Registration Dossier

Administrative data

Description of key information

In a sub-acute repeated dose toxicity study (OECD 407) in rats, the NOAEL of the test item was determined to be 100.0 mg/kg bw/day after oral administration.

Based on the results of the sub-acute inhalation toxicity study (OECD 412) in rats, the systemic NOAEC was determined to be 37.0 mg/m3, the highest dose test. In the same study a LOAEC of 3.8 mg/m3 was determined for local effects.

Key value for chemical safety assessment

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:
2008-01-15 and 2008-04-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
27 July 1995
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
30 September 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Lac Technical with a purity of 98% (substance to registered without water)
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Recognized by international guidelines as a recommended rodent test system.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, Switzerland
- Weight at study initiation: 177 to 193 g (males), 139 to 157 g (females)
- Housing: in groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding
- Diet: ad libitum (Pelleted standard Kliba Nafag 3433)
- Water: ad libitum (tap water)
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The feed batch and tap water was analyzed for contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a glass beaker on a tared Mettler balance. A small amount of vehicle was added, mixed and thereafter the remaining vehicle was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (15 -
25 °C).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
After experimental start, samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration and stability. Samples of about 2 g of each concentration were taken during week 3 after commencement of dosing to confirm homogeneity and concentration.The samples were delivered at ambient temperature to the Analytical Department and then stored at -20 ± 5 °C until analysis. The test item concentrations were determined by HPLC coupled to an UV detector and quantified with the area under the peak.
The identity of the test item was confirmed by its retention time which was similar to that measured in the working standards. The test item content in the samples, except six, was found to be within the accepted range of ± 20% of the nominal content. In addition, the homogenous distribution of the test item in PEG 300 was demonstrated. The application formulations were considered to be stable for at least 2 hours and 7 days when kept at room temperature.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 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: The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats (RCC Study Number B63448), in which slight reductions of food consumption were noted initially in both sexes at 1000 and 600 mg/kg/day, and slightly reduced body weights were noted at 1000 and 600 mg/kg/day in both sexes and at 200 mg/kg/day in females. A slight reduction in the testes-to-body weight ratio was noted in males treated with 1000 mg/kg/day.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed for clinical signs once before commencement of administration as well as twice daily on days 1 to 3, and once daily on days 4-28 (treatment period).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization, treatment and recovery periods and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule: once during the acclimatization period and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No. 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: in all animals
- Battery of functions tested: grip strength / motor activity

IMMUNOLOGY: No




Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (adrenal glands, brain (including section of medulla/pons, cerebral and cerebellar cortex), bone marrow (femur), cecum, colon, duodenum, epididymides, heart incl. auricles, ileum with Peyer's patch, Jejunum with Peyer's patch, kidneys, liver, lungs, lymph nodes (mesentric and mandibular), ovaries, prostate gland incl. coagulating glands, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus, vagina and all gross lesions)

OTHER: Organ weights (brain, heart, liver, thymus, kidneys, adrenals, spleen, testes, epidymides, ovaries)
Statistics:
The following statistical methods used to analyze body weight, clinical laboratory data, grip strength and locomotor activity, organ weights and ratios as well as macroscopic findings:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to "details on results".
Mortality:
no mortality observed
Description (incidence):
Please refer to "details on results".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to "details on results".
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
MORTALITY
All animals survived until scheduled necropsy.

CLINICAL SIGNS (DAILY AND WEEKLY)
During daily observations, soft feces were noted in rats at all dose levels. This finding is sometimes seen in studies using PEG 300 as vehicle. In the absence of similar changes in the control group, this was considered to be a possibly enhanced, non-adverse effect of the test item.
No changes or findings were noted during weekly detailed clinical observations (weeks 1-3).

FUNCTIONAL OBSERVATIONAL BATTERY
No changes or findings were noted during functional observational battery (week 4).

- Grip strength: The mean fore and hind limb grip strength values of the test item-treated males and females were considered to be unaffected by the treatment with the test item.

- Locomotor Activity: The mean locomotor activity of the test item-treated rats showed dose-unrelated and incidental differences when compared with the respective controls.

FOOD CONSUMPTION
At 1000, 300 and 100 mg/kg bw/day, the mean daily food consumption of test item-treated males and females was less than that of their respective controls during all measurement intervals.

