1,1,2,2,3,3,4-heptafluorocyclopentane

Endpoint summary

Description of key information

Oral:

The 28d test was conducted in compliance with EC B.7.

The No-Observed-Adverse-Effect Level (NOAEL) was considered to be 1000 mg/kg/day, and the NOEL for both sexes is considered to be 1000 mg/kg/day.

Inhalation:

The response of rats to repeated inhalation administration, by snout only exposure to a vapour of the test substance for 13 consecutive weeks were assessed in accordance with OECD 413. No Observed Adverse Effect Level (NOAEL): 367 ppm (2.99 mg/L).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

From 1997-09-03 to 1997-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1986

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Batch No.: 9706-1A
Purity: 98.65 %

Species:

rat

Strain:

other: CD

Details on species / strain selection:

The rat was chosen because of its acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The CD strain was used because of the historical control data available in ths laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited (Margate, Kent, England)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Ca. 38 days
- Weight at study initiation: Male: 147-162 g; Female: 127-165 g
- Fasting period before study: no
- Housing: The animals were housed five of one sex per cage, unless this number was reduced by mortality, in TR18 cages which were made of a stainless steel body with a stainless steel mesh lid and floor. The cages were suspended above absorbent paper which was changed at appropriate intervals. Cages, cage-trays, food boppers and water bottles were changed at appropriate intervals.
- Diet: Expanded rodent diet (Rat and Mouse No. 1 Maintenance Diet from Special Diets Services Limited, Witham, Essex, England), ad libitum
- Water: ad libitum
- Acclimation period: 10 days

DETAILS OF FOOD AND WATER QUALITY:
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. No contaminants that might have prejudiced the study were present in the diet or water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): at least 10
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Details on route of administration:

An appropriate quantity of dose for the animal of the group (the required amount for the animal plus 0.5 mL (the volume of the 8 choke catheter) was removed from the vial via a 16 gauge hypodermic needle; the needle was removed, the catheter attached and the excess dose was expelled. The animal was then dosed. After the first animal the catheter was removed and kept horizontal (to avoid loss of dose form contained within the catheter); the next aliquot of dose was removed from the vial (via syringe and 16 gauge needle), the catheter attached to the syringe and the animal dosed. After the last animal in the group had received its dose, the dose form contained within the catheter was sucked back and expelled into the vial (via the 16 gauge needle). A fresh syringe, needle and catheter was employed for each group of animals.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulation took place, slightly warmed (23-25°C) in a sealed unit with no air flow. Appropriate quantities of the test substance were measured volumetrically into a large glass vial containing a magnetic stirrer bar and sealed immediately. The vehicle, corn oil (warmed to 23-25°C), was added through the septum, the vial shaken and placed on a magnetic stirrer.
Bulk mixes of each test formulation were prepared weekly and aliquoted for daily administration; each aliquot was stored at 4 °C prior to use and allowed to come up to room temperature before administration to the animals.

VEHICLE
- Concentration in vehicle: 3, 30, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The formulation samples, supplied as unit doses, were diluted with toluene and dissolved test item further diluted to a concentration within a nominal range. The concentration of test item in the dilution solution was determined by GC using a flame ionization detector.

Duration of treatment / exposure:

28 d

Frequency of treatment:

once each day, seven days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: selected based on the preliminary study results (Report No. ZCE020/972390)
- Fasting period before blood sampling for clinical biochemistry: overnight
- Recovery group: A further five male and five female animals assigned to the Control and high dosage groups completed a further two weeks without treatment to assess the recovery from any treatment-related changes.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Any deviations from normal were recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing; Immediately after dosing on return of the animal to its cage; On completion of dosing of each group; Between one and two hours after completion of dosing of all groups; As late as possible in the working day.

BODY WEIGHT: Yes
- Time schedule for examinations: during the acclimatization period, on the day that treatment commenced, at twice weekly intervals throughout the treatment and recovery periods and before necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g/animal.

