Reaction mass of 3-(3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal and 3-(1,1-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal and 3-(1,1-dimethyl-2,3-dihydro-1H-inden-4-yl)propanal

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: non mutagenic (OECD 471, GLP, K, rel. 1, read-across): non mutagenic up to limit or cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

- Mouse lymphoma assay using L5178Y cells (OECD 476, GLP, K, rel.1): non mutagenic up to cytotoxic concentrations.

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:

Between 24 November 1998 and 08 January 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection date: 1998-03-23 / date of signature: 1998-07-21

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene for S. thyphimurium and tryptophan gene for E.coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (10% S9-fraction from liver of male Sprague-Dawley rat injected with Aroclor 1254)

Test concentrations with justification for top dose:

- Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
- Mutation study, Experiment 1 & 2: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: emulsion observed with water at 50 mg/mL. Miscible in DMSO at 50 mg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

see Table 7.6.1/1

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

see Table 7.6.1/1

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposition duration: 48 hours

NUMBER OF REPLICATIONS: Triplicate plate per dose level

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay was considered valid if the following criteria were met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to historical control for 1997).
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 1 to 9.9 billion bacteria per mL.
4. Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive contamination.

Rationale for test conditions:

Maximum concentration was 5000 μg/plate (the maximum recommended dose level).

Evaluation criteria:

The test material may be considered to be positive in this test system if the following criteria are met: the test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnet's method of linear regression

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:

cytotoxicity

Remarks:

initially at 150 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Remarks:

An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at 5000 µg/plate in E. coli WP2 uvrA, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: The test material was toxic to TA100 at and above 500 µg/plate, and was non toxic to E. coli strain WP2uvrA-. See Table 7.6.1/2.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. The comparison was made with the historical control ranges for 1997 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the growth of the bacterial lawn in all of the Salmonella tester strains, initially at 150 µg/plate, both with and without metabolic activation. The sensitivity of the Salmonella strains to the toxicity of the test material varied between strains and between exposures with or without S9-mix. See "Attached background material".
No toxicity was observed in E. coli strain WP2uvrA-.

Table 7.6.1/2 : Preliminary toxicity results

S9-mix	Strain	Dose (µg/plate
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
+	TA100	114	117	C	106	101	108	108	96	102S	0V	0TP
-		120	125	114	87	112	102	120	128	67V	0V	0TP
+	WP2uvrA^-	26	29	27	27	24	24	20	18	28	24	16P
-		36	33	26	16	24	14	24	22	14	23	13P

C = contaminated

P = precipitate

V = very weak background lawn

S = sparse background lawn

T = toxic, nobackground lawn

Conclusions:

The test item was not mutagenic both in the presence and absence of metabolic activation in S. thyphimurium strains TA1535, TA1537, TA98, TA100, and E.coli WP2 uvrA-.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E.coli strain WP2 uvrA- were exposed the test material diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the first experiment was determined in a preliminary toxicity assay and ranged between 1.5 and 5000 µg/plate depending on bacterial strain type and presence or absence of metabolic activation. 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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test material caused a visible reduction in the growth of the bacterial lawn in all of the Salmonella tester strains, initially at 150 µg/plate, both with and without metabolic activation. The sensitivity of the Salmonella strains to the toxicity of the test material varied between strains and between exposures with or without S9-mix. No toxicity was observed in E. coli strain WP2uvrA-. The test material was therefore tested up to either its toxic limit or the maximum recommended dose of 5000 µg/plate depending on the bacterial strain type and presence or absence of S9-mix. An oily precipitate was observed at 5000 µg/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.

