Hexafluoropropene, oxidized, oligomers, reduced, fluorinated

Endpoint summary

Description of key information

The effects of GALDEN LMW following repeated administration by oral route were evaluated in a 28-day repeated dose toxicity study (OECD TG 408, GLP). The NOEL was > 1000 mg/kg/day (male/female). No adverse effects were noted.
The effects following repeated exposure by inhalation were evaluated basing on the read across approach with the analogue substance H GALDEN, which was tested under a 28-day repeated dose toxicity study and a 90-day repeated dose toxicity study.
Under all the reported studies, no adverse effects related to toxicity were detected.
In conclusion GALDEN LMW is considered to have a low toxicity following repeated exposure both by oral route and by inhalation.

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1992 to August 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

some missing details and examinations

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(Paris, 1981)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: livestock farming
- Age at study initiation: males: 30 days, females: 25 days
- Weight at study initiation: males: 75-85 g, females: 60-70 g
- Fasting period before study: not reported
- Housing: 2 or 3 animals/sex/cage. Wire cages with stainless steel feeder. Cage size(cm):40.5 x 38.5 x 18h
- Diet (e.g. ad libitum): Certificated pelleted diet, supplied with vitamins and trace elements. Available ad libitum.
- Water (e.g. ad libitum): from municipal water main system, filtered and distributed ad libitum.
- Acclimation period: two weeks, the rats were housed in a quarantine room an their health status was assessed by daily clinical observations.

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C +- 2°C
- Humidity: 55 % +- 10%
- Air changes (per hr): approximately 20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hour cicle (7 a.m.-7 p.m.)

IN-LIFE DATES:
Males: From: 8-Jul-1992 To 5-Aug-1992
Females: From 9-Jul-1992 To 6-Aug-1992

Route of administration:

oral: gavage

Vehicle:

other: 0.5 (w/v) Methylcellulose 400 cps water solution additioned with 0.25% (w/v) Polysorbate 80 (Tween 80)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Every day appropriate amounts of substance were weighed and emulsified with vehicle in suitable containers in order to obtain the final concentration of 25, 50, 100 mg/ml to be administered, respectively, to the animals of tested groups at constant administration volume of 10 ml/kg b.w. . The suspension were kept magnetically stirred until the end of the daily administration.
Formulates were prepared just prior to the administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported
- Concentration in vehicle: 25, 50 100 mg/ml at the doses of 250, 500 and 1000 mg/kg/day, respectively.
- Amount of vehicle (if gavage): 10 ml/kg b.w.
- Lot/batch no. (if required): Methylcellulose 400 cps: Batch No.300535, Polysorbate 80 (Tween 80) Batch No.7245049
- Purity: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for concentration check were collected from formulates prepared on July 8, 1992 (week 1) and July 30, 1992 (week 4) and stored at -20 °C until dispatch to the Sponsor for analysis.

Duration of treatment / exposure:

28 days, no recovery.

Frequency of treatment:

once a day, for 7 days a week, for the entire study period.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control group

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not reported; highest dose is the highest recommended dose.
- Rationale for animal assignment: Randomization.
The 40 (20M + 20F) rats were selected from a larger goup (27M + 27F) than the one required: before commencement of treatment all the animals were weighed: animals at extreme of the body weight range or showing eye abnormalities at the pre-trial examination were discarded.
The required number of rats (20M + 20F) was allocated to the dosage groups by means of a computerized stratified sequenced randomization program.

Positive control:

Not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily Intervals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily Intervals. Physical appearance, bahaviour and clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly Intervals. Each animal was weighed prior to the beginning of the treatment period (day 0, the day before the first administration) and then at weekly intervals throughout the study period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable
Food consumption was recorded, for each cage, at weekly intervals throughout the study period. Food comsuption was calculated as the difference between the known offered amount per cage and the remainder recorded after 7 days. Individual food intake was then calculated, in g/animal/day, and reported in tables, for each 7-day 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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Water consumption was not measured
Compound intake calculation: not applicable

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment (males: day -1, females: day -2) and before the end of the dosing period (males: day 27, females day 27)
- Dose groups that were examined: All animals
Eyes were examined with a direct ophtalmoscope, after instillation of 0.5% Tropicamide.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of dosing period, day 29.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: All animals
- The following parameters were examined: Erythrocytes, Hemoglobin, Leukocytes, Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Hematocrit, Mean corpuscolar volume, Mean corpuscolar HGB conc, Mean corpuscolar HGB, Platelets, Prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of dosing period, day 29.
- Animals fasted: Yes
- How many animals: All animals
- The following parameters were examined: Glucose, Urea, Creatinine, Total bilirubin, Alkaline phosphatase, SGOT, SGPT, Total protein, Albumin, Globulin, A/G ratio, Alpha 1 globulins, Alpha 2 globulins, Beta globulins, Gamma globulins, Total cholesterol, Sodium, Potassium Calcium, Chloride, Inorganic phosphorus.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of dosing period, day 29.
- Metabolism cages used for collection of urine: Yes.
- Animals fasted: Yes (16 hours)
In order to collect urine samples, on the day before the scheduled analysis the animals received 10 ml/kg of tap water (by gavage) as water load; subsequently they were kept in metabolism cages for about 16 hours without food and water.
- The following parameters were examined: Diuresis, Specific gravity, pH, Protein, Bilirubin, Glucose, Ketones, Blood, Leukocytes, Urobilinogen, Nitrites, Epithelial cells, Leukocytes, Erythrocytes, Phosphate crystals, Urate crystals, Calcium oxalate crystals, Casts, Bacteria.

NEUROBEHAVIOURAL EXAMINATION: No (not part of the test guideline at the time of the study)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the treatment period the body weight of each animal that had been fasted overnight was recorded before the animal was killed .

The following organs were removed:
skin and mammary gland, urinary bladder, prostate, testes, epididymides, seminal vesicles, vagina, uterus, ovaries, spleen, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, mesenteric lymph nodes, pancreas, liver, kidneys, adrenals, submandibular salivary glands and lymph nodes, sternum with bone marrow, heart, thymus, lungs, aorta, trachea, esophagus, thyroid with parathyroid if present in the thyroid section, eyes, Harder's lacrimal glands, tongue, brain: coronal section at threee levels, pituitary, skeletal muscle: biceps femoris, peripheral nerve: sciatic nerve, spinal cord: thoracic, cervical and lumbar, vertebrae, femur including articular surface, gross lesions.

The following organs were removed, trigger and weighed:
testes, ovaries, spleen, liver, kidneys, adrenals, heart, brain
Individual organ weight/fasted body weight ratio was calculated.

HISTOPATHOLOGY: Yes
Histology was carried out on the following organs:
Spleen, stomach, duodenum, ileum, colon, liver, kidneys, adrenals, heart, and gross lesions found at macroscopic examination.

Statistics:

The parameters statistically examined were:
- body weight
- body weight gain
- food intake
- hematology
- blood chemistry
- urinalysis (except the semi-quantitative analysis and the microscopic examination of the sediment of the urine)
- organ weights (absolute and relative to body weight)

The above-reported data were compared and tested according a decisiontree which includes Bartlett's test, ANOVA, Dunnett's multiple test and Mann Withney's test.
Level of significance was reported for all determinations.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical changes were seen at any dose in either males or females.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No body weight changes of toxicological relevance were seen in either males or females at any dose and no variations from the control values that attained statistical relevance were recorded at any time.
A very slight retarded growth was however observed in animals treated at the highest dosage (1000 mg/kg/day). In this group, when compared to the basal body weight, a mean body weight gain slightly lower than controls was observed in males starting from week 1 onward with evidence of a time relationship (from 3% at week 1 to 8% at week 4); the same trend, even though confined to the final weighing (week 4) was seen in females for which the differential value of about 5% was mainly related to the low body weight gain of 2 out of the 5 dosed rats.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects on the quantity of food consumed that could be related to treatment were seen in either males and femals; the mean food intake data were similar in all the experimental groups and there were no individual values outside the norm.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No eye changes were seen in any animal.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes in haematologic profile of toxicological relevance were seen at any dose in either males and females at the investigation performed at the end of the dosing period. The statistical analysis did not reveal any significant difference from the control values in females treated at any dose or in male treated at 250 and 500 mg/kg/day.
In males given 1000 mg/kg/day the mean 1000 RBC number, hemoglobin, and hematocrit values were slightly higher than in controls; the changes significant at the statistical analysis, involved most of the animals of this group (Incidence: 80% ) with values above the range of the controls, but again within the normal range of variability. Moreover, since the there were no changes in the erythrocyte indices, these slight modifications were considered of no toxicological relevance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No blood chemistry changes that could be related to GALDEN LMW administration were seen at any dose in either males and females.
The statistical analysis highlighted a few significant differences from the control values in animals treated at 250 and 1000 mg/kg/day: in males sodium and chloride ion levels were slightly higher at both doses while in females a glucose level higher (at 250 mg/kg/day) or total birilubin and SGOT values lower (at 1000 mg/kg/day) were observed. Since these modifications were very slight and without any individual value outside the normal range of variability they were considered incidental in origin.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No compound-related changes were seen.
For the parameters statistically analysed (diuresis, specific gravity and pH), no significant differences from the control value were seen in either males or females treated at any dose.
Assessment of the semiquantitative analysis and sediment microscopy did not reveal any notable differences from controls in any group; the positive reactions for proteins in the urine of 3 out of the 5 male rats treated at 500 mg/kg/day were considered incidental since the findings was always slight in degree (grade 1), confined to this dose only, and without other concurent urinary parameter modifications.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

None of the noted organ weight variations were regarded as related to the compound treatment. In fact the range of these changes were included within the accumulated control backgroud data in the laboratoty.
In particular, the absolute and relative adrenal weight increase observed in all male treated groups is regarded as of no relation to treatment. In fact the individual values were included within range of individual adrenal weight seen in control rats of other experiments performed in the laboratory.
In addition no treatment related histological alteration was noted in this organ. The reduction in absolute testicular weight observed in the high dosage group was caused by an incidental reduction in tested weight of a single rat. This testicular reduction in weight was sporadically noted in control rats of the same age used in other experiments in the laboratory.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were a range of lesions observed in control and treated groups, none of them was regarded as related to test article treatment.
In fact, the incidences were comparable among treated and control rats and/or they were characteristically observed in untreated rats of the same age used as controls in other experiments carried out in the laboratory.
Examples of noted lesions included: hepatic accentuation of lobular pattern, presence of whitish areas and congestion in various organs.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

None of the observed lesions is regarded as related to the compound administration.
All observed lesions had comparable incidence in control and treated groups and/or they are characteristically noted in untreated rats of the same age used in other experiments carried out in the laboratory. Among the most frequently observed lesions were hepatocytic mononuclear cell aggregation (incidence: 30%), hepatocyte vacuolation (incidence: 23%) and renal tubular basophilia (incidence: 6%).
In particular one female rat treated with the high dose had a single focus of degeneration with inflammation in the heart (myocard). This lesion is sporadically noted in control rats of the same age. Due to its rare occurrence in the present study, it is concluded to be of no relation to the compound administration.
Moreover, congestion or hemorrhage and edema seen in the cecum of group 4 female no. 5661 and in the prostate of goup 1 male no. 5630 were considered incidentally induced at autopsy by needle trauma during the intraperitoneal injection of barbiturate to induce anesthesia.
Abdominal cavity subacute inflammation seen in group 4 female no 5664, consistent at gross pathology examination with a hernia, is an event that can sporadically occur also in untreated rats.

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed at the limit dose

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

No compound-related deaths, clinical signs or abnormalities were observed. Body weight showed only a very slight retarded growth at the highest dose which was considered of no toxicological relevance in absence of other effects. Food intake was unaffected by treatment.
Laboratory investigations and post-mortem examinations did not reveal changes attributable to GALDEN LMW administration.
In conclusion, GALDEN LMW when daily administered at the dosage of 250, 500 and 1000 mg/kg/day to Sprague Dawley by oral route for 4 weeks, was well tolerated up to and including the highest dosage administered.

Executive summary:

In this study, conducted under GLP according to OECD method No. 407, the effects of repeated administration of the test article GALDEN LMW on Sprague Dawley rats were evaluated.