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

WATER CONSUMPTION: Yes, assessed by visual observations

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 for all main study animals, Day 43 for all recovery phase animals
- Anaesthetic used for blood collection: Yes (Halothane/nitrous oxide)
- Animals fasted: Yes, overnight
- How many animals: 5 male and 5 female per group
- Parameters examined: Packed cell volume (PCV); Hemoglobin concentration (HB); Erythrocyte count (RBC); Total and differential leucocyte count (WBC); Platelet count (PLAT); Mean cell hemoglobin concentration (MCHC); Mean cell hemoglobin (MCH); Mean cell volume (MCV); Prothrombin time (PT); Activated partial thromboplastin time (PTTK); Blood film - Romanowsky stain, examined by light microscopy for abnormal morphology and unusual cell types, including normoblasts.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 for all main study animals, Day 43 for all recovery phase animals
- Animals fasted: Yes
- How many animals: 5 male and 5 female per group
- Parameters examined.: Alkaline phosphatase (ALP); Alanine aminotransferase (ALT); Aspartate aminotransferase (AST); Gamma-glutamyl transpeptidase (GGT); Total bilirubin concentration (BILT); Glucose concentration (GLUC); Urea concentration (UREA); Creatinine concentration (CREA); Total cholesterol concentration (CHOL); Total triglyceride concentration (TRlG); Total protein concentration (TP); Albumin concentration (CHEM ALB); Albumin/globulin ratio (A/G); Sodium (Na); Potassium (K); Chloride (Cl); Calcium (Ca); Inorganic phosphorus (Phos)

URINALYSIS: Yes
- Time schedule for collection of urine: after 28 days treatment and after 14 days of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Appearance (APP); Volume (VOL); pH; Specific gravity (SG); Protein (PROT); Glucose (GLUC); Ketones (KET); Bilirubin (BIL); Blood; Microscopy - the sediment from centrifugation was examined for epithelial cells (EP), polymorphonuclear leucocytes (CP), erythrocytes (RBC), crystals (CRY), spermatozoa and precursors (S) or other abnormalities (A).

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (See Terminal observations - 28d test in attached backgroud material)

HISTOPATHOLOGY: Yes (See Terminal observations - 28d test in attached backgroud material)

Statistics:

The significance of inter group differences in hematology (excluding the incidence of morphological abnormalities evident on blood smears), blood chemistry and urinalysis (volume, specific gravity and pH) were assessed by Student's t-test using a pooled error variance. Statistical significances for eosinophil, basophil, monocyte and large unstained cell counts are not reported as these data are not normally distributed.
For organ weights and bodyweight changes, homogeneity of variance was tested using Bartlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwise comparisons, otherwise a Dunnett's test was used.
Inter-group differences in macroscopic pathology and histopathology were assessed using Fisher's Exact test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Transient post-dosing salivation was evident amongst animals treated at 1000 mg/kg/day; this sign is common on studies of this tye and is not considered to be of toxicological significance

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Although when compared to the Controls, male animals treated at 1000 mg/kg/day showed a marginally low bodyweight gain, the females at this dosage showed a marginally high gain and this, therefore, was considered to be fortuitous. Bodyweight change during the recovery phase was similar to the Controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Overall food consumption during the treatment period was marginally lower in males receiving 1000 mg/kg/day when compared with the Controls, although this was likely to have been fortuitous. During the recovery phase the food consumption of animals previously given 1000 mg/kg/day was similar to the Controls.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no change that could be ascribed to treatment with test item. Inter-group variations which attained statistical significance were either minor, lacked dosage-relationship or were confined to one sex.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

After four weeks of treatment individual male and female animals receiving 150 or 1000 mg/kg/day showed, when compared with the Controls, slightly high plasma triglyceride concentrations, although a dosage-relationship was not always evident. It is likely that these isolated high values were fortuitous.
Although there were a few differences from the Control values, with some statistical significance, they either lacked dosage-relationship or were confined to one sex and are considered to be unrelated to treatment. Lower gamma glutamyl transpeptidase or total bilirubin values, when compared with the Control animals, were evident in males treated at 15 and 1000 mg/kg/day or females treated at 150 and 1000 mg/kg/day, respectively, but are not considered to be of toxicological significance.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Bodyweight-relative liver weights were marginally high in individual animals treated a 1000 mg/kg/day, when compared with the Controls, however, this was considered to be fortuitous. Absolute and bodyweight-relative adrenal weights were slightly high for females treated at this dosage but this change was minor, lacked a histopathological correlate and was not seen in the males; it is, therefore, considered to be fortuitous.
Following the recovery phase, the liver and adrenal weights were not dissimilar to the Controls.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Intra-cytoplasmic protein droplets were seen in the kidneys of male rats at all dosages

