Phthalaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential of ortho-phthalaldehyde to induce genotoxic effects was tested in an in vitro test battery. The substance was tested negative in all tester strains in a bacterial reverse gene mutation assay conducted according to OECD guideline 471. In a second reverse gene mutation assay in bacteria conducted within the US NTP programme for ortho-phthalaldehyde, the substance was tested negative in all tester strains, except for Salmonella typhimurium strain TA100.

HSE (2003) and NTP 2018 refer to an unpublished study conducted under GLP and according to OECD guidelines (Harbell, 1988). OPA did not induce mutation at the thymidine kinase (TK) locus in mouse lymphoma cells. In another mammalian cell HPRT gene mutation assay conducted in 2018 according to OECD guideline 476, ambiguous results for mutagenicity were determined for ortho-phtalaldehyde.

Link to relevant study records

Reference

Reference 1

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

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

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.

Target gene:

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

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 / 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

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

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

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

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.

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

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

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

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"

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1988

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

according to guideline

Guideline:

other: OECD guideline not specified; "mutation at the thymidine kinase (TK) locus in mouse lymphoma cells"

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In this test system, OPA did not induce gene mutation in mammalian cells.

Executive summary:

In an unpublished study conducted to GLP and OECD guidelines (1988), the potential for OPA to induce mutation at the thymidine kinase (TK) locus in mouse lymphoma cells was investigated (Harbell, 1988). Cells were incubated with OPA (99% purity) in distilled water for 5 hours without metabolic activation at 1, 5, 10, 15 and 20 μg/ml and with 3, 9, 16, 23 and 30 μg/ml with metabolic activation (5 plates per concentration). The same exposure concentrations were used for the independently repeated test. Relative cell survival was approximately 20 - 25% at the top dose both with and without activation in the first

experiment and approximately 1% in the second experiment. There was no dose-related increase in the number of mutant frequencies in either experiment. Positive and negative controls gave results in the expected region.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-03-29 to 2019-

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)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in cell culture medium (MEM + 0% FBS). The solvent was compatible with the survival of the cells and the S9 activity. The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4). Osmolality of the highest test item concentration was 309 mOsmol/kg.

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus of V79 cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins BioPharma Product Testing Munich GmbH stock cultures
- Suitability of cells: These cells are characterized by their high proliferation rate and their high cloning efficiency of untreated cells, usually more than 50%.
- Cell cycle length, doubling time or proliferation index: 12-14 h doubling time

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C incubation temperature. For purifying the cell population of pre-existing HPRT mutants cells were exposed to HAT medium containing 10 µM hypoxanthine, 3.2 µM aminopterin, 5 µM thymidine and 10 µM glycine for several cell doublings (2 - 3 days) with a subsequent recovery period in medium supplemented with 10 µM hypoxanthine and 5 µM thymidine.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

microsomal liver enzymes (S9)

Test concentrations with justification for top dose:

without metabolic activation:
0.005, 0.010, 0.025, 0.050, 0.10, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 and 0.50 mM
and with metabolic activation:
0.05, 0.10, 0.25, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.25, 1.50, 2.0, 2.5, 5.0, 7.5 and 10 mM

The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to OECD guideline 476.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium (MEM + 0% FBS)
- Justification for choice of solvent/vehicle: Based on the results of a solubility test cell culture medium was used as solvent.

Untreated negative controls:

yes

Remarks:

treatment medium (MEM medium)

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation, final concentration 300 µg/mL

Untreated negative controls:

yes

Remarks:

treatment medium (MEM medium) plus S9 mix

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation, final concentration 1 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours

- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 9 - 11 days for mutant frequency and 6 – 8 days for cloning efficiency
- Fixation time (start of exposure up to fixation or harvest of cells): Mutant frequency: 23 – 29 days, Cloning efficiency: 20 -26 days

SELECTION AGENT (mutation assays): selective medium containing 11 µg/mL 6-thioguanine


DETERMINATION OF CYTOTOXICITY
- Methods: Relative Survival, based on the cloning efficiency

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined
- one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutant frequency in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non - parametric Mann - Whitney test.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2 mM: 20% survival; 0.6 mM: 93% survival

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4).
- Effects of osmolality: Osmolality of the highest test item concentration was 309 mOsmol/kg
- Precipitation: No precipitation of the test item was noted in any of the experiments.

RANGE-FINDING/SCREENING STUDIES: The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to the OECD guideline 476.
0.25 mg/mL (without metabolic activation) and 2.0 mg/mL (with metabolic activation) were selected as the highest concentrations. The experiment with and without metabolic activation was performed as a 4 h short-term exposure assay.

POSITIVE CONTROLS:
With metabolic activation: The positive control DMBA induced a distinct increase in mutant frequency with 318.2 mutants/10^6 cells.
Without metabolic activation: The positive control EMS induced a distinct increase in mutant frequency with 298.5 mutants/10^6 cells.
In conclusion, the positive controls, DMBA (1.0 µg/mL) and EMS (300 µg/mL) demonstrating both the sensitivity and validity of the test systems.

NEGATIVE CONTROL:
With metabolic activation: In the main experiment with metabolic activation the mutant values of the negative control were within the historical control data of the test facility Eurofins Munich (about 9.6 - 44.0 mutants per 10^6 cells).
Without metabolic activation: In the experiment without metabolic activation, the mutant values of the negative controls were slightly increased (41.5 and 42.6 mutants/10^6 cells, respectively) and outside the historic control range (8.5 - 40.2 mutants/10^6 cells).
Nevertheless, the negative control values were considered acceptable for inclusion in the historical control data set as they were only slightly increased and no technical reason or human failure was determined.

