Phthalaldehyde

Endpoint summary

Description of key information

Suitable data is available for ortho-phthalaldehyde (target substance). Repeated dose inhalation toxicity studies (sub-chronic, 14 weeks) in rats and mice were conducted within the US NTP programme for ortho-phthalaldehyde. In these studies, a LOAEC (local) is considered to be 2.4 mg/L based on nasal cavity lesions indicating an irritant effect of ortho-phthalaldehyde in mice and rats. In rats, the LOAEC (systemic) is considered to be 4.8 mg/L based on clinical signs, decreased body weights, hematology findings and decreased organ weights. In the lowest concentration group, no adverse systemic effects were observed and the NOAEC (systemic) can be considered to be 2.4 mg/L. In mice, decreased body weights in males and females were observed still in the lowest concentration group. Thus, no NOAEC could be dertermined and the LOAEC (systemic) is considered to be 2.4 mg/L.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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

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

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:

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.

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).

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.

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

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

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

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

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

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

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

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%.

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.

Reference 2

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

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 rats 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; additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 23 days and evaluated for clinical pathology. 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, hematology and clinical chemistry examinations were made.

GLP compliance:

yes

Limit test:

no

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

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 (beginning of vapor generation, 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:

10 male and 10 female rats per dose group, 6 dose groups. In addition, groups of 10 male and 10 female clinical pathology rats were exposed to the same concentrations for 23 days.

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.

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: Yes
- Time schedule for collection of blood: Blood was collected via the retroorbital plexus from surviving clinical pathology rats on days 3 and 23 and from all core study rats surviving to the end of the study for hematology and clinical chemistry
- Following parameters were examined: urea nitrogen, creatinine, glucose, total protein, albumin, cholesterol, globulin, albumin/globulin ratio, triglycerides, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids

HEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected via the retroorbital plexus from surviving clinical pathology rats on days 3 and 23 and from all core study rats surviving to the end of the study for hematology and clinical chemistry
- 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 rats and 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. 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 chamber control rats, male rats exposed to 1.75, 3.5, or 7.0 ppm, and female rats 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, 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 groups.

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).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings in groups exposed to 0.88 ppm or greater generally increased with exposure concentration and 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 rats found dead in the two highest exposure groups (3.5 and 7.0 ppm), there were no clinical findings that preceded death and the probable cause of death was listed as undetermined. Based on microscopic findings, necrosis and inflammation in the respiratory tract may have led to respiratory compromise and death in animals prior to study completion. ortho-phthalaldehyde exposure also induced black discoloration on the distal regions of appendages (pinnae and/or feet) of rats exposed to 0.88 ppm or greater and caused urine and feces within the exposure chambers to turn black.

Mortality:

mortality observed, treatment-related

Description (incidence):

All rats exposed to 7.0 ppm died by the end of week 2 of the study, and seven males and two females exposed to 3.5 ppm died by week 7 of the study. In males exposed to 7.0 ppm, eight rats were found dead in week 1 and two were euthanized in weeks 1 and 2. In females exposed to 7.0 ppm, nine rats were found dead, eight in week 1 and one in week 2, and one was euthanized in week 2. In males exposed to 3.5 ppm, four were found dead in weeks 1 and 2, three were euthanized in weeks 6 and 7, and three survived to study completion. In females exposed to 3.5 ppm, two were euthanized in weeks 3 and 7, and eight 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 males and 1.75 and 3.5 ppm females were significantly less than those of the chamber controls. Final mean body weights for males was 8% (0.44 ppm), 14% (0.88 ppm), 24% (1.75 ppm) and 46% (3.5 ppm) lower and in females 10% (1.75 ppm) and 15% (3.5 ppm) lower.

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):

No hematology or clinical chemistry evaluations were performed on 7.0 ppm rats on day 23 or at week 14 due to mortality and early removal. Total leukocyte and lymphocyte counts were significantly decreased in both male and female rats in various dose groups throughout the study, but most consistently in males exposed to 0.88 ppm or greater and females exposed to 3.5 or 7.0 ppm; neutrophil counts were elevated most consistently in 0.88 ppm males and females. These combinations of leukocyte changes were consistent with a stress response; the increase in neutrophils may have also been due to inflammation. Within the erythron, the erythrocyte counts, hemoglobin concentrations, hematocrit values, and packed cell volumes were significantly elevated in both males and females at all time points and most consistently in those exposed to 0.88 ppm or greater. Reticulocytes were also observed to be significantly elevated at different time points in both sexes. Platelet numbers were significantly increased in the higher exposed groups of male and female rats early in the study, but were decreased by study termination. The increases in platelet numbers were consistent with hemoconcentration, while the decreases may have been due to altered peripheral distribution.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood urea nitrogen and total protein concentrations were significantly increased in the earlier time points. The increase in blood urea nitrogen and total protein ameliorated by the end of the study. At the end of the study, albumin levels were significantly decreased and globulin levels significantly increased in various exposed female groups, which resulted in a significant decrease in the albumin:globulin ratio. These particular biochemical changes were consistent with inflammation as albumin is a negative acute phase protein and globulin a positive acute phase protein. Decreased feed consumption, as evidenced by the decreases in body weight, may have also contributed to the decreases in albumin.
Significant decreases in alkaline phosphatase (ALP) activity, alanine aminotransferase (ALT) activity, and bile acid levels on day 3 in most male groups and female groups exposed to 0.88 ppm or greater were most likely due to decreased feed consumption or altered hepatic metabolism. At study termination, bile acid concentrations were significantly elevated and exposure concentration-dependent increases in ALT and ALP activities were observed in all exposed male groups and in females exposed to 0.88 ppm or greater. Increases in ALT activity are used as a marker of hepatocellular injury, while increases in ALP activity and bile acids are used as markers of cholestasis. Sorbitol dehydrogenase, another marker of hepatocellular injury, was unchanged. The elevations of these liver parameters were relatively mild, and no hepatic lesions were identified on histopathology. Thus, the toxicologic significance of these increases is not known. The increases may suggest mild hepatocellular injury and cholestasis, but could also be related to enzyme induction or altered hepatic function. Creatine kinase activity was significantly elevated in both the male and female 3.5 ppm rats at the end of the study, indicating skeletal or cardiac muscle injury; the reason for muscle injury is not known. Significant increases or decreases in cholesterol, glucose, and triglyceride concentrations were observed throughout the study in both male and female rats. In particular, glucose was elevated in males exposed to 1.75 ppm or greater and was most likely due to stress. Alterations in cholesterol and triglycerides were most likely due to changes in lipid metabolism or decreased food intake.

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 were significantly decreased in 0.44 ppm females (18% lower) and 0.88, 1.75, and 3.5 ppm males (14%, 31%, and 56% lower, respectively) and females (10%, 37%, and 44% lower, respectively), as were the relative thymus weights of 1.75 and 3.5 ppm females. Lymphoid atrophy of the thymus, diagnosed in the 3.5 and 7.0 ppm groups, likely contributed to the decreased thymus weights. In male rats, there were significant decreases in absolute heart, kidney, and liver weights of groups exposed to 0.44 ppm or greater. These organ weight decreases tended to parallel the mean body weight decreases, and the relative organ weights were not significantly decreased at 0.44 or 0.88 ppm for the heart and kidney, or at any exposure concentration for the liver. The decreases in absolute heart, kidney, and liver weights were not considered to be related to chemical exposure, as there were no histopathologic findings in the heart, kidney, or liver corresponding to organ weight decreases. Organ weight data were not available for the 7.0 ppm groups due to 100% mortality.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Exposure to ortho-phthalaldehyde resulted in statistically significant or biologically noteworthy histopathologic changes in the nose, larynx, trachea, lung, skin, eye, spleen, thymus, testis, and epididymis of rats. Inhalation exposure to ortho-phthalaldehyde resulted in a spectrum of lesions at sites of contact within the respiratory tract, skin, and eye that were generally consistent with an irritant effect. With increasing exposure concentration, lesions were observed at increasing depth within the respiratory tract. Another group of histopathologic changes, that were generally consistent with stress, were present in the hematopoietic system. An additional group of findings were present in the male reproductive system. In general, some lesions occurred only in the two highest exposure groups. In contrast, some lesions had lower incidences in the 3.5 and/or 7.0 ppm groups, in which case they were likely associated with the limited exposure duration due to early deaths.