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: not relevant to human

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: the intra-cytoplasmic protein droplets have no relevance to human health

Critical effects observed:

no

Conclusions:

The No-Observed-Adverse-Effect Level (NOAEL) was considered to be 1000 mg/kg/day.
Although a No-Observed-Effect Level (NOEL) in male rats has not been detailed by this investigation, the intra-cytoplasmic protein droplets have no relevance to human health, especially in the absence of any blood or urinary abnormality. With the exclusion of these findings, seen only in the kidneys of treated male rats, the NOEL for both sexes is considered to be 1000 mg/kg/day.

Executive summary:

The 28d test was conducted in compliance with EC B.7.

Three groups of rats comprising five males and five females received test by oral gavage at dosages of 15, 150 or 1000 mg/kg/day at a constant volume-dosage of 5 mL/kg bodyweight in corn oil for 28 days. A similarly constituted Control group received vehicle alone at the same volume dosage. A further five male and five female animals assigned to the Control and high dosage groups completed a further two weeks without treatment to assess the recovery from any treatment-related changes.

There was no death or sign considered to be attributable to treatment. Bodyweight gain, overall food consumption and food conversion efficiencies were considered to have been unaffected by treatment with test item. There was no change in the cellularity or chemical composition of the blood, or in the urine, which could unequivocally be attributed to treatment. There was no effect of treatment on the absolute and bodyweight- relative organ weights.

There was no macroscopic finding recorded at necropsy which could be attributed to treatment. Microscopic examination revealed intra-cytoplasmic protein droplets in the kidneys of males at all dosages; the incidence of this lesion was lower after the recovery period. This change is generally considered to be a sex- and species-specific effect and to have no human health relevance.

The No-Observed-Adverse-Effect Level (NOAEL) was considered to be 1000 mg/kg/day.

Although a No-Observed-Effect Level (NOEL) in male rats has not been detailed by this investigation, the intra-cytoplasmic protein droplets have no relevance to human health, especially in the absence of any blood or urinary abnormality. With the exclusion of these findings, seen only in the kidneys of treated male rats, the NOEL for both sexes is considered to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1 (reliable without restriction)

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1997-10-01 to 1998-02-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Batch No.: 9708-1A, 9709-1A
Purity: 98.92%, 98.98%

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was the species of choice due to regulatory requirements and the strain was selected on account of the availability of comprehensive background data, relating to clinical and pathological parameters, at our laboratories.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent, England
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Ca. 6 weeks
- Weight at study initiation: male: 221-222 g, female: 158-159 g
- Fasting period before study: no
- Housing: housed, 5 of the same sex, in suspended cages with stainless steel mesh on front, back and floor and stainless steel side panels. Each cage was 36 cm wide, 53 cm long and 26 cm high. Plastic trays, lined with absorbent paper, were placed below each cage to collect animal excreta. The paper was changed daily. Clean cages were introduced at approximately 2-weekly intervals throughout the study.
- Diet: SDS Rat and Mouse No. 1 SQC maintenance diet, Special Diets Services, Witham, Essex, ad libitum
- Water: ad libitum
- Acclimation period: Ca. 12 days

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test compound could reasonably be expected to be present in the diet.
There was no information available to indicate that any substance likely to influence the effect of the test system could reasonably be expected to be present in the drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 10
- Photoperiod: 12 hours light (0730 - 1930 hrs) and 12 hours dark per 24 hours

