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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000 to 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study according to OECD guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
The were some minor deviations from protocol that are described in the report.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report: T-7479
- Physical state: Clear colorless liquid
- Analytical purity:> 99.9% bt NMR
- Lot/batch no.: F-11925 Lot 2
- Expiration date of the lot/batch: January 2004

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 to 7 weeks old
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C):19.5 to 21.5C
- Humidity (%): 52 and 65%, (usual)
- Air changes (per hr):10
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
Animals were exposed to the test atmosphere in nose-only inhalation units, a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts. SG4 8UB, United Kingdom (see Figure 1). Each unit ­consisted of a cylindrical column, surrounded by a transparent cylinder. The column had a volume of ca. 50 L and consisted of a top assembly with the inlet of the test atmosphere, a rodent tube section and at the bottom the base assembly with the exhaust port. The rodent tube section had 20 ports for animal exposure. Several empty ports were used for test atmosphere sampling and for ­temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), posi­tioned radially through the outer cylinder around the central column. Male and female rats of each group were placed in alternating order. Animals were rotated on a weekly basis.

- Method of holding animals in test chamber:


The rats of each group were placed into their relevant holders after stabilization of the test atmospheres (minimally 10 min after start of the generation of the test atmosphere). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column.­
In our experience, the animal's body does not exactly fit in the animal holder which always results in some leak from high to low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose and dilution of test atmos­phere at the nose of the animals is pre­vented.

- Source and rate of air:
Pressurized clean air


- Method of conditioning air:
The air was humidified

- System of generating particulates/aerosols:
Vapors were generated

- Temperature, humidity, pressure in air chamber:
Standard air pressure and laboratory conditions with humidified air

- Method of particle size determination: not applicable


- Treatment of exhaust air: No reported


TEST ATMOSPHERE


- Brief description of analytical method used:

Total carbon analysis for continual sampling

- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Continual analysis for total carbon
Duration of treatment / exposure:
6 hours per day, 5 days be week for 4 weeks
Frequency of treatment:
5 days, week
Doses / concentrations
Remarks:
Doses / Concentrations:
1000, 4000, 10000, 20000 ppm (v/v)
Basis:

No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale:
- Based upon earlier screening data

Rationale for animal assignment (if not random):


- Rationale for selecting satellite groups:
To document reversibility of any effects

- Post-exposure recovery period in satellite groups:

14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

Time schedule: Each animals was observed daily in the morning. A group-wise observation was made once, about half-way during each day's exposure. All animals were checked again shortly after exposure. During weekends, only one check per day was carried out.


- Cage side observations checked in table were included.
See report.

DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule:
Each animals was observed daily in the morning. A group-wise observation was made once, about half-way during each day's exposure. All animals were checked again shortly after exposure. During weekends, only one check per day was carried out.

BODY WEIGHT:
Body weights were recorded three days before the start of the first exposure, on day before the start of the first exposure, just prior to the start of the first exposure and once per week thereafter. Animals of the recovery groups were also weighted on days 35 and 41.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):

This is not a feeding study but food consumption was recorded and reported in the study.


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):

This is not a drinking water study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes

Haematology was conducted on all rats of the main groups at the end of the exposure period. At scheduled necropsy, blood samples were taken from the abdominal aorta of the (overnight fasted) rats whilst under anaesthesia. The firs ml of blood was collected using potassium EDTA as anticoagulant. In each sample the following determinations were carried out

Haemoglobin
Packed cell volume
Red blood cell count
Reticulocytes
Total white blood cell count
Differential white blood cell count
Prothrombin time
Thrombocyte count


The following parameters were calculated:
Mean corpuscular volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration

Haematological determination were not carried out at the end of the 14-day recovery period.

