Trifluoromethanesulphonic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 July 1994 to 14 October 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Kanpogyo No. 700, Yakuhatsu No. 1039 and 61 Kikyoku No. 1014 dated 5 December 1986.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Remarks:

Kanpogyo No. 39, Yakuhatsu No. 229 and 59 Kikyoku No. 85 dated 31 March 1984 and Kankiken No. 233, Eisei No., 38 and 63 Kikyoku No. 823 dated 18 November 1988 and OECD "Principles of Good Laboratory Practice" (12 May).

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trifluoromethanesulphonic acid

EC Number:

216-087-5

EC Name:

Trifluoromethanesulphonic acid

Cas Number:

1493-13-6

Molecular formula:

CHF3O3S

IUPAC Name:

trifluoromethanesulfonic acid

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Name of test material (as cited in study report): Trifluoromethanesulfonic acid (Alternate name: PFC-MS)
- Molecular formula: CHF3O3S
- Molecular weight: 150.08
- Substance type: Clear, colourless liquid
- Physical state: Liquid
- Stability under test conditions: yes
- Storage condition of test material: Seal in a container such that there is no fear that the contents will spill or leak, and store in a cold and dark
location.
Other:
1- Solubility:
- Water: Dissolves spontaneously
- DMSO: Dissolves spontaneously
- Acetone: Dissolves spontaneously
2- Density: 1.696 g/cm2
3- Melting point: -40°C
4- Boiling point: 162°C
5- Vapour pressure: 1 mmHg

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan Inc.
- Age at study initiation: five weeks old
- Weight at study initiation: ranged from 141.2-160.1 g for male rats and 115.4-133.6 g for females
- Fasting period before study: no data
- Housing: individually reared in stainless steel wire floored cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: eight days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23+/-2°C
- Humidity (%): 55+/-10%
- Air changes (per hr): 10-15 times per hour
- Photoperiod (hrs dark / hrs light): 12-hours light/12 hours dark

IN-LIFE DATES: 6 September 1994 to 13 October 1994

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: the test material was prepared at the appropriate concentrations as a solution in distilled water. Purified water was added in small amounts and agitated to dissolve, preparing a 10w/v% preparation. 2.0, 0.4 and 0.08w/v% were also prepared by diluting the 10 w/v% preparation.
A stability test was performed on the test substance using this preparation method, and it was verified to be stable.

DIET PREPARATION
- Rate of preparation of diet (frequency): once per week
- Storage temperature of food: room temperature

VEHICLE
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): N° 914
- Purity: purified water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The PFC-MS concentration in the test material formulations was determined

Duration of treatment / exposure:

14 days (preliminary study)
28 days (main study)

Frequency of treatment:

once daily (7 days/week)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

8 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of a range-finding study.
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): no data

Positive control:

Not included

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were examined for overt signs of toxicity at least once per day. The day and week counting method was such that the first day of administration was considered day 1, the day before administration started day -1, and the week administration started week 1. Also, the day following the day administartion ended was considered day 1 (recovery), and the week recovery started considered to be week 1 (recovery)

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded in all animals before administration on day-2 (when divided into groups), during the
administration period on day 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 28 and during the recovery period on day 1, 3, 5, 8, 10, 12 and 14. Body weights
were also recorded at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
In all cases, this was measured once before administration and twice per week during the administration and recovery period.

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: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28 and day 14 (recovery period groups)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.7.5.1/2] examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28 and day 14 (recovery period groups)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.7.5.1/3] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Carried out in all animals once during the administration period (day 28) and once during the recovery period (day 14). Urine was collected for a 16-hour period inside individual metabolic cages
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: urinary volume and colour, pH, protein, ketone bodies, bilirubin, occult blood, glucose and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ Weight Measurement: in all animals, the weight of the following organs was measured: brain, liver, spleen, kidney, adrenal gland, testes (or ovaries)
HISTOPATHOLOGY: Yes

Statistics:

Absolute and relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating "F-max" test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal wallis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

* Males: salivation was observed in the 200 mg/kg group (1/6) and the 1000 mg/kg group (12/12); neck hair loss and eschar (2/12) were observed in the 1000 mg/kg group; upper incisor loss and red tears (1/6) were observed in the 200 mg/kg group.

