Benzenepropanal, .beta.-methyl-3-(1-methylethenyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key study: Lawlor (1989), OECD 471: negative

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

23rd November 1988 to 29th December 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A GLP compliant study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The read-across is considered to be suitable based on the structural and 'mechanistic action' similarities between the target substance (3-(3-isopropenylphenyl)butanal) and source substance (3-(3-isopropylphenyl)butanal) and their similar physico-chemical properties.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The study does not include a strain that can detect cross linkage.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine operon

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: The strains were kept at 5 ± 5 °C on minimal medium supplemented with excess histidine, trace biotin and ampicillin (25 µg/mL) for TA98 and TA 100. New stock cultures were made from the frozen master cultures where necessary or from single colony re-isolates that were checked for their genotype and the presence of the plasmid.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

- Type and identity of media: The strains were kept at 5 ± 5 °C on minimal medium supplemented with excess histidine, trace biotin and ampicillin (25 µg/mL) for TA98 and TA 100. New stock cultures were made from the frozen master cultures where necessary or from single colony re-isolates that were checked for their genotype and the presence of the plasmid.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal fraction

Test concentrations with justification for top dose:

0.0005 - 0.250 µL/plate (without metabolic activation); 0.001 - 0.500 µL/plate (with metabolic activation)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: quinacrine mustard, 2-anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (Vogel Bonner Medium E with 0.2 % glucose and 1.5 % agar.

In a sterile test tube containing 2 mL of molten overlay agar in a 43-45 °C water bath, 0.05 mL of the test material was combined with 0.1 mL of the tester strain. The mixture was swirled gently and then poured on the surface of minimal agar plates. After the top agar had set, the plates were incubated at 37 ± 2 °C for 48 to 72 hours. The number of his+ revertant colonies growing on the plates were counted using an automated colony counter and recorded. The S9 activation assays were run concurrently with the only difference being the addition of 0.5 mL S9 mix in place of 0.5 mL of phosphate buffer.

DURATION
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: Performed in triplicate

DETERMINATION OF CYTOTOXICITY: Appearance of microcolonies and/or clearing of the background lawn

Evaluation criteria:

TA-1535, TA-1537 and TA-1538:
Data sets will be evaluated as positive if a dose response is observed over a minimum of three test concentrations and the increase in revertants is equal to or greater than three times the solvent control value at the peak of the dose response.

TA-98 and TA-100:
Data sets will be evaluated as positive if a dose response is observed over a minimum of three test concentrations and the increase in revertants achieves a doubling of the solvent control value at the peak of the dose response.

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:

Background lawn was found to be reduced in the both experiments at the highest dose tested in both the assay with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Background lawn was found to be reduced in the both experiments at the highest dose tested in both the assay with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Does levels to be used in the mutagenesis assays were selected from a preliminary study conducted on the test material at does from 150 to 0.018 µL/plate using strain TA-100. Cytotoxicity was observed at 0.146 µL/plate and above in the presence of S9 and at 0.073 µL/plate in the absence of S9 as evidence by the reduced number of revertants and/or the appearance of microcolonies on the minimal media plates. These results were used to select seven doses for the mutagenicity assays.

ANALOGUE APPROACH JUSTIFICATION:
- See “Justification for read-across” document attached in section 13 for full details.
- In summary, important considerations for the use of read-across for acute toxicity are: i) 3-(3-isopropenylphenyl)butanal (the target substance) has similar physico-chemical properties as 3-(3-isopropylphenyl)butanal (the source substance), ii) there are structural similarities between the two substances, iii) the OECD QSAR Toolbox assigns very similar toxicity profiles to both substances, with any differences indicating that the source substance may be representative of a worst case scenario. The information reported in this summary is included to demonstrate comparability between the source (3-(3-isopropylphenyl)butanal) and target substance (3-(3-isopropenylphenyl)butanal).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