CLINICAL CHEMISTRY:

After the last exposure, blood was collected from the abdominal aorta of all overnight fasted animals of the mail groups while under ether anesthesia. The following measurements were made in the plasma:

ALP
ASAT
ALAT
GGT
Total protein
Albumin
urea
creatine
glucose
bilirubin
cholesterol
triglycerides
phospholipids
Calcium
Sodium
Chloride
Inorganic phosphate

URINALYSIS: Yes

Urine was collected from selected animals after the last 6 hours exposure for 16 hours.

Urine was analyzed for total fluoride, and creatinine


Sacrifice and pathology:
GROSS PATHOLOGY: Yes


The following organs were examined:
adrenals
brain
heart
kidneys
liver
spleen
stomach
urinary bladder
all gross lesions
testes
lungs
ovaries
bone marrow
eye
pituitary
thyroids
nasal passages

Histopathology was performed on the lungs and liver of all animals of all groups. The other organs were examined in animals of the main control group the high concentration group only.
Statistics:
Statistics were performed using ANOVA, ANCOVA, Kruskal-Wallis and Mann-Whitney U-test, Fisher's exact probability test.

Results and discussion

Results of examinations

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


Few minor effects were noted. All rats survived until their scheduled necropsy

BODY WEIGHT AND WEIGHT GAIN

A decrease in body weight gain developed in males of the high concentration groups which was statistically significant on days 21 and in the recovery group on days 28 and 35. In female animals, such an effect was not seen.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)

Not a feeding study

FOOD EFFICIENCY
Food conversion efficiency was lower in the males of the high concentration group.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)


OPHTHALMOSCOPIC EXAMINATION:
Not performed


HAEMATOLOGY
At the end of the exposure period, changes in red blood cell parameters were limited to an increase in mean corpuscular volume in females of the upper mind concentration group. This was considered unrelated to treatment.


CLINICAL CHEMISTRY

Glucose content was significantly increased in males and females of the upper mid and high concentration groups and also in males of the lower-mid concentration group. Albumin was significantly increased in male animals of the mower mid, upper mid and high concentration groups and the albumin/globulin ratio was significantly increased in the males animals of all exposed groups. Similar increases were not significant in female animals.

Cholesterol levels were decreased in all male exposure groups.
Phospholipid levels were increased in all female exposure groups reaching a significant degree in the upper mid concentration group only. Triglycerides were increased in all exposure groups and statistically significant in the male animals of the lower and upper mid concentration groups. Creatinine was significantly decreased in female animals of the high concentration group. Bilirubin was decreased in female of the lower mid and high concentrations groups.

All findings are recorded in the attached report.

URINALYSIS:

Urinary fluoride concentrations were increased in a dose level response.


NEUROBEHAVIOUR:

No gross behavioral effects were noted


ORGAN WEIGHTS:

Liver weights were effected in the male and female animals at the higher levels. relative lung weights were concentration-related increase in male and female animals of the upper mid and high concentrations groups.


GROSS PATHOLOGY:

No abnormal effects were noted


HISTOPATHOLOGY: NON-NEOPLASTIC

Minor but significant changes in the lung and liver were reported.
The effects are discussed in detail in the report


HISTOPATHOLOGY: NEOPLASTIC (if applicable)

Not Applicable
HISTORICAL CONTROL DATA (if applicable)
OTHER FINDINGS





Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 ppm
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL/LOAEL is 1000 ppm. The effects at this level are due to peroxisome proliferation and are not relevant to human quantitative risk assessment.
Executive summary:

The inhalation toxicity of the test substance was studied in a sub-acute (28-day) study in Wistar rats. Groups of 5 rats per sex were exposed to target concentrations of 0 (control), 1,000, 4,000, 10,000 or 20,000 ppm T-7479 for six hours a day, 5 days a week during a period of 28 days, with a total of 20 exposure days. The rats were necropsied the day after the last exposure. To examine the toxicity of the test material clinical signs, body weights, food consumption, food conversion efficiency, haematology, clinical chemistry, urinalysis and determination of liver peroxisome proliferation were used. In addition, a full necropsy was performed and a selection of organs including the respiratory tract, was examined microscopically.