* Females: Salivation was observed in the 1000 mg/kg group (12/12); decreased locomotor activity, abnormal breathing, and abnormal respiratory sound were observed in one animal in the 1000 mg/kg bw/day group; head hair loss and eschar were observed in one animal in the 8 mg/kg group

Salivation observed in the male 200mg/kg group and the male and female 1,000mg/kg groups during the administration period appeared to be temporary directly after administration.

The cases of neck hair loss and eschar often accompany forced oral administration, so they were not thought to be changes caused by PFC-MS.
For the case of decreased locomotor activity, abnormal breathing, and abnormal respiratory sound observed in the 1,000mg/kg group, the animal died two days after. In addition, these kinds of symptoms were not observed in other animals. Due to the symptoms and the fact that blood congestion, dropsy, and intra-alveolar foam cells were observed in the histopathological examination of the lungs, this case was not considered due to the effects of PFC-MS, but rathe to a a mistake in its administration.

No abnormalities were observed in either the males or the females during the recovery period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female treated at 1000 mg/kg bw/day was found died, however, the cause of death was due to a mistake in its administration

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

* Males: a slight decrease but not statistically significant in body weight gain was observed in the 1000 mg/kg group
* Females: no abnormalities were observed at all doses tested.

During the recovery period, no abnormalities were observed in either the males or the females

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No abnormalities were observed in males or females during the administration period or the recovery period.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

* Males:
In the 1000 mg/kg group, a slight decrease but not statistically significant in haemoglobin concentration, haematocrit values, mean corpuscular volume (MCV), and mean corpuscular haemoglobin (MCH) was observed.
In the 8 mg/kg group, an increase in platelet count was observed.
These haematological changes were reversible after a 14-day recovery period except a decrease in mean corpuscular haemoglobin observed in Males in the 1000 mg/kg group at the end of the recovery period.

* Females: an increase in platelet count was observed in the 1000 mg/kg group.
No abnormalities were observed at the end of the recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

* Males:
An increase in cholinesterase was observed in the 200 mg/kg group.
At the end fo the recovery period, an increase in total cholesterol, triglycerides, calcium, inorganic phosphhorus reduction, A/G ratio and chlorine was observed in the 1000 mg/kg group.

* Females:
A statistically significant reduction in total protein was observed in the 1000 mg/kg group. In addition, an increase in GOT, GPT, alkaline phosphatase, γ-GTP, urea nitrogen, and creatinine was observed in one case in this group.
No abnormalities was observed at the end of the recovery period.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

*Males: a reduction in urinary volume was observed in the 8 mg/kg and 200 mg/kg groups. This change was considered as not treatment related.

*Females: an increase in ketone bodies and glucose was observed in one animal at 1000 mg/kg bw/day. We cannot deny that it is not an effect of PFC-MS. However, it is thought that the toxicological significance is small due to the fact that no changes were observed in the kidney or bladder in the histopathological examination.
No abnormalities were observed at the end of the recovery period.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

*Males: an increase in relative kidney weight was observed in the 200 mg/kg group but not statisitacally significant.
At the end of the recovery period, a reduction in relative liver weight and kidney weight was observed in the 1,000mg/kg group but not statistically significant.

*Females: no abnormalities were observed in all treated animals during the administration period and after a 14-day recovery period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
In the 1000 mg/kg group, boundary ridge protrusion in the forestomach (6/6) and reddening of the mucosa in the glandular stomach (1/6) were observed.
In the control group, blackening of the mucosa in the glandular stomach was observed (1/6).
No abnormalities were observed after a 14-day recovery period.

Females:
In the 1000 mg/kg group, adhesion of the spleen to the peritoneum (1/6), kidney fading (1/6), boundary ridge protrusion in the forestomach (6/6), atrophy/roughening of the glandular stomach mucosa (2/6), and blackening of the mucosa (1/6) were observed.
In the 200 mg/kg group, Bbackening of the mucosa in the glandular stomach (1/6) was observed.
In the control group, blackening of the mucosa in the glandular stomach (1/6) was observed.
At the end of the recovery period, pyelectasis (1/5) was observed in the 1,000mg/kg group and blackening of the mucosa in the glandular stomach (1/6) was observed in the conrtol group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males:
> In the 1000 mg/kg group, squamous epithelium hyperplasia in the forestomach (6/6), glandular stomach parietal cell reduction (4/6), surface epithelial cell hyperplasia (3/6), submucosal bleeding (1/6), and loss of villi in the duodenum (2/6) were observed.
> In the 200 mg/kg group, squamous epithelium hyperplasia in the forestomach (1/6) was observed.
> In the control group, liver microgranuloma (2/6) and glandular stomach mucosal necrosis (1/6) were observed.
After a 14-day recovery, liver peripheral hepatocyte enlargement (1/6), and squamous epithelium hyperplasia in the forestomach (1/6) were observed in the 1000 mg/kg group. Liver microgranuloma was observed in the control group (2/6), and in the 1,000 mg/kg group (1/6).