TOXICITY:
A biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in the experiment with and without metabolic activation.
In the experiment without metabolic activation the relative survival was 19% for the highest concentration (0.25 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2.0 mM with a relative survival of 20%.

For individual results see Tables 2-9 in box "Any other information on results incl. tables".

Remarks on result:

other: no significant dose-response relationship was observed

MUTANT FREQUENCIES:

With metabolic activation: The mutant frequency of the highest concentration evaluated (2.0 mM with a Mutant Frequency of 66.2 mutants/10⁶ cells) and one lower concentration (0.60 mM with a Mutant Frequency of 53.4 mutants/10⁶ cells) were clearly above the historical data (44.0 mutants per 10⁶ cells) with a relative survival of 20% and 93%, respectively. Additionally, a statistical analysis displayed that these increases were statistically significant. However a significant dose-response relationship was not determined in the Chi-Square test for trend. Thus, the results are considered to be ambiguous for mutagenicity under the reported experimental conditions.

Without metabolic activation: The mutant frequencies induced by the test item did not show a biologically relevant increase. None of the observed mutant frequencies was statistically significantly increased over those of the negative controls.The highest mutant frequency was observed at a concentration of 0.05 mM (44.7 mutants per 10⁶ cells) with a relative survival of 75%. Since no statistically significant increase and no dose-response relationship was observed this increase was considered as not biological.

Table 2: Pre-Experiment for Toxicity, without metabolic activation

Dose Group	Concentration	Number of cells at the		Number of colonies per flask			CE [%]	Adjusted CE [%]	Relative Survival (RS)
	[mM]	beginning of treatment	end of treatment	I	II	mean			[%]
NC1	0	10000000	12529000	132	132	132	66	83	100
NC2		10000000	13243000	170	182	176	88	117
1	0.10	10000000	8194000	70	57	64	32	26	26
2	0.25	10000000	7021000	1	1	1	1	0	0
3	0.50	10000000	9248000	0	0	0	0	0	0
4	1.0	10000000	2295000	0	0	0	0	0	0
5	2.5	10000000	2278000	0	0	0	0	0	0
6	5.0	10000000	3689000	0	0	0	0	0	0
7	7.5	10000000	1579300	0	0	0	0	0	0
8	10	10000000	1632000	0	0	0	0	0	0

NC: negative control

CE: cloning efficiency

RS: relative survival 

Table 3:  Pre-Experiment for Toxicity, with metabolic activation

Dose Group	Concentration	Number of cells at the		Number of colonies per flask			CE [%]	Adjusted CE [%]	Relative Survival (RS)
	[mM]	beginning of treatment	end of treatment	I	II	mean			[%]
NC1	0	10000000	11730000	157	155	156	78	91	100
NC2		10000000	11985000	142	150	146	73	87
1	0.05	10000000	11577000	152	163	158	79	91	102
2	0.10	10000000	12172000	163	176	170	85	103	115
3	0.25	10000000	12138000	150	144	147	74	89	100
4	0.50	10000000	11849000	115	137	126	63	75	83
5	1.0	10000000	9316000	21	22	22	11	10	11
6	2.5	10000000	2635000	0	1	1	0	0	0
7	5.0	10000000	4063000	0	0	0	0	0	0
8	10	10000000	1630300	0	0	0	0	0	0

NC: negative control

CE: cloning efficiency

RS: relative survival 

Table 4:  Main Experiment – Toxicity, without metabolic activation

Dose Group	Concen-tration	Number of cells at the		Number of colonies per flask			CE [%]	Adjusted CE [%]	Relative Survival (RS)
	[mM]	beginning of treatment	end of treatment	I	II	mean			[%]
NC1	0	10000000	14603000	89	83	86	43	63	100
NC2		10000000	14212000	104	100	102	51	72
1	0.005	10000000	12801000	139	126	133	66	85	125
2	0.01	10000000	13651000	104	102	103	52	70	104
3	0.03	10000000	14943000	111	93	102	51	76	113
4	0.05	10000000	12954000	72	84	78	39	51	75
5	0.10	10000000	12155000	28	37	33	16	20	29
6	0.20	10000000	8959000	3	5	4	2	2	3
7	0.25	10000000	10778000	26	22	24	12	13	19
8	0.30	10000000	9622000	9	11	10	5	5	7
9	0.35	10000000	9860000	1	1	1	1	0	1
10	0.40	10000000	10285000	0	1	1	0	0	0
11	0.45	10000000	9826000	0	0	0	0	0	0
12	0.50	10000000	10132000	0	0	0	0	0	0
EMS	300 µg/mL	10000000	16269000	69	89	79	40	64	95

NC: negative control

EMS: Ethylmethanesulfonate

a: number of cells plated: 200 cells/flask

b: cloning efficiency: CE [%] = [(number of colonies / number of cells plated) x 100]

c: adjusted CE [%] = [CE x (number of cells at the end of treatment / number of cells at the beginning of treatment)]

d: relative survival: RS [%] = [(adjusted CE in treated culture / adjusted CE in the negative control) x 100]