GALDEN LMW, emulsified in 0.5% (w/v) Methylcellulose 400 cps water solution, additioned with 0.25% (w/v) Tween 80, was administered by oral route (gavage) once a day for 4 consecutive weeks to groups of Sprague Dawley Crl:CD rats at the doses of 250, 500 and 1000 mg/kg/day.

The test article formulates were kept under magnetic stirring during administration.

The administration volume was kept constant at 10 mg/kg bw in all treated groups, the test article concentration in the vehicle being varied accordingly (25, 50 and 100 mg/ml, respectively). The volume administered was adjusted on the basis of most recently recorded body weight (individual values).

Control animals received 10 ml/kg/day of 0.5 % (w/v) Methylcellulose 400 cps water solution, additioned with 0.25% (w/v) Tween 80.

Each experimental group consisted of 5 males and 5 females.

At the end of the 4-week dosing period, all animals were killed for pathology studies.

 

No animals died during the study.

No clinical signs were seen in any animal.

No body weight changes of toxicological relevance were seen at any dose. Moreover, at the highest dose, a minor body weight variation consistent with a slightly retarded growth was observed in males from week 1 onward and in females at the final (week 4) measurement.

Food consumption was unaffected by GALDEN LMW oral administration at all the dose levels applied.

No eye abnormalities were observed at ophthalmoscopic examination.

No changes of toxicological relevance were seen in the hematological profile of either males and females at any dose.

No blood chemistry or urinary changes that could be related to treatment were seen at any dose.

No changes deemed of compound-related origin were seen at post-mortem examination.

 

In conclusion GALDEN LMW, when daily administered at the dosage of 250, 500 and 1000 mg/kg/day to Sprague Dawley rats by oral route for 4 weeks, was well tolerated up to and including the highest dosage administered.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

No adverse effects were observed at the limit dose of 1000 mg/kg/day

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

Study period:

19 July 2001 to 01 October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline-conform study conducted under GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: livestock farming
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males: 266-324 g, females: 185-245 g
- Fasting period before study: no. Animals had no access to food and water during the 6-hour exposure period and overnight prior to sampling for laboratory investigations.
- Housing: 5 animals/sex/cage
- Diet (e.g. ad libitum): pellet diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: approximately 2 weeks, between arrival of animals and the commencement of exposure.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22 (measured)
- Humidity (%): 46-75 (measured)
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hour cicle (6 a.m.-6 p.m.)

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: Whole body exposure chambers
- Method of holding animals in test chamber: Animals were placed into individual compartment of the exposure chamber. A separate exposure chamber was used for each group.
- Method of conditioning air: Air supplied to the vapour generators and secondary dilution vessels was filtered to remove any residual particulate and was dried.
- System of generating vapour: The chamber atmospheres were produced by metering the liquid test substance into glass vapour generators through which dried and heated air was passed at a group dependent flow rate ranging from 90 to 150 l/minute and subsequently 60 to 100 l/minute from exposure 40. The flow rate was decrease to conserve test material. For all exposed groups, the vapour/air mixture produced in the vapour generators was passed into the base of a secondary dilution vessel. A further supply of clean and dry air was supplied to Groups 2 and 3. The air supply to Group 4 was provided solely by the vapour generation system.
- Temperature, humidity, pressure in air chamber: The mean temperatures in chambers ranged between 22.6 °C +-1.13 (Group 1 Chamber) and 24.3 22.6 °C +- 0.99 (Group 4 Chamber). The mean relative humidity (RH) in chambers ranged between 30% +- 6.2 (Group 4 Chamber) and 42% +- 6.6 (Group 1 Chamber) .
- Air flow rate: about 150 l/minute initially and about 100 l/minute from exposure 40 onward to give the final chamber conconcentrations.
- Air change rate: not reported
- Method of particle size determination: not relevant
- Treatment of exhaust air: not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Gas Chromatograph was used to measure the concentration of H GALDEN in the test atmospheres within the four inhalation chambers.
- Samples taken from breathing zone: yes. Chamber atmosphere was sampled in sequence from each of the four exposure chambers and from one point within each chamber. Air from each chamber was continually drawn through a transfer line, which was therefore equilibrated with the mean concentration from each chamber. Every seven minutes, air from the transfer lines was swiched to the injection loop pf the gas chromatograph for automated analysis and data logging. When not being sampled, these transfer lines were pumped to waste.

VEHICLE (if applicable)
- Justification for use and choice of vehicle: air
- Composition of vehicle: air
- Type and concentration of dispersant aid (if powder): not relevant
- Concentration of test material in vehicle: 1014, 3297, 10075 ppm
- Lot/batch no. of vehicle (if required): not relevant
- Purity of vehicle: not relevant

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples of chamber atmosphere were injected into a gas chromatograph, which was calibrated using vapour standards prepared in gas sampling bags.

Vapour samples are collected using an automated system fitted with electrically controlled valves, which are manipulated using Chamber Environment Monitoring System (CEMS-2) software.
The test atmosphere is drawn directly from the inhalation chamber through the sample line to the gas-sampling valve located on the GC. Initially, the gas-sampliong valve of the GC is set to the "load" position after 60 seconds. Simultaneously, the GC activates the start of the run sequence.

Chromatographic conditions:
Analytical column: DB.1, 5 micrometer film thickness, 30 m x 0.53 mm i.d.
Carrier gas: Helium (2.1 ml/minute)
Split ratio 1:25
Oxidant: Air ( 330 ml/minute)
Fuel: Hydrogen (33 ml/minute)
Injection volume: 500 microliter via an automated gas valve
Injection temperature: 200 °C
Detector temperature: 200 °C
Range: 1000
Gas sample valve: Off at 2.5 minute
Retention time H GALDEN: approximately 0.6-2.3 minutes (analysis for major peak at 0.9 minutes)

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days/week for 13 consecutive weeks.

Dose / conc.:

0 ppm (analytical)

Dose / conc.:

1 014 ppm (analytical)

Remarks:

Target: 1000 ppm

Dose / conc.:

3 297 ppm (analytical)

Remarks:

Target: 3300 ppm

Dose / conc.:

10 075 ppm (analytical)

Remarks:

Target: 10000 ppm

No. of animals per sex per dose:

20 (10 males + 10 females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels (1000, 3300, 10000 ppm) were selected in consultation with the sponsor, on the results obtained in a 14-day preliminary toxicity study performed in the same laboratory.
- Rationale for animal assignment (if not random): random

Positive control:

no

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
Dated and signed records of appearence, change and disappearence of clinical signs were maintened in clinical history sheets for individual animals.
Sign of ill health, together with any behavioural change or response to tratment, were recorded.
In addition detailed observations were made according to the following time schedule:
-Daily during exposure period as following:
1. Pre-exposure observation, 2. During exposure, 3. As each animal is returned to its home cage, 4. As late as possible in the working day.
Throughout the study, checks were made early in the working day and again in the afternoon to look for dead or moribund animals.

DETAILED PHYSICAL EXAMINATION AND ARENA OBSERVATION
Once prior to the study start, in each week of exposures, a detailed physical examination was performed on all animals. The observations took place, at the weekend, when there were no exposures, at approximately the same time of day on each occasion. After removal from the cage, the animal was assessed for physical condition and behaviour during handling and after being placed in a standard arena. Particular attention was made to possible signs of neurotoxicity, such as convulsions, tremor and abnormal gait or behaviour, as well as to any audible respiratory noise. Any deviations from normal were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: one week before the start of exposure, on the day that treatment commenced, each week thereafter and also prior to necropsy.

FOOD CONSUMPTION: Yes
The quantity of food consumed by each cage of rats was recorded on a weekly basis, commencing 1 week before the start of exposures.
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each cage in each group and the number of rats in each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
The quantity of water consumed by each cage of rats was recorded on a daily basis, commencing 1 week before the start of exposures.
Water intake per rat (g/rat/day) was calculated using the total amount of water given to and left by each cage in each group and the number of rats in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all rats were examined once before the start of dosing.
All control and high dose group rats were examined during Week 13 of dosing (prior to dosing on the day).
The following ocular structures were examined: adnexa, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and ocular fundus.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Week 13 of exposure from all rats.
- Animals fasted: Yes, overnight
- How many animals: 80
- The following parameters were examined: Haematocrit, Haemoglobin, Erythrocyte count, Mean cell haemoglobin concentration , Mean cell volume, Mean cell haemoglobin, Total leucocyte count (Neutrophilis, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Plateled count, Reticulocyte count, Cell morphology, Prothrombin time, Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Week 13 of exposure from all rats.
- Animals fasted: Yes, overnight
- How many animals: 80
- The following parameters were examined: Glucose, Total protein, Albumin/Globulin (A/G) ratio, Urea, Creatinine, Alkaline phosphatase, Alanine amino-transferase, Aspartate amino-transferase, Gamma Glutamyl transpeptidase, Creatine phosphokinase, Total bilirubin, Sodium, Potassium, Calcium, Inorganic phosphorus, Chloride, Cholesterol, Triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: on Week 13 of exposure from all rats
- Metabolism cages used for collection of urine: Yes. Rats were placed overnight in individual urine cages.
- Animals fasted: Food and water were withheld overnight
- The following parameters were examined: Appearance, Volume, pH, Specific gravity, Protein, Sodium, Potassium, Chloride, Total reducing substances, Glucose, Ketones, Bilirubin, Blood pigments.

For microscopic examination, a portion of the urine sample was centrifuged and the resulting deposit was examined for the presence of the following: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Renal tubule casts, Spermatozoa and precursors, Other abnormal constituents

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 12; prior to the start of exposure; during the weekend, when no exposures were in progress.
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity, grip strength, motor activity
Sensory reactivity: Approach response, touch response, auditory startle response, tail pinch response.
Grip strength: forelimb and hindlimb grip strength was measured. Two trials were performed.
Motor activity: motor activity was recorded using an infra-red detector. The following categories were recorded: time spent in locomotory activity, non-locomotor activity, in no movement. The number of occurrences (events) of each category is also recorded. The test session for each animal was one hour, with data being collected every 2 minutes.

Sacrifice and pathology:

Following 13 weeks of exposure the rats were sacrificed. The examinations took place over 2 days, and, for animals sacrifieced on the second day, exposure were performed on Day 1 of Week 14.

GROSS PATHOLOGY: Yes
All rats were subjected to a detailed macroscopic examination.
The following organs from all animals were dissected free of fat and weighed: adrenals, kidney, ovaries, thymus, brain, liver, spleen, uterus with cervix, epididymides, lungs including bronchi, testes, heart.

HISTOPATHOLOGY: Yes
The following tissues were examined: abnormalities*, adrenals, aorta (thoracic), brain, caecum, colon, duodenum, epididymides, eyes, femur (longitudinal section through joint), heart, ileum jejunum, kidneys, larynx (2 levels), liver (section from 2 lobes)*, lungs (section from 4 major lobes including bronchi)*, lymph nodes (mandibular, mesenteric and tracheobronchial), nasal turbinates, oesophagus, ovaries, pancreas, pituitary, rectum, salivary grands, sciatic nerves, seminal vesicles, skeletal muscle (thigh), spinal cord, spleen, sternum, stomach, testes (PAS staining), thymus, thyroid with parathyroids, trachea (including bifurcation), urinary bladder, uterus with cervix.
Groups 1 and 4: all above listed tissues.
Groups 2 and 3: only star(*)marked tissues and, due to treatment-related changes, liver

Other examinations:

BONE MARROW EXAMINATION
Prior the post mortem examination, a tibial/femoral bone marrow sample was obtained from each animal. Each smear was air-dried and held pending a possible future requirements for examination (a visual assessment for cellularity, distribution and morphology).

Statistics:

All statistical analysis were performed separately for males and females.
Data relating to food and water consumption were analyzed fon a cage basis. For all other parameters the analysis were performed using the individual animal as experimental unit. Bodyweight data were analysed using weight gains.
A sequence of statistical analysis was used for bodyweight, food and water consumption, clinical pathology, organ weight and grip strength data. Details on the sequence of statistical analysis are reported in the section "Any other information on materials and method".