Nose: A main target of ortho-phthalaldehyde exposure was the respiratory tract. The first site of contact, the nose, was most affected. All exposed males and females (except one 0.44 ppm female) exhibited suppurative inflammation of the nose that, in general, increased in severity with increasing exposure concentration. The incidences of turbinate atrophy were significantly increased in males exposed to 0.88 or 1.75 ppm and females exposed to 0.88, 1.75, or 3.5 ppm. Turbinate atrophy is a chronic change that likely did not occur in 3.5 and 7.0 ppm males or 7.0 ppm females due to early deaths.
Microscopically, suppurative inflammation was primarily present in the level I and II histologic sections of the nose and was characterized by variable numbers of viable and degenerate neutrophils within the lamina propria and the epithelium, and neutrophils often accumulated in necrotic debris within the nasal lumen. Turbinate atrophy was present as blunted, thinned, or misshapen turbinates and was most prominent in the level I and II histologic sections and occasionally appeared as shortening of the ethmoid turbinates in the level III section. Several lesions in the olfactory epithelium of the nose were noted in exposed male and female rats. Mild necrosis of the olfactory epithelium was present in a few males exposed to 1.75 or 3.5 ppm, although these incidences were not statistically significant. Hyaline droplet accumulation of the olfactory epithelium, a common background finding, was present in chamber control rats and in rats exposed to 1.75 ppm or less, but absent at the two highest exposure concentrations in males and females, likely due to the early deaths. Alternatively, detection or formation of hyaline droplets could have been obscured or decreased by inflammation or reparative or adaptive changes in the higher exposure groups. Reparative or adaptive changes observed in the olfactory epithelium of the nose included hyperplasia, atrophy, metaplasia, and regeneration. There were low numbers of exposed males and females with minimal hyperplasia of the glands of the olfactory epithelium of the nose. All exposed groups of rats, except 7.0 ppm males, had significantly increased incidences of olfactory epithelium atrophy. The incidences of respiratory metaplasia of the olfactory epithelium in males exposed to 0.88 or 1.75 ppm and females exposed to 1.75 or 3.5 ppm were significantly increased. A few male rats exposed to 0.88 ppm or greater had squamous metaplasia or regeneration of the olfactory epithelium of the nose; the incidence of regeneration was significantly increased in 3.5 ppm males. Microscopically, olfactory epithelial necrosis was noted as focal to focally extensive areas of shrunken, fragmented, or partially sloughed epithelium along either the dorsal aspect in level II histologic sections of the nose or along the dorsal aspect and ethmoid turbinates of level III. Hyaline droplet accumulation was microscopically noted as bright, eosinophilic material in the cytoplasm of olfactory epithelial cells. Hyperplasia of the glands of the olfactory epithelium, present along the nasal septum and the dorsal aspect of level II, was characterized by Bowman’s glands within the lamina propria that were increased in size and number, with increased cytoplasm and cytoplasmic basophilia. Olfactory epithelium atrophy was noted as thinning or reduced numbers of layers of the olfactory epithelium, most often along the dorsal meatus of level II, but occasionally present along the ethmoid turbinates of level III. Respiratory metaplasia of the olfactory epithelium was noted as replacement of the normal olfactory epithelial cells by cuboidal to tall columnar ciliated epithelium, generally along the lateral edges of the dorsal aspect of level II. Squamous metaplasia of the olfactory epithelium was characterized by replacement of the normal olfactory epithelial cells with multiple layers of stratified squamous epithelium that often progressed to keratinization along the superficial surface, primarily in the dorsal aspect of level II. Regeneration was characterized by a single layer of flattened cells that replaced the denuded olfactory epithelium. Several lesions were also diagnosed in the respiratory epithelium of the nose of rats. Respiratory epithelium necrosis increased in severity with increasing exposure concentration in males and females, and incidences were significantly increased in females exposed to 0.88 ppm or greater and in males exposed to 1.75 ppm or greater. Significant increases in the incidences of respiratory epithelium hyperplasia were observed in males and females exposed to 0.44 or 0.88 ppm and in females exposed to 1.75 ppm. There were significantly increased incidences of respiratory epithelium goblet cell hyperplasia in male rats exposed to 0.44 or 0.88 ppm. All exposed females and males, except two males in the 7.0 ppm group, had respiratory epithelium squamous metaplasia; this finding was not present in chamber controls. The incidence of regeneration was significantly increased in males exposed to 7.0 ppm. Microscopically, respiratory epithelium necrosis was comprised of shrunken, fragmented, or partially sloughed epithelial cells covering nasal turbinates and the dorsolateral walls in level I and II histologic sections of the nose. Often, the necrotic foci were associated with fibrin, debris, and neutrophils. Respiratory epithelium hyperplasia was characterized by focal to extensive areas of thickening of the epithelium with nuclear crowding, primarily lining the nasal septum. Goblet cell hyperplasia was primarily present in the respiratory epithelium along the nasal septum at levels I and II. This change was characterized by the proliferation of enlarged, tall epithelial cells containing abundant clear to pale basophilic cytoplasm. Respiratory epithelium squamous metaplasia was characterized by replacement of the normal ciliated respiratory epithelial cells with multiple layers of stratified squamous epithelium that often progressed to keratinization along the superficial surface. Respiratory epithelium regeneration was present as a thin single layer of elongate squamous epithelium that replaced the normal respiratory epithelium.

Larynx: Exposure-related lesions occurred in the laryngeal epithelium and included chronic active inflammation, squamous metaplasia, and necrosis. The incidences of chronic active inflammation were significantly increased in males exposed to 0.88 ppm or greater and females exposed to 1.75 ppm or greater. The incidences of squamous metaplasia were significantly increased in males and females exposed to 0.88 ppm or greater. The incidences of necrosis of the larynx were significantly increased in males exposed to 1.75 ppm or greater and in females exposed to 3.5 or 7.0 ppm. The incidence of regeneration in the larynx was significantly increased in 7.0 ppm males. In general, the severity of the laryngeal lesions increased with increasing exposure concentration.
Microscopically, chronic active inflammation was characterized by numerous neutrophils, lymphocytes, plasma cells, and macrophages that frequently infiltrated the submucosa subjacent to the areas of necrosis. Squamous metaplasia was characterized by replacement of the normal respiratory epithelium of the larynx with multiple thickened layers of cuboidal to flattened epithelium, which often progressed to keratinization along the luminal 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: Exposure-related lesions occurred in the trachea and included chronic active inflammation, squamous metaplasia, necrosis, regeneration, and fibrosis. The incidences of chronic active inflammation and squamous metaplasia were significantly increased in males exposed to 0.88 ppm or greater and females exposed to 1.75 ppm or greater. The incidences of necrosis were significantly increased in male rats exposed to 3.5 ppm or greater and in females exposed to 7.0 ppm. The incidences of regeneration were significantly increased in males and females exposed to 1.75 ppm or greater. The incidences of fibrosis were significantly increased in males exposed to 1.75 ppm and females exposed to 3.5 ppm. Tracheal ulcers were noted in the two highest exposure groups; however, the incidences were not statistically significant. In rats exposed to 3.5 ppm, there was one male and one female with tracheal ulceration; both were moribund sacrifices as a result of abnormal breathing. In rats exposed to 7.0 ppm, there were two males and two females with tracheal ulceration; one male was a moribund sacrifice due to abnormal breathing and the remaining male and both females were found dead. Microscopically, in the trachea, chronic active inflammation was characterized by mixed submucosal infiltrates of lymphocytes, plasma cells, neutrophils, and macrophages. Squamous metaplasia consisted of replacement of the normal respiratory epithelium of the trachea with multiple layers of flattened to cuboidal epithelial cells with increasing cytoplasmic eosinophilia (keratinisation) towards the lumen. Necrosis was seen as hypereosinophilia and loss of differential staining in focal to diffuse areas of respiratory epithelium that often sloughed into the tracheal lumen. 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. Ulcer was characterized by a focal to focally extensive area of epithelial loss that lacked necrotic epithelial layers, fibrin, or necrotic debris overlying the denuded area.