Table 5:  Main Experiment – Mutagenicity, without metabolic activation

		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration	Number of colonies per flask			CE [%]	Number of colonies per flask							CE [%]	Mutant Frequency per 10^6cells
	[mM]	I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	169	168	169	84	14	11	8	24	13	14.0	5.4	0.0035	41.5
NC2		175	168	172	86	21	14	10	10	18	14.6	4.4	0.0037	42.6
2	0.01	181	146	164	82	15	13	13	10	13	12.8	1.6	0.0032	39.1
3	0.03	160	165	163	81	12	10	16	5	12	11.0	3.6	0.0028	33.8
4	0.05	184	174	179	90	16	16	12	16	21	16.2	2.9	0.0041	45.3
5	0.10	155	154	155	77	9	9	4	3	11	7.2	3.1	0.0018	23.3
7	0.25	150	141	146	73	14	6	6	8	11	9.0	3.1	0.0023	30.9
EMS	300 µg/mL	173	166	170	85	97	102	107	100	100	101.2	3.3	0.0253	298.5

NC: negative control

EMS: Ethylmethanesulfonate

a: number of cells plated: 200 cells/flask

b: cloning efficiency: CE [%] = [(number of colonies / number of cells plated) x 100]

c: number of cells plated: 400000 cells/petri dish

d: mutant frequency (per 10⁶ cells): MF = [CE of mutant colonies in selective medium / CE in non-selective medium) x 10⁶]

Table 6: Main Experiment – Toxicity, without metabolic activation

Dose Group	Concentration	Number of cells at the		Number of colonies per flask			CE[%]	Adjusted CE [%]	Relative Survival (RS) [%]
	[mM]	beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	11866000	145	138	142	71	84	100
NC2		10000000	12852000	133	126	130	65	83
1	0.05	10000000	13311000	113	115	114	57	76	91
2	0.10	10000000	13600000	97	143	120	60	82	98
3	0.25	10000000	13039000	148	156	152	76	99	119
4	0.50	10000000	10693000	127	133	130	65	70	83
5	0.60	10000000	12801000	125	118	122	61	78	93
6	0.70	10000000	12852000	84	102	93	47	60	71
7	0.80	10000000	13974000	91	84	88	44	61	73
8	0.90	10000000	13413000	89	103	96	48	64	77
9	1.00	10000000	11781000	87	92	90	45	53	63
10	1.25	10000000	10693000	96	98	97	49	52	62
11	1.50	10000000	9775000	61	69	65	33	32	38
12	2.00	10000000	8721000	37	38	38	19	16	20
13	2.50	10000000	9350000	21	8	15	7	7	8
14	5.00	10000000	2669000	0	0	0	0	0	0
15	7.50	10000000	2006000	0	0	0	0	0	0
16	10.00	10000000	1972000	0	0	0	0	0	0
DMBA	1.0 µg/mL	10000000	12223000	102	95	99	49	60	72

NC: negative control

DMBA: 7,12-dimethylbenz(a)anthracene

a: number of cells plated: 200 cells/flask

b: cloning efficiency: CE [%] = [(number of colonies / number of cells plated) x 100]

c: adjusted CE [%] = [CE x (number of cells at the end of treatment / number of cells at the beginning of treatment)]

d: relative survival: RS [%] = [(adjusted CE in treated culture / adjusted CE in the negative control) x 100]

Table 7: Main Experiment – Mutagenicity, with metabolic activation

		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration	Number of colonies per flask			CE              [%]	Number of colonies per flask							CE [%]	Mutant Frequency per 10^6cells
	[mM]	I	II	mean		I	II	III	IV	V	mean	SD			%RS
NC1	0	167	151	159	80	6	6	7	6	7	6.4	0.5	0.0016	20.1	100
NC2		180	175	178	89	10	13	14	9	13	11.8	1.9	0.0030	33.2	100
3	0.25	152	167	160	80	12	13	10	8	11	10.8	1.7	0.0027	33.9	119
5	0.60	165	142	154	77	26	11	17	12	16	16.4	5.3	0.0041	53.4	93
8	0.90	162	175	169	84	16	14	10	10	15	13.0	2.5	0.0033	38.6	77
11	1.50	141	151	146	73	4	5	6	6	8	5.8	1.3	0.0015	19.9	38
12	2.00	153	158	156	78	23	21	22	17	20	20.6	2.1	0.0052	66.2	20
DMBA	1.0 µg/mL	165	153	159	80	97	102	107	100	100	101.2	3.3	0.0253	318.2

NC: negative control

DMBA: 7,12-dimethylbenz(a)anthracene

a: number of cells plated: 200 cells/flask

b: cloning efficiency: CE [%] = [(number of colonies / number of cells plated) x 100]

c: number of cells plated: 400000 cells/petri dish

d: mutant frequency (per 10⁶ cells): MF = [CE of mutant colonies in selective medium / CE in non-selective medium) x 10⁶]

Table 8: Statistical Analysis–Main Experiment, without metabolic activation

Dose Group	Concentration	Mutant Frequency	p-value	Statistical Significance
	[mM]
NC1	0	41.5	/	/
NC2		42.6	/	/
2	0.01	39.1	> 0.9999	-
3	0.03	33.8	0.4209	-
4	0.05	44.7	0.3546	-
5	0.10	23.3	0.0170	+*
7	0.25	30.9	0.1212	-
EMS	300 µg/mL	298.5	0.0007	+

NC: Negative control

S: Solvent Control

EMS: Ethylmethanesulfonate

+: significant

-: not significant

+*: significantly decreased compared to negative control, therefore not relevant for interpretation of results

Table 9: Statistical Analysis–Main Experiment, with metabolic activation

Dose Group	Concentration	Mutant Frequency	p-value	Statistical Signi-ficance
	[mM]
NC1	0	20.1	/	/
NC2		33.2	/	/
3	0.25	33.9	0.1205	-
5	0.60	53.4	0.0073	+
8	0.90	38.6	0.0157	+
11	1.50	19.9	0.1202	-
12	2.00	66.2	0.0007	+
DMBA	1.0 µg/mL	318.2	0.0007	+

NC: Negative control

S: Solvent Control

DMBA:7,12-dimethylbenz(a)anthracene

+: significant

-: not significant

Conclusions:

Under the experimental conditions reported, ambiguous results were determined for mutagenicity of the test item ortho-phtalaldehyde in the HPRT gene mutation assay using V79 chinese hamster cells.