Females:
> In the one case of the female that died in the 1000 mg/kg group, blood congestion of the lungs, dropsy and intra-alveolar foam cells were observed. Autolysis was observed in the forestomach, glandular stomach, duodenum, jejunum, ileum, cecum, colon and rectum.
> In the 1000 mg/kg group, cellular infiltration of the spleen membrane (1/6), liver microgranuloma (1/6), kidney renal tubular necrosis (1/6), urinary casts (1/6), squamous epithelium hyperplasia in the forestomach (1/6, +;2/6), glandular stomach parietal cell reduction (6/6), surface epithelial cell hyperplasia (3/6), mucosal necrosis (1/6), and loss of villi in the duodenum (1/6) were observed.
> In the 200 mg/kg group, glandular stomach mucosal necrosis (1/6) was observed.
> In the control group, glandular stomach mucosal necrosis (1/6) was observed.
At the end of the recovery period, pyelectasis was observed in the 1,000mg/kg group, and kidney sacculation (1/6) and glandular stomach mucosal necrosis (1/6) were observed in the control group.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
MORTALITY:
There were no deaths related to treatment during the study. One female treated at 1000 mg/kg bw/day was found died (day-2), however, the cause of death was not due to the effects of PFC-MS, but rather a mistake in its administration.

CLINICAL SIGNS:
* Males: salivation was observed in the 200 mg/kg group (1/6) directly after administration on day 24 and the 1000 mg/kg group (12/12) either continuously or sporadically only directly after administration starting between day 2 and day 5. In addition, neck hair loss and eschar (2/12) were
observed in the 1000 mg/kg group. Also, upper incisor loss and red tears (1/6) were observed in the 200 mg/kg group.

* Females: Salivation was observed in the 1000 mg/kg group (12/12) either continuously or sporadically only directly after administration starting between day 1 and day 11. In addition, decreased locomotor activity, abnormal breathing, and abnormal respiratory sound were observed in one animal in the 1000 mg/kg bw/day group, these female died two days after the onset of symptoms. Also head hair loss and eschar were observed (1/6) in the 8 mg/kg group.
The salivation observed in the male 200mg/kg group and the male and female 1,000mg/kg groups during the administration period appeared to be
temporary directly after administration, and, as no other related symptoms were observed, it was thought that the toxicological significance was low. Two cases of neck hair loss and eschar were observed in the male 1,000mg/kg group, but these are findings that often accompany forced oral administration, so they were not thought to be changes caused by PFC-MS. Also, one case of decreased locomotor activity, abnormal breathing, and abnormal respiratory sound was observed in the female 1,000mg/kg group, and the animal died two days after the onset of these symptoms. These kinds of symptoms were not observed in other animals, and due to the symptoms in this case and the fact that blood congestion, dropsy, and intra-alveolar foam cells were observed in the histopathological examination of the lungs, it is thought that the cause of death was not due to the effects of PFC-MS, but rather a mistake in its administration.
It was considered that none of these signs of toxicity were related to treatement with the test item.
No abnormalities were observed in either the males or the females during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
* Males: a slight decrease but not statistically significant in body weight gain was observed in the 1000 mg/kg group from day 22 to day 28
* Females: no abnormalities were observed at all doses tested.
No abnormalities were observed in either the males or the females during the recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE
No abnormalities were observed in feed intake for either the males or females during the administation or recovery periods.

HAEMATOLOGY
* Males: in the 1000 mg/kg group, a slight decrease but not statistically significant in haemoglobin concentration, haematocrit values, mean corpuscular volume (MCV), and mean corpuscular haemoglobin (MCH) was observed. In addition, an increase in platelet count was observed in the 8 mg/kg group. These haematological changes were reversible after a 14-day recovery period except a decrease in mean corpuscular haemoglobin (MCH) that was observed in the 1000 mg/kg group at the end of the recovery period.
* Females: an increase in platelet count was observed in the 1000 mg/kg group. No abnormalities were observed at the end of the recovery period.