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related effects during the 13 weeks of the treatment period. No observations were observed during or immediately post exposure in any group.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were considered to be no effects of treatment on the variable pattern of bodyweight change between the groups.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There were considered to be no effect of treatment on water consumption of males during the 13 weeks of treatment.
Group mean water intake of Group 4 (High dose) females was slightly greater that the concurrent controls over the 13 weeks of exposure, although the differences did not attain statistical significance. There were no dosage related effects on water consumption amongst Low and Intermediate dose females.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings observed amongst High dose animals during the ophthalmic examinations in Week 13.

Haematological findings:

no effects observed

Description (incidence and severity):

There were considered to be no treatment-related findings in the haematological parameters investigated in Week 13.

Occasional values were statistically different from Controls (i.e. mean cell haemoglobin concentration (MCHC) levels of High dose female and prothrombin time (PT) and activated partial thromboplastin time (APTT) of treated females). In the absence of dosage-responses and correlation amongst sexes, these findings are considered not to be of toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean creatine phosphokinase (CPK) levels were lower in all treated groups (statistically significantly lower for all treated females) compared with the control in Week 13. However, the Control values for males and females were remarkably high (expected values are in the region of 150 U/L) and as such these changes are of no toxicological importance.
Group mean urea levels were slightly lower than Control amongst High dose animals, with statistical significance being attained for males. In the absence of a dose response this change is considered to be of no toxicological significance.
Group mean total protein levels (due to higher albumin levels) for Intermediate and High dose males and females were slightly higher than the Controls with the values for females attaining statistical significance. In the absence of a dose response this change is considered to be of no toxicological significance.
Although other slight inter-group differences in the biochemical parameters investigated in Week 13 occasionally attained statistical significance, these findings were considered incidental and of no toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Group mean urinary volume was lower in High dose males when compared with the Controls with a consequent lower pH and higher specific gravity. However, in the absence of similar findings amongst females, and no effects on water consumption, these changes are no of toxicological significance.
There were considered to be non effects of treatment on the remaining urinalysis parameters investigated.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

SENSORY REACTIVITY and GRIP STRENGTH: In week 12 group mean forelimb grip strength of Intermediate dose and High dose female rats was slightly but statistically significantly lower than the Controls. In the absence of effects in males, no dosage-relationship and no similar effect on hindlimb grip strength, this change is considered to be of no toxicological significance.
There were considered to be no effects of treatment on group mean hindlimb grip strength.
Sensory reactivity was considered unaffected by treatment in Week 12.

MOTOR ACTIVITY: There were considered to be no treatment-related effects in the time spent in locomotory activity in Week 12.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Following 13 weeks of treatment, group mean body weight-adjusted kidney weight were statistically significantly higher than Controls, for Intermediate dose females and High dose males and females. However, in the absence of any pathological changes, this change is considered to be of no toxicological significance.

Group mean body weight-adjusted liver weights were statistically significantly higher in High dose animals following 13 weeks of treatment.

Group mean thymus weights ( bodyweight-adjusted fro females) were higher in High dose animals, with statistically significance being attained for female. However, since no dosage related trend was apparent the change is considered to be of no toxicological significance.
There were considered to be no further effects on the organ weight investigated.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examinations performed at termination revealed no changes attributable to treatment with HFPE.
The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic changes.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings considered to be related with treatment were reported in the liver:
Centrilobular hepatocyte hypertrophy was reported in all Group 4 males, the majority of Group 4 female and a proportion of Group 3 males and females, but no in Group 2 or Control animals.

All other findings were considered to be incidental and of no toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOEC

Effect level:

1 014 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEC

Effect level:

10 075 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

RESULTS of MICROSCOPIC EXAMINATION

Treatment related findings

		MALES				FEMALES
Group / (Concentration)		1 Control group	2 (1014 ppm)	3 (3297 ppm)	4 (10075 ppm)	1 Control group	2 (1014 ppm)	3 (3297 ppm)	4 (10075 ppm)
Centrilobular hepatocyte hypertrophy	Total	0	0	5a	10c	0	0	3	9c
	Minimal	0	0	5	3	0	0	3	6
	Slight	0	0	0	7	0	0	0	3
Number of livers examined		10	10	10	10	10	10	10	10
Statistical significance compared to Control Group 1:a: p < 0,05 ; c : p < 0.001

Conclusions:

The only biologically significant change seen in this study on H GALDEN was centrilobular hypertrophy of the liver in the majority of High dose animals (correlating with an increased bodyweight-adjusted liver weight) and a proportion of Intermediate animals. There was no concurrent alteration of biochemical parameters (e.g., hepatic enzymes). As this is considered to be an adaptive response to exposure of the test material rather than an indication of toxicity it is concluded that No Adverse Effect Concentration (NOAEC) was 10075 ppm (115209 mg/m3) and that the No Effect Concentration (NOEC) was 1014 ppm (11595 mg/m3).

Executive summary:

H GALDEN was administered to rats by whole body inhalation exposure for 13 weeks. Three groups (each of 10 males and 10 females) were exposed to target concentrations of 1000, 3300 and 10000 ppm, 6 hours a day, 5 days a week for 13 consecutive weeks. A fourth group acting as a control was exposed to air alone.

During the study clinical signs, physical examination, arena observations were recorded. Bodyweight and food consumption were recorded weekly. Water consumption was recorded daily. Sensory reactivity, grip strength and motor activity were recorded for all animals in Week 12. Opthalmoscopy was performed on all animals pre-dose and in Week 13. In Week 13, haematology, biochemistry and urinalysis was undertaken. Following the 13-week period, all animals were sacrificed on the day following the last exposure

and subjected to a full macroscopic examination and a range of organ weights recorded. Histopathological examinations were carried out on selected tissues from all animals (dependent upon dose group).

Mean analysed vapour concentrations of HFPE were 1014, 3297 and 10075 ppm. These levels were in good agreement with the target exposure levels of 1000, 3300 and 10000 ppm.

The only biologically significant change seen in this study was centrilobular hypertrophy of the liver in the majority of high dose animals (correlating with an increased in bodyweight-adjusted liver weight) and a proportion of intermediate dose animals. As this is considered to be an adaptive response to exposure of the test material rather than an indication of toxicity it is concluded that the No Adverse Effect Concentration (NOAEC) was 10075 ppm (115209 mg/m3) and that the No Effect Concentration (NOEC) was 1014 ppm (11595 mg/m3).

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from 19 July 2001 to 01 October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline-conform study conducted under GLP on analogue substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: livestock farming
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males: 263-327 g, females: 196-237 g

- Fasting period before study: no
- Housing: 5 animals/sex/cage
- Diet (e.g. ad libitum): pellet diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: approximately 2 weeks, between arrival of animals and the commencement of exposure.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-24 (measured)
- Humidity (%): 50-74 % (measured)
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hour cicle (6 a.m.-6 p.m.)

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: Whole body exposure chambers
- Method of holding animals in test chamber: Animals were placed into individual compartment of the exposure chamber. A separate exposure chamber was used for each group.
- Source and rate of air: about 150 l/minute
- Method of conditioning air: Air supplied to the vapour generators and secondary dilution vessels was filtered to remove any residual particulate and was dried.
- System of generating vapour: The chamber atmospheres were produced by metering the liquid test substance into glass vapour generators through which dried and heated air was passed at a group dependent flow rate ranging from 90 to 150 l/minute. For all exposed groups, the vapour/air mixture produced in the vapour generators was passed into the base of a secondary dilution vessel. A further supply of clean and dry air was supplied to Groups 2 and 3. The air supply to Group 4 was provided solely by the vapour generation system.
- Temperature, humidity, pressure in air chamber: The mean temperatures in chambers ranged between 22.6 °C +-1.13 (Group 2 Chamber) and 24.9 °C +- 1.05 (Group 4 Chamber). The mean relative humidity (RH) in chambers ranged between 25% +- 1.9 (Group 4 Chamber) and 39% +- 5.1 (Group 1 Chamber). The low value of RH probably arose from generation and dilution of the chamber atmospheres with air that was supplied from a compressor system incorporating a refrigerant drier. This deviation from the target conditions had no discernible effect upon the animals and is not considered to have affected the outcome of the study.
- Air flow rate: 150 litre/minute
- Air change rate: not reported
- Method of particle size determination: not relevant
- Treatment of exhaust air: not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Gas Chromatograph was used to measure the concentration of H GALDEN in the test atmospheres within the four inhalation chambers.
- Samples taken from breathing zone: yes. Chamber atmosphere was sampled in sequence from each of the four exposure chambers and from one point within each chamber. Air from each chamber was continually drawn through a transfer line, which was therefore equilibrated with the mean concentration from each chamber. Every seven minutes, air from the transfer lines was swiched to the injection loop pf the gas chromatograph for automated analysis and data logging. When not being sampled, these transfer lines were pumped to waste.

VEHICLE (if applicable)
- Justification for use and choice of vehicle: air
- Composition of vehicle: air
- Type and concentration of dispersant aid (if powder): not relevant
- Concentration of test material in vehicle: 1016, 3323, 9842 ppm
- Lot/batch no. of vehicle (if required): not relevant
- Purity of vehicle: not relevant

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples of chamber atmosphere were injected into a gas chromatograph, which was calibrated using vapour standards prepared in gas sampling bags.

Vapour samples were collected using an automated system fitted with electrically controlled valves, which were manipulated using Chamber Environment Monitoring System (CEMS-2) software.
The test atmosphere was drawn directly from the inhalation chamber through the sample line to the gas-sampling valve located on the GC. Initially, the gas-sampliong valve of the GC was set to the "load" position and the valve was automatically switched to the "inject position" after 60 seconds. Simultaneously, the GC activates the start of the run sequence.

Chromatographic conditions:
Analytical column: DB-1, 5 micrometer film thickness, 30 m x 0.53 mm i.d.
Carrier gas: Helium (2.1 ml/minute)
Split ratio 1:25
Oxidant: Air ( 330 ml/minute)
Fuel: Hydrogen (33 ml/minute)
Injection volume: 500 microliter via an automated gas valve
Injection temperature: 200 °C
Detector temperature: 200 °C
Range: 1000
Gas sample valve: Off at 2.5 minute
Retention time H GALDEN: approximately 0.6-2.3 minutes (analysis for major peak at 0.9 minutes)

Precision data showed coefficient of variation for H GALDEN of less than0.7% with solutions in the range of 11000 to 550.

Duration of treatment / exposure:

6 hours/day for 4 consecutive weeks,
Two additional groups (high dose and control groups) were included with a 2-week recovery period without exposure.

Frequency of treatment:

7 days/week

Dose / conc.:

0 ppm (analytical)

Dose / conc.:

1 016 ppm (analytical)

Remarks:

Target: 1000 ppm

Dose / conc.:

3 323 ppm (analytical)

Remarks:

Target: 3300 ppm

Dose / conc.:

9 842 ppm (analytical)

Remarks:

Target: 10000 ppm

No. of animals per sex per dose:

10: (5 females + 5 males)/dose
+ 10 additional animals (5 females + 5 males) were assigned to the high dose group (Group 4) and to the control group (Group 1). These additional animals were retained after exposure for 2 additional weeks.

See table below for details on groups.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: the dose levels (1000, 3300, 10000 ppm) were selected basing on the results obtained in a 14-day preliminary toxicity study performed in the same laboratory.
- Rationale for animal assignment : random
- Rationale for selecting satellite groups: control group and higher dose group
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

no

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
Dated and signed records of appearence, change and disappearence of clinical signs were maintened in clinical history sheets for individual animals.
Sign of ill health, together with any behavioural change or response to tratment, were recorded.
In addition detailed observations were made according to the following time schedule:
-Daily during exposure period as following:
1. Pre-exposure observation, 2. During exposure, 3. As each animal is returned to its home cage, 4. As late as possible in the working day.
-During the recovery phase:
once daily during week 1; at least once a week during week 2.

BODY WEIGHT: Yes
- Time schedule for examinations:
one week before the start of exposure, on the day that treatment commenced, each week thereafter and also prior to necropsy.