Executive summary:

In a mammalian cell HPRT gene mutation assay, V79 cells cultured in vitro were exposed to ortho-phthalaldehyde (99.85% purity) at concentrations of 0, 0.1, 0.03, 0.05, 0.10, 0.25 mM in the absence of mammalian metabolic activation and 0, 0.25, 0.60, 0.90, 1.50 and 2.00 mM in the presence of metabolic activation for 4 hours.

Ortho-phthalaldehyde was tested up to cytotoxic concentrations. In the experiment without metabolic activation the relative survival was 19% for the highest concentration (0.25 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2.0 mM with a relative survival of 20%.

In the experiment without metabolic activation, the mutant frequencies induced by the test item did not show a biologically relevant increase. None of the observed mutant frequencies was statistically significantly increased over those of the negative controls. The highest mutant frequency was observed at a concentration of 0.05 mM (44.7 mutants per 10⁶ cells) with a relative survival of 75%. Since no statistically significant increase and no dose-response relationship was observed this increase was considered as not biological.

In the experiment with metabolic activation, the mutant frequency of the highest concentration evaluated (2.0 mM with a Mutant Frequency of 66.2 mutants/10⁶ cells) and one lower concentration (0.60 mM with a Mutant Frequency of 53.4 mutants/10⁶cells) were clearly above the historical data (44.0 mutants per 10⁶ cells) with a relative survival of 20% and 93%, respectively. Additionally, a statistical analysis displayed that these increases were statistically significant. However a significant dose-response relationship was not determined in the Chi-Square test for trend. Thus, the results are considered to be ambiguous for mutagenicity under the reported experimental conditions.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data. 

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2000-04-25 to 2000-07-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted: 21st July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name used in the report: o-PHTHALALDEHYDE
- Chemical name: 1,2-Benzenedicarboxaldehyde
- Trade name: OPA
- CAS no.: 643-79-8
- Batch no.: FKGP908
- Appearance: Slight yellow sloid with scaly appearance
- Conditions of storage: at room temperature, in the dark
- Date of expiry: December 2000

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Prof. Bruce N. Ames, Berkeley, California
The actual batch of the strains was tested for ampicillin resistance (TA102: ampicillin/tetracycline resistance), UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances in November 1997.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver microsomal fraction S9 Mix

Test concentrations with justification for top dose:

The concentrations for the first experiment were set according to a preliminary toxicity test. The test substance prevented the growth of strain TA100 at concentrations of 62 µg/plate and above. At 21 µg/plate a reduced growth was obtained. It was therefore decided to use 36 µg/plate as the highest concentration which would probably be toxic and to use 5 more lower concentrations which should allow a useful evaluation.

Test concentrations: 36, 12, 4.0, 1.3, 0.44 and 0.15 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (90%)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylene-diamine

Remarks:

TA97a (10µg), without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

TA97a (5 µg), with S9

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 (2 µg), without S9

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:

TA98 (1 µg), TA100 (2 µg) and TA1535 (2 µg), with S9

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 (2 µg) and TA1535 (1 µg), without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,8-Dihydroxy-anthraquinone

Remarks:

TA102 (50 µg), with S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: t-Butyl-hydroperoxide

Remarks:

TA102 (50 µg), without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation

EXPERIMENTAL PERFORMANCE
The exposure was performed according to the ‘Plate Incorporation Assay‘, in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. The number of viable cells in the overnight-cultures is in the range of 2 x 10^8 cells per mL.

For each sample the following solutions were combined:
0.1 ml of the overnight culture of the bacteria,
0.5 ml of S9~mix (or phosphate buffered saline for samples without metabolic activation),
0.1 ml of the appropriate test- or reference substance solution and
2 ml of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

Counting of colonies
The plates with less than about 50 revertant colonies, i.e. the plates of TA98 and TA1535 with the exception of the positive controls, were counted visually by marking the colonies with a felt tipped pen. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.

NUMBER OF REPLICATIONS: 3 plates/strain/concentration level including the controls

DETERMINATION OF CYTOTOXICITY
Cytotoxicity is considered either as a clearing or diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

CONDITIONS OF CULTIVATION
One day before the Ames test was performed, a small amount from each of the frozen bacterial cultures was transferred to nutrient broth. The liquid cultures were incubated overnight at 37 °C and then used for the exposure.

Evaluation criteria:

The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2.5 fold of the amount of the spontaneous revertants.
- For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 12/3 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of our historic data of the Ames test.

Statistics:

Means and standard deviation were calculated for concentration group.