Lung: A spectrum of lesions occurred in the lungs of exposed rats. The incidences of alveolar histiocytic cellular infiltration and alveolar suppurative inflammation were significantly increased in males and females exposed to 3.5 ppm and males and females exposed to 7.0 ppm, respectively. There were two occurrences of interstitial granulomatous inflammation in the lung of female rats exposed to 7.0 ppm. A significant increase in the incidence of perivascular chronic active inflammation occurred in females exposed to 3.5 ppm.
Microscopically, alveolar histiocytic cellular infiltration was comprised of focal accumulations of plump histiocytes in alveolar spaces, whereas alveolar suppurative inflammation was comprised of accumulations of low numbers of neutrophils. Interstitial granulomatous inflammation was characterized by multifocal accumulations of neutrophils, macrophages, and multinucleated giant cells within the interstitium of the lung. Perivascular chronic active inflammation was seen as variable expansion of perivascular spaces by minimal to mild numbers of neutrophils, lymphocytes, plasma cells, and macrophages. In the bronchi, exposure-related lesions occurred in male and female rats only at exposures of 1.75 ppm or greater. Bronchus necrosis and regeneration were only seen in males and females at the two highest concentrations, 3.5 and 7.0 ppm. Significantly increased incidences of bronchus lesions included chronic active inflammation in males and females exposed to 3.5 or 7.0 ppm, necrosis in males exposed to 3.5 ppm and in males and females exposed to 7.0 ppm, and regeneration in females exposed to 7.0 ppm. A few males exposed to 3.5 or 7.0 ppm also had regeneration, but the incidences were not significantly increased. Significantly increased lesion incidences in the bronchus also included hyperplasia in males and females exposed to 3.5 ppm and squamous metaplasia in males exposed to 1.75 or 3.5 ppm and females exposed to 3.5 ppm. Bronchus fibrosis was present in one male and one female exposed to 1.75 ppm, two males and one female exposed to 3.5 ppm, and one male and three females exposed to 7.0 ppm. Microscopically, in the bronchi, chronic active inflammation was seen as variable numbers of neutrophils, lymphocytes, plasma cells, and macrophages within mucus in the bronchi and within the peribronchial connective tissue. Necrosis in the bronchus was noted as epithelium with loss of differential staining with accumulation of fibrin or necrotic cellular debris. Regeneration of the bronchus consisted of a single layer of elongate, thin squamous cells that stretched to cover an area of epithelial loss. In the bronchus, hyperplasia was noted as segmental areas of crowded epithelium with plump epithelial cells and prominent goblet cells. Squamous metaplasia consisted of replacement of the normal respiratory epithelial cells by non-ciliated cuboidal to flattened squamous cells that rarely keratinized and were several layers thick. Bronchus fibrosis included intraluminal and intramural changes. Intraluminal fibrosis included large inflammatory fibrotic polyps or polyploid structures extending into and partially occluding the bronchial lumen, whereas intramural fibrosis was thickening of the bronchial wall by similar connective tissue without projection into the lumen.

Skin: Clinical observations of the skin included black discoloration of pinnae and/or feet in rats exposed to 0.88 ppm or greater. Although skin from pinnae and feet were not available for evaluation in rats, routine inguinal skin sections from all exposure groups were reviewed. Exposure-related lesions in the skin of males and females included adnexa degeneration and hair follicle epithelium parakeratosis. Significant increases in the incidences of adnexa degeneration were noted in males exposed to 3.5 ppm and males and females exposed to 7.0 ppm. Significantly increased incidences of hair follicle epithelium parakeratosis occurred in 0.44, 0.88, and 1.75 ppm males and 7.0 ppm males and females.
Microscopically, skin adnexa degeneration, which was less pronounced in rats than in mice, was seen primarily within the hair follicle epithelium and adnexal structures and was characterized by the intracytoplasmic accumulation of pale eosinophilic to amphophilic material that compressed the nucleus of hair follicle epithelial cells, or by an increase in the amount of individual cell death seen as multiple small, round, and dark pyknotic bodies (apoptotic debris) within areas of single cell epithelial loss. Hair follicle epithelium parakeratosis was minimal to mild and characterized by thickened plaques of keratinizing squamous epithelium with retention of keratinocyte nuclei that often extended down into hair follicles. In interfollicular areas, smaller plaques of material were seen that often did not contain nuclei.

Eye: The eye was another site of contact affected by ortho-phthalaldehyde exposure. Exposure-related lesions included anterior chamber suppurative inflammation, corneal suppurative inflammation, and corneal necrosis. The incidences of anterior chamber suppurative inflammation in 3.5 ppm males and 7.0 ppm males and females and suppurative inflammation and necrosis of the cornea in 7.0 ppm males and females were significantly increased in comparison to the chamber controls. In addition, there were two occurrences of corneal hyperplasia in male rats exposed to 7.0 ppm.
Microscopically, anterior chamber suppurative inflammation was characterized by the presence of neutrophils within the anterior chamber of the eye that often collected subjacent to the corneal endothelium. Suppurative inflammation of the cornea was seen as few to numerous neutrophils present within the corneal epithelium and in rats was often accompanied by anterior chamber suppurative inflammation. Necrosis of the cornea was seen as focal to multifocal thinning of the corneal epithelium due to erosion or ulceration. Corneal hyperplasia was present as focal corneal thickening due to increased layers.

