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Toxicological information

Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
waiting for details (final report)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed in compliance with the GLP. The purpose of this acute inhalation study was focalized on the determination of the irritant potential of TFA on the upper respiratory tract in rat rather than the determination of the LC50 parameter.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
not specified
Principles of method if other than guideline:
An extended acute toxicity study by inhalation has been performed including three concentrations of exposure for NOAEC determination and relevant additional examinations including broncho-alveolar lavage analysis and upper respiratory tract histopathology.
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Trifluoroacetic acid (TFA)
- Physical state: colorless to yellow liquid
- Lot/batch No.: SFA1010430
- Expiration date of the lot/batch: 13 April 2011
- Stability under test conditions: assumed to be stable during the test (sponsor responsibility)
- Storage condition of test material: ambient temperature (15-25°C)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: first and second range finding studies: 10 weeks; main study: 9 weeks
- Weight at study initiation:
first range finding study: 291.8 g and 199.1 g for the males and females, respectively
Second range finding study: 330.8 g and 189.5 g for the males and females, respectively
Main study: 263.8 g (male animals only)
- Fasting period before study: no
- Housing: macrolon cages with bedding of sterilized wood shavings and strips of paper as environmental enrichment. Three (range finding study) or five (main study) rats were housed to a cage. During the 4-hour exposure periods the rats were individually housed in the inhalation unit.
- Diet (e.g. ad libitum): ad libitum (except during exposure), Rodent diet as pellets (Rat & Mouse No. 3 Breeding Diet, RM3; SDS Special Diets Services, England)
- Water (e.g. ad libitum): ad libitum (except during exposure), domestic main tap-water suitable for human consumption.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs light/ 12 hrs dark

IN-LIFE DATES: From: To:
Arrival of the first group of animals for RF study : 26 May 2010
Exposure of the first group of animals for RF study : 15 June 2010
Sacrifice of the first group of animals for RF study : 16 June 2010
Arrival of the second group of animals for RF study : 23 June 2010
Exposure of the second group of animals for RF study : 14 July 2010
Sacrifice of the second group of animals for RF study : 15 July 2010
Arrival of the animals for the main study : 25 September 2010
Exposure of the animals of the main study : 6 September 2010
Sacrifice of the animals of the main study : 20 September 2010

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed to the test atmosphere in nose-only exposure units consisting of a cylindrical column with a volume of ca. 50 L, surrounded by a transparent hood. The test atmosphere was introduced at the top of the central column, and was exhausted at the bottom. Each column includes two rodent tube sections of 20 ports each for animal exposure. Several empty ports will be used for test atmosphere sampling and measurement of temperature and relative humidity. The remaining ports will be closed. The animals were placed in the exposure unit after stabilization of the test atmosphere.

- Vapor generator: The inhalation equipment is designed to expose the rats to a continuous supply of fresh test atmosphere. To generate the test atmosphere, the test material was evaporated in a tiny flow of dry nitrogen gas and subsequently diluted in a main flow of dry compressed air.

- Exposure chamber volume: 50 L
- Method of holding animals in test chamber: The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Only the nose of the rats protruded into the interior of the column.
- Source and rate of air:, Source: Filtered and dried compressed air; Airflow 36, 17 and 32 L/min in first range finding study, second range finding study and main study, respectively.
- Method of conditioning air: To generate the test atmosphere, the test material was evaporated in
dry air. The resulting test atmosphere will be led to the top of the inhalation chamber and from there to the noses of the animals. The test atmosphere will be exhausted at the bottom.

- System of generating particulates/aerosols: not applicable, because the test material was evaporated in a tiny flow of dry nitrogen gas.
- Method of particle size determination: Although the test material was generated as a vapour, an aerosol mist may be formed due to the hygroscopic character of the test material and the water vapour exhaled by the animals. Therefore, a particle size distribution measurements was carried out.