Key result

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility: No precipitation of the test substance was seen in any of the concentration groups.
Toxicity: In the preliminary test, the test substance prevented the growth of strain TA100 at concentrations of 62 µg/plate and above. In the main test without metabolisation, strong toxicity (no bacterial background lawn or only microcolonies instead of the lawn) was observed in all plates of the 36 µg/plate group and in some samples of the 12 µg/plate group. Metabolic activation reduced the toxicity in all strains.
Positive control substances: All positive control substances increased the mutation frequency to more than the above mentioned threshold values. As 2-aminoanthracene, 1,8-dihyclroxy-anthraquinone and 7,12-dimethyl-benz[a]anthracene require metabolic activation for mutagenicity, the results of these substances demonstrate also the efficiency of the metabolising system.

Table 1: Results on mutation frequency

	without metabolisation with metabolisation (S9-mix)		without metabolisation with metabolisation (S9-mix)
Strain	Substance, concentration (µg / plate)	Revertants/plate (Mean)	Substance, concentration (µg / plate)	Revertants/plate (Mean)
TA97a	Control	82	Control	111
	4-NOPD, 10 µg	534	DMBA, 5 µg	378
TA98	Control	8.6	Control	11.6
	2-Nitro-fluorene, 2 µg	227	2-Aminoanthracene, 1 µg	469
TA100	Control	57	Control	71
	Na-Azide, 2 µg	516	2-Aminoanthracene,2 µg	1413
TA102	Control	172	Control	235
	tBHPO, 50 µg	489	DHA, 50 µg	707
TA1535	Control	7.5	Control	9.0
	Na-Azide, 1 µg	252	2-Aminoanthracene, 2 µg	173

Conclusions:

Under the experimental conditions reported, ortho-phthalaldehyde did not cause gene mutations in an Ames Test conducted according to OECD 471. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse gene mutation assay.

Executive summary:

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, strains TA97a, TA98, TA100, TA102 and TA1535 of Salmonella typhimurium were exposed to ortho-phthalaldehyde in DMSO at concentrations of 36, 12, 4.0, 1.3, 0.44 and 0.15 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains in both experiments (plate incorporation and pre-incubation). Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo data is available to assess the genotoxic potential of ortho-phthalaldehyde. In a peripheral blood micronucleus assay in mice and rats conducted within the US NTP programme, treatment with the test item did not increase the frequencies of micronucleated PCEs in male or female rats and in female mice exposed for 3 months by inhalation. In male mice, the result of the micronucleus test was judged to be equivocal. Based on the weight of evidence, the test item is not considered to induce chromosome damage in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

- Principle of test: Mice were exposed to atmospheres containing ortho-phthalaldehyde vapors at concentrations of 0.44, 0.88, 1.75, 3.5, or 7.0 parts per million of air (ppm) 6 hours per day, 5 days per week for 3 months. Control groups were not exposed to the chemical. At the end of the studies, blood samples were collected and analysed for frequency of micronucleated reticulocytes (PCEs) and erythrocytes (NCEs) using a flow cytometer both the mature erythrocyte population and the immature reticulocyte population can be accurately distinguished by employing special cell surface markers to differentiate the two cell types. Approximately 20,000 reticulocytes and 1 million erythrocytes were analyzed per animal; the percentage of reticulocytes among circulating erythrocytes was also determined as a measurement of bone marrow toxicity.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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.

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Biosciences, Inc. (Germantown, NY)
- Age at study initiation: 5 to 6 weeks
- Housing: Animals were housed individually in stainless steel wire-bottom (Lab Products, Inc., Seaford, DE), changed and rotated weekly. Cageboard:Untreated paper (Shepherd Specialty Papers, Watertown, TN), changed daily
- Diet (e.g. ad libitum): ad libitum, NTP-2000 irradiated wafer diet (Zeigler Brothers, Inc., Gardners, PA)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 75 ± 3 °F
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 ± 2/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Vehicle:

- Vehicle(s)/solvent(s) used: none

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The study laboratory designed the inhalation exposure chamber (Harford Systems Division of Lab Products, Inc., Aberdeen, MD) so that uniform vapor concentrations could be maintained throughout the chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m3.
- System of generating particulates/aerosols: o-Phthalaldehyde vapor was generated from a flask heated with a heating mantle, purged by a heated nitrogen flow entering above the flask area, blended with heated dilution air to obtain the vapor concentration desired, and transported into a distribution manifold located above the generator.
- Temperature, humidity, pressure in air chamber:
Temperature: 75 ° ± 3 °F
Relative humidity: 55% ± 15%
Room fluorescent light: 12 hours/day
Room air changes: 15 ± 2/hour

- Air change rate: At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapor generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapor generation was terminated (T10) was approximately 9.2 minutes.
- Method of particle size determination: A small particle detector (Model 3022A; TSI Inc., St. Paul, MN) was used with and without animals in the exposure chambers to ensure that o-phthalaldehyde vapor, and not aerosol, was produced. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.

TEST ATMOSPHERE
- Brief description of analytical method used: Concentrations of o-phthalaldehyde in exposure chambers were monitored by an on-line GC/FID system. Samples were drawn from all exposure and control chambers approximately every 20 minutes during each exposure period using Hasteloy-C stream-select and gas-sampling valves (VALCO Instruments Company, Houston, TX) in a separate, heated oven. The sampling lines composing the sample loop were made from Teflon® tubing and were connected to the exposure chamber relative humidity sampling lines near the gas chromatograph. A vacuum regulator maintained a constant vacuum in the sample loop to compensate for variations in sample line pressure. An in-line flow meter between the vacuum regulator and the gas chromatograph allowed digital measurement of sample flow.