 

Under the test condition, ST 07 C 98 was not mutagenic to S. typhimurium strains TA1535, TA1537 TA98, TA100, andE.coliWP2 uvrA-, in the presence and absence of metabolic activation.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 16 to March 24, 2015

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on July 01-03, 2014/ signed on September 15, 2014)

Type of assay:

mammalian cell gene mutation assay

Target gene:

The test involves detection of mutation of mouse lymphoma L5178Y cells. The cells are heterozygous at the thymidine kinase (tk) locus (TK+/-), and forward mutation (TK-/-) is detected by the ability of these cells to divide and form colonies in the presence of trifluorothymidine (TFT), a toxic analogue of thymidine.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source: L5178Y mouse lymphoma (3.7.2c) cells were obtained from American Type Culture Collection (ATCC), Virginia.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- These cells are heterozygous at the thymidine kinase locus, TK +/-. Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (5% v/v); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 3.95, 7.89, 15.78, 31.56, 63.13, 126.25, 252.5, 505, 1010 and 2020 µg/mL, with and without S9 mix (3 hours exposure); without S9 mix (24 hours exposure)

Mutation tests:
-S9 mix (3 hours): 5, 10, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL
+S9 mix (3 hours): 20, 40, 50, 60, 65, 70, 75, 80, 100 and 120 µg/mL
-S9 mix (24 hours): 2, 6, 10, 14, 18, 20, 22, 24 and 28 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Test item was dissolved and diluted in DMSO (ACS reagent grade), shortly before dosing. The final volume of DMSO added to the cultures was 1% v/v. Test item was confirmed to be soluble at 202 mg/mL (1M) in DMSO as part of the preliminary toxicity test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 1% v/v

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 1% v/v

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: RPMI 1640 medium
R0: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.
R10p: R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R30p: R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.
R10p medium was used for cell culture unless otherwise specified.
R20p medium was used for the cloning efficiency plating. This was prepared by mixing equal volumes of R10p and R30p.

DURATION
- Exposure duration:
Preliminary toxicity test: 3 hours in the absence and presence of S9 mix and 24 hours in the absence of S9 mix
Main mutation assay: 3 hours in the absence and presence of S9 mix and 24 hours in the absence of S9 mix
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 7 days for viability plates and approximately 10 to 14 days for mutant plates
- All incubations were performed at 37 °C in a humidified atmosphere of 5 % CO2 in air.

SELECTION AGENT (mutation assays): Selective medium consisted of R10p containing 4 μg/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item and 2 cultures for vehicle control
- Main test: 4 cultures for vehicle control, 2 cultures/dose for test item and positive controls

NUMBER OF CELLS EVALUATED: 1.6 and 2000 cells per well plated for assessing cloning efficiency (CE) and mutant frequency (MF), respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth (RSG), Cloning efficiency (CE), Relative cloning efficiency (RCE) and Relative total growth (RTG)
RSG = (Individual SG x 100) / Mean solvent control SG
CE = - InP(0) / No. of cells per well
RCE = (Individual CE x 100) / Mean vehicle control CE
RTG = (RSG x Day2 RCE) / 100

OTHER:
Mutant frequency per 10^6 survivors (MF) was calculated as:: CE selective medium / CE non-selective medium

Rationale for test conditions:

The selection of the maximum dose level was based on toxicity.

Evaluation criteria:

Criteria for assessing mutagenic potential:
The following criteria were applied for assessment of individual assay results using data for Mutant Frequency (MF) where the Relative total growth (RTG) normally exceeded 10%.

The assay was considered valid in accordance with the assay acceptance criteria.

The test agent was regarded as negative if:
- The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
- If the linear trend test was negative, the result was regarded as negative.
- If the linear trend test was positive, this indicated a positive, biologically relevant response.
Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989). Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend (p<0.05).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: The osmolality of the test substance in medium was tested at 2020 µg/mL (10mM); no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control. No fluctuations in pH of the medium were observed at 2020 µg/mL of more than 1.0 unit compared with the vehicle control.

PRELIMINARY TOXICITY TEST:
- Precipitate (observed by eye at the end of treatment) was observed at concentrations of 126.25 µg/mL and greater in both the absence and presence of S9 mix following a 3-hour exposure.
- Exposure to test item at concentrations from 3.95 to 2020 µg/mL in the absence and presence of S9 mix (3-hour exposure) resulted in relative suspension growth (RSG) values from 100 to 0% and from 107 to 0% respectively.
- Following a continuous exposure for 24 hours, precipitation (assessed by eye at the end of treatment) was observed at concentrations of 126.25 µg/mL and greater. Exposure to concentrations from 3.95 to 2020 µg/mL resulted in RSG values from 90 to 0%.