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each exposure system employed consisted of an ADG snout-only inhalation chamber, rat restraining tubes and all glass vaporiser to produce a vapour from the liquid test material. Accessories included an elutriator, air supply and extract lines which attached to the top and bottom of the chamber respectively.
- Inhalation chamber: ADG snout-only inhalation chamber (ADG Developments Ltd, Hitchin, Hertfordshire, England). A modular apparatus of aluminium alloy construction comprising a base unit, a variable number of animal exposure sections, each having 20 exposure ports and a top section incorporating a central aerosol inlet with a tangential air inlet. Each chamber used on the present study consisted of 3 animal sections identified as levels 1 (top) to 3 (bottom) and formed an internal chamber volume of approximately 47 litres. During dosing the chamber was housed in an enclosed cabinet to avoid cross contamination.
- Method of holding animals in test chamber:
Moulded polycarbonate tubes tapered at one end to allow the snout only to project from the tapered end. The other end is normally closed by insertion of an expanded plastic bung. A push-rod by passes through the centre of the bung and is adjustable to maintain the position of the rat during restraint. Tubes are attached to a chamber by means of push fit "0" ring seals located in the exposure ports of the animal exposure sections. The restraining tubes holding the rats were attached to chamber level 2 (middle) for Groups 2 &3 and chamber levels 2 & 3 (bottom) for Groups 1 & 4. All exposure ports not in use were sealed with blanking plugs.
- Vapour generator and infusion pump:
An all glass vaporiser was used to generate the vapour. The vaporiser was housed in a water bath at approximately 60 °C. The liquid to be administered was supplied to the vaporiser from a polypropylene syringe driven at a controlled rate by an infusion pump (Precidor type 5003).
The generation apparatus was positioned under a heating lamp used to maintain the ambient temperature greater than 23 °C, to prevent solidification ofthe test substance.
The vapour was carried to a glass elutriator attached to the central inlet at the top of the chamber by flexible tubing. The air supply was provided by compressor. The air was filtered to remove any residual particulate and was dried.
Each air supply was monitored by an in-line tapered tube rotameter. The air flow was calibrated (29 L/min) prior to exposures using a high quality tapered tube rotameter at the point of attachment to the elutriator and the in-line rotameter f10w readings were monitored during exposures.
- Extract lines:
The inhalation system was operated with an air extract attached to the base of the inhalation chamber. The extracted air passed through a trapping system comprising of glass wool, a glass fibre filter and silica gel.
Air extract was provided by vacuum pumps. Air extracts were monitored using in-line tapered tube rotameters. Each inhalation chamber used on the present study was connected to an air extract of 30L/minute. The air extract was calibrated prior to exposures using a high quality tapered tube rotameter at the point of attachment to the chamber.


TEST ATMOSPHERE
- Brief description of analytical method used: The samples of chamber atmosphere were injected directly into a gas chromatograph calibrated using vapour standards prepared in gas bags.
- Samples taken from breathing zone: yes, Chamber atmosphere was sampled in sequence from each of the four exposure chambers (Chambers 4 - 1 sampled sequentially) from chamber level 2 using gas tight polypropylene syringes at approximately hourly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Comparison of room air blanks, standards and test samples showed no interfering peaks.
Precision data showed coefficients of variation of 4.2% or less for test item standards in the range of 5000 to 100 ppm.
Unweighted Least squares regression of the peak area response against concentration of standards (5000 to 100 ppm) produced a correlation coefficient of 0.99991 and relative errors less than 4.7%. Limit of Quantification (LOQ) in any batch was the level of the lowest valid QC sample, however, the potential Limit of Detection (LOD) and LOQ have been calculated statistically as 4 ppm and 16 ppm respectively using the standard deviation obtained at a concentration of 100 ppm. Standards of test item in air in the range 5000 to 100 ppm stored at room temperature for 6 days and subsequently analysed against fresh standards showed concentrations within 2.7% of their nominal concentrations. The integrity of syringe samples was investigated and it was found that a drop in response of less than 1.3% was observed for samples of test item of 5000 and 100 ppm held in a polypropylene syringe at room temperature for 10 minutes.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours a day, 5 days a week

Dose / conc.:

0 ppm (nominal)

Remarks:

Group 1, Air control

Dose / conc.:

350 ppm (nominal)

Remarks:

Group 2, Low dose

Dose / conc.:

1 000 ppm (nominal)

Remarks:

Group 2, Inter dose

Dose / conc.:

3 500 ppm (nominal)

Remarks:

