Phthalaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

- Principle of test: The test item ortho-phthalaldehyde was incubated with Salmonella typhimurium tester strains TA98 and TA100 and an Escherichia coli strain WP2 uvrA/pKM101, either in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague Dawley rat liver) for 20 minutes at 37 ºC. Top agar supplemented with L-histidine (for the S. typhimurium strains) or tryptophan (for the E. coli strain) and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine- or tryptophan-independent mutant colonies arising on these plates were counted following 2 days incubation at 37 ºC.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phthalaldehyde

EC Number:

211-402-2

EC Name:

Phthalaldehyde

Cas Number:

643-79-8

Molecular formula:

C8H6O2

IUPAC Name:

phthalaldehyde

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MP Biomedicals, LLC (Solon, OH), Lot No. 8674J
- Appearance: pale-yellow, coarse, crystalline material
- Purity: >99 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test chemical was stored refrigerated in the original sealed plastic containers.

Method

Target gene:

Histidine locus (S. typhimurium), tryptophan locus (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media: Top agar supplemented with L-histidine and d-biotin poured onto surfaces of minimal glucose agar plates

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media: Top agar supplemented with tryptophan and d-biotin poured onto surfaces of minimal glucose agar plates

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

n.a.

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation mix

Test concentrations with justification for top dose:

The highest concentration tested was limited by toxicity.
Test concentrations: (TA100): 0, 0.5, 1, 5, 10, 25, 50, 100, 200, 300 and 400 µg/plate
Test concentrations: (TA98 and E.coli WP2 uvrA/pKM101): 0, 0.5, 1, 5, 10, 25, 50, 100, 200 µg/plate

Vehicle / solvent:

- Solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98, without S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E.coli, without S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains, with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar
- Cell density at seeding (if applicable): not specified

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 3

Rationale for test conditions:

n.a.

Evaluation criteria:

In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose-related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold-increase required for a chemical to be judged positive or weakly positive, although positive calls are typically reserved for increases in mutant colonies that are at least twofold over background.

Statistics:

n.a.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

For individual results see Table 1 in box "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1: Mutagenicity of o-Phthalaldehyde in Bacterial Tester Strains

Strain	Dose (µg/plate)	Without S9	Without S9	With 10% rat S9	With 10% rat S9
TA100	0	98 ± 1	104 ± 4	116 ± 7	145 ± 4
	0.5		100 ± 11
	1		108 ± 3
	5	124 ± 2	110 ± 8
	10	147 ± 8	155 ± 5	139 ± 5
	25	191 ± 15	224 ± 2	129 ± 3
	50	58 ± 6		127 ± 3	140 ± 2
	100	0 ± 0		152 ± 3	157 ± 2
	200			162 ± 15	136 ± 3
	300				62 ± 8
	400				1 ± 1
Trial summary		Positive	Positive	Negative	Negative
Positive control		443 ± 15	599 ± 29	771 ± 34	680 ± 15
TA98	0	40 ± 4	37 ± 1	41 ± 2	46 ± 4
	0.5	40 ± 4
	1	47 ± 3
	5	55 ± 1	40 ± 1
	10	53 ± 0	39 ± 1	36 ± 3	62 ± 3
	25	19 ± 3	31 ± 5	37 ± 3	49 ± 2
	50		43 ± 4	32 ± 2	61 ± 3
	100		0 ± 0	27 ± 1	55 ± 4
	200			28 ± 2	25 ± 4
Trial summary		Negative	Negative	Negative	Negative
Positive control		560 ± 36	564 ± 11	1061 ± 146	861 ± 71
E.coli WP2 uvrA/pKM101	0	176 ± 15	228 ± 20	205 ± 8	194 ± 22
	0.5		230 ± 6
	1		254 ± 20
	5	175 ± 12	244 ± 7
	10	200 ± 5	285 ± 8	248 ± 8	194 ± 14
	25	207 ± 4	77 ± 7	241 ± 15	194 ± 25
	50	252 ± 8		245 ± 13	198 ± 5
	100	81 ± 13		251 ± 8	224 ± 28
	200			264 ± 14	223 ± 9
Trial summary		Negative	Negative	Negative	Negative
Positive control		799 ± 52	667 ± 29	641 ± 25	913 ± 64

Applicant's summary and conclusion

Conclusions:

In conclusion, the test item, ortho-phthalaldehyde was found to be mutagenic in Salmonella typhimurium strain TA100 in the absence of S9 metabolic activation system but was non-mutagenic in the presence of S9 metabolic activation. In Salmonella typhimurium strain TA98 and E.coli WP2 uvrA/pKM101, the test item was found to be non-mutagenic in the presence and absence of S9 metabolic activation system.

Executive summary:

In a reverse gene mutation assay in bacteria conducted similar to guideline OECD 471, strains of S. typhimurium (TA98, TA100) and E.coli WP2 uvrA/pKM101 were exposed to the test item, ortho-phthalaldehyde in DMSO at concentrations of 0, 0.5, 1, 5, 10, 25, 50, 100, 200 µg/plate for TA98 and E. coli WP2 uvrA/pKM101 and at concentrationa of 0, 0.5, 1, 5, 10, 25, 50, 100, 200, 300 and 400 µg/plate for TA100. The positive controls induced the appropriate responses in the corresponding strains. Under the conditions of this study, the test item was found to be mutagenic in S. typhimurium strain TA100 in the absence of S9 metabolic activation system but non-mutagenic in the presence of S9 metabolic activation. In S. typhimurium strain TA98 and E. coli WP2 uvrA/pKM101, the test item was found to be non-mutagenic in the presence and absence of S9 metabolic activation system.