Two additional groups of 5 rats per sex were exposed to either 0 (control) or 20,000 ppm T-7479 as described above and were kept for a post-treatment period of 14 days to examine reversibility of effects.

The mean actual concentrations ± standard deviation of T-7479 in the test atmospheres were 997 ± 0 ppm, 3,984 ± 1 ppm, 10,018 ± 3 ppm and 19,644 ± 1252 ppm, for the low, lower mid, upper mid and high concentration test atmospheres, respectively. No treatment-related abnormalities in clinical signs or behaviour were seen during the exposure and recovery period. Reduced body weight gain, food intake and reduced food conversion efficiency were observed in male rats of the high concentration group. No treatment-related changes in haematological parameters were noted.

Clinical chemistry parameters revealed an increase, which was not concentration-related, in glucose content in males and females of the upper mid and high concentration groups and in males of the lower mid concentration group. Significantly increased albumin levels were observed in males of the lower mid, upper mid and high concentration groups. The albumin/globulin ratio was significantly, but not concentration-related, increased in all male exposure groups. GGT was significantly increased in females of the high concentration group. Further, a significant increase in triglyceride levels was found in males of the lower and upper mid concentration groups and in females of the upper mid and high concentrations. Cholesterol levels were significantly, but not concentration-related, decreased in males of the low, lower mid and high concentration groups. A tendency towards a decreased cholesterol content was also seen in males of the upper mid concentration group. Phospholipid levels were increased in all female exposure groups reaching a statistically significant degree in females of the upper mid concentration group only. Finally, creatinine was significantly decreased in female animals of the high concentration group.

At the end of the 14-day recovery period, glucose was increased in the exposed animals and the increase was significant in males. Albumin was slightly though significantly increased in exposed male animals, a similar increase in female animals was not significant. The albumin/globulin ratio was significantly increased in exposed male animals and significantly decreased in exposed female animals. Cholesterol was significantly increased in exposed female animals. Phospholipids were increased in both male and female exposed animals. Creatinine was decreased in male exposed animals, a similar decrease in female exposed animals did not reach significance.

An increased mean urinary fluoride concentration was observed in males and females of the upper mid and high concentration groups, and in males of the lower mid concentration group at the end of the treatment period. Accordingly, total fluoride excretion in urine and the urinary fluoride/creatinine ratio were also significantly increased in a concentration-related fashion in these male and female groups. These parameters were still slightly increased when compared to controls at the end of the recovery period.

A strong induction of liver peroxisome proliferation was observed in all male exposure groups, which practically saturated at 4,000 ppm for both lauric acid hydroxylase activity and acyl-CoA oxydase activity. A much weaker effect was observed in female rats of these groups. At the end of the recovery period, the effects of strongly induced peroxisome proliferation had clearly diminished in exposed males and was absent in exposed females.

A concentration-related increase in absolute and relative liver weights was observed in all male exposure groups, and in females of the upper mid and high concentration groups. Increases in absolute and relative lung weights were observed in males and females of the upper mid and high concentration groups. Absolute and relative spleen weights were reduced in female exposure groups and in male animals of the upper mid concentration group, but a concentration-response relationship was not observed. At the end of the recovery period, increases in relative liver and lung weights were still observed in males and females of the high concentration group.

Inhalation of T-7479 induced histopathological changes in the lungs and liver. Alveolar macrophage accumulation was observed in the lungs of males and females of the upper mid and high concentration group. Nucleolar enlargement was observed in the livers of all male exposure groups. A concentration-response relationship was not obtained. At the end of the recovery period, accumulation of alveolar macrophages was still present albeit to a slightly lesser degree. Liver changes were no longer present.

From the results of the present study in rats, it was concluded that exposure to T-7479 even at the lowest concentration used (1,000 ppm) induced hepatic effects, especially, but not exclusively in male rats.

NOAEL/LOAEL is 1000 ppm. The effects at this level are due to peroxisome proliferation and are not relevant to human quantitative risk assessment.