CLINICAL CHEMISTRY
* Males: an increase in cholinesterase was observed in the 200 mg/kg group. An increase in total cholesterol, triglycerides, calcium, inorganic phosphhorus reduction, A/G ratio and chlorine was observed in the 1000 mg/kg group at the end of the recovery period.
* Females: a statistically significant reduction in total protein was observed in the 1000 mg/kg group. In addition, an increase in GOT, GPT, alkaline phosphatase, y-GTP, urea nitrogen and creatinine was observed in one animal at 1000 mg/kg bw/day. No abnormalities was observed at the end of the recovery period.

URINALYSIS
*Males: a reduction in urinary volume was observed in the 8 mg/kg and 200 mg/kg groups. This change was considered as not treatment related.
*Females: an increase in ketone bodies and glucose was observed in one animal at 1000 mg/kg bw/day. No abnormalities were observed at the end of the recovery period.

ORGAN WEIGHTS
*Males: an increase in relative kidney weight was observed in the 200 mg/kg group but not statisitacally significant. A reduction in relative liver weight and kidney weight was observed in the 1,000mg/kg group but not statistically significant.
*Females: no abnormalities were observed in all treated animals during the administration period and after a 14-day recovery period.
Therefore, no abnormalities were observed at the end of the administration period that were thought to be due to the effects of PFC-MS for either the males or females.

GROSS PATHOLOGY
*Males: Boundary ridge protrusion in the forestomach (6/6) and reddening of the mucosa in the glandular stomach (1/6) were observed in the 1,000mg/kg group, and blackening of the mucosa in the glandular stomach was observed in the control group (1/6). No abnormalities were observed after a 14-day recovery period.
*Females: Blackening of the mucosa in the glandular stomach (1/6) in the 200mg/kg group, adhesion of the spleen to the peritoneum (1/6), kidney fading (1/6), boundary ridge protrusion in the forestomach (6/6. One case in the 1,000mg/kg group presented boundary ridge protrusion in the forestomach, died during the administration period), atrophy/roughening of the glandular stomach mucosa (2/6), and blackening of the mucosa (1/6) were observed in the 1,000mg/kg group. In addition, blackening of the mucosa in the glandular stomach (1/6) was observed in the control group. Pyelectasis (1/5) was observed in the 1,000mg/kg group, and blackening of the mucosa in the glandular stomach (1/6) was observed
in the control group after a 14-day recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC
*Males:
Squamous epithelium hyperplasia in the forestomach (±;1/6) was observed in the 200mg/kg group, and squamous epithelium hyperplasia in the forestomach (±;1/6, +;1/6, ++;2/6, +++;2/6), glandular stomach parietal cell reduction (±;1/6, +;2/6, ++;1/6), surface epithelial cell hyperplasia (+;3/6), submucosal bleeding (+;1/6), and loss of villi in the duodenum (+;2/6) were observed in the 1,000mg/kg group. In addition, liver microgranuloma (+; 2/6) and glandular stomach mucosal necrosis (+;1/6) were observed in the control group.
After a 14-day recovery, In the 1,000mg/kg group, liver peripheral hepatocyte enlargement (+;1/6), and squamous epithelium hyperplasia in the forestomach (±;1/6) were observed. In addition, liver microgranuloma (+;2/6) was observed in the control group, and liver microgranuloma (+;1/6) were observed in the 1,000mg/kg group.

*Females:
In the one case of the female that died in the 1000 mg/kg group during the administration period, blood congestion of the lungs (++), dropsy (+) and intra-alveolar foam cells (+) were observed. Autolysis was observed in the forestomach, glandular stomach, duodenum, jejunum, ileum, cecum, colon and rectum.
Glandular stomach mucosal necrosis (+;1/6) was observed in the 200mg/kg group, and cellular infiltration of the spleen membrane (++;1/6), liver microgranuloma (+;1/6), kidney renal tubular necrosis (++;1/6), urinary casts (++;1/6), squamous epithelium hyperplasia in the forestomach (±;1/6, +;2/6), glandular stomach parietal cell reduction (±;2/6, +;1/6, ++;3/6), surface epithelial cell hyperplasia (+;3/6), mucosal
necrosis (++;1/6), and loss of villi in the duodenum (+;1/6) were observed in the 1,000mg/kg group. In addition, glandular stomach mucosal necrosis (+;1/6) was observed in the control group.
Kidney sacculation (1/6) and glandular stomach mucosal necrosis (+;1/6) were observed in the control group, and pyelectasis was observed in the
1,000mg/kg group at the end of the recovery period.