Group 4, High dose

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale: The levels were selected taking account of all available data,
including the results of a previous 14-day inhalation toxicity study in rats performed (Huntingdon Life Sciences report number: ZCE 023/973806).
- Animal assignment: The rats were weighed individually and the bodyweight data used to allocate the animals to experimental groups. The allocation procedure involved, for each sex, initial calculation ofthe population mean followed by rejection ofthose animals with bodyweights furthest from the mean.
- Fasting period before blood sampling for clinical biochemistry: approximately 16 hours

Positive control:

Not performed

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: in the moming and again at the end on the normal working day
- Dated and signed records of appearance, change and disappearance of clinical signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly intervals when special attention was given to the detection of audible respiratory noise.

BODY WEIGHT: Yes
- Time schedule for examinations: Each rat was weighed for allocation to groups, and weekly thereafter, commencing one week before the first exposure. During the period of exposures, the bodyweights were recorded before exposure on the day. In addition, the weight of each rat was recorded at necropsy.

FOOD CONSUMPTION :
- Mean intake (g/rat/week) was caIculated for each cage or group from the food consumed weekly and the number of rats in each cage.

WATER CONSUMPTION: Yes
- Time schedule for examinations: recorded daily and reported on a weekly basis commencing 1 week prior to the start of exposures until the end of the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of aIl rats submitted for allocation were examined once before the start of dosing using a binocular indirect ophthalmoscope. Similarly, aIl rats in Groups 1 and 4 were examined during Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 13 from all main study rats, during Week 17 (Week 4 of withdrawal) from all withdrawal rats
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes
- How many animals: all main study rats and all withdrawal rats
- Parameters examined: See "Laboratory Investigations and statistical analysis" in attached background material.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Week 13 from all main study rats, during Week 17 (Week 4 of withdrawal) from all withdrawal rats
- Animals fasted: Yes
- How many animals: all main study rats and all withdrawal rats
- Parameters examined: See "Laboratory Investigations and statistical analysis" in attached background material.

URINALYSIS: Yes
- Time schedule for collection of urine: during Week 13 from all main study rats, during Week 17 (Week 4 of withdrawal) from all withdrawal rats
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Food and water were withheld from rats during urine collection.
- Parameters examined: See "Laboratory Investigations and statistical analysis" in attached background material.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and Dose groups examined: A full functional observational battery (FOB) and quantitative assessment of motor activity (MAT) using Coulbourn infra red activity system was performed on 10 male and 10 female rats from each group during Week -1 and at the end of Week 12. For Groups 1 and 4, the 10 rats of each sex examined consisted of 5 main study and 5 withdrawal. At the end of Weeks 4 and 8 the same rats were subjected to a reduced version of the FOB testing. A further assessment of motor activity was performed on all withdrawal rats during Week 4 of withdrawal.
- Battery of functions tested: motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

Sacrifice:
Main groups: Following 13 weeks of dosing all surviving rats were killed on the Monday and Tuesday following the last exposure on the previous Friday. On each day an equal number of rats from each sex/ group were sacrificed. Exposure continued up to the day of sacrifice for main group rats.
Withdrawal groups: Following a 4-week withdrawal period all surviving rats were killed during Week 17 of the study.
The rats were kiIIed by intraperitoneal injection of pentobarbitone sodium followed by exsanguination from the brachial blood vessels. Immediately prior to exsanguination the terminal bodyweight of each animal was recorded.

GROSS PATHOLOGY: Yes (See "Laboratory Investigations and statistical analysis" in attached background material.)
HISTOPATHOLOGY: Yes (See "Laboratory Investigations and statistical analysis" in attached background material.)