Hematopoietic System: The incidences of lymphoid atrophy of the spleen and thymus were significantly increased in males and females exposed to 3.5 and 7.0 ppm; these lesions were not noted in the three lowest exposure groups (except one 1.75 ppm female). The lesions were more severe in the 7.0 ppm groups than in the 3.5 ppm groups. Five of seven male rats exposed to 3.5 ppm that died within the first 7 weeks of the study had both splenic and thymic atrophy. Lymphoid atrophy of the spleen and thymus are likely attributable to glucocorticoid release-induced lymphocyte death because they occurred in the two highest exposure groups in which there were early deaths. Microscopically, lymphoid atrophy in the spleen was seen as decreased numbers of lymphocytes within follicles, periarteriolar lymphoid sheaths, and mantle zones. Lymphoid atrophy in the thymus was characterized by a diffuse reduction in cortical lymphocytes resulting in an overall decrease in organ size, shrinkage of thymic lobules, and loss of the corticomedullary junction. Decreased thymus weights corresponded to histologic findings of lymphoid atrophy in males and females exposed to 3.5 ppm.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Male Reproductive System: Male rats in the 0.88 and 1.75 ppm groups exhibited lower cauda epididymis (16% and 23% lower), epididymis (14% and 19% lower) and testis (12% and 13% lower) weights compared to chamber controls. Total sperm/cauda exhibited a negative trend (14% and 18% lower in the 0.88 and 1.75 ppm groups, respectively). Sperm motility was lower (10% to 21% lower) in all exposed groups evaluated; the 3.5 and 7.0 ppm groups were not evaluated due to excessive mortality. No histopathologic correlates were identified at exposure concentrations below 3.5 ppm that could explain the observed responses in sperm parameters and decreased testicular and epidydimal weights. At higher exposure concentrations (where morbidity and mortality were observed), testicular and epidydimal histopathologic findings were noted as described below.
Because rats are not sexually mature at 6 weeks, which was the approximate age of rats at the start of the study, male rats exposed to 3.5 or 7.0 ppm that died or were euthanized in the first 10 days of the study (four 3.5 ppm males and all 7.0 ppm males) displayed some microscopic features consistent with sexual immaturity, prompting enhanced evaluations of the testes and epididymides to distinguish any treatment-related findings from findings consistent with sexual immaturity. Some of the features observed in rats that died early were consistent with peripuberty, as observed in the sample of age-matched (45-day-old) untreated rats. These peripubertal features included low numbers of elongated spermatids, especially in stage VII and VIII tubules; relatively small testicular size and tubular lumen diameters as compared to adults; no or negligible sperm in the epididymis; relatively small lumen diameters of the ducts in the distal corpus and cauda of the epididymis; and sloughed germ cells and debris throughout the epididymis. Spermatogenic development in the rats that died early in the two highest exposure groups was also consistent with that described by Picut et al. (2015) for 46-day-old rats. Biologically relevant or statistically significant treatment-related microscopic findings were present in the testis or epididymis of rats in the two highest exposure groups (3.5 and 7.0 ppm). Because animals in the two highest exposure groups died within the first 2 weeks, in addition to the aforementioned peripubertal findings, they exhibited some stage- or cell-specific findings in the testis. Therefore specific diagnoses for findings in the germinal epithelium were favored over the more general diagnosis of germinal epithelium degeneration. The diagnosis of germinal epithelium degeneration encompasses a spectrum of degenerative changes typically seen in animals that survive to the end of a study; such changes are not stage or cell specific. In the testes of rats, incidences of germinal epithelium apoptosis and interstitial cell atrophy were significantly increased in the 3.5 and 7.0 ppm groups. Interstitial cell atrophy occurred in seven 3.5 ppm and all 7.0 ppm males. The incidence of elongated spermatid degeneration was significantly increased in the 3.5 ppm group, occurring in four males. Three 3.5 ppm males that survived to at least 39 days of exposure had unilateral or bilateral degenerative changes in the testes that were characterized by seminiferous tubule cytoplasmic vacuolation, although this increase was not statistically significant. One 3.5 ppm and two 7.0 ppm males had focal germ cell exfoliation in seminiferous tubules near the rete testis. Microscopically, germinal epithelium apoptosis was minimal to mild and characterized by low numbers of round spermatids and pachytene spermatocytes with shrunken, deeply basophilic pyknotic nuclei and condensed hypereosinophilic cytoplasm, almost exclusively in stage VII and VIII seminiferous tubules. Interstitial cell atrophy was generally moderate in severity and noted as decreased numbers and size of interstitial cells; this finding exceeded low numbers/sizes expected due to sexual immaturity, based on comparison to a sample of age-matched untreated animals. Elongated spermatid degeneration was noted as minimal to mild decreases in numbers of elongated spermatids and with clubbing and/or misshapen heads. Seminiferous tubule cytoplasmic vacuolation was minimal to mild and seen as one or more large vacuoles near the periphery of the seminiferous tubule. The vacuolation was often accompanied by focal loss of germ cells from the Sertoli cell cytoplasm and/or evidence of degeneration/depletion of elongated spermatids. These two degenerative changes, seminiferous tubule vacuolation and elongated spermatid degeneration, were sometimes accompanied by exfoliated germ cells and debris in the ductular lumen of the epididymis. Focal germ cell exfoliation in the testis, which was characterized by the presence of rounded, isolated germ cells in the lumen in a few seminiferous tubules adjacent to the rete testis, was also accompanied in one 3.5 ppm rat and two 7.0 ppm rats by exfoliated germ cells and debris in the epididymis. However, the low incidence, focal distribution, and the frequently unilateral nature of this lesion near the rete testis make its relationship to ortho-phthalaldehyde administration uncertain. In the epididymis, there were significant increases in the incidences of exfoliated germ cell within the duct lumen in males exposed to 3.5 or 7.0 ppm. The ductular lumen of the caput epididymis from five 3.5 ppm males and four 7.0 ppm males contained increased numbers of exfoliated germ cells and cell debris, a change that generally reflects the exfoliation of germ cells from the testis. Also within the epididymis, there were significantly increased incidences of epithelial apoptosis in 7.0 ppm males. Apoptosis of the epithelium of the epididymis was sometimes accompanied by two changes in the testis, germinal epithelium apoptosis and interstitial cell atrophy.
Microscopically, exfoliated germ cell within the epididymal duct lumen was seen as mild to moderate amounts of rounded germ cells and debris in the caput epididymis. Apoptosis of the epithelium of the epididymis was minimal to mild and characterized by single shrunken cells lining the duct of the epididymis with deeply basophilic, pyknotic nuclei and condensed, hypereosinophilic cytoplasm.
Some findings in rats exposed to ortho-phthalaldehyde are of unknown toxicologic significance or considered incidental findings. Two 7.0 ppm rats had degeneration of the round spermatids, noted as deeply basophilic ring-shaped nuclei, which indicates acute spermatid degeneration. However, because the change was only present in two rats and both rats were found dead, the change may be associated with moribundity rather than ortho-phthalaldehyde administration. One 1.75 ppm rat had mild testis germinal epithelium degeneration accompanied by mild sloughed germ cells/debris in the epididymis. The diagnosis of germinal epithelium degeneration, rather than specific degenerative changes as diagnosed for males in the two highest exposure groups with early deaths, is more appropriate in this case because degeneration in males surviving to study completion typically encompasses a spectrum of degenerative changes. One 0.88 ppm rat had marked germinal epithelium atrophy associated with absence of sperm in the epididymis. Based on the single occurrences of germinal epithelium degeneration or atrophy in these two exposure groups and the absence of germinal epithelium atrophy in higher exposure groups, these are both considered to be incidental findings.

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

Key result

Dose descriptor:

LOAEC

Remarks:

systemic

Effect level:

0.88 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

haematology

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

0.44 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose

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

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

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

Critical effects observed:

yes

Lowest effective dose / conc.:

0.88 ppm

System:

male reproductive system

Organ:

testes

other: epididymis

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

3.5 ppm

System:

eye

Organ:

cornea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

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%.

Conclusions:

In a sub-chronic inhalation toxicity study, there were treatment-related effects in male and female rats exposed with ortho-phthalaldehyde. The major targets from ortho-phthalaldehyde exposure in rats included the respiratory system, skin, eye, testis and epididymis. The most sensitive measure of ortho-phthalaldehyde inhalation toxicity in male and female rats was significantly increased incidences of nasal cavity lesions (lowest-observed-effect concentration = 0.44 ppm). In this study, the LOAEC (local) for rats is considered to be 0.44 ppm, which is equal to 2.4 mg/L. No local NOAEC could be observed. The LOAEC (systemic) is considered to be 0.88 ppm, which is equal to 4.8 mg/L based on clinical signs, decreased body weights, hematology findings and decreased organ weights. As a result, the NOAEC (systemic) is 0.44 ppm.

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 Sprague Dawley rats 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 of the rats exposed to 7.0 ppm died within the first 2 weeks of the study. Among animals that were exposed to 3.5 ppm, 70% of the male rats and 20% of the female rats died in the first 7 weeks of exposure.

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 rats. 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 rats. From 0.88 ppm, systemic effects as clinical signs, decreased body weights, hematology findings and decreased organ weights were observed.

Based on these results, the LOAEC (local) for ortho-phthalaldehyde is determined to be 0.44 ppm which is equal to 2.4 mg/L. No local NOAEC could be observed. The LOAEC (systemic) is considered to be 0.88 ppm, which is equal to 4.8 mg/L. As a result, the NOAEC (systemic) is 0.44 ppm.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

2 400 mg/m³

Study duration:

subchronic

Species:

mouse

Quality of whole database:

similar to guideline study, GLP study

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

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

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 rats 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; additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 23 days and evaluated for clinical pathology. 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, hematology and clinical chemistry examinations were made.

GLP compliance:

yes

Limit test:

no

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

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 (beginning of vapor generation, 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:

10 male and 10 female rats per dose group, 6 dose groups. In addition, groups of 10 male and 10 female clinical pathology rats were exposed to the same concentrations for 23 days.

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.