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on local effects observed in forestomach.

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the test conditions of this study, the NOAEL (males+females) rat is 1000 mg/kg bw/day based on the absence of toxicologically relevant systemic effects. Only minor changes were observed in hematological and biochemical parameters at 1000 mg/kg bw/day. However these observations showed no dose- relation effects and there was no correlation between observations in males and females. Moreover, most changes were reversible after 14 days of recovery and were not accompanied by histopathological changes.

Executive summary:

In a 28 day sub-acute toxicity study (Kanji Yamasaki, 1995), trifluoromethanesulfonic acid (PFC-MS) has been tested for oral toxicity in Crj: CD (SD) rats, according to the Chemical Substance Control Law of Japan (Kanpogyo n° 700, Yakuhatsu n° 1039 and Kikyoku n° 1014; 1986), to OECD guideline n° 407 and in compliance with Good Laboratory Practice.

The test article dissolved in purified water was administered by gavage at dose levels of 0 (vehicle), 8, 40, 200 and 1000 mg/kg bw/day to groups of 6 male and 6 female rats. Six males and six females each were included in supplement in the control and 1000 mg/kg bw/day for a 14-day recovery period. Examinations for mortality, clinical signs, and body weight gain and food consumption were performed. Laboratory investigations (hematology, blood biochemistry and urinalysis) were carried out on Day 29 of the study and Day 15 of the recovery period. All surviving animals were subjected to a complete histopathology examination (organ weight, macro and microscopy) at the end of the treatment and recovery periods.

There were no deaths related to treatment during the study. Salivation was seen in 1/6 male of the 200 mg/kg bw/day groups and nearly all the male and female of the 1000 mg/kg bw/day groups. This clinical sign was considered as not significant and not specific to trifluoromentanesulfonic acid toxicity. No treatment related changes were detected for body weight and food consumption. A slight decrease but not statistically significant in haemoglobin concentration, haematocrit values, mean corpuscular volume (MCV), and mean corpuscular haemoglobin (MCH) was observed in males treated at 1000 mg/kg bw/day. These haematological changes were reversible after a 14-day recovery period except a decrease in mean corpuscular haemoglobin (MCH) that was observed in the 1000 mg/kg group at the end of the recovery period. In Females, an increase in platelet count was observed in the 1000 mg/kg group. No abnormalities were observed at the end of the recovery period. In blood chemistry tests, a reduction in total protein and an increase in urea nitrogen and creatinine were observed in the female 1000 mg/kg bw/day group after the administration period had ended. In urinalysis, an increase in ketone bodies and glucose was obsrevde in the female 1000 mg/kg group. No abnormalities were observed at the end of the recovery period. No abnormalities on organ weight were observed in all treated animals (males + females) during the administration period and after a 14-day recovery period. At necropsy, boundary ridge protrusion in the forestomach and reddening of the mucosa in the glandular stomach were observed in the male at 1000 mg/kg bw/day and boundary ridge protrusion in the forestomach, roughening and blackening of the glandular stomach mucosa, and fading of the kidney were observed in the female group. In the histopathological examination after the administration period had ended, squamous epithelium hyperplasia in the forestomach was observed in male groups of 200 mg/kg bw/day or higher, and a reduction in glandular stomach parietal cells, surface epithelial cell hyperplasia, submucosal bleeding, and loss of villi in the duodenum were observed in the 1000 mg/kg bw/day group. In the females treated at 1000 mg/kg bw/day, squamous epithelium hyperplasia in the forestomach, a reduction in glandular stomach parietal cells, surface epithelial cell hyperplasia, mucosal necrosis, loss of villi in the duodenum, renal tubular necrosis, and urinary casts were observed. In the recovery test, these changes were only observed in the male 1000 mg/kg group.

Based on the experimental conditions of this study:

- The NOEL/male and female rats was to be 40 mg/kg bw/day based on microscopic local effects observed in the forestomach in male groups of 200 mg/kg or higher.

- The NOAEL/male and female rats was to be 1000 mg/kg bw/day based on the absence of toxicologically relevant systemic effects. Only minor changes were observed in hematological and biochemical parameters at 1000 mg/kg bw/day. However these observations showed no dose- relation effects and there was no correlation between observations in males and females. Moreover, most changes were reversible after 14 days of recovery and were not accompanied by histopathological changes.

Based on these results, no classification is required accroding to the EU criteria.