MAIN MUTATION TEST
3-hour treatment in the absence of S9 mix: No precipitate was observed by eye at the end of treatment. Cultures exposed to test item at concentrations from 5 to 27.5 µg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 111 to 15% were obtained relative to the vehicle control.

3-hour treatment in the absence of S9 mix: No precipitate was observed by eye at the end of treatment. Cultures exposed to test item at concentrations from 20 to 70 µg/mL were assessed for determination of mutation frequency. RTG values from 113 to 12% were obtained relative to the vehicle control.

24-hour treatment in the absence of S9 mix: No precipitate was observed by eye at the end of treatment. Cultures exposed to test item at concentrations from 2 to 22 µg/mL were assessed for determination of mutation frequency. RTG values from 85 to 15% were obtained relative to the vehicle control.

There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical control data (2013 - 2015).

Table 7.6.1/1: Main mutation test - results

 

Test Substance	Concentration mg/mL	3 hour Treatment ‑S9‑mix		3 hour Treatment +S9‑mix		24 hour Treatment ‑S9‑mix
		Mean Relative Total Growth (%)	Mean Mutant Freq. (x10^‑6)	Mean Relative Total Growth (%)	Mean Mutant Freq. (x10^‑6)	Mean Relative Total Growth (%)	Mean Mutant Freq. (x10^‑6)
DMSO	0	100	119	100	118	100	94
Test item	2	NT	NT	NT	NT	85	104
	5	111	113	NT	NT	NT	NT
	6	NT	NT	NT	NT	71	78
	10	NT	NT	NT	NT	44	95
	18	NT	NT	NT	NT	23	84
	20	58	107	113	93	NT	NT
	22	NT	NT	NT	NT	15	78
	22.5	28	108	NT	NT	NT	NT
	25	24	94	NT	NT	NT	NT
	27.5	15	118	NT	NT	NT	NT
	50	NT	NT	102	75	NT	NT
	60	NT	NT	65	91	NT	NT
	65	NT	NT	33	118	NT	NT
	70	NT	NT	12	93	NT	NT
Methyl methanesulphonate	10	114	722^a	NT	NT	NT	NT
	5	NT	NT	NT	NT	29	1944^a
Benzo[a]pyrene	1	NT	NT	93	440^a	NT	NT

^a Positive control induced an acceptable response

NT:  Not Tested

Conclusions:

Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk^+/- (3.7.2C) mouse lymphoma cells were exposed to test item at the following concentrations:

 

Preliminary toxicity test: 3.95, 7.89, 15.78, 31.56, 63.13, 126.25, 252.5, 505, 1010 and 2020 µg/mL, with and without S9 mix (3 hours exposure); without S9 mix (24 hours exposure)

 

Mutation tests:

-S9 mix (3 hours): 5, 10, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL

+S9 mix (3 hours): 20, 40, 50, 60, 65, 70, 75, 80, 100 and 120 µg/mL

-S9 mix (24 hours): 2, 6, 10, 14, 18, 20, 22, 24 and 28 µg/mL

 

Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 5 % (v/v) S9 mix; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone.

 

Toxicity was observed in the preliminary toxicity test. Following a 3-hour exposure to test item at concentrations from 3.95 to 2020µg/mL, relative suspension growth (RSG) was reduced from 100 to 0% and from 107 to 0% in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 90 to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG). Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix were 27.5 and 70µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 15 and 12% respectively. In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 22µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The RTG was reduced to 15%.

 

In all tests the concurrent vehicle and positive control were within acceptable ranges.

 

Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Reference 3

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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[further information is included as attachment to Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their composition similarity

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and the source substances are reaction mass, composed of three and two dimethylindanylpropanal isomers, respectively.