- Treatment of exhaust air: exhaust air is filtered for particulates
- Temperature, humidity, pressure in air chamber: see details in table 7.2.2/1.
TEST ATMOSPHERE
- Brief description of analytical method used: HPLC analysis
- Samples taken from breathing zone: yes
VEHICLE: not applicable
TEST ATMOSPHERE (if not tabulated)
Not applicable
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Range finding study: 0; 30 and 300 mg/m3
Main study: 0; 30; 100 and 300 mg/m3 (see details in Tables 7.2.2/2 and 7.2.2/3)
No. of animals per sex per dose:
Range finding study: 3 animals/sex/concentration
Main study: 10 animals/concentration (only male rats were tested)
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 1 group (5 males) was sacrificed on the day following the exposure while the other group (5 males) was kept for an observation period of 14 days before sacrifice.
- Frequency of observations and weighing: The general health status of the rats was checked before, during and after the exposure. During the recovery period, the rats were inspected at least once daily. All abnormalities, signs of ill health, reactions to treatment and mortality were recorded. The body weight of each animal was recorded pre-treatment, just prior to exposure (day 0), on days 1, 3, 7 and 14, and at death (if it occurred).
- Necropsy of survivors performed: yes. On the first day following the exposure for all animals of the range finding study and for half of the animals (5 males/concentration) of the main study. On Day 14 for the other half of the animals of the main study.
- Other examinations performed: Macroscopic observations of the thymus and of the testes, Histopathology (see below for details), biochemical and cellular determinations after broncho-alveolar lavage (BAL) (see below for details). Food consumption was not measured.
Histopathology: Lung and the upper respiratory tract including the trachea, the larynx and the nasopharyngeal tissue, liver, testes and kidneys.
Biochemistry in the BAL: Total protein content, alkaline phosphatase (ALP), lactate Deshydrogenase (LDH), N-acetylglucosaminidase (NAG), gamma-glutamyltransferase (gamma-GT).
Cellular determinations in the BAL: total white blood cell numbers
Statistics:
Body weight data were analysed by one-way analysis of variance (ANOVA) at each time point.
Total cell counts, absolute and differential cell counts, relative differential bronchoalveolar cell counts and viability, and, biochemical parameters in broncho-alveolar lavage, were analysed by one-way analysis of variance (ANOVA) after checking for homogeneity of variance (Bartlett test) and normality of distribution (Shapiro-Wilks test). If variances were not homogeneous or data not normally distributed, the data were stepwise log or rank transformed prior to the ANOVA. If the ANOVA yielded a significant effect (p<0.05), inter group comparisons with the control group were made by Dunnett’s multiple comparison test.
Arithmetic means and standard errors of the means or standard deviations were given in the reports of continuous and semi-continuous data. Tests were performed as twosided tests with results taken as significant where the probability of the results was p<0.05 (*) or p<0.01 (**). Because numerous variables were subjected to statistical analysis, the overall false positive rate (Type I errors) was greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results were significant in the light of other biological and pathological findings.

