Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
23 October 2006 to 7 March 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary, guideline and GLP compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Terephthalic acid, obtained from Sigma-Aldrich, purity 99.9%, expiry 30.06.2007.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
The animals were male and female Sprague-Dawley rats (Crl:CD(SD)), approximately 4 weeks of age at delivery. They were purchased from Charles River Deutschland. The rats underwent a 5 week acclimatisation period. Starting in the first week of the acclimatisation period, a training programme was performed to familiarise the animals with the exposure tubes for increasing period of time. The rats were observed daily. Body weight and food and water consumption were measured in the last week of the acclimatisation period.
Rats were housed two per cage in Makrolon Type III cages with absorbent softwood as bedding material. Tap water was offered fresh weekly, ad libitum. Food (Sniff R/M-Z, sniff GmbH, Germany) was offered ad libitum. The temperature was maintained at 22±2°C and the relative humidity at 30-70%. Light was provided on a 12 hour light/dark cycle.
Rats were uniquely identified by ID numbers held on the cages. They were placed into groups by weight using a computer-generated randomisation program. The weight variation per sex within or between groups did not exceed ±20% of the mean weight at randomisation.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: The MMAD was 3.25, 2.87 and 2.94 μm for the low, mid and high dose group.
Details on inhalation exposure:
The rats were placed around the exposure cylinder in tapered acrylic glass tubes with adjustable backstops. The animal's snout protruded in the anterior end of the tube, which was connected to the exposure cylinder by way of a push fit. The aerosol entered the nasal region of the animal through a small tube. The exposure cylinder was operated at slightly positive pressure with respect to the surrounding air. This ensured that a continuous air flow was passing through the animal's breathing zone. The exposure units were placed in separate chambers in the animal room to avoid cross contamination between the groups.
The exposure air was supplied by the animal house air conditioning system. The test substance was aerosolized using a microfeeding system and a dispersion nozzle operated with pressurised air. A pre-classifier removing bigger particles was tested and used to adjust the size distribution of the aerosols to MMAD’s about 3 μm. The aerosol concentration was monitored continuously using aerosol photometers developed by Fraunhofer ITEM. The signal was used to control the feed rate of the micro feeder used in order to keep the aerosol concentration in the inhalation unit constant.
Mean temperature in the exposure chamber ranged from 22.4 to 23.4°C. Mean relative humidity ranged from 45.8 to 50.9%.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Actual concentrations were measured in the breathing zone of the animals. For calibration of the photometers the aerosol concentration in each exposure chamber was determined gravimetrically two times per week using filter samples. Additionally the exposure concentration of terephthalic acid was determined two times per week by chemical analysis of these filter samples. The mass median aerodynamic diameter (MMAD) of the aerosol was determined twice during the exposure period for each exposure concentration using a cascade impactor. The mass of each size fraction was determined gravimetrically. All samples were taken through a special sampling port of the inhalation chamber.
Duration of treatment / exposure:
6 hours/day, 5 days/week for 4 weeks
Frequency of treatment:
5 days/week for 4 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1, 3 and 10 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 1.03, 2.93 and 10.05 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
There were 10 males and 10 females per dose, with an additional 5 per sex in the control and high dose groups.
Control animals:
yes, concurrent vehicle
Details on study design:
Four groups were exposued to the substance (or air alone) for 28 days. Two additional groups (one control and one high dose) were allowed a 14 day recovery period after the 28 day exposure period.
Positive control:
Not examined: not required