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: Yes
- Time schedule for collection of blood: Blood was collected via the retroorbital plexus from surviving clinical pathology rats on days 3 and 23 and from all core study rats surviving to the end of the study for hematology and clinical chemistry
- Following parameters were examined: urea nitrogen, creatinine, glucose, total protein, albumin, cholesterol, globulin, albumin/globulin ratio, triglycerides, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids

HEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected via the retroorbital plexus from surviving clinical pathology rats on days 3 and 23 and from all core study rats surviving to the end of the study for hematology and clinical chemistry
- 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 rats and 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. 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 chamber control rats, male rats exposed to 1.75, 3.5, or 7.0 ppm, and female rats 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, 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 groups.

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).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings in groups exposed to 0.88 ppm or greater generally increased with exposure concentration and 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 rats found dead in the two highest exposure groups (3.5 and 7.0 ppm), there were no clinical findings that preceded death and the probable cause of death was listed as undetermined. Based on microscopic findings, necrosis and inflammation in the respiratory tract may have led to respiratory compromise and death in animals prior to study completion. ortho-phthalaldehyde exposure also induced black discoloration on the distal regions of appendages (pinnae and/or feet) of rats exposed to 0.88 ppm or greater and caused urine and feces within the exposure chambers to turn black.

Mortality:

mortality observed, treatment-related

Description (incidence):

All rats exposed to 7.0 ppm died by the end of week 2 of the study, and seven males and two females exposed to 3.5 ppm died by week 7 of the study. In males exposed to 7.0 ppm, eight rats were found dead in week 1 and two were euthanized in weeks 1 and 2. In females exposed to 7.0 ppm, nine rats were found dead, eight in week 1 and one in week 2, and one was euthanized in week 2. In males exposed to 3.5 ppm, four were found dead in weeks 1 and 2, three were euthanized in weeks 6 and 7, and three survived to study completion. In females exposed to 3.5 ppm, two were euthanized in weeks 3 and 7, and eight 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 males and 1.75 and 3.5 ppm females were significantly less than those of the chamber controls. Final mean body weights for males was 8% (0.44 ppm), 14% (0.88 ppm), 24% (1.75 ppm) and 46% (3.5 ppm) lower and in females 10% (1.75 ppm) and 15% (3.5 ppm) lower.

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):

No hematology or clinical chemistry evaluations were performed on 7.0 ppm rats on day 23 or at week 14 due to mortality and early removal. Total leukocyte and lymphocyte counts were significantly decreased in both male and female rats in various dose groups throughout the study, but most consistently in males exposed to 0.88 ppm or greater and females exposed to 3.5 or 7.0 ppm; neutrophil counts were elevated most consistently in 0.88 ppm males and females. These combinations of leukocyte changes were consistent with a stress response; the increase in neutrophils may have also been due to inflammation. Within the erythron, the erythrocyte counts, hemoglobin concentrations, hematocrit values, and packed cell volumes were significantly elevated in both males and females at all time points and most consistently in those exposed to 0.88 ppm or greater. Reticulocytes were also observed to be significantly elevated at different time points in both sexes. Platelet numbers were significantly increased in the higher exposed groups of male and female rats early in the study, but were decreased by study termination. The increases in platelet numbers were consistent with hemoconcentration, while the decreases may have been due to altered peripheral distribution.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood urea nitrogen and total protein concentrations were significantly increased in the earlier time points. The increase in blood urea nitrogen and total protein ameliorated by the end of the study. At the end of the study, albumin levels were significantly decreased and globulin levels significantly increased in various exposed female groups, which resulted in a significant decrease in the albumin:globulin ratio. These particular biochemical changes were consistent with inflammation as albumin is a negative acute phase protein and globulin a positive acute phase protein. Decreased feed consumption, as evidenced by the decreases in body weight, may have also contributed to the decreases in albumin.
Significant decreases in alkaline phosphatase (ALP) activity, alanine aminotransferase (ALT) activity, and bile acid levels on day 3 in most male groups and female groups exposed to 0.88 ppm or greater were most likely due to decreased feed consumption or altered hepatic metabolism. At study termination, bile acid concentrations were significantly elevated and exposure concentration-dependent increases in ALT and ALP activities were observed in all exposed male groups and in females exposed to 0.88 ppm or greater. Increases in ALT activity are used as a marker of hepatocellular injury, while increases in ALP activity and bile acids are used as markers of cholestasis. Sorbitol dehydrogenase, another marker of hepatocellular injury, was unchanged. The elevations of these liver parameters were relatively mild, and no hepatic lesions were identified on histopathology. Thus, the toxicologic significance of these increases is not known. The increases may suggest mild hepatocellular injury and cholestasis, but could also be related to enzyme induction or altered hepatic function. Creatine kinase activity was significantly elevated in both the male and female 3.5 ppm rats at the end of the study, indicating skeletal or cardiac muscle injury; the reason for muscle injury is not known. Significant increases or decreases in cholesterol, glucose, and triglyceride concentrations were observed throughout the study in both male and female rats. In particular, glucose was elevated in males exposed to 1.75 ppm or greater and was most likely due to stress. Alterations in cholesterol and triglycerides were most likely due to changes in lipid metabolism or decreased food intake.

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 were significantly decreased in 0.44 ppm females (18% lower) and 0.88, 1.75, and 3.5 ppm males (14%, 31%, and 56% lower, respectively) and females (10%, 37%, and 44% lower, respectively), as were the relative thymus weights of 1.75 and 3.5 ppm females. Lymphoid atrophy of the thymus, diagnosed in the 3.5 and 7.0 ppm groups, likely contributed to the decreased thymus weights. In male rats, there were significant decreases in absolute heart, kidney, and liver weights of groups exposed to 0.44 ppm or greater. These organ weight decreases tended to parallel the mean body weight decreases, and the relative organ weights were not significantly decreased at 0.44 or 0.88 ppm for the heart and kidney, or at any exposure concentration for the liver. The decreases in absolute heart, kidney, and liver weights were not considered to be related to chemical exposure, as there were no histopathologic findings in the heart, kidney, or liver corresponding to organ weight decreases. Organ weight data were not available for the 7.0 ppm groups due to 100% mortality.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Exposure to ortho-phthalaldehyde resulted in statistically significant or biologically noteworthy histopathologic changes in the nose, larynx, trachea, lung, skin, eye, spleen, thymus, testis, and epididymis of rats. Inhalation exposure to ortho-phthalaldehyde resulted in a spectrum of lesions at sites of contact within the respiratory tract, skin, and eye that were generally consistent with an irritant effect. With increasing exposure concentration, lesions were observed at increasing depth within the respiratory tract. Another group of histopathologic changes, that were generally consistent with stress, were present in the hematopoietic system. An additional group of findings were present in the male reproductive system. In general, some lesions occurred only in the two highest exposure groups. In contrast, some lesions had lower incidences in the 3.5 and/or 7.0 ppm groups, in which case they were likely associated with the limited exposure duration due to early deaths.