Statistics:

See "Laboratory Investigations and statistical analysis" in attached background material.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During exposures rats from Group 4 (High dose) appeared to be less responsive to tapping on the restraining tube than control rats. However, it should be noted that assessment of clinical signs was severely limited due to this restraint.
The upper incisors were discernibly white in a total of 18 out of 40 rats (6 male and 12 female) from Group 4 (High dose) and a single male rat from Group 3 (Intermediate dose). The sign was first noted during Week 11. During the withdrawal period white upper incisors were noted for up to 9 males and 8 females out of 10 rats/sex of Group 4 (High dose). By the end of the withdrawal period the sign was recorded in 5 rats of each sex.
Missing upper incisors with associated overgrown lower incisors (subsequently clipped) were observed in 3 male rats from Group 4 (High dose) during Week 10 of exposures. The upper incisors grew back white in one animal. ln view of the finding of white upper incisors, the low incidence of missing teeth confined to High dose males may be also related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control animal was killed early during the withdrawal period and, therefore, the death was unrelated to treatment with test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Over 13 weeks of exposures a lower bodyweight gain was noted in all treated groups compared with control, although only for male Groups 3 (Intermediate dose) and 4 (High dose) were the differences statistically significant. There was no evidence of a dose-relationship.
Over the 4 weeks of withdrawal, bodyweight gain in both sexes of Group 4 (High dose) was higher than control, statistically significantly so for males. These findings show complete regression of the treatment-related findings observed during the exposure period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Over 13 weeks of exposures the cumulative food consumption for all treated groups was slightly lower than control, although only for female Groups 3 (Intermediate dose) and 4 (High dose) were the differences statistically significant. There was no clear dose-relationship although, for both sexes, Group 2 (Low dose) was least affected.
Over the 4 weeks of withdrawal there were no statistically significant differences in cumulative food consumption between Group 4 (High dose) and control. These findings are considered to show a regression of the minimal effect of treatment observed during the exposure period.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Over 13 weeks of exposures the cumulative water consumption for all treated groups was lower than control, statistically significantly so for female Group 3 (Intermediate dose) and both sexes of Group 4 (High dose). There was no evidence of a dose-relationship and similar differences between treated and control groups were apparent pre-dose. For these reasons an effect due to the administration of test item is discounted.
Over the 4 weeks of withdrawal there were no statistically significant differences between Group 4 (High dose) and control in the cumulative amount of water consumed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The eyes of all rats in Group 1 (Air control) and 4 (High dose) were examined during Week 13. No treatment-related abnormalities were observed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

During Week 13 of exposures packed cell volume for all treated male groups, haemoglobin concentration for male Group 3 (Intermediate dose) and both sexes of Group 4 (High dose) and the red cell count for male Group 4 were statistically significantly higher than the corresponding controls. There was some evidence of a dose-relationship. The differences in the females generally did not attain statistical significance although there was a tendency towards higher values for the same parameters. In addition, reticulocytes for treated male groups were more abundant achieving statistical significance in Groups 3 and 4.
During Week 4 of withdrawal, the red cell count for male Group 4 (High dose) was statistically significantly lower than control and packed cell volume and haemoglobin (both sexes) and female reticulocytes were similar to control indicating regression of the treatment-related findings observed during the exposure period. Reticulocytes in male Group 4 (High dose) remained statistically significantly higher than control sustaining the difference noted during the exposure period.
The thrombotest time for male Groups 3 (Intermediate dose) and 4 (High dose) and the activated partial thromboplastin time for Group 4 were statistically significantly lower than control. However the differences were minimal and considered to be of no toxicological importance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

During Week 13 of exposures potassium, calcium and inorganic phosphorus concentrations for both sexes of all treated groups were mostly higher than control. The differences were statistically significant on all occasions for the females and for the male Groups 3 (Intermediate dose) and 4 (High dose) calcium levels. The differences may reflect a minimal, treatment-related disturbance in electrolyte levels.
During Week 4 of withdrawal potassium and inorganic phosphorus levels in female Group 4 (High dose) were statistically significantly higher than control while calcium in females and all three electrolytes measured in males were similar to control. Therefore, the minimal treatment-related disturbance in electrolyte levels recorded during the exposure period had fully regressed in males but only partially regressed in females.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- Quantitative parameters:
The pH of the urine for male Group 4 (High dose) was statistically significantly lower than control. The difference was small, absent in the females and all individual values were within the normal background range for rats of the age and strain used. For these reasons the observed differences were considered to be unrelated to the treatment with test item.
- Qualitative parameters:
The finding of trace or small amounts of ketones in male rats is consistent with this laboratory's database for the age and strain of rat used.
- Urinary inorganic fluoride:
During Week 13 of exposures the total urinary inorganic fluoride output for both sexes of all treated groups was statistically significantly higher than control. The differences were marked and a clear dose-relationship was evident.
During Week 4 of withdrawal total urinary inorganic fluoride output for both sexes of Group 4 (High dose) were statistically significantly higher than control. However a degree of regression was evident since the values showed a five- to six-fold decrease compared to those recorded at Week 13.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