Examinations

Observations and examinations performed and frequency:
All animals were observed daily before, during and after exposure. In addition, all animals were removed from their cages once a week and carefully examined for abnormalities. Individual body weight, food and water consumption were recorded throughout the study starting 1 week prior to exposures.
Ophthalmologic evaluations were performed with a slit lamp for all groups predose and during the fourth week of exposure for the non-recovery groups, and during week 6 for the recovery groups.
Spontaneous locomotor activity was assessed during the last week of treatment, over 90 minutes using a computerised light-beam system. The total values for distance, time in rest, time in movement, rearing time and number of rearings were determined.
A functional observational battery (FOB) was carried out during the last week of treatment. The following endpoints were determined: forelimb grip strength, righting reflex, body temperature, salivation, startle response, respiration, urination, mouth breathing, convulsions, pineal response, piloerection, diarrhea, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire maneuver, hind-leg splay, tremors, extensor thrust, positive geotropism, activity and limb rotation.
Hematological, clinical chemistry analyses (hematology and serum chemistry), and urinalysis were performed before final sacrifice for all animals (see below).
Sacrifice and pathology:
Animals were anaesthetised with CO2, exsanguinated and necropsied. The physical condition of the animal prior to euthanasia and the examination of the internal organs was described in detail on individual necropsy protocol sheets. In addition to the terminal body weight, selected organs (lung including 2/3 of trachea, heaert, liver, spleen, brain, adrenals, kidneys, testes and ovaries) were weighed (paired organs separately). Relative organ weight data was also computed.
The following organs and tissues were collected and fixed in 10% neutral buffered formalin: adrenals, aorta, bone, brain, cecum, colon, duodenum, epididymides (fixed in modified Davidson's fixative), oesophagus, eyes with optic nerve, forestomach, femur including joint, glandular stomach, harderian glands, heart, ileum, jejunum, submandibular lymph nodes (unilateral), lacrimal glands, larynx, liver, lungs, lung associated lymph nodes (tracheobronchial), mammary glands, mandibular lymph nodes, mesentery and lymph nodes, nasal and paranasal cavities, ovaries (fixed in modified Davidson's fixative), pancreas, parathyroids, peripheral nerve (Sciatic nerve), pharynx, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, testes (fixed in modified Davidson's fixative), thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina and ureters.
Tissues were fixed, embedded in paraffin, sectioned and stained with haematoxylin and eosin. Slides were examined by light microscopy.
Other examinations:
None reported.
Statistics:
All evaluations were done separately for males and females. Differences between groups were considered case by case as statistically significant for p < 0.05. Data were analysed using analysis of variance. If the group means differed significantly according to the analysis of variance, the means of the treatment groups were compared with the means of the control group, using DUNNETT's modification of the t-test. Kruskal-Wallis ANOVA and Mann-Whitney U-test were applied in the case of non - homogeneous data. The statistical evaluation of the histopathological findings was done with the two-tailed FISHER test using the P.L.A.C.E.S system. For comparisons of semi-quantitative data, the chi-square test was used.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality

No deaths occurred.

Signs of toxicity

No adverse clinical signs were observed in the rats during the course of the study.

Bodyweights, food and water consumption

Body weight development was not affected by exposure. There were no compound-related effects on food consumption throughout the course of the study. Total water consumption was significantly lower in the female mid and high dose groups compared to the control group. This effect was not seen in males. The male and female high dose recovery groups showed a higher (not significant) total water consumption compared to the recovery control group. The effect was considered to be of low toxicological relevance.

Ophthalmoscopy

There were no compound-related ophthalmologic findings.

FOB

There were some (mainly transient) effects in some of the locomotor activity parameters of all treated groups, without any dose-response relationship and differently directed in males and females. They were considered to be of low toxicological relevance. Functional Observational Battery: In females, body temperature was significantly lower in the low dose group compared to the control group. In males, forelimb grip strength was significantly increased in the low dose group and the righting reflex score was significantly increased in the high dose group compared to the control group. A similar, but statistically nonsignificant effect was observed for the righting reflex in females of the high dose group. No other treatment induced effects were observed in any of the investigated endpoints. Since only one parameter was affected in the high dose group, this finding may be considered of low toxicological relevance.

Clinical investigations

Relevant differences in the total and differential blood counts were not observed. After the recovery period, the partial thromboplastin time was significantly elongated in the high dose group in both sexes. Male rats in the high dose group showed decreased cholinesterase levels, which can be indicative for decreased synthesis in the liver. Bilirubin levels were significantly decreased in the low dose group in males and females but slightly increased in female rats in the high dose group. In male rats, plasma glucose levels were slightly increased in the low dose group whereas glucose was mildly decreased in the mid and high dose groups. The same trend was seen in female rats, with the decrease in the male and female high dose groups being statistically significant. The changes listed were all reversed in the recovery group and were considered to be of limited toxicological relevance. All other changes were considered to be secondary to biological variance. Furthermore, all values were in the range expected for this species, strain, sex and age.

Relevant changes were not observed in the urinalysis parameters. All values were in the range expected for this species, strain, sex and age.