VAPOR GENERATION AND EXPOSURE SYSTEM:
Vapor concentration was determined by the reservoir temperature, nitrogen flow rate, and dilution air flow rate. Pressure in the distribution manifold was fixed to ensure constant flows through the manifold and into the chambers.
Due to the high boiling point of o-phthalaldehyde, all vapor transport lines and the on-line GC transport sample line of the 7.0 ppm chambers were heated above the minimum temperature needed to transport vapor without condensation. Individual Teflon- delivery lines carried the vapor from the distribution manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor from the metering valves to exposure chamber exhaust until the generation system stabilized and exposure could proceed; an additional 60 minutes was added to the prestart stabilization time to purge residual toluene present in the test chemical to less than 1% before exposures began. To initiate exposure, the chamber exposure valves were rotated to allow the vapor to flow to each chamber exposure duct where it was diluted with conditioned chamber air to achieve the desired exposure concentration.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

6 hours plus T90 (17 minutes) per day, 5 days per week for 14 weeks

Dose / conc.:

0 ppm

Dose / conc.:

0.44 ppm

Dose / conc.:

0.88 ppm

Dose / conc.:

1.75 ppm

Dose / conc.:

3.5 ppm

Dose / conc.:

7 ppm

No. of animals per sex per dose:

5 male and 5 female mice per dose group, 6 dose groups.

Control animals:

yes, concurrent no treatment

Positive control(s):

n.a.

Tissues and cell types examined:

micronucleated reticulocytes (PCEs) and erythrocytes (NCEs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The highest exposure concentration was selected based on NTP evaluations of the maximum achievable concentration without aerosolization (MACWA) under normal chamber environmental specifications. The lowest concentration was similar to the experimental limit of quantitation for the online monitor used in these studies. Due to the high mortality at 7.0 ppm, no micronucleus assay could be perfomed at this concentration. Although a lower limit of quantitation may have been achievable using this online monitor or available offline methods, exposure of animals to lower concentrations was not feasible under the conditions of these studies due to reactivity of the aldehyde moieties of o-phthalaldehyde with amines resulting from the presence of animals.

Statistics:

The statistical tests selected for trend and for pairwise comparisons with the chamber control group depend on whether the variances among the groups are equal. Levene’s test at α=0.05 is used to test for equal variances. In the case of equal variances, linear regression is used to test for a linear trend with dose and Williams’ test is used to test for pairwise differences between each treatment group and the control group. In the case of unequal variances, Jonckheere’s test is used to test for linear trend and Dunn’s test is used for pairwise comparisons of each treatment group with the control group. To correct for multiple pairwise comparisons, the P value for each comparison with the control group is multiplied by the number of comparisons made. In the event that this product is greater than 1.00, it is replaced with 1.00. Trend tests and pairwise comparisons with the controls are considered statistically significant at P ≤ 0.025.

Key result

Sex:

male

Genotoxicity:

ambiguous

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Key result

Sex:

female

Genotoxicity:

negative

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

o-Phthalaldehyde (0.44 to 3.5 ppm) did not increase the frequencies of micronucleated PCEs or micronucleated NCEs in female B6C3F1/N mice exposed to the chemical for 3 months by inhalation. In male mice, results of the micronucleus test were judged to be equivocal, based on an increased frequency of micronucleated PCEs in the 3.5 ppm group, which resulted in a significant trend (P=0.005). A pairwise comparison (William’s test) of this treatment group to the chamber control group was not significant (P=0.0289 with 0.025 required for significance), and the increase in micronucleated PCEs seen in the male mice was not confirmed in the NCE population, where no increase in micronucleated NCEs was seen at any dose level. This difference in response in the male mice between the immature (PCE) and mature (NCE) erythrocyte populations is difficult to reconcile because the frequency of micronucleated erythrocytes reaches steady state in peripheral blood of mice after about 30 days of exposure. The %PCE was elevated (trend P=0.001) in female mice exposed to o-phthalaldehyde but not in male mice.

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

Table 1: Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Exposure to o-Phthalaldehyde by Inhalation for 3 Months

Sex	Concentration (ppm)	Number of Mice with Erythrocytes Scored	Micronucleated PCEs/1000 PCEs	P Value(c)	Micronucleated NCEs/1000 NCEs(b)	P Value(d)	PCEs(b) (%)	P Value(c)
Male	0 (air)(e)	5	2.31 ± 0.17		1.40 ± 0.02		1.413 ± 0.03
	0.44	5	2.02 ± 0.15	0.6297	1.40 ± 0.01	1.000	1.251 ± 0.01	0.1137
	0.88	5	2.16 ± 0.21	0.7169	1.35 ± 0.03	1.000	1.446 ± 0.05	1.000
	1.75	5	2.25 ± 0.10	0.6720	1.29 ± 0.04	1.000	1.378 ± 0.05	1.000
	3.5	5	3.24 ± 0.61	0.0289	1.44 ± 0.13	1.000	1.521 ± 0.21	1.000
			P = 0.005(f)		P = 0.992(g)		P = 0.151(g)
Female	0 (air)(e)	5	2.39 ± 0.19		0.98 ± 0.02		1.400 ± 0.16
	0.44	5	2.06 ± 0.17	0.9680	0.99 ± 0.02	1.000	1.323 ± 0.04	1.000
	0.88	5	1.85 ± 0.08	0.9873	1.02 ± 0.02	0.3124	1.398 ± 0.06	1.000
	1.75	5	1.88 ± 0.13	0.9918	0.98 ± 0.02	1.000	1.460 ± 0.04	1.000
	3.5	5	1.79 ± 0.12	0.9942	0.98 ± 0.03	1.000	1.920 ± 0.11	0.0351
			P = 0.989(f)		P = 0.424(g)		P = 0.001(g)