3. ANALOGUE APPROACH JUSTIFICATION
No (reverse) gene mutations were detected in the Ames test performed on the source substance. The study design (OECD 471, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
The target substance is considered as negative for inducing gene mutations at the TK locus in L5178Y.
Taken together, all data confirm the absence of mutagenic potential of the source and target substances.
Therefore, based on the considerations above, it can be concluded that the result of the Ames test conducted with the source substance is likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.1.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

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:

cytotoxicity

Remarks:

initially at 150 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Remarks:

An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at 5000 µg/plate in E. coli WP2 uvrA, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: The test material was toxic to TA100 at and above 500 µg/plate, and was non toxic to E. coli strain WP2uvrA-. See Table 7.6.1/2.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. The comparison was made with the historical control ranges for 1997 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the growth of the bacterial lawn in all of the Salmonella tester strains, initially at 150 µg/plate, both with and without metabolic activation. The sensitivity of the Salmonella strains to the toxicity of the test material varied between strains and between exposures with or without S9-mix. See "Attached background material".
No toxicity was observed in E. coli strain WP2uvrA-.

Table 7.6.1/2 : Preliminary toxicity results

S9-mix	Strain	Dose (µg/plate
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
+	TA100	114	117	C	106	101	108	108	96	102S	0V	0TP
-		120	125	114	87	112	102	120	128	67V	0V	0TP
+	WP2uvrA^-	26	29	27	27	24	24	20	18	28	24	16P
-		36	33	26	16	24	14	24	22	14	23	13P

C = contaminated

P = precipitate

V = very weak background lawn

S = sparse background lawn

T = toxic, nobackground lawn

Conclusions:

The source substance and target substances are not mutagenic both in the presence and absence of metabolic activation in S. thyphimurium strains TA1535, TA1537, TA98, TA100, and E.coli WP2 uvrA-.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E.coli strain WP2 uvrA- were exposed the source substance diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the first experiment was determined in a preliminary toxicity assay and ranged between 1.5 and 5000 µg/plate depending on bacterial strain type and presence or absence of metabolic activation. 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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The source substance caused a visible reduction in the growth of the bacterial lawn in all of the Salmonella tester strains, initially at 150 µg/plate, both with and without metabolic activation. The sensitivity of the Salmonella strains to the toxicity of the test material varied between strains and between exposures with or without S9-mix. No toxicity was observed in E. coli strain WP2uvrA-. The source substance was therefore tested up to either its toxic limit or the maximum recommended dose of 5000 µg/plate depending on the bacterial strain type and presence or absence of S9-mix. An oily precipitate was observed at 5000 µg/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 source substance, either with or without metabolic activation.

 

The source and target substances are not mutagenic to S. thyphimurium strains TA1535, TA1537 TA98, TA100, andE.coliWP2 uvrA-, in the presence and absence of metabolic activation.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°	Substance tested	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   SafePharm, 1998	Supporting substance	Ames Test (OECD 471) K, rel. 1	Gene mutation	S. thyphimurium TA 1535, TA 1537, TA 98, TA 100 & E. coli WP2 uvrA-	-S9 +S9	Up to limit or cytotoxic concentration	-S9 : non mutagenic +S9 : non mutagenic
2   HLS, 2015	Registered substance	MLA (OECD 476) K, rel. 2	Gene mutation	L5178Y mouse lymphoma cells	-S9 +S9	Up to cytotoxic concentrations	-S9 : non mutagenic +S9 : non mutagenic

 

Gene mutation Assays (Tests n° 1 -2):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with the supporting substance (see the corresponding ESR for read-across justification). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. The supporting substance does not induce gene mutations in bacteria under the test condition whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The supporting substance and the registered substance are therefore considered as non-mutagenic according to the Ames test.

- Inability to produce gene mutation was confirmed in mammal cells using an in vitro gene mutation assay in L5178Y tk+/-(3.7.2C) mouse lymphoma cells. None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore, the registered substance is considered as negative for inducing gene mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the registered substance to mammalian cells.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.