Nose: A main target of ortho-phthalaldehyde exposure was the respiratory tract. The first site of contact, the nose, was most affected. All exposed males and females (except one 0.44 ppm female) exhibited suppurative inflammation of the nose that, in general, increased in severity with increasing exposure concentration. The incidences of turbinate atrophy were significantly increased in males exposed to 0.88 or 1.75 ppm and females exposed to 0.88, 1.75, or 3.5 ppm. Turbinate atrophy is a chronic change that likely did not occur in 3.5 and 7.0 ppm males or 7.0 ppm females due to early deaths.
Microscopically, suppurative inflammation was primarily present in the level I and II histologic sections of the nose and was characterized by variable numbers of viable and degenerate neutrophils within the lamina propria and the epithelium, and neutrophils often accumulated in necrotic debris within the nasal lumen. Turbinate atrophy was present as blunted, thinned, or misshapen turbinates and was most prominent in the level I and II histologic sections and occasionally appeared as shortening of the ethmoid turbinates in the level III section. Several lesions in the olfactory epithelium of the nose were noted in exposed male and female rats. Mild necrosis of the olfactory epithelium was present in a few males exposed to 1.75 or 3.5 ppm, although these incidences were not statistically significant. Hyaline droplet accumulation of the olfactory epithelium, a common background finding, was present in chamber control rats and in rats exposed to 1.75 ppm or less, but absent at the two highest exposure concentrations in males and females, likely due to the early deaths. Alternatively, detection or formation of hyaline droplets could have been obscured or decreased by inflammation or reparative or adaptive changes in the higher exposure groups. Reparative or adaptive changes observed in the olfactory epithelium of the nose included hyperplasia, atrophy, metaplasia, and regeneration. There were low numbers of exposed males and females with minimal hyperplasia of the glands of the olfactory epithelium of the nose. All exposed groups of rats, except 7.0 ppm males, had significantly increased incidences of olfactory epithelium atrophy. The incidences of respiratory metaplasia of the olfactory epithelium in males exposed to 0.88 or 1.75 ppm and females exposed to 1.75 or 3.5 ppm were significantly increased. A few male rats exposed to 0.88 ppm or greater had squamous metaplasia or regeneration of the olfactory epithelium of the nose; the incidence of regeneration was significantly increased in 3.5 ppm males. Microscopically, olfactory epithelial necrosis was noted as focal to focally extensive areas of shrunken, fragmented, or partially sloughed epithelium along either the dorsal aspect in level II histologic sections of the nose or along the dorsal aspect and ethmoid turbinates of level III. Hyaline droplet accumulation was microscopically noted as bright, eosinophilic material in the cytoplasm of olfactory epithelial cells. Hyperplasia of the glands of the olfactory epithelium, present along the nasal septum and the dorsal aspect of level II, was characterized by Bowman’s glands within the lamina propria that were increased in size and number, with increased cytoplasm and cytoplasmic basophilia. Olfactory epithelium atrophy was noted as thinning or reduced numbers of layers of the olfactory epithelium, most often along the dorsal meatus of level II, but occasionally present along the ethmoid turbinates of level III. Respiratory metaplasia of the olfactory epithelium was noted as replacement of the normal olfactory epithelial cells by cuboidal to tall columnar ciliated epithelium, generally along the lateral edges of the dorsal aspect of level II. Squamous metaplasia of the olfactory epithelium was characterized by replacement of the normal olfactory epithelial cells with multiple layers of stratified squamous epithelium that often progressed to keratinization along the superficial surface, primarily in the dorsal aspect of level II. Regeneration was characterized by a single layer of flattened cells that replaced the denuded olfactory epithelium. Several lesions were also diagnosed in the respiratory epithelium of the nose of rats. Respiratory epithelium necrosis increased in severity with increasing exposure concentration in males and females, and incidences were significantly increased in females exposed to 0.88 ppm or greater and in males exposed to 1.75 ppm or greater. Significant increases in the incidences of respiratory epithelium hyperplasia were observed in males and females exposed to 0.44 or 0.88 ppm and in females exposed to 1.75 ppm. There were significantly increased incidences of respiratory epithelium goblet cell hyperplasia in male rats exposed to 0.44 or 0.88 ppm. All exposed females and males, except two males in the 7.0 ppm group, had respiratory epithelium squamous metaplasia; this finding was not present in chamber controls. The incidence of regeneration was significantly increased in males exposed to 7.0 ppm. Microscopically, respiratory epithelium necrosis was comprised of shrunken, fragmented, or partially sloughed epithelial cells covering nasal turbinates and the dorsolateral walls in level I and II histologic sections of the nose. Often, the necrotic foci were associated with fibrin, debris, and neutrophils. Respiratory epithelium hyperplasia was characterized by focal to extensive areas of thickening of the epithelium with nuclear crowding, primarily lining the nasal septum. Goblet cell hyperplasia was primarily present in the respiratory epithelium along the nasal septum at levels I and II. This change was characterized by the proliferation of enlarged, tall epithelial cells containing abundant clear to pale basophilic cytoplasm. Respiratory epithelium squamous metaplasia was characterized by replacement of the normal ciliated respiratory epithelial cells with multiple layers of stratified squamous epithelium that often progressed to keratinization along the superficial surface. Respiratory epithelium regeneration was present as a thin single layer of elongate squamous epithelium that replaced the normal respiratory epithelium.

Larynx: Exposure-related lesions occurred in the laryngeal epithelium and included chronic active inflammation, squamous metaplasia, and necrosis. The incidences of chronic active inflammation were significantly increased in males exposed to 0.88 ppm or greater and females exposed to 1.75 ppm or greater. The incidences of squamous metaplasia were significantly increased in males and females exposed to 0.88 ppm or greater. The incidences of necrosis of the larynx were significantly increased in males exposed to 1.75 ppm or greater and in females exposed to 3.5 or 7.0 ppm. The incidence of regeneration in the larynx was significantly increased in 7.0 ppm males. In general, the severity of the laryngeal lesions increased with increasing exposure concentration.
Microscopically, chronic active inflammation was characterized by numerous neutrophils, lymphocytes, plasma cells, and macrophages that frequently infiltrated the submucosa subjacent to the areas of necrosis. Squamous metaplasia was characterized by replacement of the normal respiratory epithelium of the larynx with multiple thickened layers of cuboidal to flattened epithelium, which often progressed to keratinization along the luminal 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: Exposure-related lesions occurred in the trachea and included chronic active inflammation, squamous metaplasia, necrosis, regeneration, and fibrosis. The incidences of chronic active inflammation and squamous metaplasia were significantly increased in males exposed to 0.88 ppm or greater and females exposed to 1.75 ppm or greater. The incidences of necrosis were significantly increased in male rats exposed to 3.5 ppm or greater and in females exposed to 7.0 ppm. The incidences of regeneration were significantly increased in males and females exposed to 1.75 ppm or greater. The incidences of fibrosis were significantly increased in males exposed to 1.75 ppm and females exposed to 3.5 ppm. Tracheal ulcers were noted in the two highest exposure groups; however, the incidences were not statistically significant. In rats exposed to 3.5 ppm, there was one male and one female with tracheal ulceration; both were moribund sacrifices as a result of abnormal breathing. In rats exposed to 7.0 ppm, there were two males and two females with tracheal ulceration; one male was a moribund sacrifice due to abnormal breathing and the remaining male and both females were found dead. Microscopically, in the trachea, chronic active inflammation was characterized by mixed submucosal infiltrates of lymphocytes, plasma cells, neutrophils, and macrophages. Squamous metaplasia consisted of replacement of the normal respiratory epithelium of the trachea with multiple layers of flattened to cuboidal epithelial cells with increasing cytoplasmic eosinophilia (keratinisation) towards the lumen. Necrosis was seen as hypereosinophilia and loss of differential staining in focal to diffuse areas of respiratory epithelium that often sloughed into the tracheal lumen. 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. Ulcer was characterized by a focal to focally extensive area of epithelial loss that lacked necrotic epithelial layers, fibrin, or necrotic debris overlying the denuded area.