Results and discussion

Preliminary study:
At 30 mg/m3, no significant differences were observed in the evaluated parameters (clinical signs, body weight, BAL) between controls and exposed animals, males or females. Effects after BAL were observed in male and female rats exposed to 300 mg/m3. Indeed, in male animals ALP was increased which showed in the ANOVA as an interaction of the factor sexe. The total protein dosage was increased in the highest tested concentration (300 mg/m3). Cellular parameters showed an increase in neutrophils in the broncho-alveolar lavage fluid of male animals of group animal treated with 300 mg/m3reflected in significant group and sex factors in the ANOVA (p<0.05), a tendency for an interaction. No other parameters were affected. As male animals seemed more sensitive to the effects of exposure to trifluoroacetic acid, they were used for the main study. Based on these results, the concentrations for the main study were chosen to be 30, 100 and 300 mg/m3. Furthermore, it was assumed that at this highest concentration of 300 mg/m3, histopathological findings would be observed.
Effect levelsopen allclose all
Sex:
male
Dose descriptor:
other: NOAEC
Effect level:
ca. 300 mg/m³ air (nominal)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: Irritation effects on the upper respiratory tract
Sex:
male
Dose descriptor:
other: NOAEC
Effect level:
>= 327.7 - <= 344.1 mg/m³ air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: Irritation effects on the upper respiratory tract
Mortality:
No mortality was observed at the tested concentrations during the study (see table 7.2.2/4)
Clinical signs:
other: No clinical abnormalities were found in any of the animals during the study.
Body weight:
No differences in body weight were found between exposed animals and control animals (see table 7.2.2/5).
Gross pathology:
The macroscopic examination 1 day after exposure and after the recovery period of 14 days, could not establish any treatment-related changes. All changes observed were common findings for rats of this strain and age. There were no effect neither on testes nor on thymus.
Other findings:
- Organ weights: not measured
- Histopathology: At the microscopic level, the examination revealed treatment-related histopathological changes at the second and third levels of the rat nasal cavity within the day following the end of exposure (see attached document: Scheme of the rat hard palate region). The histopathological lesion consisted of very slight focal degeneration of the respiratory epithelium lining the dorsal part of the septum. This lesion was only observed at the highest tested concentration in 4/5 animals for the second level of the nasal cavity and for 1/5 animal for the third level. Furthermore, this effect was reversible as there was no more irritation of the nasal cavity (all levels considered) in the animals necropsied 14 days after treatment. All other changes observed were about equally distributed amongst the various groups, or they occured in one or a few animals only. Moreover, these changes were considered common for rats of the strain and age used.
- Potential target organs: no effect
- Other observations: No significant differences were found between the control group and the exposed groups in any of the biochemical parameters in broncho-alveolar lavage on any necrospsy day. In the animals necropsied on day 1, total cells (both relative and absolute) were decreased in the mid concentration group, but not in the high concentration group. The number of mononuclear macrophages was also decreased in animals of the mid concentration group. These differences were likely to be related.Since this effect was not seen in animals of the high concentration group (no dose-response), this effect was not considered to be treatment related. In the animals necropsied on day 14, no significant differences between control group and the exposed groups were found in the cellular parameters in broncho-alveolar lavage.

Any other information on results incl. tables

Table 7.2.2/4: Mortality after a four-hour inhalation exposure

Sex

Exposure group

Mortality (Number of dead animals/Number of animals)

Time of death

Male

Low dose

0/10

NA

Middle dose

0/10

NA

High dose

0/10

NA

NA: Not Applicable

Table 7.2.2/5:Body weight (in g) after a four-hour inhalation exposure (Mean of the body weight of the 10 males/[TFA] for the measurement at day -3, 0 and 1 and Mean of the body weight of the 5 animals/[TFA] for the measurement at day 3, 7 and 14)

 

Day numbers relative to start date

Exposure group

Day -3

Day 0

Day 1

Day 3

Day 7

Day 14

Control

250.05 ± 11.36

263.03 ± 12.45

261.99 ± 11.02

266.54 ± 9.06

287.20 ± 10.12

307.86 ± 10.73

Low dose

249.36 ± 8.22

262.42 ± 8.56

258.53 ± 8.87

262.84 ± 12.59

279.12 ± 13.01

297.24 ± 13.66

Middle dose

251.28 ± 9.05

266.37 ± 10.01

262.92 ± 11.22

271.24 ± 12.43

290.28 ± 11.82

311.52 ± 15.43

High dose

251.56 ± 7.84

263.35 ± 9.99

261.90 ± 10.28

263.10 ± 5.32

281.30 ± 9.65

302.94 ± 10.63

 

Applicant's summary and conclusion

Interpretation of results:
other: NOAEC determination
Remarks:
Criteria used for interpretation of results: expert judgment
Conclusions:
Under the test conditions, TFA induced transient irritation effects of the nasal cavity epithelium at the highest tested concentration (300 mg/m3). Therefore the NOAEC was considered to be 300 mg/m3.
Executive summary:

An extended acute inhalation toxicity study was performed similarly to the OECD guideline No. 403, including three concentrations of exposure for NOAEC determination, and relevant additional examinations including broncho-alveolar lavage (BAL) analysis, upper respiratory tract histopathology including the lung analysis, and histopathology of the liver, the kidneys, the testis and the thymus. This study was conducted in compliance with the GLP.