(a) Study was performed at ILS, Inc. The detailed protocol is presented by Witt et al. (2008). PCE=polychromatic erythrocyte; NCE=normochromatic erythrocyte

(b) Mean ± standard error

(c) Pairwise comparison with the chamber control group; exposed group values are significant at P≤0.025 by William’s test

(d) Pairwise comparison with the chamber control group; exposed group values are significant at P≤0.025 by Dunn’s test

(e) Chamber control

(f) Exposure concentration-related trend; significant at P≤0.025 by linear regression

(g) Exposure concentration-related trend; significant at P≤0.025 by Jonckheere’s test

Conclusions:

In this study, under the given conditions treatmend with ortho-phthalaldehyde did not increase the frequencies of micronucleated PCEs or micronucleated NCEs in female B6C3F1/N mice exposed to the chemical for 3 months by inhalation. In male mice, results of the micronucleus test were judged to be equivocal, based on an increased frequency of micronucleated PCEs in the 3.5 ppm group, which resulted in a significant trend (P=0.005).

Executive summary:

In a B6C3F1/N mouse peripheral blood micronucleus assay conducted equivalent to OECD guideline 474, 10/sex/dose were treated by inhalation with ortho-phthalaldehyde (>99% purity) at doses of 0.44, 0.88, 1.75, 3.5, and 7.0 ppm 6 hours per day, 5 days per week for 3 months. At the end of the study, thawed blood samples were analysed for frequency of micronucleated reticulocytes (PCEs) and erythrocytes (NCEs) using a flow cytometer. Due to the high mortality at 7.0 ppm, no micronucleus assay could be perfomed at this concentration.

The test substance,ortho-phthalaldehyde (0.44 to 3.5 ppm) did not increase the frequencies of micronucleated PCEs or micronucleated NCEs in female B6C3F1/N mice exposed to the chemical for 3 months by inhalation. In male mice, results of the micronucleus test were judged to be equivocal, based on an increased frequency of micronucleated PCEs in the 3.5 ppm group, which resulted in a significant trend (P=0.005). 

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

- Principle of test: Rats were exposed to atmospheres containing ortho-phthalaldehyde vapors at concentrations of 0.44, 0.88, 1.75, 3.5, or 7.0 parts per million of air (ppm) 6 hours per day, 5 days per week for 3 months. Control groups were not exposed to the chemical. At the end of the studies, blood samples were collected and analysed for frequency of micronucleated reticulocytes (PCEs) and erythrocytes (NCEs) using a flow cytometer both the mature erythrocyte population and the immature reticulocyte population can be accurately distinguished by employing special cell surface markers to differentiate the two cell types. Approximately 20,000 reticulocytes and 1 million erythrocytes were analyzed per animal; the percentage of reticulocytes among circulating erythrocytes was also determined as a measurement of bone marrow toxicity.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, Inc., (Livermore, CA)
- Age at study initiation: 6 weeks old on the first day of the studies
- Housing: individual
- Diet (e.g. ad libitum): ad libitum, NTP-2000 irradiated wafer diet (Zeigler Brothers, Inc., Gardners, PA)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 12 (males) or 13 (females) days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 75 ± 3° F
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 ± 2/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Vehicle:

- Vehicle(s)/solvent(s) used: none

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The study laboratory designed the inhalation exposure chamber (Harford Systems Division of Lab Products, Inc., Aberdeen, MD) so that uniform vapor concentrations could be maintained throughout the chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m3.
- System of generating particulates/aerosols: o-Phthalaldehyde vapor was generated from a flask heated with a heating mantle, purged by a heated nitrogen flow entering above the flask area, blended with heated dilution air to obtain the vapor concentration desired, and transported into a distribution manifold located above the generator.
- Temperature, humidity, pressure in air chamber:
Temperature: 75 ° ± 3 °F
Relative humidity: 55% ± 15%
Room fluorescent light: 12 hours/day
Room air changes: 15 ± 2/hour

- Air change rate: At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapor generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapor generation was terminated (T10) was approximately 9.2 minutes.
- Method of particle size determination: A small particle detector (Model 3022A; TSI Inc., St. Paul, MN) was used with and without animals in the exposure chambers to ensure that o-phthalaldehyde vapor, and not aerosol, was produced. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.

TEST ATMOSPHERE
- Brief description of analytical method used: Concentrations of o-phthalaldehyde in exposure chambers were monitored by an on-line GC/FID system. Samples were drawn from all exposure and control chambers approximately every 20 minutes during each exposure period using Hasteloy-C stream-select and gas-sampling valves (VALCO Instruments Company, Houston, TX) in a separate, heated oven. The sampling lines composing the sample loop were made from Teflon® tubing and were connected to the exposure chamber relative humidity sampling lines near the gas chromatograph. A vacuum regulator maintained a constant vacuum in the sample loop to compensate for variations in sample line pressure. An in-line flow meter between the vacuum regulator and the gas chromatograph allowed digital measurement of sample flow.