 Table 1: Summary of results 1^stexperiment

Dose (µL/plate)	Mean Revertants per Plate with Standard Deviations
	TA 1535	TA 1537	TA 1538	TA 98	TA 100
Without S9
Vehicle	11 ± 3	9 ± 2	9 ± 3	18 ± 6	112 ± 11
0.0005	8 ± 5	12 ± 2	10 ± 3	19 ± 6	122 ± 8
0.001	11 ± 3	11 ± 4	8 ± 3	16 ± 3	107 ± 13
0.010	10 ± 3	9 ± 4	9 ± 1	13 ± 1	123 ± 2
0.025	9 ± 4	6 ± 2	11 ± 3	12 ± 4	108 ± 12
0.050	11 ± 1	3 ± 3	6 ± 2	12 ± 3	100 ± 10
0.100	7 ± 2	2 ± 2	6 ± 3	12 2) 3	79 ± 10
0.250	4 ± 2	0 ± 0	0 ± 0	3 ± 2	54 ± 7
Positive control*	1514 ± 101	651 ± 96	1306 ± 166	1001 ± 30	1469 ± 117
With S9
Vehicle	11 ± 2	12 ± 2	15 ± 3	23 ± 3	125 ± 6
0.001	10 ± 3	13 ± 5	15 ± 3	28 ± 6	149 ± 9
0.010	11 ± 4	12 ± 3	18 ± 4	24 ± 3	153 ± 4
0.025	14 ± 2	7 ± 5	17 ± 5	22 ± 2	141 ± 9
0.050	12 ± 1	11 ± 3	16 2) 4	24 ± 2	134 ± 9
0.100	12 ± 1	10 ± 2	14 ± 4	26 ± 2	134 ± 15
0.250	9 ± 5	3 ± 2	10 ± 2	19 ± 2	86 ± 3
0.500	11 ± 1	3 ± 0	8 ± 1	11 ± 2	80 ± 10
Positive control**	200 ± 8	163 ± 5	989 ± 66	1318 ± 98	1673 ± 84
* TA 1535 and TA 100 10 µg/plate sodium azide; TA 1537 5 µg/plate quinacrine mustard; TA 1538 and TA 98 10 µg/plate 2-nitrofluorene ** 2.5 µg/plate 2-antramine all strains

 

Table 2: Summary of results 2^ndexperiment

Dose (µL/plate)	Mean Revertants per Plate with Standard Deviations
	TA 1535	TA 1537	TA 1538	TA 98	TA 100
Without S9
Vehicle	6 ± 5	8 ± 4	6 ± 4	22 ± 2	98 ± 12
0.0005	13 ± 2	9 ± 3	9 ± 3	20 ± 3	85 ± 5
0.001	9 ± 2	10 ± 2	8 ± 2	21 ± 3	93 ± 10
0.010	10 ± 4	10 ± 2	7 ± 3	23 ± 2	88 ± 9
0.025	11 ± 4	12 ± 1	5 ± 1	27 ± 1	99 ± 5
0.050	14 ± 1	7 ± 1	6 ± 2	22 ± 6	90 ± 19
0.100	10 ± 4	9 ± 3	9 ± 3	18 ± 10	85 ± 3
0.250	7 ± 2	5 ± 1	7 ± 2	8 ± 2	55 ± 8
Positive control*	11693 ± 27	965 ± 91	1039 ± 79	808 ± 106	1060 ± 37
With S9
Vehicle	14 ± 3	14 ± 8	13 ± 3	32 ± 3	137 ± 11
0.001	15 ± 6	16 ± 4	10 ± 5	35 ± 10	141 ± 12
0.010	14 ± 6	13 ± 3	10 ± 3	31 ± 10	127 ± 8
0.025	15 ± 7	15 ± 3	15 ± 3	39 ± 4	139 ± 10
0.050	16 ± 3	14 ± 4	14 ± 2	38 ± 5	139 ± 3
0.100	17 ± 1	14 ± 3	14 ± 3	34 ± 11	122 ± 11
0.250	13 ± 3	15 ± 6	17 ± 3	35 ± 3	131 ± 7
0.500	14 ± 5	7 ± 2	19 ± 8	18 ± 2	97 ± 14
Positive control**	239 ± 17	155 ± 22	1311 ± 69	954 ± 156	1176 ± 18
* TA 1535 and TA 100 10 µg/plate sodium azide; TA 1537 5 µg/plate quinacrine mustard; TA 1538 and TA 98 10 µg/plate 2-nitrofluorene ** 2.5 µg/plate 2-antramine all strains

 

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of the test, the test material did not exhibit genetic activity in any of the assays conducted and was not mutagenic to Salmonella typhimurium indicator organisms under the test conditions.

Executive summary:

The test material was examined for mutagenic activity in the Ames Salmonella/Microsome assays using Salmonella typhimurium strains TA-1535, TA-1537, TA-1538, TA-98 and TA-100. The assays were conducted using three plates per dose level in the presence and absence of a metabolic activation system. The entire assay was performed twice, confirming the negative response observed. The test material was found to be non-mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The test material was examined for mutagenic activity in the Ames Salmonella/Microsome assays using Salmonella typhimurium strains TA-1535, TA-1537, TA-1538, TA-98 and TA-100. The assays were conducted using three plates per dose level in the presence and absence of a metabolic activation system. The entire assay was performed twice. The test material did not exhibit genetic activity in these assays and was considered to be non-mutagenic under the test conditions.

Justification for selection of genetic toxicity endpoint
Only one key study available.

Justification for classification or non-classification

According to Regulation 1272/2008 and Directive 67/548/EEC, the substance does not require classification for genotoxicity.

On the basis that the test material for the genotoxicity study (3-(3-isopropylphenyl)butanal, tradename Florhydral) is being used to support 3-(3-isopropenylphenyl)butanal (Dehydro Florhydral) on the basis of read-across, Dehydro Florhydral is also considered to be unclassified for genotoxicity.