Necropsy

No compound-related effects were observed during necropsy. Statistical significant differences observed in organ weights and relative organ weights are declared as incidental and therefore of no toxicological relevance.

Statistically significant test substance related histopathological findings were not observed.

Some histopathological lesions were detected in the nasal and paranasal cavities but they were regarded as incidental as they were also seen in some control rats. Inflammatory cell infiltration in the larynx was distributed equally over control and treated groups, and may be mild irritation due to the route of exposure (nose-only inhalation). There were some pulmonary findings such as a relatively high incidence of alveolar macrophage accumulations (alveolar histiocytosis), however the findings were within the limits of normal variation for this strain. Lesions observed in other organs, such as retrobulbar haemorrhage of the eyes, necrosis and inflammation in the Harderian glands or tubular mineralisation in the kidneys were either related to the blood sampling procedure or spontaneous in origin and not unusual for rats of this strain and age.

Effect levels

Dose descriptor:
NOAEL
Effect level:
> 10.05 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were seen at the highest concentration in this study

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The 28 day NOAEL was >10.05 mg/m3

Applicant's summary and conclusion

Conclusions:
No significant inflammatory or other lesions were observed in the terephthalic acid treatment groups under the present experimental conditions.The 28 day NOAEL was >10.05 mg/m3
Executive summary:

The objective of this study was to evaluate the possible toxicity of terephthalic acid after inhalation in rats for 28 days. In addition the effect (degeneration of the tracheal lining epithelium) observed in the previous 28-day study performed by the IIT Research Institute (Leach & Hatoum, 1987) was investigated.

Fifty male and fifty female Sprague Dawley rats (Crl:CD(SD)) , approximately 8 weeks of age at study start were exposed for 6 hrs/day, 5 days/week over a period of 4 weeks to clean air or terephthalic acid at concentrations of 1.03 mg/m³, 2.93 mg/³ and 10.05 mg/m³ for the low, mediu, and high dose group. The mass aerodynamic diameter (MMAD) was 3..25, 2.87, and 2.94 μm for the low, medium, and high dose group. Additionally the study included a control and high dose group with a 14 day recovery period.

No adverse compound-related effects were observed in clinical observations, body weight, food consumption, water consumption, organ weights, and gross pathology findings.

Male rats in the high dose group showed decreased cholinesterase levels, which can be indicative for decreased synthesis in the liver. Bilirubin levels were significantly decreased in the low dose group in males and females but slightly increased in females rats in the high dose group. In male rats, plasma glucose levels were slightly increased in the low dose group whereas glucose was mildly decreased in the medium and high dose groups. The same trend was seen in female rats, with the decrease in the high dose group being statistically significant. The changes listed above were all reversed in the recovery group and are thus considered to be of limited toxicological relevance. Statistically significant treatment-related histopathological lesions were not observed in the main or recovery groups, either in the target organs or in any other organ. All findings in the nasal and paranasal cavities were regarded as incidental. The incidence of inflammatory/mononuclear cell infiltration in the larynx was distributed equally over control and treatment groups. The relatively high incidence of this change might be attributed to mild local irritation due to the experimental setting (nose only inhalation), as the lesion was also frequently observed in the control groups. In almost all cases the cellular infiltrates were located at level 1 of the larynx, i.e. in close vicinity to the nasal and oral cavities. The assumption, that this change might be caused by the experimental setting is also supported by the fact that the incidences of this lesion decreased in the recovery groups as compared to the main groups. The observed changes in the trachea were minimal and all considered to be unrelated to inhalation of TPA. The degenerative lesions observed in a previous inhalation study (LEach & Hatoum, 1987) could consistently not be reproduced in the this study. The pulmonary findings, especially the relatively high incidence of alveolar macrophage accumulations (alveolar histiocytosis) were still within the limits of normal variation in Sprague Dawley (SD) rats, which are known to develop an age-related high incidence of spontaneous alveolar histiocytosis. Lesions observed in other organs, such as retrobulbar haemorrhage of the eyes, necrosis and inflammation in the Harderian glands or tubular mineralization in the kidneys were either related to the blood sampling procedure or spontaneous in origin and not unusual for rats of this strain and age.

In summary, no significant inflammatory or other lesions were observed in the terephthalic acid treatment groups under the present experimental conditions.