After twelve weeks of treatment, only a few changes in behaviour were observed: a statistically significantly increased grip strength among treated females in Group 4 (High dose) and a statistically significantly decreased locomotor activity in treated males in Group 4 with a tendency for lower activity among treated females in Group 4.
The apparent change in grip strength in Group 4 (High dose) females was not associated with any other changes. An examination of the pre-dose values suggested a slightly increased grip strength among this group. The adjusted values using an analysis of covariance with the pre-dose data did not show a statistically significant change. In the absence of supporting data, the change in grip strength was considered not to be of toxicological importance
The change in locomotor activity could possibly be due to treatment, however, following a 4 week withdrawal period there were no significant differences in activity and the pattern of activity over time was similar for treated and control animals. The change in locomotor activity in itself is not an indicator of neurotoxicity as general toxicity may aIso resuIt in altered activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Following 13 weeks of exposure, kidney weights (adjusted for bodyweight) in all treated groups were higher than control with the differences achieving statistical significance in all but male Group 2 (Low dose). Following 4 weeks of withdrawal male Group 4 (High dose) values were similar to control while female values had not regressed and remained statistically significantly higher than control.
Following 13 weeks of exposures and 4 weeks of withdrawal liver weights (adjusted for bodyweight) for Group 4 (High dose) males were statistically significantly higher than control. The finding following the withdrawal period was considered to be associated with differences in bodyweight since absolute values were lower than controls. As such, the higher liver weight recorded after 13 weeks of exposure was considered to have regressed following 4 weeks of withdrawal.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed at termination revealed white upper incisors in 3/10 male rats and 6/10 female rats from Group 4 (High dose) and 1/10 male rats from Group 3 (Intermediate dose) compared with no control rats. The finding of white incisors had not regressed following 4 weeks of withdrawal with 7/10 male and 5/10 female rats of Group 4 (High dose) showing this sign. The three remaining Group 4 (High dose) males and 3 of the remaining Group 4 females had white areas on the incisors. The toxicological importance of this finding is uncertain in view of the 4 female control rats also showing this sign.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence and degree of ameloblastic dysplasia, generally focal, in the roots of the upper incisor teeth was seen in male and female rats of Group 3 (Intermediate dose) and Group 4 (High dose), compared with rats of Group 1 (Air control).
No ameloblastic dysplasia was seen in the upper incisors following 4 weeks of withdrawal.
A slightly increased degree of haemosiderosis was noted in the spleen of male rats of Group 3 (Intermediate dose) and Group 4 (High dose). Following 4 weeks of withdrawal there were no significant differences in the degree of haemosiderosis between rats of Group 4 (High dose) and Group 1 (Air control).

Dose descriptor:

NOAEL

Effect level:

367 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Remarks on result:

other: equal to 2.99 mg/L

Critical effects observed:

no

Chamber concentration:

The study mean analysed vapour concentrations were 367, 1054 and 3519 ppm (approximately 2.99, 8.59 and 28.7 mg/L at 20 °C) and close to the target levels of 350, 1000 and 3500 ppm.

Over 13 weeks of exposures the study mean chamber concentrations were within 5% of target for Group 2 (Low dose), 6% of target for Group 3 (Intermediate dose) and 1 % of target for Group 4 (High dose).

The coefficients of variation for daily exposure means were 3.8 - 8.7% indicating good control over the exposure system.