FOOD CONSUMPTION: Yes
The quantity of food consumed by each cage of rats was recorded on a weekly basis, commencing 1 week before the start of exposures.
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each cage in each group and the number of rats in each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
The quantity of water consumed by each cage of rats was recorded on a daily basis, commencing 1 week before the start of exposures.
Water intake per rat (g/rat/day) was calculated using the total amount of water given to and left by each cage in each group and the number of rats in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
All rats were examined once before the start of dosing.
All control and high dose main study rats were examined during week 4 of dosing prior to dosing of the day.
The following ocular structures were examined: adnexa, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and ocular fundus.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 29 of exposure from all main study rats (40) ; on day 15 of the recovery period for all recovery animals (20).
- Anaesthetic used for blood collection: Yes, light (Isoflurane)
- Animals fasted: Yes, overnight
- How many animals: 60
- The following parameters were examined: Haematocrit, Haemoglobin, Erythrocyte count, Mean cell haemoglobin concentration , Mean cell volume, Mean cell haemoglobin, Total leucocyte count (Neutrophilis, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Plateled count, Reticulocyte count, Cell morphology, Prothrombin time, Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 29 of exposure from all main study rats (40) ; on day 15 of the recovery period for all recovery animals (20).
- Animals fasted: Yes, overnight
- How many animals: 60
- The following parameters were examined: Glucose, Total protein, Albumin, Globulin, A/G ratio, Urea, Creatinine, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma Glutamyl transferase, Creatine phosphokinase, Total bilirubin, Sodium, Potassium, Calcium, Inorganic phosphorus, Chloride, Cholesterol, Triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: on day 29 of exposure from all main study rats (40) ; on day 15 of the recovery period for all recovery animals (20).
- Metabolism cages used for collection of urine: Yes. Rats were placed overnight in individual urine cages.
- Animals fasted: Food and water were withheld overnight
- The following parameters were examined: Appearance, Volume, pH, Specific gravity, Protein, Sodium, Potassium, Chloride, Total reducing substances, Glucose, Ketones, Bilirubin, Blood pigments.

For microscopic examination, a portion of the urine sample was centrifuged. The deposit was examined for the presence of the following: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Renal tubule casts, Sperm, Other abnormal constituents

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 4 of treatment and in recovery Week 2.
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity, grip strength, motor activity
Sensory reactivity: Approach response, touch response, auditory startle response, tail pinch response.
Grip strength: forelimb and hindlimb grip strength was measured. Two trials were performed.
Motor activity: motor activity was recorded using an infra-red detector. The following categories were recorded: time spent in locomotory activity, non-locomotor activity, in no movement. The number of occurrences (events) of each category is also recorded. The test session for each animal was one hour, with data being collected every 2 minutes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All rats were subjected to a detailed macroscopic examination.
The following organs from all animals were dissected free of fat and weighed: adrenals, kidney, ovaries, thymus, brain, liver, spleen, uterus with cervix, epididymides, lungs including bronchi, testes, heart.

HISTOPATHOLOGY: Yes
The following tissues were examined: abnormalities*, adrenals*, aorta (thoracic), brain, caecum, colon, duodenum, epididymides* eyes, femur (longitudinal section through joint)*, heart*, ileum jejunum, kidneys*, larynx (2 levels), liver*, lungs*, lymph nodes (mandibular, mesenteric and tracheobronchial), nasal turbinates, nasopharynx, oesophagus, ovaries, pancreas, pituitary, rectum, salivary grands, sciatic nerves, seminal vesicles, skeletal muscle (thigh), spinal cord, spleen*, sternum, stomach, testes (PAS staining)*, thymus, thyroid with parathyroids, trachea (including bifurcation), urinary bladder, uterus with cervix.
Groups 1 and 4: all above listed tissues.
Groups 2 and 3: only star(*)marked tissues

Other examinations:

URINARY FLUORIDE
Following assessment of the urinary parameters, the remaining urine was analysed for level of inorganic fluoride by ion-specific electrode.

BONE MARROW EXAMINATION
Prior the post mortem examination, a tibial/femoral bone marrow sample was obtained from each animal. The smears from all animals were examined to assess the cellularity, distribution andmorphology of the marrow.

Statistics:

All statistical analysis were performed separately for males and females.
Data relating to food and water consumption, normally analysed one a cage basis, were not analyzed for this study since there was only one cage of animal/sex/group. For all other parameters the analysis were performed using the individual animal as experimental unit. Bodyweight data were analysed using weight gains.
A sequence of statistical analysis was used for bodyweight, clinical pathology organ weight and grip strength data. Details on the sequence of statistical analysis are reported in the section "Any other information on materials and method".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical effects considered treatment-related.

One Group 4 female showed signs of fast respiration during the weekly physical examinations during Weeks 2-5. During the second week of the recovery period this sign was no longer apparent. In isolation, this findings is thought to be not consistent with exposure to H GALDEN .
There were no other treatment-related effects during the 4 weeks of the treatment period or in any of the recovery animals during the recovery period.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweight was unaffected by treatment.

There were no effects considered related to treatment on the pattern of bodyweight change.
The apparent group differences in weight over the 4 weeks of exposure were not dosage-related and are considered unlikely to be of any toxicological significance.
Following 2 weeks recovery Group 4 males previously exposed to the high concentration had higher bodyweight gains than control males, but since a similar effect was not seen in female bodyweight gains this change is considered to be of no toxicological significance

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean food consumption of intermediate dose females and high dose males and females was lower than the controls during the 4 weeks of treatment.

Group mean food consumption was slightly lower than the controls for Group 3 females and Group 4 males and females during the 4-week exposure period. Food intake during the recovery period was unaffected by previous treatment.
There were no treatment-related effects on group mean food consumption of intermediate dose males or low dose males and females.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related effects on group mean water consumption of males during the first 4 weeks of treatment of during a 2-week recovery period.
Group mean water intake of Group 4 females was greater than that of the concurrent Controls over the 4 weeks of exposure, and remained slightly, but to a lesser magnitude, higher over the 2-week recovery period.
There were no dosage related effects on water consumption amongst low and intermediate dose females.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Group mean haemoglobin levels and monocyte counts were statistically significantly higher in treated females compared with concurrent Control in Week 4. There were considered to be no effects on these parameters amongst females previously exposed to the high concentration following a 2-week recovery period. However, since these differences were not dose related and independent of sex, they are considered unlikely to be related to treatment.
Although other slight inter-group differences in the haematological parameters investigated in Week 4, or following a 2-week recovery period, attained statistical significance, these findings were considered incidental and of no toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Group mean alkaline phosphatase levels were statistically significantly lower for Group 4 females compared with the Control in Week 4, and remained slightly, but not significantly lower following a 2-week recovery period. Since the activity was lower than the controls this is considered to be of no toxicological significance.
Cholesterol levels were significantly reduced for Group 4 males compared with the Control. Potassium levels were significantly hogher in Group 4 males compared with the Control. These changes were not markedly present following a 2-week recovery period and since they showed no correlation between the sexes are considered to be of no toxicological significance.
There were no other treatment-related effects.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related effects on any of the urinalysis parameters investigated at Week 4 and recovery Week 2.

Group mean urinary fluoride levels were increased following 4 weeks of treatment in animals of the intermediate and high dose levels. The effect showed partial regression after 2 weeks withdrawal from treatment in females and full regression amongst males.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

SENSORY REACTIVITY and GRIP STRENGTH:
In Week 4, group mean forelimb and hindlimb grip strength of males of the high concentration group was lower than the controls, although the difference did not attained statistical significance. It remained slightly lower following a 2-week recovery period.
No effects were observed on mean group strength for males of the low and intermediate concentrations, and in any females treated groups.

Sensory reactivity was considered unaffected by treatment in Week 4 or recovery Week 2.

MOTOR ACTIVITY:
There was a statistically significant increase in time spent in locomotor activity of males in the high concentration group at Week 4 which persisted in recovery Week 2. However the absence of a dosage-related response in males and the absence of any response in treated females complicates interpretation of these observations.
Group mean time spent in locomotory activity of females in Week 4 and recovery Week 2 was similar in all groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights were increased in group 3 (inter dose) and group 4 (high dose) animals, following 4 weeks of treatment, and remained higher following a 2-week recovery period for males.

Following 4 weeks of treatment, group mean bodyweight-adjusted liver weights were higher than controls in Group 3 and Group 4 animals, with statistical significance being attained for all but Group 3 females. Following a 2-week recovery period group mean bodyweight-adjusted liver weights of recovery group animals remained higher for males but not females previously exposed to the high concentration.

Group mean bodyweight-adjusted heart weights were reduced in Group 4 female animals following 4 weeks of treatment. However, since no dosage related trend was apparent, the effect was confined to one sex, was not apparent following 2 weeks recovery and was not associated with any pathlogical changes, the change is considered to be of no toxicological significance.
There were no other effects on the organ weights investigated.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Centrilobular hepatocyte hypertrophy was reported in all males and some females of the high concentration group and a portion of the males in the intermediate concentration group killed following 4 weeks of treatment. The animals of the other treated and control groups were not affected.
All other findings were considered to be incidental and of no toxicological relevance.
The centrilobular hepatocyte hypertrophy reported in the terminal animals was not reported in any animals after the 2-week recovery period.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOEC

Effect level:

1 016 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed

Key result

Dose descriptor:

NOAEC

Effect level:

9 842 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

no

Incidence of centrilobular hepatocyte hypertrophy in males and females

	Male				Female
group	1	2	3	4	1	2	3	4
total incidence	0	0	3*	5*	0	0	0	3
Minimal severity	0	0	3	2	0	0	0	3
Slight severity	0	0	0	3	0	0	0	0
Number of liver examined	5	5	5	5	5	5	5	5

* comparison with the control group, p< 0.05

Urinary Fluoride - group mean values

	Day 29		Recovery day 15
target dose	Males	Females	Males	Females
0 ppm	2.44	2.36	2.64	2.40
1000 ppm	2.56	2.22
3300 ppm	3.46	4.20
10000 ppm	4.12	4.18	2.74	3.12

Conclusions:

The only biologically significant effects seen in this study were elevation of urinary fluoride levels, elevation of liver weights and centrilobular hepatocyte hypertrophy in rats exposed at 3323 or 9842 ppm together with slightly increased water consumption and reduced food consumption in rats exposed at 9842 ppm. The effects on the liver were considered to be a "work hypertrophy" associated with the metabolism of the test substance. The increased urinary fluoride level and increased water consumption were considered to be associated with metabolic breakdown of the test material releasing free fluoride but in the absence of any histopathology in the kidney this is considered not to be indicative of overt toxicity. The effects on food consumption was small but was probably treatment related. It is concluded that 9842 ppm (116735 mg/m3) is close to, if not coincident with, the NOAEC for H GALDEN.
The NOEC is considered to be 1016 ppm (11618 mg/m3).

Executive summary:

Under the reported study H GALDEN was administered to rats by whole body inhalation exposure for 4 weeks. Three groups (each of 5 males and 5 females) were exposed to target concentrations of 1000, 3300 and 10000 ppm, 6 hours a day, 7 days a week for 4 consecutive weeks. A fourth group acting as a control was exposed to air alone.

Additional 5 males and 5 females were assigned to the control and high dose groups which were exposed for 4 weeks and then retained unexposed for a further 2 weeks to assess recovery of any treatment-related effects.

During the study clinical signs, physical examination, sensory reactivity, grip strength and motor activity were recorded. Bodyweight and food and water consumption were recorded weekly. Ophthalmoscopy was performed on Control and High dose animals in week 4. At the end of the 4-week treatment period and 2-week recovery period, haematology, biochemistry and urinalysis was undertaken, and the animals then killed. All animals were subjected to a full macroscopic examination and a range of organ weights recorded. Histopathological examinations were carried out on selected tissues from all animals (dependant upon dose group). Main study rats were killed on the day following the last expoure and the recovery rats were killed on the day 16 of recovery.

The study mean analysed vapour concentrations of H GALDEN were 1016, 3323 and 9842 ppm. These levels were in good agreement with the target exposure concentrations of 1000, 3300 and 10000 ppm.

The only biologically significant effects seen in this study were elevation of urinary fluoride levels, elevation of liver weights and centrilobular hepatocyte hypertrophy in rats exposed at 3323 or 9842 ppm together with slightly increased water consumption and reduced food consumption in rats exposed to 9842 ppm. The effects on the liver were considered to be a "work hypertrophy" associated with the metabolism of the test substance. The increased urinary fluoride level and increased water consumption were considered to be associated with metabolic breakdown of the test material releasing free fluoride but in the absence of any histopathology in the kidney this is considered not to be indicative of overt toxicity. The effects on food consumption was small but was probably treatment related. It is concluded that 9842 ppm (112545 mg/m3) is closed to, if not coincident with, the No Adverse Effect Concentration for H-GALDEN.