Lung: A spectrum of lesions occurred in the lungs of exposed rats. The incidences of alveolar histiocytic cellular infiltration and alveolar suppurative inflammation were significantly increased in males and females exposed to 3.5 ppm and males and females exposed to 7.0 ppm, respectively. There were two occurrences of interstitial granulomatous inflammation in the lung of female rats exposed to 7.0 ppm. A significant increase in the incidence of perivascular chronic active inflammation occurred in females exposed to 3.5 ppm.
Microscopically, alveolar histiocytic cellular infiltration was comprised of focal accumulations of plump histiocytes in alveolar spaces, whereas alveolar suppurative inflammation was comprised of accumulations of low numbers of neutrophils. Interstitial granulomatous inflammation was characterized by multifocal accumulations of neutrophils, macrophages, and multinucleated giant cells within the interstitium of the lung. Perivascular chronic active inflammation was seen as variable expansion of perivascular spaces by minimal to mild numbers of neutrophils, lymphocytes, plasma cells, and macrophages. In the bronchi, exposure-related lesions occurred in male and female rats only at exposures of 1.75 ppm or greater. Bronchus necrosis and regeneration were only seen in males and females at the two highest concentrations, 3.5 and 7.0 ppm. Significantly increased incidences of bronchus lesions included chronic active inflammation in males and females exposed to 3.5 or 7.0 ppm, necrosis in males exposed to 3.5 ppm and in males and females exposed to 7.0 ppm, and regeneration in females exposed to 7.0 ppm. A few males exposed to 3.5 or 7.0 ppm also had regeneration, but the incidences were not significantly increased. Significantly increased lesion incidences in the bronchus also included hyperplasia in males and females exposed to 3.5 ppm and squamous metaplasia in males exposed to 1.75 or 3.5 ppm and females exposed to 3.5 ppm. Bronchus fibrosis was present in one male and one female exposed to 1.75 ppm, two males and one female exposed to 3.5 ppm, and one male and three females exposed to 7.0 ppm. Microscopically, in the bronchi, chronic active inflammation was seen as variable numbers of neutrophils, lymphocytes, plasma cells, and macrophages within mucus in the bronchi and within the peribronchial connective tissue. Necrosis in the bronchus was noted as epithelium with loss of differential staining with accumulation of fibrin or necrotic cellular debris. Regeneration of the bronchus consisted of a single layer of elongate, thin squamous cells that stretched to cover an area of epithelial loss. In the bronchus, hyperplasia was noted as segmental areas of crowded epithelium with plump epithelial cells and prominent goblet cells. Squamous metaplasia consisted of replacement of the normal respiratory epithelial cells by non-ciliated cuboidal to flattened squamous cells that rarely keratinized and were several layers thick. Bronchus fibrosis included intraluminal and intramural changes. Intraluminal fibrosis included large inflammatory fibrotic polyps or polyploid structures extending into and partially occluding the bronchial lumen, whereas intramural fibrosis was thickening of the bronchial wall by similar connective tissue without projection into the lumen.

Skin: Clinical observations of the skin included black discoloration of pinnae and/or feet in rats exposed to 0.88 ppm or greater. Although skin from pinnae and feet were not available for evaluation in rats, routine inguinal skin sections from all exposure groups were reviewed. Exposure-related lesions in the skin of males and females included adnexa degeneration and hair follicle epithelium parakeratosis. Significant increases in the incidences of adnexa degeneration were noted in males exposed to 3.5 ppm and males and females exposed to 7.0 ppm. Significantly increased incidences of hair follicle epithelium parakeratosis occurred in 0.44, 0.88, and 1.75 ppm males and 7.0 ppm males and females.
Microscopically, skin adnexa degeneration, which was less pronounced in rats than in mice, was seen primarily within the hair follicle epithelium and adnexal structures and was characterized by the intracytoplasmic accumulation of pale eosinophilic to amphophilic material that compressed the nucleus of hair follicle epithelial cells, or by an increase in the amount of individual cell death seen as multiple small, round, and dark pyknotic bodies (apoptotic debris) within areas of single cell epithelial loss. Hair follicle epithelium parakeratosis was minimal to mild and characterized by thickened plaques of keratinizing squamous epithelium with retention of keratinocyte nuclei that often extended down into hair follicles. In interfollicular areas, smaller plaques of material were seen that often did not contain nuclei.

Eye: The eye was another site of contact affected by ortho-phthalaldehyde exposure. Exposure-related lesions included anterior chamber suppurative inflammation, corneal suppurative inflammation, and corneal necrosis. The incidences of anterior chamber suppurative inflammation in 3.5 ppm males and 7.0 ppm males and females and suppurative inflammation and necrosis of the cornea in 7.0 ppm males and females were significantly increased in comparison to the chamber controls. In addition, there were two occurrences of corneal hyperplasia in male rats exposed to 7.0 ppm.
Microscopically, anterior chamber suppurative inflammation was characterized by the presence of neutrophils within the anterior chamber of the eye that often collected subjacent to the corneal endothelium. Suppurative inflammation of the cornea was seen as few to numerous neutrophils present within the corneal epithelium and in rats was often accompanied by anterior chamber suppurative inflammation. Necrosis of the cornea was seen as focal to multifocal thinning of the corneal epithelium due to erosion or ulceration. Corneal hyperplasia was present as focal corneal thickening due to increased layers.

