Phthalaldehyde

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Principles of method if other than guideline:

- Principle of test: Groups of 10 male and 10 female mice were exposed to ortho-phthalaldehyde at concentrations of 0, 0.44, 0.88, 1.75, 3.5, or 7.0 ppm, 6 hours plus T90 (17 minutes) per day, 5 days per week for 14 weeks. At the end of the studies, samples were collected for hematology, histopathology and clinical chemistry. In addition, sperm samples were collected from males in the control and three lowest exposure groups.
- Parameters analysed / observed: Detailed clinical observations, body weight, organ weight, histopathology and hematology examinations were made.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phthalaldehyde

EC Number:

211-402-2

EC Name:

Phthalaldehyde

Cas Number:

643-79-8

Molecular formula:

C8H6O2

IUPAC Name:

phthalaldehyde

Specific details on test material used for the study:

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

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

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

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation 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 m^3.
- 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/cm^3) 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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 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.

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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

0.44 ppm

Doses / concentrations 3

Dose / conc.:

0.88 ppm

Doses / concentrations 4

Dose / conc.:

1.75 ppm

Doses / concentrations 5

Dose / conc.:

3.5 ppm

Doses / concentrations 6

Dose / conc.:

7 ppm

No. of animals per sex per dose:

10 male and 10 female mice per dose group, 6 dose groups.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: 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.
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.
- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights

Positive control:

n.a.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings were recorded on day 8, weekly, and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: weighed initially, on day 8, weekly, and at the end of the studies

CLINICAL CHEMISTRY: No

HEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital sinus of mice at the end of the study for hematology.

- Following parameters were examined: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte, leukocyte, and platelet morphology; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte count and differentials, and packed cell volume

OTHER: Sperm motility: At the end of the studies, spermatid and sperm samples were collected from male mice in the 0, 0.44, 0.88, and 1.75 ppm groups. The following parameters were evaluated: spermatid heads per testis and per gram testis, sperm motility, and sperm per cauda epididymis and per gram cauda epididymis. The left cauda, left epididymis, and left testis were weighed.

Sacrifice and pathology:

GROSS PATHOLOGY: Necropsies were performed on core study animals. Organs weighed in the chamber control, 0.44, 0.88, 1.75, and 3.5 ppm groups were heart, right kidney, liver, lung, right testis, and thymus.
HISTOPATHOLOGY: Yes. Histopathology was performed on core study chamber control rats and mice, male mice exposed to 1.75, 3.5, or 7.0 ppm, and female mice exposed to 3.5 or 7.0 ppm. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eye, Harderian gland, gallbladder, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung, lymph nodes (mandibular, mesenteric, bronchial, and mediastinal), mammary gland, nose, ovary, pancreas, parathyroid gland, pharynx, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), thymus, thyroid gland, trachea, urinary bladder, and uterus. Tissues were examined to a no-effect level in the remaining core study groups. In addition, the pinna was examined in chamber control and 7.0 ppm mice and the testis with epididymis was examined in all male mice.

Other examinations:

Organ weights

Statistics:

The incidences of lesions are presented as numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically. The Fisher exact test, a procedure based on the overall proportion of affected animals, was used to determine significance between exposed and chamber control animals, and the Cochran-Armitage trend test was used to test for significant trends. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epidydimal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings in males and females exposed to 3.5 or 7.0 ppm included abnormal breathing, sneezing, and thinness. One or more of these clinical findings were present in animals that were euthanized prior to study completion. Of the mice that were found dead, there were no clinical findings that preceded death. Further, animal urine and feces within the exposure chambers to turn black. Black discoloration of the appendages was not noted in ortho-phthalaldehyde-exposed mice, possibly due to the normal dark skin pigmentation and coat color of B6C3F1/N mice.