In a preliminary study, male and female Wistar rats (3 animals/sex/concentration) were exposed to vapours of TFA (0; 30 or 300 mg/m3) according to a nose only exposure. At 30 mg/m3, no significant differences were observed in the evaluated parameters (clinical signs, body weight, BAL) between controls and exposed animals, males or females. Effects after BAL were observed in male and female rats exposed to 300 mg/m3. Indeed, in male animals ALP was increased which showed in the ANOVA as an interaction of the factor sexe. The total protein dosage was increased in the highest tested concentration (300 mg/m3). Cellular parameters showed an increase in neutrophils in the broncho-alveolar lavage fluid of male animals of group animal treated with 300 mg/m3reflected in significant group and sex factors in the ANOVA (p<0.05), a tendency for an interaction. No other parameters were affected. As male animals seemed more sensitive to the effects of exposure to trifluoroacetic acid, they were used for the main study. Based on these results, the concentrations for the main study were chosen to be 30, 100 and 300 mg/m3. Furthermore, it was assumed that at this highest concentration of 300 mg/m3, histopathological findings would be observed.

In the main study, the exposure method was unchanged compared to the preliminary study. Two groups of 5 males/concentration were therefore exposed to TFA vapours at concentrations of 0; 30; 100 or 300 mg/m3for 4 hours. The animals of the first group/concentration were sacrificed and necropsied one day after the end of the exposure, while the animals of the second group/concentration were sacrificed after a 14-day observation period. This second group was designed in order to study the reversibility of the effects which would be observed at day 1.

Clinical signs were observed, body weight (at day 0, 1, 3, 7, and 14) was measured, gross pathology (at the sacrifice or death of animal) and histopathology were analysed. In addition a broncho-alveolar lavage was performed on the animals of the both groups in order to analyse biochemical parameters such as the total protein content, the alkaline phosphatase (ALP), the lactate Deshydrogenase (LDH), the N-acetylglucosaminidase (NAG) and the gamma-glutamyltransferase (gamma-GT). Furthermore thetotal white blood cellnumber was determined in the BAL.

No mortality nor clinical signs or effect on body weight gain were observed at the tested concentrations during the study. The macroscopic examination of the testes and the thymus performed both one day after exposure and after the 14-day recovery period, could not establish any treatment-related changes. All changes observed were common findings for rats of this strain and age.

At the microscopic level, the examination revealed treatment-related histopathological changes at the second and third levels of the rat nasal cavity within the day following the end of exposure. The histopathological lesions consisted of a very slight focal degeneration of the respiratory epithelium lining the dorsal part of the septum. It was considered as the consequence of irritating local effects of the TFA vapours. This lesion was only observed at the highest tested concentration (300 mg/m3) in 4/5 animals for the second level of the nasal cavity and in 1/5 animal for the third level. Furthermore, this irritating local effect was reversible as there was no irritation of the nasal cavity (all levels considered) in the animals necropsied 14 days after treatment. The histopathological analyses revealed no effect of TFA vapours on the trachea, larynx, lungs, kidneys, liver and testes. Furthermore, no treatment-related differences were recorded between the control group and the TFA exposed groups in any of the biochemical and cellular parameters after the broncho-alveolar lavage on both day 1 and 14 recovery periods.

In conclusion, TFA induced no mortality at the tested concentrations (up to 300 mg/m3). However, at the highest concentration, TFA induced local irritation of the nasal cavity epithelium of the rats. This effect is reversible since no irritation of the nasal cavity was noted in the animals necropsied at the end of the observation period. The NOAEC for local effects based on the nasal cavity epithelium irritation is therefore 300 mg/m3.