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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HSE (2003) and NTP 2018 refer to an unpublished study conducted under GLP and according to OECD guidelines (Harbell, 1988).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

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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.

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.

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
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 106 cells
[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 106cells   
[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. 

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

Referenceopen allclose all

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). 

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)

Additional information

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.