Mortality:

mortality observed, treatment-related

Description (incidence):

All mice exposed to 7.0 ppm died during week 1 of the study, and five males and four females exposed to 3.5 ppm died by week 6 of the study. In males exposed to 7.0 ppm, eight mice were found dead and two were euthanized. In females exposed to 7.0 ppm, nine mice were found dead and one was euthanized. In males exposed to 3.5 ppm, five were euthanized (during weeks 5 and 6), and five survived to study completion. In females exposed to 3.5 ppm, four were euthanized (in weeks 1, 3, and 6), and six survived to study completion.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The final mean body weights and body weight gains of all surviving exposed groups of mice were significantly less than those of the chamber controls, and 3.5 ppm males lost weight during the study. Final body weights relative to controls of all surviving mice were 17% (0.44 ppm), 24% (0.88 ppm), 27% (1.75 ppm) and 39% (3.5 ppm) lower in exposed males and 14% (0.44 ppm), 22% (0.88 ppm), 23% (1.75 ppm) and 30% (3.5 ppm) in exposed females, respectively.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology data were not available for the 7.0 ppm groups due to 100% mortality. The total leukocyte counts were significantly increased in all exposed male groups and in the 3.5 ppm female group. Lymphocyte numbers were significantly increased in all exposed male groups, while segmented neutrophil and eosinophil counts were significantly increased in groups exposed to 1.75 ppm or greater. In female mice, the segmented neutrophil count was significantly increased in the 3.5 ppm group and the eosinophil counts increased in groups exposed to 1.75 ppm or greater. These alterations were consistent with an inflammatory leukogram. Hemoglobin concentrations, erythrocyte counts, hematocrit values, and packed cell volumes were significantly decreased in 1.75 and 3.5 ppm male mice. All other statistically significant changes were sporadic or minimal, and not considered toxicologically relevant.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute thymus weights of surviving groups of exposed mice were significantly lower than those of the chamber controls (up to 51% lower in males and 46% lower in females); the relative thymus weight of 3.5 ppm females was also significantly decreased. In males and females, there were significant decreases in absolute heart, kidney, and liver weights at all exposure concentrations that were, in general, unaccompanied by significant decreases in relative organ weights. There were no histopathologic findings in the heart, kidney, or liver corresponding to organ weight decreases. Because body weights were significantly decreased at all exposure concentrations in male and female mice, these organ weight decreases were considered to be related to decreased body weights rather than exposure to ortho-phthalaldehyde.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant or biologically relevant histopathologic changes were noted in the nose, larynx, trachea, lung, skin, eye, spleen, thymus, bone marrow, testis, and epididymis of mice. Effects included lesions at sites of contact within the respiratory system, skin, and eye that were generally consistent with an irritant effect; changes in the hematopoietic system that may be attributed to stress and inflammation; and changes in the male reproductive system. Microscopically, olfactory epithelium hyaline droplet accumulation, atrophy, respiratory metaplasia, squamous metaplasia, and necrosis were similar to the same lesions observed in rats. Hyperplasia of the glands in the olfactory epithelium occurred predominantly in level III sections of the nose, subjacent to atrophic olfactory epithelium, and was seen as clusters of Bowman’s glands with increased cell numbers with more densely staining cytoplasm and nuclei, often arranged around a distinct lumen, which sometimes contained neutrophils.