 The No Effect Concentration (NOEC) is considered to be 1016 ppm (11618 mg/m3).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

115 209.37 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

All the reported studies are OECD guideline conformed and were conducted under GLP.

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

Study period:

19 July 2001 to 01 October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline-conform study conducted under GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: livestock farming
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males: 266-324 g, females: 185-245 g
- Fasting period before study: no. Animals had no access to food and water during the 6-hour exposure period and overnight prior to sampling for laboratory investigations.
- Housing: 5 animals/sex/cage
- Diet (e.g. ad libitum): pellet diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: approximately 2 weeks, between arrival of animals and the commencement of exposure.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22 (measured)
- Humidity (%): 46-75 (measured)
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hour cicle (6 a.m.-6 p.m.)

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: Whole body exposure chambers
- Method of holding animals in test chamber: Animals were placed into individual compartment of the exposure chamber. A separate exposure chamber was used for each group.
- Method of conditioning air: Air supplied to the vapour generators and secondary dilution vessels was filtered to remove any residual particulate and was dried.
- System of generating vapour: The chamber atmospheres were produced by metering the liquid test substance into glass vapour generators through which dried and heated air was passed at a group dependent flow rate ranging from 90 to 150 l/minute and subsequently 60 to 100 l/minute from exposure 40. The flow rate was decrease to conserve test material. For all exposed groups, the vapour/air mixture produced in the vapour generators was passed into the base of a secondary dilution vessel. A further supply of clean and dry air was supplied to Groups 2 and 3. The air supply to Group 4 was provided solely by the vapour generation system.
- Temperature, humidity, pressure in air chamber: The mean temperatures in chambers ranged between 22.6 °C +-1.13 (Group 1 Chamber) and 24.3 22.6 °C +- 0.99 (Group 4 Chamber). The mean relative humidity (RH) in chambers ranged between 30% +- 6.2 (Group 4 Chamber) and 42% +- 6.6 (Group 1 Chamber) .
- Air flow rate: about 150 l/minute initially and about 100 l/minute from exposure 40 onward to give the final chamber conconcentrations.
- Air change rate: not reported
- Method of particle size determination: not relevant
- Treatment of exhaust air: not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Gas Chromatograph was used to measure the concentration of H GALDEN in the test atmospheres within the four inhalation chambers.
- Samples taken from breathing zone: yes. Chamber atmosphere was sampled in sequence from each of the four exposure chambers and from one point within each chamber. Air from each chamber was continually drawn through a transfer line, which was therefore equilibrated with the mean concentration from each chamber. Every seven minutes, air from the transfer lines was swiched to the injection loop pf the gas chromatograph for automated analysis and data logging. When not being sampled, these transfer lines were pumped to waste.

VEHICLE (if applicable)
- Justification for use and choice of vehicle: air
- Composition of vehicle: air
- Type and concentration of dispersant aid (if powder): not relevant
- Concentration of test material in vehicle: 1014, 3297, 10075 ppm
- Lot/batch no. of vehicle (if required): not relevant
- Purity of vehicle: not relevant

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples of chamber atmosphere were injected into a gas chromatograph, which was calibrated using vapour standards prepared in gas sampling bags.

Vapour samples are collected using an automated system fitted with electrically controlled valves, which are manipulated using Chamber Environment Monitoring System (CEMS-2) software.
The test atmosphere is drawn directly from the inhalation chamber through the sample line to the gas-sampling valve located on the GC. Initially, the gas-sampliong valve of the GC is set to the "load" position after 60 seconds. Simultaneously, the GC activates the start of the run sequence.

Chromatographic conditions:
Analytical column: DB.1, 5 micrometer film thickness, 30 m x 0.53 mm i.d.
Carrier gas: Helium (2.1 ml/minute)
Split ratio 1:25
Oxidant: Air ( 330 ml/minute)
Fuel: Hydrogen (33 ml/minute)
Injection volume: 500 microliter via an automated gas valve
Injection temperature: 200 °C
Detector temperature: 200 °C
Range: 1000
Gas sample valve: Off at 2.5 minute
Retention time H GALDEN: approximately 0.6-2.3 minutes (analysis for major peak at 0.9 minutes)

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days/week for 13 consecutive weeks.

Dose / conc.:

0 ppm (analytical)

Dose / conc.:

1 014 ppm (analytical)

Remarks:

Target: 1000 ppm

Dose / conc.:

3 297 ppm (analytical)

Remarks:

Target: 3300 ppm

Dose / conc.:

10 075 ppm (analytical)

Remarks:

Target: 10000 ppm

No. of animals per sex per dose:

20 (10 males + 10 females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels (1000, 3300, 10000 ppm) were selected in consultation with the sponsor, on the results obtained in a 14-day preliminary toxicity study performed in the same laboratory.
- Rationale for animal assignment (if not random): random

Positive control:

no

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
Dated and signed records of appearence, change and disappearence of clinical signs were maintened in clinical history sheets for individual animals.
Sign of ill health, together with any behavioural change or response to tratment, were recorded.
In addition detailed observations were made according to the following time schedule:
-Daily during exposure period as following:
1. Pre-exposure observation, 2. During exposure, 3. As each animal is returned to its home cage, 4. As late as possible in the working day.
Throughout the study, checks were made early in the working day and again in the afternoon to look for dead or moribund animals.

DETAILED PHYSICAL EXAMINATION AND ARENA OBSERVATION
Once prior to the study start, in each week of exposures, a detailed physical examination was performed on all animals. The observations took place, at the weekend, when there were no exposures, at approximately the same time of day on each occasion. After removal from the cage, the animal was assessed for physical condition and behaviour during handling and after being placed in a standard arena. Particular attention was made to possible signs of neurotoxicity, such as convulsions, tremor and abnormal gait or behaviour, as well as to any audible respiratory noise. Any deviations from normal were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: one week before the start of exposure, on the day that treatment commenced, each week thereafter and also prior to necropsy.

FOOD CONSUMPTION: Yes
The quantity of food consumed by each cage of rats was recorded on a weekly basis, commencing 1 week before the start of exposures.
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each cage in each group and the number of rats in each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
The quantity of water consumed by each cage of rats was recorded on a daily basis, commencing 1 week before the start of exposures.
Water intake per rat (g/rat/day) was calculated using the total amount of water given to and left by each cage in each group and the number of rats in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all rats were examined once before the start of dosing.
All control and high dose group rats were examined during Week 13 of dosing (prior to dosing on the day).
The following ocular structures were examined: adnexa, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and ocular fundus.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Week 13 of exposure from all rats.
- Animals fasted: Yes, overnight
- How many animals: 80
- The following parameters were examined: Haematocrit, Haemoglobin, Erythrocyte count, Mean cell haemoglobin concentration , Mean cell volume, Mean cell haemoglobin, Total leucocyte count (Neutrophilis, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Plateled count, Reticulocyte count, Cell morphology, Prothrombin time, Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Week 13 of exposure from all rats.
- Animals fasted: Yes, overnight
- How many animals: 80
- The following parameters were examined: Glucose, Total protein, Albumin/Globulin (A/G) ratio, Urea, Creatinine, Alkaline phosphatase, Alanine amino-transferase, Aspartate amino-transferase, Gamma Glutamyl transpeptidase, Creatine phosphokinase, Total bilirubin, Sodium, Potassium, Calcium, Inorganic phosphorus, Chloride, Cholesterol, Triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: on Week 13 of exposure from all rats
- Metabolism cages used for collection of urine: Yes. Rats were placed overnight in individual urine cages.
- Animals fasted: Food and water were withheld overnight
- The following parameters were examined: Appearance, Volume, pH, Specific gravity, Protein, Sodium, Potassium, Chloride, Total reducing substances, Glucose, Ketones, Bilirubin, Blood pigments.

For microscopic examination, a portion of the urine sample was centrifuged and the resulting deposit was examined for the presence of the following: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Renal tubule casts, Spermatozoa and precursors, Other abnormal constituents

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 12; prior to the start of exposure; during the weekend, when no exposures were in progress.
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity, grip strength, motor activity
Sensory reactivity: Approach response, touch response, auditory startle response, tail pinch response.
Grip strength: forelimb and hindlimb grip strength was measured. Two trials were performed.
Motor activity: motor activity was recorded using an infra-red detector. The following categories were recorded: time spent in locomotory activity, non-locomotor activity, in no movement. The number of occurrences (events) of each category is also recorded. The test session for each animal was one hour, with data being collected every 2 minutes.

Sacrifice and pathology:

Following 13 weeks of exposure the rats were sacrificed. The examinations took place over 2 days, and, for animals sacrifieced on the second day, exposure were performed on Day 1 of Week 14.

GROSS PATHOLOGY: Yes
All rats were subjected to a detailed macroscopic examination.
The following organs from all animals were dissected free of fat and weighed: adrenals, kidney, ovaries, thymus, brain, liver, spleen, uterus with cervix, epididymides, lungs including bronchi, testes, heart.

HISTOPATHOLOGY: Yes
The following tissues were examined: abnormalities*, adrenals, aorta (thoracic), brain, caecum, colon, duodenum, epididymides, eyes, femur (longitudinal section through joint), heart, ileum jejunum, kidneys, larynx (2 levels), liver (section from 2 lobes)*, lungs (section from 4 major lobes including bronchi)*, lymph nodes (mandibular, mesenteric and tracheobronchial), nasal turbinates, oesophagus, ovaries, pancreas, pituitary, rectum, salivary grands, sciatic nerves, seminal vesicles, skeletal muscle (thigh), spinal cord, spleen, sternum, stomach, testes (PAS staining), thymus, thyroid with parathyroids, trachea (including bifurcation), urinary bladder, uterus with cervix.
Groups 1 and 4: all above listed tissues.
Groups 2 and 3: only star(*)marked tissues and, due to treatment-related changes, liver

Other examinations:

BONE MARROW EXAMINATION
Prior the post mortem examination, a tibial/femoral bone marrow sample was obtained from each animal. Each smear was air-dried and held pending a possible future requirements for examination (a visual assessment for cellularity, distribution and morphology).

Statistics:

All statistical analysis were performed separately for males and females.
Data relating to food and water consumption were analyzed fon a cage basis. For all other parameters the analysis were performed using the individual animal as experimental unit. Bodyweight data were analysed using weight gains.
A sequence of statistical analysis was used for bodyweight, food and water consumption, clinical pathology, organ weight and grip strength data. Details on the sequence of statistical analysis are reported in the section "Any other information on materials and method".

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related effects during the 13 weeks of the treatment period. No observations were observed during or immediately post exposure in any group.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were considered to be no effects of treatment on the variable pattern of bodyweight change between the groups.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There were considered to be no effect of treatment on water consumption of males during the 13 weeks of treatment.
Group mean water intake of Group 4 (High dose) females was slightly greater that the concurrent controls over the 13 weeks of exposure, although the differences did not attain statistical significance. There were no dosage related effects on water consumption amongst Low and Intermediate dose females.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings observed amongst High dose animals during the ophthalmic examinations in Week 13.

Haematological findings:

no effects observed

Description (incidence and severity):

There were considered to be no treatment-related findings in the haematological parameters investigated in Week 13.

