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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

3-(3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal

Cas Number:

173445-65-3

Molecular formula:

C14H18O

IUPAC Name:

3-(3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal

Constituent 2

Reference substance name:

3-(1,1-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal

Cas Number:

173445-44-8

Molecular formula:

C14H18O

IUPAC Name:

3-(1,1-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): ST 07 C 98
- Physical state: colourless slightly viscous liquid
- Stability under test conditions: stable under normal temperature and pressure
- Storage condition of test material: room temperature, under nitrogen in the dark

Method

Target gene:

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

Species / strain

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.

Controls

Controls 1

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

Controls 2

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

Results and discussion

Test results

Test results 1

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

Test results 2

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-.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.