Nose: A main target of o-phthalaldehyde exposure was the respiratory system. In the nose, mice had many lesions occurring within the lowest exposure group. All exposed male and female mice (except four 7.0 ppm females) exhibited minimal to marked suppurative inflammation. Significantly increased incidences of turbinate atrophy occurred in all exposed groups of mice, except 7.0 ppm males and females. Turbinate atrophy, a chronic change, may have been absent at 7.0 ppm due to decreased exposure duration because of the early deaths.
Several lesions were noted in the olfactory epithelium of the nose in exposed male and female mice. The incidences of olfactory epithelium hyaline droplet accumulation and glands hyperplasia were significantly increased in all exposed groups, except 7.0 ppm males and females. All exposed groups of male and female mice had increased incidences of olfactory epithelium atrophy as compared to the chamber controls. Incidences of respiratory metaplasia of the olfactory epithelium in 0.88, 1.75, and 3.5 ppm males and 0.88 and 3.5 ppm females were significantly increased. A few males in the 0.88, 1.75, and 3.5 ppm groups also had squamous metaplasia of the olfactory epithelium. The incidence of necrosis of the olfactory epithelium of the nose in males exposed to 7.0 ppm was significantly increased compared to that in the chamber controls.
Microscopically, olfactory epithelium hyaline droplet accumulation, atrophy, respiratory metaplasia, squamous metaplasia, and necrosis were observed. Hyperplasia of the glands in the olfactory epithelium occurred predominantly in level III sections of the nose, subjacent to atrophic olfactory epithelium, and was seen as clusters of Bowman’s glands with increased cell numbers with more densely staining cytoplasm and nuclei, often arranged around a distinct lumen, which sometimes contained neutrophils.
Lesions were present in the respiratory epithelium of the nose. Exposed male and female mice exhibited significantly increased incidences of respiratory epithelium hyaline droplet accumulation, except in the highest exposure group (7.0 ppm), likely due to their limited exposure duration due to early deaths. Many of the mice with hyaline droplet accumulation in the respiratory epithelium also had significantly increased incidences of hyaline droplet accumulation in the underlying glands. All exposed groups, except for the 7.0 ppm females, exhibited significantly increased incidences of respiratory epithelium squamous metaplasia. There were significantly increased incidences in necrosis of the respiratory epithelium in males and females exposed to 0.88 ppm or greater compared to the chamber controls. The incidences of respiratory epithelium regeneration were significantly increased in 7.0 ppm males and females. There were two incidences of respiratory epithelium ulcer in 7.0 ppm females, but the increases were not statistically significant.
Microscopically, respiratory epithelium hyaline droplet accumulation was similar to that observed in the olfactory epithelium. Hyaline droplet accumulation in the glands of the respiratory epithelium was characterized in levels I and II sections of the nose by robust enlargement of the glands in the lamina propria of the dorsal meatus and nasal septum by accumulations of bright eosinophilic, hyaline material that often compressed the nucleus. These glands were arranged around distinct lumens that were often filled with degenerate neutrophils. Necrosis and regeneration of the respiratory epithelium were also observed. Ulcers were noted as focal to focally extensive areas of loss of the normal respiratory epithelium.

Larynx: In the larynx, male and female mice exhibited significantly increased incidences of chronic active inflammation, squamous metaplasia, and necrosis (Tables 10, A3, and A4). Significantly increased incidences of chronic active inflammation occurred in 1.75 ppm males as well as in almost all 3.5 and 7.0 ppm males and females, and severity increased with exposure concentration. Significantly increased incidences of squamous metaplasia occurred in males and females exposed to 1.75 ppm or greater. Significant increases in the incidences of necrosis occurred in male and female mice exposed to 7.0 ppm. Three 7.0 ppm females exhibited regeneration in the larynx, but this incidence was not statistically significant compared to the chamber control incidence.
Microscopically, chronic active inflammation of the larynx varied in that the animals with more necrosis demonstrated an infiltrate that was predominantly neutrophilic, whereas animals that developed squamous metaplasia had increased numbers of lymphocytes, plasma cells, and macrophages, in addition to neutrophils. Squamous metaplasia was characterized as replacement of the normal respiratory epithelium with multiple layers of flattened squamous epithelium that sometimes progressed to keratinization along the superficial surface. Necrosis was characterized as partial to complete loss of the epithelium with partial involvement of the underlying lamina propria in some cases. Regeneration was characterized by focal to focally extensive loss of the normal respiratory epithelial cells in the larynx and replacement by a single layer of elongate cells that stretched to cover the denuded area.
Trachea: While lesions were noted in the nose beginning in the lowest exposure group, 0.44 ppm, and in the larynx beginning at 0.88 ppm, findings in the trachea were only noted beginning at exposures of 1.75 ppm for males and 3.5 ppm for females. In the trachea, male and female mice exhibited significantly increased incidences of chronic active inflammation, squamous metaplasia, necrosis, and regeneration (males only). The incidences of chronic active inflammation were significantly increased in 3.5 and 7.0 ppm males and females. Significant increases in the incidences of squamous metaplasia were observed in males and females exposed to 3.5 ppm. Significantly increased incidences of necrosis occurred in 7.0 ppm males and females, and a significantly increased incidence of regeneration occurred in 7.0 ppm males. Only one occurrences of tracheal ulceration noted in mice. Tracheal ulceration was present in a 3.5 ppm female, which was a moribund sacrifice noted to have abnormal breathing. Microscopically, regeneration was noted as focal to focally extensive areas of epithelial loss with replacement by a single layer of elongated thin epithelial cells that stretched to cover the denuded area. Fibrosis was noted as an increase in spindloid cells within the lamina propria of the trachea that distorted and partially occluded the tracheal lumen in some cases.