Occasional values were statistically different from Controls (i.e. mean cell haemoglobin concentration (MCHC) levels of High dose female and prothrombin time (PT) and activated partial thromboplastin time (APTT) of treated females). In the absence of dosage-responses and correlation amongst sexes, these findings are considered not to be of toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean creatine phosphokinase (CPK) levels were lower in all treated groups (statistically significantly lower for all treated females) compared with the control in Week 13. However, the Control values for males and females were remarkably high (expected values are in the region of 150 U/L) and as such these changes are of no toxicological importance.
Group mean urea levels were slightly lower than Control amongst High dose animals, with statistical significance being attained for males. In the absence of a dose response this change is considered to be of no toxicological significance.
Group mean total protein levels (due to higher albumin levels) for Intermediate and High dose males and females were slightly higher than the Controls with the values for females attaining statistical significance. In the absence of a dose response this change is considered to be of no toxicological significance.
Although other slight inter-group differences in the biochemical parameters investigated in Week 13 occasionally attained statistical significance, these findings were considered incidental and of no toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Group mean urinary volume was lower in High dose males when compared with the Controls with a consequent lower pH and higher specific gravity. However, in the absence of similar findings amongst females, and no effects on water consumption, these changes are no of toxicological significance.
There were considered to be non effects of treatment on the remaining urinalysis parameters investigated.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

SENSORY REACTIVITY and GRIP STRENGTH: In week 12 group mean forelimb grip strength of Intermediate dose and High dose female rats was slightly but statistically significantly lower than the Controls. In the absence of effects in males, no dosage-relationship and no similar effect on hindlimb grip strength, this change is considered to be of no toxicological significance.
There were considered to be no effects of treatment on group mean hindlimb grip strength.
Sensory reactivity was considered unaffected by treatment in Week 12.

MOTOR ACTIVITY: There were considered to be no treatment-related effects in the time spent in locomotory activity in Week 12.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Following 13 weeks of treatment, group mean body weight-adjusted kidney weight were statistically significantly higher than Controls, for Intermediate dose females and High dose males and females. However, in the absence of any pathological changes, this change is considered to be of no toxicological significance.

Group mean body weight-adjusted liver weights were statistically significantly higher in High dose animals following 13 weeks of treatment.

Group mean thymus weights ( bodyweight-adjusted fro females) were higher in High dose animals, with statistically significance being attained for female. However, since no dosage related trend was apparent the change is considered to be of no toxicological significance.
There were considered to be no further effects on the organ weight investigated.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examinations performed at termination revealed no changes attributable to treatment with HFPE.
The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic changes.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings considered to be related with treatment were reported in the liver:
Centrilobular hepatocyte hypertrophy was reported in all Group 4 males, the majority of Group 4 female and a proportion of Group 3 males and females, but no in Group 2 or Control animals.

All other findings were considered to be incidental and of no toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOEC

Effect level:

1 014 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEC

Effect level:

10 075 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

RESULTS of MICROSCOPIC EXAMINATION

Treatment related findings

		MALES				FEMALES
Group / (Concentration)		1 Control group	2 (1014 ppm)	3 (3297 ppm)	4 (10075 ppm)	1 Control group	2 (1014 ppm)	3 (3297 ppm)	4 (10075 ppm)
Centrilobular hepatocyte hypertrophy	Total	0	0	5a	10c	0	0	3	9c
	Minimal	0	0	5	3	0	0	3	6
	Slight	0	0	0	7	0	0	0	3
Number of livers examined		10	10	10	10	10	10	10	10
Statistical significance compared to Control Group 1:a: p < 0,05 ; c : p < 0.001

Conclusions:

The only biologically significant change seen in this study on H GALDEN was centrilobular hypertrophy of the liver in the majority of High dose animals (correlating with an increased bodyweight-adjusted liver weight) and a proportion of Intermediate animals. There was no concurrent alteration of biochemical parameters (e.g., hepatic enzymes). As this is considered to be an adaptive response to exposure of the test material rather than an indication of toxicity it is concluded that No Adverse Effect Concentration (NOAEC) was 10075 ppm (115209 mg/m3) and that the No Effect Concentration (NOEC) was 1014 ppm (11595 mg/m3).

Executive summary:

H GALDEN was administered to rats by whole body inhalation exposure for 13 weeks. Three groups (each of 10 males and 10 females) were exposed to target concentrations of 1000, 3300 and 10000 ppm, 6 hours a day, 5 days a week for 13 consecutive weeks. A fourth group acting as a control was exposed to air alone.

During the study clinical signs, physical examination, arena observations were recorded. Bodyweight and food consumption were recorded weekly. Water consumption was recorded daily. Sensory reactivity, grip strength and motor activity were recorded for all animals in Week 12. Opthalmoscopy was performed on all animals pre-dose and in Week 13. In Week 13, haematology, biochemistry and urinalysis was undertaken. Following the 13-week period, all animals were sacrificed on the day following the last exposure

and subjected to a full macroscopic examination and a range of organ weights recorded. Histopathological examinations were carried out on selected tissues from all animals (dependent upon dose group).

Mean analysed vapour concentrations of HFPE were 1014, 3297 and 10075 ppm. These levels were in good agreement with the target exposure levels of 1000, 3300 and 10000 ppm.

The only biologically significant change seen in this study was centrilobular hypertrophy of the liver in the majority of high dose animals (correlating with an increased in bodyweight-adjusted liver weight) and a proportion of intermediate dose animals. As this is considered to be an adaptive response to exposure of the test material rather than an indication of toxicity it is concluded that the No Adverse Effect Concentration (NOAEC) was 10075 ppm (115209 mg/m3) and that the No Effect Concentration (NOEC) was 1014 ppm (11595 mg/m3).

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from 19 July 2001 to 01 October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline-conform study conducted under GLP on analogue substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: livestock farming
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males: 263-327 g, females: 196-237 g

- Fasting period before study: no
- Housing: 5 animals/sex/cage
- Diet (e.g. ad libitum): pellet diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: approximately 2 weeks, between arrival of animals and the commencement of exposure.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-24 (measured)
- Humidity (%): 50-74 % (measured)
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hour cicle (6 a.m.-6 p.m.)

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: Whole body exposure chambers
- Method of holding animals in test chamber: Animals were placed into individual compartment of the exposure chamber. A separate exposure chamber was used for each group.
- Source and rate of air: about 150 l/minute
- Method of conditioning air: Air supplied to the vapour generators and secondary dilution vessels was filtered to remove any residual particulate and was dried.
- System of generating vapour: The chamber atmospheres were produced by metering the liquid test substance into glass vapour generators through which dried and heated air was passed at a group dependent flow rate ranging from 90 to 150 l/minute. For all exposed groups, the vapour/air mixture produced in the vapour generators was passed into the base of a secondary dilution vessel. A further supply of clean and dry air was supplied to Groups 2 and 3. The air supply to Group 4 was provided solely by the vapour generation system.
- Temperature, humidity, pressure in air chamber: The mean temperatures in chambers ranged between 22.6 °C +-1.13 (Group 2 Chamber) and 24.9 °C +- 1.05 (Group 4 Chamber). The mean relative humidity (RH) in chambers ranged between 25% +- 1.9 (Group 4 Chamber) and 39% +- 5.1 (Group 1 Chamber). The low value of RH probably arose from generation and dilution of the chamber atmospheres with air that was supplied from a compressor system incorporating a refrigerant drier. This deviation from the target conditions had no discernible effect upon the animals and is not considered to have affected the outcome of the study.
- Air flow rate: 150 litre/minute
- Air change rate: not reported
- Method of particle size determination: not relevant
- Treatment of exhaust air: not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Gas Chromatograph was used to measure the concentration of H GALDEN in the test atmospheres within the four inhalation chambers.
- Samples taken from breathing zone: yes. Chamber atmosphere was sampled in sequence from each of the four exposure chambers and from one point within each chamber. Air from each chamber was continually drawn through a transfer line, which was therefore equilibrated with the mean concentration from each chamber. Every seven minutes, air from the transfer lines was swiched to the injection loop pf the gas chromatograph for automated analysis and data logging. When not being sampled, these transfer lines were pumped to waste.

VEHICLE (if applicable)
- Justification for use and choice of vehicle: air
- Composition of vehicle: air
- Type and concentration of dispersant aid (if powder): not relevant
- Concentration of test material in vehicle: 1016, 3323, 9842 ppm
- Lot/batch no. of vehicle (if required): not relevant
- Purity of vehicle: not relevant

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples of chamber atmosphere were injected into a gas chromatograph, which was calibrated using vapour standards prepared in gas sampling bags.

Vapour samples were collected using an automated system fitted with electrically controlled valves, which were manipulated using Chamber Environment Monitoring System (CEMS-2) software.
The test atmosphere was drawn directly from the inhalation chamber through the sample line to the gas-sampling valve located on the GC. Initially, the gas-sampliong valve of the GC was set to the "load" position and the valve was automatically switched to the "inject position" after 60 seconds. Simultaneously, the GC activates the start of the run sequence.

Chromatographic conditions:
Analytical column: DB-1, 5 micrometer film thickness, 30 m x 0.53 mm i.d.
Carrier gas: Helium (2.1 ml/minute)
Split ratio 1:25
Oxidant: Air ( 330 ml/minute)
Fuel: Hydrogen (33 ml/minute)
Injection volume: 500 microliter via an automated gas valve
Injection temperature: 200 °C
Detector temperature: 200 °C
Range: 1000
Gas sample valve: Off at 2.5 minute
Retention time H GALDEN: approximately 0.6-2.3 minutes (analysis for major peak at 0.9 minutes)

Precision data showed coefficient of variation for H GALDEN of less than0.7% with solutions in the range of 11000 to 550.

Duration of treatment / exposure:

6 hours/day for 4 consecutive weeks,
Two additional groups (high dose and control groups) were included with a 2-week recovery period without exposure.

Frequency of treatment:

7 days/week

Dose / conc.:

0 ppm (analytical)

Dose / conc.:

1 016 ppm (analytical)

Remarks:

Target: 1000 ppm

Dose / conc.:

3 323 ppm (analytical)

Remarks:

Target: 3300 ppm

Dose / conc.:

9 842 ppm (analytical)

Remarks:

Target: 10000 ppm

No. of animals per sex per dose:

10: (5 females + 5 males)/dose
+ 10 additional animals (5 females + 5 males) were assigned to the high dose group (Group 4) and to the control group (Group 1). These additional animals were retained after exposure for 2 additional weeks.

See table below for details on groups.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: the dose levels (1000, 3300, 10000 ppm) were selected basing on the results obtained in a 14-day preliminary toxicity study performed in the same laboratory.
- Rationale for animal assignment : random
- Rationale for selecting satellite groups: control group and higher dose group
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

no

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
Dated and signed records of appearence, change and disappearence of clinical signs were maintened in clinical history sheets for individual animals.
Sign of ill health, together with any behavioural change or response to tratment, were recorded.
In addition detailed observations were made according to the following time schedule:
-Daily during exposure period as following:
1. Pre-exposure observation, 2. During exposure, 3. As each animal is returned to its home cage, 4. As late as possible in the working day.
-During the recovery phase:
once daily during week 1; at least once a week during week 2.

BODY WEIGHT: Yes
- Time schedule for examinations:
one week before the start of exposure, on the day that treatment commenced, each week thereafter and also prior to necropsy.

FOOD CONSUMPTION: Yes
The quantity of food consumed by each cage of rats was recorded on a weekly basis, commencing 1 week before the start of exposures.
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each cage in each group and the number of rats in each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
The quantity of water consumed by each cage of rats was recorded on a daily basis, commencing 1 week before the start of exposures.
Water intake per rat (g/rat/day) was calculated using the total amount of water given to and left by each cage in each group and the number of rats in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
All rats were examined once before the start of dosing.
All control and high dose main study rats were examined during week 4 of dosing prior to dosing of the day.
The following ocular structures were examined: adnexa, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and ocular fundus.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 29 of exposure from all main study rats (40) ; on day 15 of the recovery period for all recovery animals (20).
- Anaesthetic used for blood collection: Yes, light (Isoflurane)
- Animals fasted: Yes, overnight
- How many animals: 60
- The following parameters were examined: Haematocrit, Haemoglobin, Erythrocyte count, Mean cell haemoglobin concentration , Mean cell volume, Mean cell haemoglobin, Total leucocyte count (Neutrophilis, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Plateled count, Reticulocyte count, Cell morphology, Prothrombin time, Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 29 of exposure from all main study rats (40) ; on day 15 of the recovery period for all recovery animals (20).
- Animals fasted: Yes, overnight
- How many animals: 60
- The following parameters were examined: Glucose, Total protein, Albumin, Globulin, A/G ratio, Urea, Creatinine, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma Glutamyl transferase, Creatine phosphokinase, Total bilirubin, Sodium, Potassium, Calcium, Inorganic phosphorus, Chloride, Cholesterol, Triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: on day 29 of exposure from all main study rats (40) ; on day 15 of the recovery period for all recovery animals (20).
- Metabolism cages used for collection of urine: Yes. Rats were placed overnight in individual urine cages.
- Animals fasted: Food and water were withheld overnight
- The following parameters were examined: Appearance, Volume, pH, Specific gravity, Protein, Sodium, Potassium, Chloride, Total reducing substances, Glucose, Ketones, Bilirubin, Blood pigments.

