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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Phthalaldehyde
EC Number:
211-402-2
EC Name:
Phthalaldehyde
Cas Number:
643-79-8
Molecular formula:
C8H6O2
IUPAC Name:
phthalaldehyde
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MP Biomedicals, LLC (Solon, OH), Lot No. 8674J
- Appearance: pale-yellow, coarse, crystalline material
- Purity: >99 %

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

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Test resultsopen allclose all
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".

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

Applicant's summary and conclusion

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).