Hematopoietic System: The incidences of lymphoid atrophy of the spleen and thymus were significantly increased in males and females exposed to 3.5 and 7.0 ppm; these lesions were not noted in the three lowest exposure groups (except one 1.75 ppm female). The lesions were more severe in the 7.0 ppm groups than in the 3.5 ppm groups. Five of seven male rats exposed to 3.5 ppm that died within the first 7 weeks of the study had both splenic and thymic atrophy. Lymphoid atrophy of the spleen and thymus are likely attributable to glucocorticoid release-induced lymphocyte death because they occurred in the two highest exposure groups in which there were early deaths. Microscopically, lymphoid atrophy in the spleen was seen as decreased numbers of lymphocytes within follicles, periarteriolar lymphoid sheaths, and mantle zones. Lymphoid atrophy in the thymus was characterized by a diffuse reduction in cortical lymphocytes resulting in an overall decrease in organ size, shrinkage of thymic lobules, and loss of the corticomedullary junction. Decreased thymus weights corresponded to histologic findings of lymphoid atrophy in males and females exposed to 3.5 ppm.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Male Reproductive System: Male rats in the 0.88 and 1.75 ppm groups exhibited lower cauda epididymis (16% and 23% lower), epididymis (14% and 19% lower) and testis (12% and 13% lower) weights compared to chamber controls. Total sperm/cauda exhibited a negative trend (14% and 18% lower in the 0.88 and 1.75 ppm groups, respectively). Sperm motility was lower (10% to 21% lower) in all exposed groups evaluated; the 3.5 and 7.0 ppm groups were not evaluated due to excessive mortality. No histopathologic correlates were identified at exposure concentrations below 3.5 ppm that could explain the observed responses in sperm parameters and decreased testicular and epidydimal weights. At higher exposure concentrations (where morbidity and mortality were observed), testicular and epidydimal histopathologic findings were noted as described below.
Because rats are not sexually mature at 6 weeks, which was the approximate age of rats at the start of the study, male rats exposed to 3.5 or 7.0 ppm that died or were euthanized in the first 10 days of the study (four 3.5 ppm males and all 7.0 ppm males) displayed some microscopic features consistent with sexual immaturity, prompting enhanced evaluations of the testes and epididymides to distinguish any treatment-related findings from findings consistent with sexual immaturity. Some of the features observed in rats that died early were consistent with peripuberty, as observed in the sample of age-matched (45-day-old) untreated rats. These peripubertal features included low numbers of elongated spermatids, especially in stage VII and VIII tubules; relatively small testicular size and tubular lumen diameters as compared to adults; no or negligible sperm in the epididymis; relatively small lumen diameters of the ducts in the distal corpus and cauda of the epididymis; and sloughed germ cells and debris throughout the epididymis. Spermatogenic development in the rats that died early in the two highest exposure groups was also consistent with that described by Picut et al. (2015) for 46-day-old rats. Biologically relevant or statistically significant treatment-related microscopic findings were present in the testis or epididymis of rats in the two highest exposure groups (3.5 and 7.0 ppm). Because animals in the two highest exposure groups died within the first 2 weeks, in addition to the aforementioned peripubertal findings, they exhibited some stage- or cell-specific findings in the testis. Therefore specific diagnoses for findings in the germinal epithelium were favored over the more general diagnosis of germinal epithelium degeneration. The diagnosis of germinal epithelium degeneration encompasses a spectrum of degenerative changes typically seen in animals that survive to the end of a study; such changes are not stage or cell specific. In the testes of rats, incidences of germinal epithelium apoptosis and interstitial cell atrophy were significantly increased in the 3.5 and 7.0 ppm groups. Interstitial cell atrophy occurred in seven 3.5 ppm and all 7.0 ppm males. The incidence of elongated spermatid degeneration was significantly increased in the 3.5 ppm group, occurring in four males. Three 3.5 ppm males that survived to at least 39 days of exposure had unilateral or bilateral degenerative changes in the testes that were characterized by seminiferous tubule cytoplasmic vacuolation, although this increase was not statistically significant. One 3.5 ppm and two 7.0 ppm males had focal germ cell exfoliation in seminiferous tubules near the rete testis. Microscopically, germinal epithelium apoptosis was minimal to mild and characterized by low numbers of round spermatids and pachytene spermatocytes with shrunken, deeply basophilic pyknotic nuclei and condensed hypereosinophilic cytoplasm, almost exclusively in stage VII and VIII seminiferous tubules. Interstitial cell atrophy was generally moderate in severity and noted as decreased numbers and size of interstitial cells; this finding exceeded low numbers/sizes expected due to sexual immaturity, based on comparison to a sample of age-matched untreated animals. Elongated spermatid degeneration was noted as minimal to mild decreases in numbers of elongated spermatids and with clubbing and/or misshapen heads. Seminiferous tubule cytoplasmic vacuolation was minimal to mild and seen as one or more large vacuoles near the periphery of the seminiferous tubule. The vacuolation was often accompanied by focal loss of germ cells from the Sertoli cell cytoplasm and/or evidence of degeneration/depletion of elongated spermatids. These two degenerative changes, seminiferous tubule vacuolation and elongated spermatid degeneration, were sometimes accompanied by exfoliated germ cells and debris in the ductular lumen of the epididymis. Focal germ cell exfoliation in the testis, which was characterized by the presence of rounded, isolated germ cells in the lumen in a few seminiferous tubules adjacent to the rete testis, was also accompanied in one 3.5 ppm rat and two 7.0 ppm rats by exfoliated germ cells and debris in the epididymis. However, the low incidence, focal distribution, and the frequently unilateral nature of this lesion near the rete testis make its relationship to ortho-phthalaldehyde administration uncertain. In the epididymis, there were significant increases in the incidences of exfoliated germ cell within the duct lumen in males exposed to 3.5 or 7.0 ppm. The ductular lumen of the caput epididymis from five 3.5 ppm males and four 7.0 ppm males contained increased numbers of exfoliated germ cells and cell debris, a change that generally reflects the exfoliation of germ cells from the testis. Also within the epididymis, there were significantly increased incidences of epithelial apoptosis in 7.0 ppm males. Apoptosis of the epithelium of the epididymis was sometimes accompanied by two changes in the testis, germinal epithelium apoptosis and interstitial cell atrophy.
Microscopically, exfoliated germ cell within the epididymal duct lumen was seen as mild to moderate amounts of rounded germ cells and debris in the caput epididymis. Apoptosis of the epithelium of the epididymis was minimal to mild and characterized by single shrunken cells lining the duct of the epididymis with deeply basophilic, pyknotic nuclei and condensed, hypereosinophilic cytoplasm.
Some findings in rats exposed to ortho-phthalaldehyde are of unknown toxicologic significance or considered incidental findings. Two 7.0 ppm rats had degeneration of the round spermatids, noted as deeply basophilic ring-shaped nuclei, which indicates acute spermatid degeneration. However, because the change was only present in two rats and both rats were found dead, the change may be associated with moribundity rather than ortho-phthalaldehyde administration. One 1.75 ppm rat had mild testis germinal epithelium degeneration accompanied by mild sloughed germ cells/debris in the epididymis. The diagnosis of germinal epithelium degeneration, rather than specific degenerative changes as diagnosed for males in the two highest exposure groups with early deaths, is more appropriate in this case because degeneration in males surviving to study completion typically encompasses a spectrum of degenerative changes. One 0.88 ppm rat had marked germinal epithelium atrophy associated with absence of sperm in the epididymis. Based on the single occurrences of germinal epithelium degeneration or atrophy in these two exposure groups and the absence of germinal epithelium atrophy in higher exposure groups, these are both considered to be incidental findings.

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

Key result

Dose descriptor:

LOAEC

Remarks:

systemic

Effect level:

0.88 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

haematology

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

0.44 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose

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

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

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

Critical effects observed:

yes

Lowest effective dose / conc.:

0.88 ppm

System:

male reproductive system

Organ:

testes

other: epididymis

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

3.5 ppm

System:

eye

Organ:

cornea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

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%.

Conclusions:

In a sub-chronic inhalation toxicity study, there were treatment-related effects in male and female rats exposed with ortho-phthalaldehyde. The major targets from ortho-phthalaldehyde exposure in rats included the respiratory system, skin, eye, testis and epididymis. The most sensitive measure of ortho-phthalaldehyde inhalation toxicity in male and female rats was significantly increased incidences of nasal cavity lesions (lowest-observed-effect concentration = 0.44 ppm). In this study, the LOAEC (local) for rats is considered to be 0.44 ppm, which is equal to 2.4 mg/L. No local NOAEC could be observed. The LOAEC (systemic) is considered to be 0.88 ppm, which is equal to 4.8 mg/L based on clinical signs, decreased body weights, hematology findings and decreased organ weights. As a result, the NOAEC (systemic) is 0.44 ppm.

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 Sprague Dawley rats 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 of the rats exposed to 7.0 ppm died within the first 2 weeks of the study. Among animals that were exposed to 3.5 ppm, 70% of the male rats and 20% of the female rats died in the first 7 weeks of exposure.

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 rats. 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 rats. From 0.88 ppm, systemic effects as clinical signs, decreased body weights, hematology findings and decreased organ weights were observed.

Based on these results, the LOAEC (local) for ortho-phthalaldehyde is determined to be 0.44 ppm which is equal to 2.4 mg/L. No local NOAEC could be observed. The LOAEC (systemic) is considered to be 0.88 ppm, which is equal to 4.8 mg/L. As a result, the NOAEC (systemic) is 0.44 ppm.

Reference 2

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

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

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

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

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:

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.

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).

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.

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

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

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

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

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

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

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

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%.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

2 400 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

similar to guideline study, GLP study

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Suitable data is available for ortho-phthalaldehyde (target substance).

Sub-chronic repeated dose toxicity studies are available, which were conducted within the US NTP programme for ortho-phthalaldehyde. They do not fully comply with the current respective guidelines due to the lack of e.g. urinalysis data. Instead, clinical signs (rats only), hematology, body weight development, histopathology and sperm motility examinations were performed. The studies were assessed as of high quality, since they were peer-reviewed by a panel nominated by the US NTP.

 

In a sub-chronic toxicity study conducted similar to OECD 413, ortho-phthalaldehyde (>99% purity) was administered to 10 males and 10 female Sprague Dawley rats 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 of the rats exposed to 7.0 ppm died within the first 2 weeks of the study. Among animals that were exposed to 3.5 ppm, 70% of the male rats and 20% of the female rats died in the first 7 weeks of exposure.

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 rats. 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 rats. From 0.88 ppm, systemic effects as clinical signs, decreased body weights, hematology findings and decreased organ weights were observed.

Based on these results, the LOAEC (local) for ortho-phthalaldehyde is determined to be 0.44 ppm which is equal to 2.4 mg/L. No local NOAEC could be observed. The LOAEC (systemic) is considered to be 0.88 ppm, which is equal to 4.8 mg/L. As a result, the NOAEC (systemic) is 2.4 mg/L.

 

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 LOAEC (systemic/local) for ortho-phthalaldehyde is determined to be 0.44 ppm which is equal to 2.4 mg/L. No local/systemic NOAEC could be observed in mice.

Justification for classification or non-classification

Based on the available data, classification as H335 (may cause respiratory irritation) is proposed in accordance to CLP, since the target substance produced irritation of the respiratory tract (nose, larynx, trachea and lung) in a 14-week inhalation study in rats and mice. No classification is warranted for specific target organ toxicity.