Conclusions:

Exposure of rats to test item vapour in air for 6 hours a day, 5 days a week at 367, 1054 or 3519 ppm for 13 weeks resulted in clinical and pathological effects at all levels of exposure. The incidence and severity of the effects was dose-related.
The effects included reduced food consumption, reduced bodyweight gain, ameloblastic changes in the incisor teeth, disturbances in some clinical pathology parameters and increased haemosiderosis in the spleen. Elevated kidney and liver weights were not supported by microscopic changes in these organs.
The findings are consistent with the consequences of release of inorganic fluoride from test item. This conclusion is supported by the finding of markedly elevated urinary fluoride levels at all exposure levels.
Changes seen in rats exposed at 367 ppm (Low dose) were minimal and consisted of a statistically significant elevation of urinary fluoride levels and a low incidence of ameloblastic dysplasia in the upper incisor teeth. The elevation of urinary fluoride is considered to be a consequence of a breakdown of the test article. The minimal ameloblastic dysplasia seen in the upper incisors consisted of focal loss of ameloblasts and similar changes were seen in the untreated controls. The dental lesion, even at the higher exposure level, was reversible on withdrawal of treatment.
Thus, although an effect on the incidence of ameloblastic dysplasia in the upper incisor teeth was observed at 367 ppm (Low dose), it is concluded that, considering the nature of the lesion, 367 ppm can be regarded as a No Observed Adverse Effect Level (NOAEL) in this study.

Executive summary:

Three groups of rats (each of 10 males and 10 females) of the Cr:l CD( (SD) BR strain were exposed to a vapour of test item, 6 hours a day, 5 days a week for 13 weeks at 367, 1054 or 3519 ppm for 13 weeks using a snout only exposure system. A fourth group acting as control was exposed to air only. An additional 10 male and 10 female rats in the control and High dose groups were retained following the final exposure for a further 4 weeks of withdrawal (recovery) to assess the reversibility of any adverse findings.The study was designed in accordance with OECD 413.

Exposure of rats to test item vapour in air resulted in clinical and pathological effects at all levels of exposure. The incidence and severity of the effects was dose-related.

The effects included reduced food consumption, reduced bodyweight gain, ameloblastic changes in the incisor teeth, disturbances in some clinical pathology parameters and increased haemosiderosis in the spleen. Elevated kidney and liver weights were not supported by microscopic changes in these organs.

The findings are consistent with the consequences of release of inorganic fluoride from test item. This conclusion is supported by the finding of markedly elevated urinary fluoride levels at all exposure levels.

Changes seen in rats exposed at 367 ppm (Low dose) were minimal and consisted of a statistically significant elevation of urinary fluoride levels and a low incidence of ameloblastic dysplasia in the upper incisor teeth. The elevation of urinary fluoride is considered to be a consequence of a breakdown of the test article. The minimal ameloblastic dysplasia seen in the upper incisors consisted of focal loss of ameloblasts and similar changes were seen in the untreated controls. The dental lesion, even at the higher exposure level, was reversible on withdrawal of treatment.

Thus, although an effect on the incidence of ameloblastic dysplasia in the upper incisor teeth was observed at 367 ppm (Low dose), it is concluded that, considering the nature of the lesion, 367 ppm can be regarded as a No Observed Adverse Effect Level (NOAEL) in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

2 990 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

1 (reliable without restriction)

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

28d repeated oral: NOAEL: 1000 mg/kg/day

Effects observed:

Microscopic examination revealed intra-cytoplasmic protein droplets in the kidneys of males at all dosages; the incidence of this lesion was lower after the recovery period. This change is generally considered to be a sex- and species-specific effect and to have no human health relevance.

 

90 d repeated inhalation, OECD 413: NOAEL 367 ppm (2.99 mg/L)

Effects observed:

Changes seen in rats exposed at 367 ppm (Low dose) were minimal and consisted of a statistically significant elevation of urinary fluoride levels and a low incidence of ameloblastic dysplasia in the upper incisor teeth. The elevation of urinary fluoride is considered to be a consequence of a breakdown of the test article. The minimal ameloblastic dysplasia seen in the upper incisors consisted of focal loss of ameloblasts and similar changes were seen in the untreated controls. The dental lesion, even at the higher exposure level, was reversible on withdrawal of treatment.

 

Classification:

These effects are not considered to support classification for specific target organ toxicity following repeated exposure. Therefore, this substance should not be classified for STOT-RE according to Regulation (EC) 1272/2008 Table 3.9.1 and section 3.9.2.7.