Lung: Lesions in the lung in mice were notable only in the two highest exposure groups, 3.5 and 7.0 ppm. Thus, inflammatory and degenerative or reparative findings in mice increased in depth within the respiratory tract from nose to lung with increased exposure concentration of ortho-phthalaldehyde. The occurrences of bronchiole goblet cell hyperplasia in 3.5 ppm males and females, chronic active inflammation of the bronchus in 3.5 ppm females and 7.0 ppm males and females, and necrosis in the bronchus of 7.0 ppm males and females were significantly increased compared to those in the chamber controls. Although not statistically significant, ulcer of the bronchus occurred in two female mice in the 7.0 ppm group. Microscopically, goblet cell hyperplasia in the lung was characterized by epithelial cells with increased cytoplasm expanded by pale basophilic material, with mucus and occasional inflammatory cells in the lumen of bronchioles. Chronic active inflammation of the bronchus was characterized by neutrophils with rare plasma cells and lymphocytes within bronchi and peribronchial connective tissue. Rarely, this infiltrate extended into bronchioles. Bronchus necrosis was seen as a focal to focally extensive area of epithelium that displayed loss of differential staining and was associated with fibrin or necrotic cellular debris. Ulcer of the bronchus was noted when the epithelium was absent with no associated necrotic cellular debris or fibrin overlying the denuded epithelium.

Skin: In addition to the respiratory tract, the skin was also a target of ortho-phthalaldehyde inhalation exposure. In standard inguinal skin sections, male and female mice exhibited significantly increased incidences of squamous hyperplasia, chronic active inflammation, adnexa degeneration, epidermis necrosis (females only), and hair follicle epithelium parakeratosis. The incidences of squamous hyperplasia were significantly increased in 3.5 ppm males and females and 7.0 ppm males. The incidences of chronic active inflammation were significantly increased in females exposed to 1.75 ppm and males and females exposed to 3.5 or 7.0 ppm. Significant increases in the incidences of adnexa degeneration occurred in 1.75 ppm males and in 7.0 ppm males and females. Significantly increased incidences of epidermal necrosis occurred in 7.0 ppm females and significantly increased incidences of hair follicle epithelium parakeratosis occurred in males exposed to 0.88 or 7.0 ppm and in females exposed to 0.88 ppm or greater. Microscopically, squamous hyperplasia in routine inguinal skin sections was observed as thickening of the epithelium that exceeded two to three cell layers. Chronic active inflammation consisted of neutrophils, plasma cells, and lymphocytes that were scattered throughout the superficial dermis and rarely extended into the overlying epidermis. In a few cases, small clusters of degenerate neutrophils (pustules) accumulated within the epidermis. Adnexa degeneration was characterized by the accumulation of pale basophilic homogenous material in the cytoplasm of multiple follicular and adnexal epithelial cells that often surrounded and compressed the nucleus. Electron microscopy, performed on an affected skin sample, showed that the cytoplasmic accumulations were inconsistently membrane bound and varied from diffusely electron lucent particles to small dense bodies that resemble glycogen. In addition to the cytoplasmic accumulations, adnexa degeneration also encompassed an increase in apoptosis, seen as small cells with dark eosinophilic cytoplasm and multiple small round, dark pyknotic bodies (apoptotic debris) within scattered individual hair follicle epithelial cells. Necrosis in the epidermis was noted as decreased staining and loss of cellular detail involving the entire thickness of the epidermis, with occasional cleft formation between the epidermis and dermis. Hair follicle epithelium parakeratosis was characterized by increased layers of brightly eosinophilic material (keratin) with retention of nuclei (parakeratosis) that collected into plaques along the epithelial surface, with preferential involvement of follicular regions over interfollicular areas.
Evaluation of skin included additional evaluation of the left pinna in control and 7.0 ppm male and female mice. Squamous hyperplasia, chronic active inflammation, adnexa degeneration, and hair follicle epithelium parakeratosis were present in the pinnae of all male and female mice exposed to 7.0 ppm.
Microscopically, within skin of the pinna, squamous hyperplasia was characterized by diffuse thickening of the epithelium, primarily involving the stratum spinosum, in some cases up to 10 cell layers thick. Chronic active inflammation was noted as minimal to mild collections of neutrophils, lymphocytes, plasma cells, and mast cells primarily within the superficial dermis. Adnexa degeneration was noted as irregular, poorly defined sebaceous glands with increased pyknotic and karyorrhectic debris evident both within the follicular epithelium and adnexal structures. Hair follicle epithelium parakeratosis was described as diffuse thickening of the stratum corneum with increased keratin within follicular regions predominantly characterized by retention of nuclei (parakeratosis), whereas interfollicular regions lacked nuclei (orthokeratosis).

