of marked toxicity following exposure to the test item in the absence of metabolic activation, as indicated by the %RSG and RTG values (Table 9). Moderate levels of test item toxicity were observed in the presence of metabolic activation as indicated by the %RSG and RTG values (Table 9). Based on the %RSG and/or RTG values it was considered that optimum levels of toxicity were achieved in the absence of metabolic activation. Whilst optimum levels of toxicity were not achieved in the presence of metabolic activation, the test item was exposed to the cultures to the maximum achievable concentration due to formulation issues above this concentration. There was no evidence of reductions in viability (%V) in either the absence or presence of metabolic activation; therefore indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had no significant effect on the toxicity of the test item.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test item did not induce a statistically significant dose related (linear-trend) response in either the absence or presence of metabolic activation (Table 9 and 12). Precipitate of the test item was observed at and above 64.77 µg/ml.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals NO.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9 final concentration). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9 final concentration) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test and for Experiment 1 and 2 was 64.77 to 1036.25 µg/ml in both the absence and presence of metabolic activation.

Results

The maximum dose level used in the Mutagenicity Test was limited by the formulation of the test item and the maximum achievable dose level was 1036.25 µg/ml. Precipitate of the test item was observed at and above 64.77 µg/ml in the Mutagenicity Test.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus.

The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion

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

Reference 10

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical:
1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-glucitol (EC 413-110-2, CAS 135861-56-2)

Taget chemical:
2,6-bis(4-ethylphenyl)perhydro-1,3,5,7-tetraoxanaphth-4-ylethane-1,2-diol (EC 406-176-9, CAS 79072-96-1)

3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see 'Read-across justification to support the REACH registration of EC 406-176-9' document attached section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

optimum levels of toxicity were achieved in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item (source substance EC 413-110-2) on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals NO.476"In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9 final concentration). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation(1% S9final concentration) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test and for Experiment 1 and 2 was 64.77 to 1036.25 µg/ml in both the absence and presence of metabolic activation.

Results

The maximum dose level used in the Mutagenicity Test was limited by the formulation of the test item and the maximum achievable dose level was 1036.25 µg/ml. Precipitate of the test item was observed at and above 64.77 µg/ml in the Mutagenicity Test.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus.

The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion

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

It is proposed that this result can be used in the assessment of the target substance (EC 406 -176 -9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was found to be:

- non-mutagenic in a bacterial reverse mutation assay (Ames test).

- non-clastogenic in in-vitro chromosome aberration study

- non-mutgenic to L5178Y cells in a mouse lymphoma assay (based on results read-across from two structural analogues).

Based on the above, the substance has been assessed as not requiring classification as a germ cell mutagen.