For microscopic examination, a portion of the urine sample was centrifuged. The deposit was examined for the presence of the following: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Renal tubule casts, Sperm, Other abnormal constituents

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 4 of treatment and in recovery Week 2.
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity, grip strength, motor activity
Sensory reactivity: Approach response, touch response, auditory startle response, tail pinch response.
Grip strength: forelimb and hindlimb grip strength was measured. Two trials were performed.
Motor activity: motor activity was recorded using an infra-red detector. The following categories were recorded: time spent in locomotory activity, non-locomotor activity, in no movement. The number of occurrences (events) of each category is also recorded. The test session for each animal was one hour, with data being collected every 2 minutes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All rats were subjected to a detailed macroscopic examination.
The following organs from all animals were dissected free of fat and weighed: adrenals, kidney, ovaries, thymus, brain, liver, spleen, uterus with cervix, epididymides, lungs including bronchi, testes, heart.

HISTOPATHOLOGY: Yes
The following tissues were examined: abnormalities*, adrenals*, aorta (thoracic), brain, caecum, colon, duodenum, epididymides* eyes, femur (longitudinal section through joint)*, heart*, ileum jejunum, kidneys*, larynx (2 levels), liver*, lungs*, lymph nodes (mandibular, mesenteric and tracheobronchial), nasal turbinates, nasopharynx, oesophagus, ovaries, pancreas, pituitary, rectum, salivary grands, sciatic nerves, seminal vesicles, skeletal muscle (thigh), spinal cord, spleen*, sternum, stomach, testes (PAS staining)*, thymus, thyroid with parathyroids, trachea (including bifurcation), urinary bladder, uterus with cervix.
Groups 1 and 4: all above listed tissues.
Groups 2 and 3: only star(*)marked tissues

Other examinations:

URINARY FLUORIDE
Following assessment of the urinary parameters, the remaining urine was analysed for level of inorganic fluoride by ion-specific electrode.

BONE MARROW EXAMINATION
Prior the post mortem examination, a tibial/femoral bone marrow sample was obtained from each animal. The smears from all animals were examined to assess the cellularity, distribution andmorphology of the marrow.

Statistics:

All statistical analysis were performed separately for males and females.
Data relating to food and water consumption, normally analysed one a cage basis, were not analyzed for this study since there was only one cage of animal/sex/group. For all other parameters the analysis were performed using the individual animal as experimental unit. Bodyweight data were analysed using weight gains.
A sequence of statistical analysis was used for bodyweight, clinical pathology organ weight and grip strength data. Details on the sequence of statistical analysis are reported in the section "Any other information on materials and method".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical effects considered treatment-related.

One Group 4 female showed signs of fast respiration during the weekly physical examinations during Weeks 2-5. During the second week of the recovery period this sign was no longer apparent. In isolation, this findings is thought to be not consistent with exposure to H GALDEN .
There were no other treatment-related effects during the 4 weeks of the treatment period or in any of the recovery animals during the recovery period.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweight was unaffected by treatment.

There were no effects considered related to treatment on the pattern of bodyweight change.
The apparent group differences in weight over the 4 weeks of exposure were not dosage-related and are considered unlikely to be of any toxicological significance.
Following 2 weeks recovery Group 4 males previously exposed to the high concentration had higher bodyweight gains than control males, but since a similar effect was not seen in female bodyweight gains this change is considered to be of no toxicological significance

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean food consumption of intermediate dose females and high dose males and females was lower than the controls during the 4 weeks of treatment.

Group mean food consumption was slightly lower than the controls for Group 3 females and Group 4 males and females during the 4-week exposure period. Food intake during the recovery period was unaffected by previous treatment.
There were no treatment-related effects on group mean food consumption of intermediate dose males or low dose males and females.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related effects on group mean water consumption of males during the first 4 weeks of treatment of during a 2-week recovery period.
Group mean water intake of Group 4 females was greater than that of the concurrent Controls over the 4 weeks of exposure, and remained slightly, but to a lesser magnitude, higher over the 2-week recovery period.
There were no dosage related effects on water consumption amongst low and intermediate dose females.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Group mean haemoglobin levels and monocyte counts were statistically significantly higher in treated females compared with concurrent Control in Week 4. There were considered to be no effects on these parameters amongst females previously exposed to the high concentration following a 2-week recovery period. However, since these differences were not dose related and independent of sex, they are considered unlikely to be related to treatment.
Although other slight inter-group differences in the haematological parameters investigated in Week 4, or following a 2-week recovery period, attained statistical significance, these findings were considered incidental and of no toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Group mean alkaline phosphatase levels were statistically significantly lower for Group 4 females compared with the Control in Week 4, and remained slightly, but not significantly lower following a 2-week recovery period. Since the activity was lower than the controls this is considered to be of no toxicological significance.
Cholesterol levels were significantly reduced for Group 4 males compared with the Control. Potassium levels were significantly hogher in Group 4 males compared with the Control. These changes were not markedly present following a 2-week recovery period and since they showed no correlation between the sexes are considered to be of no toxicological significance.
There were no other treatment-related effects.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related effects on any of the urinalysis parameters investigated at Week 4 and recovery Week 2.

Group mean urinary fluoride levels were increased following 4 weeks of treatment in animals of the intermediate and high dose levels. The effect showed partial regression after 2 weeks withdrawal from treatment in females and full regression amongst males.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

SENSORY REACTIVITY and GRIP STRENGTH:
In Week 4, group mean forelimb and hindlimb grip strength of males of the high concentration group was lower than the controls, although the difference did not attained statistical significance. It remained slightly lower following a 2-week recovery period.
No effects were observed on mean group strength for males of the low and intermediate concentrations, and in any females treated groups.

Sensory reactivity was considered unaffected by treatment in Week 4 or recovery Week 2.

MOTOR ACTIVITY:
There was a statistically significant increase in time spent in locomotor activity of males in the high concentration group at Week 4 which persisted in recovery Week 2. However the absence of a dosage-related response in males and the absence of any response in treated females complicates interpretation of these observations.
Group mean time spent in locomotory activity of females in Week 4 and recovery Week 2 was similar in all groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights were increased in group 3 (inter dose) and group 4 (high dose) animals, following 4 weeks of treatment, and remained higher following a 2-week recovery period for males.

Following 4 weeks of treatment, group mean bodyweight-adjusted liver weights were higher than controls in Group 3 and Group 4 animals, with statistical significance being attained for all but Group 3 females. Following a 2-week recovery period group mean bodyweight-adjusted liver weights of recovery group animals remained higher for males but not females previously exposed to the high concentration.

Group mean bodyweight-adjusted heart weights were reduced in Group 4 female animals following 4 weeks of treatment. However, since no dosage related trend was apparent, the effect was confined to one sex, was not apparent following 2 weeks recovery and was not associated with any pathlogical changes, the change is considered to be of no toxicological significance.
There were no other effects on the organ weights investigated.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Centrilobular hepatocyte hypertrophy was reported in all males and some females of the high concentration group and a portion of the males in the intermediate concentration group killed following 4 weeks of treatment. The animals of the other treated and control groups were not affected.
All other findings were considered to be incidental and of no toxicological relevance.
The centrilobular hepatocyte hypertrophy reported in the terminal animals was not reported in any animals after the 2-week recovery period.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOEC

Effect level:

1 016 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed

Key result

Dose descriptor:

NOAEC

Effect level:

9 842 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

no

Incidence of centrilobular hepatocyte hypertrophy in males and females

	Male				Female
group	1	2	3	4	1	2	3	4
total incidence	0	0	3*	5*	0	0	0	3
Minimal severity	0	0	3	2	0	0	0	3
Slight severity	0	0	0	3	0	0	0	0
Number of liver examined	5	5	5	5	5	5	5	5

* comparison with the control group, p< 0.05

Urinary Fluoride - group mean values

	Day 29		Recovery day 15
target dose	Males	Females	Males	Females
0 ppm	2.44	2.36	2.64	2.40
1000 ppm	2.56	2.22
3300 ppm	3.46	4.20
10000 ppm	4.12	4.18	2.74	3.12

Conclusions:

The only biologically significant effects seen in this study were elevation of urinary fluoride levels, elevation of liver weights and centrilobular hepatocyte hypertrophy in rats exposed at 3323 or 9842 ppm together with slightly increased water consumption and reduced food consumption in rats exposed at 9842 ppm. The effects on the liver were considered to be a "work hypertrophy" associated with the metabolism of the test substance. The increased urinary fluoride level and increased water consumption were considered to be associated with metabolic breakdown of the test material releasing free fluoride but in the absence of any histopathology in the kidney this is considered not to be indicative of overt toxicity. The effects on food consumption was small but was probably treatment related. It is concluded that 9842 ppm (116735 mg/m3) is close to, if not coincident with, the NOAEC for H GALDEN.
The NOEC is considered to be 1016 ppm (11618 mg/m3).

Executive summary:

Under the reported study H GALDEN was administered to rats by whole body inhalation exposure for 4 weeks. Three groups (each of 5 males and 5 females) were exposed to target concentrations of 1000, 3300 and 10000 ppm, 6 hours a day, 7 days a week for 4 consecutive weeks. A fourth group acting as a control was exposed to air alone.

Additional 5 males and 5 females were assigned to the control and high dose groups which were exposed for 4 weeks and then retained unexposed for a further 2 weeks to assess recovery of any treatment-related effects.

During the study clinical signs, physical examination, sensory reactivity, grip strength and motor activity were recorded. Bodyweight and food and water consumption were recorded weekly. Ophthalmoscopy was performed on Control and High dose animals in week 4. At the end of the 4-week treatment period and 2-week recovery period, haematology, biochemistry and urinalysis was undertaken, and the animals then killed. All animals were subjected to a full macroscopic examination and a range of organ weights recorded. Histopathological examinations were carried out on selected tissues from all animals (dependant upon dose group). Main study rats were killed on the day following the last expoure and the recovery rats were killed on the day 16 of recovery.

The study mean analysed vapour concentrations of H GALDEN were 1016, 3323 and 9842 ppm. These levels were in good agreement with the target exposure concentrations of 1000, 3300 and 10000 ppm.

The only biologically significant effects seen in this study were elevation of urinary fluoride levels, elevation of liver weights and centrilobular hepatocyte hypertrophy in rats exposed at 3323 or 9842 ppm together with slightly increased water consumption and reduced food consumption in rats exposed to 9842 ppm. The effects on the liver were considered to be a "work hypertrophy" associated with the metabolism of the test substance. The increased urinary fluoride level and increased water consumption were considered to be associated with metabolic breakdown of the test material releasing free fluoride but in the absence of any histopathology in the kidney this is considered not to be indicative of overt toxicity. The effects on food consumption was small but was probably treatment related. It is concluded that 9842 ppm (112545 mg/m3) is closed to, if not coincident with, the No Adverse Effect Concentration for H-GALDEN.

 The No Effect Concentration (NOEC) is considered to be 1016 ppm (11618 mg/m3).

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH: see RAAF document attached

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEC

Effect level:

10 075 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Conclusions:

The only biologically significant change seen in the study on the structural analogue H GALDEN was centrilobular hypertrophy of the liver in the majority of High dose animals (correlating with an increased bodyweight-adjusted liver weight) and a proportion of Intermediate animals. This was considered to be an adaptive response to exposure of the test material rather than an indication of toxicity.
Based on an analogue approach, the target substance GALDEN LMW is anticipated to have a similar toxicological profile, as a worst case, i.e. with a low toxicity.
The NOAEC of the analogue substance is considered a reasonable value as a starting point for the DNEL derivation for the inhalation route.

Executive summary:

The read-across approach with the analogue substance H-GALDEN was applied in order to assess the systemic toxicity of GALDEN LMW following repeated dose exposure by inhalation. 