Eye: In addition to the skin, the eye was another target of ortho-phthalaldehyde inhalation exposure in mice. In the eye, the incidence of cornea suppurative inflammation was significantly increased in 7.0 ppm males. Necrosis of the cornea was present in two 7.0 ppm females.

Hematopoietic System: A change observed in mice, was significantly increased incidences of bone marrow hyperplasia in 3.5 ppm males and females and 7.0 ppm males. Microscopically, bone marrow hyperplasia was seen as hypercellularity with a subjective increase in the myeloid:erythroid ratio, most noticeably within the diaphysis of the femur, but also within sections of the skull. The increase in the myeloid lineage was likely in response to inflammation in the respiratory tract, skin, and/or eye.
Lymphoid atrophy occurred in the spleen and thymus of male and female mice. Lymphoid atrophy of the spleen was significantly increased in 7.0 ppm males and females. The incidence of lymphoid atrophy of the thymus was significantly increased in 3.5 and 7.0 ppm males and females.
Microscopically, lymphoid atrophy in the spleen was similar to what was seen in rats. In the thymus, lymphoid atrophy consisted of a diffuse reduction in cortical lymphocytes with a resulting decrease in organ size, shrinkage of thymic lobules, loss of the corticomedullary junction, and increased prominence of centrilobular septae. Less affected animals had subtle lesions that often consisted only of increased numbers of apoptotic bodies and tingible body macrophages. In mice exposed to ortho-phthalaldehyde, lymphoid atrophy in the spleen and thymus are likely attributable to glucocorticoid release because they were present in the two highest exposure groups, which had early deaths.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Male Reproductive System: Mice exposed to 0.44, 0.88, or 1.75 ppm ortho-phthalaldehyde exhibited lower sperm motility (10% to 21% lower). No histopathologic lesions were attributed to exposure concentrations less than 3.5 ppm. However, as was observed in rats, at the two highest exposure concentrations (at which morbidity and mortality were observed), testicular and epididymal histopathologic lesions were noted. Sperm parameter data were not available in the 3.5 and 7.0 ppm groups due to excessive mortality.
A significantly increased incidence of cellular depletion of the germinal epithelium of the testis occurred in 3.5 ppm males. In addition, three of the early death mice from the 3.5 ppm group had mild interstitial cell atrophy. There was a significantly increased incidence of exfoliated germ cell in the epididymal duct in males exposed to 3.5 ppm. These changes in the testis and epididymis in the 3.5 ppm group (partial depletion of germ cells, interstitial cell atrophy, and exfoliated germ cells in the epididymis) could be due to decreased body weight gain secondary to treatment, but an effect of ortho-phthalaldehyde cannot be ruled out.
Microscopically, germinal epithelium cellular depletion of the testis was minimal to mild and characterized by small numbers of seminiferous tubules with partial depletion of one or more generations of germ cells. Four of the seven mice with germinal epithelium cellular depletion also had minimal to mild accumulations of exfoliated germ cells and debris present in the duct of the epididymis. Interstitial cell atrophy resembled was less severe in mice.