VAPOR GENERATION AND EXPOSURE SYSTEM:
Vapor concentration was determined by the reservoir temperature, nitrogen flow rate, and dilution air flow rate. Pressure in the distribution manifold was fixed to ensure constant flows through the manifold and into the chambers.
Due to the high boiling point of o-phthalaldehyde, all vapor transport lines and the on-line GC transport sample line of the 7.0 ppm chambers were heated above the minimum temperature needed to transport vapor without condensation. Individual Teflon- delivery lines carried the vapor from the distribution manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor from the metering valves to exposure chamber exhaust until the generation system stabilized and exposure could proceed; an additional 60 minutes was added to the prestart stabilization time to purge residual toluene present in the test chemical to less than 1% before exposures began. To initiate exposure, the chamber exposure valves were rotated to allow the vapor to flow to each chamber exposure duct where it was diluted with conditioned chamber air to achieve the desired exposure concentration.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

6 hours plus T90 (17 minutes) per day, 5 days per week for 14 weeks

Dose / conc.:

0 ppm

Dose / conc.:

0.44 ppm

Dose / conc.:

0.88 ppm

Dose / conc.:

1.75 ppm

Dose / conc.:

3.5 ppm

Dose / conc.:

7 ppm

No. of animals per sex per dose:

5 male and 5 female rats per dose group, 6 dose groups

Control animals:

yes, concurrent no treatment

Positive control(s):

n.a.

Tissues and cell types examined:

micronucleated reticulocytes (PCEs) and erythrocytes (NCEs)

Details of tissue and slide preparation:

n.a.

Evaluation criteria:

CRITERIA FOR DOSE SELECTION: The highest exposure concentration (7 ppm) was selected based on NTP evaluations of the maximum achievable concentration without aerosolization (MACWA) under normal chamber environmental specifications. Due to the high mortality at 7.0 ppm, no micronucleus assay could be perfomed at this concentration.
The lowest concentration was similar to the experimental limit of quantitation for the online monitor used in these studies. Although a lower limit of quantitation may have been achievable using this online monitor or available offline methods, exposure of animals to lower concentrations was not feasible under the conditions of these studies due to reactivity of the aldehyde moieties of o-phthalaldehyde with amines resulting from the presence of animals.

Statistics:

The statistical tests selected for trend and for pairwise comparisons with the chamber control group depend on whether the variances among the groups are equal. Levene’s test at α=0.05 is used to test for equal variances. In the case of equal variances, linear regression is used to test for a linear trend with dose and Williams’ test is used to test for pairwise differences between each treatment group and the control group. In the case of unequal variances, Jonckheere’s test is used to test for linear trend and Dunn’s test is used for pairwise comparisons of each treatment group with the control group. To correct for multiple pairwise comparisons, the P value for each comparison with the control group is multiplied by the number of comparisons made. In the event that this product is greater than 1.00, it is replaced with 1.00. Trend tests and pairwise comparisons with the controls are considered statistically significant at P ≤ 0.025.

Key result

Sex:

male/female

Genotoxicity:

negative

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

o-Phthalaldehyde (0.44 to 3.5 ppm) did not increase the frequencies of micronucleated PCEs in male or female Sprague Dawley rats exposed to o-phthalaldehyde for 3 months by inhalation. No increases in micronucleated erythrocytes (NCEs) were seen in male or female rats following exposure to o-phthalaldehyde, and that is expected, because the rat spleen efficiently removes damaged reticulocytes soon after they emerge from the bone marrow. In addition, no significant effects on the percentage of PCEs (%PCE) were seen in either male or female rats. It should be noted that in the 3.5 ppm group of male rats, only two animals survived; the data from this group were not included in the pairwise comparisons or trend tests because a minimum of three animals is required for a valid data point.

Conclusions:

In this study, under the given conditions, the test item, ortho-phthalaldehyde did not increase the frequencies of micronucleated PCEs or micronucleated NCEs in male and female Sprague Dawley rats.

Executive summary:

In a Sprague Dawley rat peripheral blood micronucleus assay conducted equivalent to OECD guideline 474, 5/sex/dose were treated by inhalation with ortho-phthalaldehyde (>99% purity) at doses of 0.44, 0.88, 1.75, 3.5, and 7.0 ppm 6 hours per day, 5 days per week for 3 months. At the end of the study, thawed blood samples were analysed for frequency of micronucleated reticulocytes (PCEs) and erythrocytes (NCEs) using a flow cytometer. Evaluation of peripheral blood micronuclei was not performed at 7 ppm due to the high mortality in the animals.

The test substance, ortho-phthalaldehyde (0.44 to 3.5 ppm) did not increase the frequencies of micronucleated PCEs in male or female Sprague Dawley rats exposed to ortho-phthalaldehyde for 3 months by inhalation. In this study, ortho-phthalaldehyde is not considered to induce cytogenicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Ortho-phthalaldehyde was tested negative in a bacterial reverse gene mutation assay. In a second Ames test, a positive result was reported in a single strain (TA100), but not in TA98 and E. coli WP2 uvrA. Ambiguous results were observed in one in vitro HPRT test (2018), while the NTP 2018 report refers to a negative HGPRT (Harbell, 1988). Here, in vivo cytogenicity was tested negative in a peripheral blood micronucleus assay in rat and female mice. The result in male mice was equivocal.

Overall, there is no strong indication of genotoxicity. However, to finally decide the relevance of the equivocal results, an additional experiment of the mammalian cell HPRT mutation test (OECD 476) will be conducted. After receiving the results the classification will be re-assessed.