GALDEN LMW and H-GALDEN have a similar chemical structure and common characteristic which are typical of compound having C-F bounds. However the presence of –OCF2H as terminal groups in H-GALDEN, gives to the product slight more reactivity and more polarity than those observed for GALDEN, characterised by neutral, non functional terminal groups –OCF3.

The available experimental data show that GALDEN LMW and H-GALDEN have the same profile regarding physico-chemical reactivity, skin and eye irritation and skin sensitization.

The only biologically significant change seen in this study on the structural analogue H GALDEN was centrilobular hypertrophy of the liver in the majority of High dose animals (correlating with an increased bodyweight-adjusted liver weight) and a proportion of Intermediate animals. As this is considered to be an adaptive response to exposure of the test material rather than an indication of toxicity it is concluded that No Adverse Effect Concentration (NOAEC) was 10075 ppm (115209 mg/m3) and that the No Effect Concentration (NOEC) was 1014 ppm (11595 mg/m3).

Based on an analogue approach, the target substance GALDEN LMW is anticipated to have a similar toxicological profile, as a worst case. As discussed in the read-across justification document, both the source and the target substances are in the same range of molecular weight and contain similar perfluoroether constituents. H GALDEN is expected to display a higher reactivity due to the presence of hydrogen on the terminal groups and thus constitutes a conservative surrogate for the neutral, non-functional GALDEN LMW.

 

Reference 4

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH: see RAAF document attached

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

NOAEC

Effect level:

9 842 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Critical effects observed:

no

Conclusions:

The only biologically significant effects seen in the study performed with the structural analogue substance were elevation of urinary fluoride levels, increased liver weights and centrilobular hepatocyte hypertrophy in rats exposed at 3323 or 9842 ppm together with slightly increased water consumption and reduced food consumption in rats exposed at 9842 ppm. The effects on the liver were considered to be "work hypertrophy" associated with the metabolism of the test substance. The increased urinary fluoride level and increased water consumption were considered to be associated with metabolic breakdown of the test material releasing free fluoride but in the absence of any histopathology in the kidney this is considered not to be indicative of overt toxicity. It is concluded that 9842 ppm (116735 mg/m3) is close to, if not coincident with, the NOAEC for H GALDEN. The No Effect Concentration (NOEC) is considered to be 1016 ppm (11618 mg/m3).
Based on an analogue approach, the target substance GALDEN LMW is anticipated to have a similar toxicological profile, as a worst case. As discussed in the read-across justification document, both the source and the target substances are in the same range of molecular weight and contain similar perfluoroether constituents. H GALDEN is expected to display a higher reactivity due to the presence of hydrogen on the terminal groups and thus constitutes a conservative surrogate for the neutral, non-functional GALDEN LMW.

Reference 5

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

Sighting study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

March 1999 to January 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Preliminary study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

(5 day exposure. Pregnant females included.)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

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: whole-body inhalation chamber with volume of 0.75m3
- Source and rate of air: compressed air
- Method of conditioning air: The chamber atmospheres were produced by metering the liquid test substance into copper coil vaporizers and then mixing the H GALDEN vapour produced in a stream of heated air. The atmosphere produced by the generation system was further diluted with air to give the final chamber concentrations of vapour.

- Temperature, humidity, pressure in air chamber: 20 - 20.5°C, 41-55%; internal pressure maintained slightly below ambient.
- Air flow rate: 75 l/min
- Air change rate: 12-15 ACH

TEST ATMOSPHERE
- Brief description of analytical method used: Concentration of H GALDEN in the chambers was analysed by Gas Chromatography.
- Samples taken from breathing zone: not specified

A separate exposure chamber was used for each group. The control animals were exposed using an identical exposure chamber to that used for the test group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling on at least 6 occasions at approximately hourly intervals. The samples collected into a 20 ml gas-tight polypropylene syrings were injected directly into sample loop of the gas chromatograph.

Duration of treatment / exposure:

6 Hours

Frequency of treatment:

5 consecutive days

Dose / conc.:

0 ppm (analytical)

Dose / conc.:

4 470 ppm (analytical)

Remarks:

Target: 5000 ppm

Dose / conc.:

10 020 ppm (analytical)

Remarks:

Target : 10000 ppm

Dose / conc.:

18 190 ppm (analytical)

Remarks:

Target: 20000 ppm

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during transfer to exposure cage (prior to exposure), on return to holding cages following exposure and during daily checks

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: before exposure
Males and non-pregnant females: daily beginning 1 week before treatment.
Pregnant females: daily beginning on day 3 of presumed pregnancy.

FOOD CONSUMPTION:
- Food consumption for each cage determined and mean daily diet consumption : Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION : Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No.

URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following organs were dissected from each rat and weighted: lungs, adrenals, liver, ovaries, spleen, heart, kidneys, brain, testes.

HISTOPATHOLOGY: No

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related signs detected during exposure comprised partially closed eyes in Group 3 (Inter dose) and 4 (High dose).
Lowered response to external stimulus, unsteady gait, piloerection and hunched posture were seen in Group 4 (High dose) only.
During no-exposure period, there were no treatment-related signs.

These effects suggest a low anaesthetic effect at 18190 ppm.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Weight gain by male and pregnant female rats from Group 3 (Inter dose) and 4 (High dose) was significantly lower than that of control rats over the 1 week exposure period.
(Male rats: 32.6% lower in Inter dose group and 8.3 % lower in High dose group. Pregnant female rats: 43.2% lower in Inter dose group, 33,3% lower in High dose group)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Consumption by male rats from all exposed groups was slightly lower than that of controls.
male rats: -11,9 % in Low dose group, -9,7% in Inter dose group, -8,0% in High dose group. The differences are statistically significant (p < 0.01)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Adrenal weights of all rats from Group 4 (High dose) were greater controls.
Increases in group mean values, compared to the controls: males, High dose group = 16.4%; non-pregnant females, high dose group = 16.8%; pregnant females, high dose group = 27.2 %.
The difference was statistically significant in pregnant and non-pregnant females. The findings may be related to exposure to the test substance.

Other difference from control values attaining statistical significance comprised
- lower spleen weight in all non-pregnant female groups exposed to the test substance
- lower brain weight in Group 4 (High dose) non-pregnant females.
Altough the differences were statistically significant, they were small, not dose related and considered to be of no toxicological importance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings. All presumed pregnant rats were pregnant. Fetal development appeared to be normal.

Histopathological findings: non-neoplastic:

not examined

Dose descriptor:

NOAEC

Effect level:

4 470 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

Conclusions:

As conclusion of this study the exposure levels of 5000, 10000 and 20000 ppm were considered suitable for the 14-day study.

Executive summary:

The study was performed to assess the response of rats to repeated exposure by inhalation (whole body) to the vapour of H GALDEN. It was designed to be a sighting study for a subsequent 14 -day preliminary study.

In this study, the exposure levels were selected in consultation with the Sponsor, following a review of available data. The highest exposure level in this study was considered likely to cause anaesthetic like effects. It was necessary to establish the level at which such side effects may occur since the clinical signs of anaesthesia may be the only guide for selection of exposure levels for the 14-day study if there were no signs of respiratory irritation or systemic toxicity. Pregnant and non-pregnant animals were included in the study in case the anaesthetic effects were more pronounced in pregnant animals.

Three groups of rats each comprising male, females and pregnant females were exposed by inhalation (whole-body) to a vapour generated from H GALDEN 6 hours each day on 5 consecutive days. The pregnant females were exposed on days 6 -10 of presumed pregnancy. The target vapour concentrations were 5000, 10000 and 20000 ppm. A fourth group acting as control was exposed to air only.

The achieved study mean analysed vapour concentrations of H GALDEN were 4470, 10020 and 18190 ppm for the Low, Intermediate and High dose groups respectively.

Treatment-related signs comprised partially closed eyes in Group 3 (Inter dose) and 4 (High dose). Lowered response to external stimulus, unsteady gait, piloerection and hunched posture were seen in Group 4 only.

Weight gain by rats exposed to 18190 ppm was lower than that of controls.

Greater adrenal weights were recorded in rats exposed to 18190 ppm. The toxicological importance of higher adrenal weights is unclear.

Clinical signs during exposure suggested a low level anaesthetic effect at 18190 ppm. The lower bodyweight gain seen over the one week exposure period in rats exposed to 18190 ppm was principally due to an initial weight loss following the first exposure. By the end of the exposure period rate of weight gain by Control and High dose rats was similar.

There were no observation during the study indicating a difference in effects of exposure on pregnant and non-pregnant females.

As conclusion of this study the exposure levels of 5000, 10000 and 20000 ppm were considered suitable for the 14-day study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

All the reported studies are OECD guideline conformed and were conducted under GLP.

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

Mode of Action Analysis / Human Relevance Framework

Not relevant as no adverse effects were observed in the available studies.

Additional information

EXPOSURE by ORAL ROUTE

The effects of GALDEN LMW following repeated administration by oral route were evaluated in a 28-day Subacute inhalation toxicity study on rats according to OECD 407.

Under the study GALDEN LMW was administered at the dosage of 250, 500 and 1000 mg/kg/day.

Neither mortality nor adverse effects related to toxicity were detected, even at highest tested doses and therefore the NOEL was established to be 1000 mg/kg/day.

EXPOSURE by INHALATION

The effects of GALDEN LMW following repeated exposure by inhalation were evaluated basing on the Read Across approach with the analogue substance H GALDEN.

 

H GALDEN is a hydrofluoropolyether with similar chain structure and similar molecular weight of GALDEN LMW. The differences between the two substances are the monomer units (the monomer unit in H GALDEN is (C2F4O) while the monomer unit of GALDEN is (C3F6O) ) and the presence of a single hydrogen atom segregated in the terminal groups of the molecule H GALDEN. The pendant group -CF3 in the monomer unit of GALDEN LMW is not expected to affect the chemical reactivity and most of the physical-chemical properties since it is a non-reactive group. The available experimental data show that GALDEN LMW and H GALDEN have the same profile regarding physico-chemical reactivity, skin and eye irritation and skin sensitization, although, probably due to the presence of -OCF2H as terminal groups, H GALDEN is more reactive than GALDEN LMW .

With respect to the acute toxicity, experimental results show that H GALDEN is slightly more reactive than GALDEN following inhalation exposure since some clinical signs were noted during exposure at the highest concentration (26411 ppm) , whereas GALDEN LMW was tested at the saturated concentration of 95000 ppm and neither mortality nor clinical signs were noted.

The slightly higher reactivity of H GALDEN is hypothesized deriving from –OCF2H as terminal groups. This allows to consider this read across a worst case approach in regard to the potential hazard of GALDEN LMW following repeated dose exposure by inhalation.

 

H GALDEN was tested under GLP in a 28-day Subacute inhalation toxicity study according to OECD 412 and in a 90-day Subchronic inhalation toxicity study according to OECD 413.

No adverse effects related to toxicity were detected.

The only biologically significant observed changes were centrilobular hypertrophy of the liver, seen under both the studies, and increases in the urinary fluoride level and in water consumption observedunder the 28-day toxicity study (under the 90-day toxicity study urinary fluoride level was not analysed).

The centrilobular hypertrophy of the liver was considered to be an adaptive response to exposure of the test material, as well as the increases in the urinary fluoride level and in water consumption were considered to be associated with metabolic breakdown of the test material releasing free fluoride.

NOECs and NOAECs determined under the 28-day and 90-day studies are similar:

NOEC~1000 ppm

NOAEC~10000 ppm

The outcomes of these studies are consistent with the results of the preliminary 5-day and 14-day repeated dose toxicity studies.

 

Basing on the analogue approach it can be concluded that GALDEN LMW is expected to have the same toxicological profile as H GALDEN.

Moreover it should be underline that, because of the greater reactivity of H GALDEN, the reported read across represents a worst case approach and therefore it allows to be on the safe side in regard to the potential hazard of GALDEN LMW following repeated dose exposure by inhalation.

EXPOSURE by the DERMAL ROUTE

No repeated dose toxicity study is available for the dermal route. However, based on the physico-chemical properties and IH SkinPerm model predictions it is anticipated that the majority of the substance would not remain on the skin upon contact and amounts potentially absorbed are predicted to be very low.

Justification for classification or non-classification

GALDEN LMW does not meet the classification criteria for hazard classes related to repeated dose exposure according to CLP Regulation (EC) 1272/2008.