Effect levels

1

Key result

Dose descriptor:

LOAEC

Remarks:

systemic

Effect level:

0.44 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

2

Key result

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

0.44 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

0.44 ppm

System:

respiratory system: upper respiratory tract

Organ:

larynx

nasal cavity

trachea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

2

Critical effects observed:

yes

Lowest effective dose / conc.:

3.5 ppm

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

3

Critical effects observed:

yes

Lowest effective dose / conc.:

0.88 ppm

System:

integumentary

Organ:

skin

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

4

Critical effects observed:

yes

Lowest effective dose / conc.:

7 ppm

System:

eye

Organ:

cornea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

5

Critical effects observed:

yes

Lowest effective dose / conc.:

0.44 ppm

System:

male reproductive system

Organ:

testes

other: epididymis

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Results on analytical chemistry: Karl Fischer titration indicated approximately 0.12% water; elemental analyses for carbon and hydrogen were in agreement with the theoretical values for o-phthalaldehyde. GC/FID by system A indicated one major peak that was 99% of the total peak area and three impurities that were each over 0.1% of the total peak area, with a combined total of approximately 1% of the total peak area. Two of the impurities were tentatively identified as toluene and phthalide by comparison of GC retention times to a chromatogram obtained from a standard solution containing possible impurities or degradation products that included toluene, phthalide, benzaldehyde, phthalan, N-hydroxyphthalimide, naphthalene, isophthalaldehyde, and terepthaldicarboxaldehyde. The third peak was not identified. GC/MS by system B indicated that the phthalide peak also contained phthalic acid, which eluted at the same retention time. Different GC columns of varying polarity with FID were used but failed to resolve these two compounds. To ensure the absence of certain degradation products, GC/FID by system A was used to determine the presence of acetonitrile, GC/MS by system B was used for chloroform, and high-performance liquid chromatography with ultraviolet detection (HPLC/UV) was used for 2-carboxybenzaldehyde. Acetonitrile and chloroform were less than 0.1%, and 2-carboxybenzaldehyde was approximately 0.4% by weight. The overall purity of lot 8674J was determined to be greater than 99%. The purity relative to the commercial standard was greater than 99.7%.

Applicant's summary and conclusion

Conclusions:

In a sub-chronic inhalation toxicity study, there were treatment-related effects in male and female mice exposed with ortho-phthalaldehyde. The major targets from ortho-phthalaldehyde exposure in mice included the respiratory system, skin, eye, testis and epididymis. The most sensitive measure of ortho-phthalaldehyde inhalation toxicity in male and female mice was significantly increased incidences of nasal cavity lesions (lowest-observed-effect concentration = 0.44 ppm). In this study, the sytemic and local LOAEC for mice is considered to be 0.44 ppm, which is equal to 2.4 mg/L. No local NOAEC could be observed. The LOAEC is based on the described histopathological findings in the respiratory system and decreased body weights in male and female mice.

Executive summary:

In a sub-chronic toxicity study conducted similar to OECD 413, ortho-phthalaldehyde (>99% purity) was administered to 10 males and 10 female B6C3F1 mice at concentrations of 0, 0.44, 0.88, 1.75, 3.5 and 7.0 ppm via vapor inhalation for 6 h a day, 5 days a week for 14 weeks.

Exposure to ortho-phthalaldehyde caused overt toxicity at the two highest exposure concentrations, resulting in clinical findings of toxicity and death. All mice exposed to 7.0 ppm died during week 1 of the study, and five males and four females exposed to 3.5 ppm died by week 6 of the study. Significantly reduced body weights were observed in male and female mice from 0.44 ppm.

The most significant toxic response to ortho-phthalaldehyde inhalation occurred within the respiratory tract, including the nose, larynx, trachea, and lung. The first site of contact, the nose, was most affected, with many lesions occurring at the lowest exposure concentration (0.44 ppm) in male and female mice. These nasal changes and observed lesions at skin and eye were generally consistent with an irritant effect of ortho-phthalaldehyde. Further major targets from ortho-phthalaldehyde exposure in rats included the testis and epididymis in male mice.

Based on these results, the local and systemic LOAEC for ortho-phthalaldehyde is determined to be 0.44 ppm which is equal to 2.4 mg/L. No local NOAEC could be observed.