BODY WEIGHTS
A test item-related reduction in mean absolute body weights and mean body weight gain were noted in males treated with 1000 mg/kg bw/day from day 8 of treatment onwards. Minor differences seen in the females at this dose level were considered to be insufficient to be a test item-related effect. The mean absolute body weights and mean body weight gain of the groups treated with 100
mg/kg bw/day or 300 mg/kg bw/day were generally similar to those of the controls.

HEMATOLOGY
The males and females treated with 1000 mg/kg bw/day had elevated white blood cell counts when compared with the respective controls, which resulted primarily from elevated lymphocyte counts in both sexes and in elevated neutrophils in males. These differences, although they remained within the ranges of the historical control values, were considered to be indicative of a mild inflammatory reaction. The remaining differences seen in the hematology parameters were considered to be unrelated to the treatment with the test item.

CLINICAL BIOCHEMISTRY
Liver-specific changes included elevations of aspartate aminotransferase activity in both sexes at 1000 mg/kg bw/day when compared with controls. Alanine aminotransferase activity was elevated in males at 300 mg/kg bw/day and in both sexes at 1000 mg/kg bw/day. The slight elevation in the glutamate dehydrogenase activity in both sexes at 1000 mg/kg bw/day was also considered to be an indication of hepatic involvement. These changes were considered to indicate elevated metabolism in the liver as a consequence of the test item. The slight reduction in blood glucose seen in rats treated with 1000 mg/kg bw/day may be related to the reductions in food consumption or a mild hepatic insufficiency.
Markedly elevated creatine kinase levels in rats treated with 1000 mg/kg bw/day were considered to be related to the test item as a possible secondary effect of muscle mass loss which culminated in the body weight changes. The elevated creatinine noted in rats at 1000 mg/kg/day was also considered to be related to this finding.
Elevated potassium levels were noted in males at 300 mg/kg bw/day and in both sexes at 1000 mg/kg bw/day; phosphorus levels were elevated in females at 1000 mg/kg bw/day.
Protein was reduced in both sexes at 1000 mg/kg bw/day, primarily as a result of reduced globulin, and with a concomitant increase in albumin/globulin ratios.

ORGAN WIGHTS
Test item-related changes in the mean absolute organ weights and/or ratios were noted at 1000 mg/kg bw/day in the heart, liver, thymus, adrenal and spleen. At 300 mg/kg bw/day, changes in liver weight and/or ratios were considered to be test item-related.

MACROSCOPIC/MICROSCOPIC FINDINGS
Test item-related macroscopical changes included crateriform retractions in the stomachs of both sexes at 1000 mg/kg bw/day. Liver enlargement was restricted to males treated with 300 mg/kg bw/day or 1000 mg/kg bw/day, but was considered to be test item related.

Microscopically, hepatocellular hypertrophy (adaptive in nature) was noted in the livers of males treated with 300 mg/kg bw/day and in both sexes treated with 1000 mg/kg bw/day.
In the stomach of females treated with 300 mg/kg bw/day, squamous cell hyperplasia and hyperkeratosis were noted. These changes were also seen in both sexes treated with 1000 mg/kg bw/day. Parakeratosis, submucosal inflammation, submucosal edema, and pustules were recorded in male and females treated with 1000 mg/kg bw/day, whereas forestomach ulceration and erosion were recorded in females. These findings were considered to be adverse.
Thymus atrophy was noted with increased incidence in both sexes treated with 1000 mg/kg bw/day, whereas the affected females also showed a minimal increase of severity grade of this finding, likely due to stress.
Atrophy of adrenal cortices was recorded in one female treated with 1000 mg/kg bw/day. The reason of this single case of adrenal atrophy remains unclear.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
immune system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
no
Conclusions:
Based on the results of this study, 100 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL). Based upon the microscopical changes in the stomachs of rats treated with 300 mg/kg/day or 1000 mg/kg/day, the no-observed-adverse-effect-level (NOAEL) was considered to be 100 mg/kg/day.
Executive summary:

In this sub-acute toxicity study, the test item was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest and the treatment periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, as well as liver, stomach, thymus of both sexes, adrenal glands of females of the mid- and low dose group, and all gross lesions from all animals.

No mortality, no changes during daily observations or weekly detailed observations (weeks 1-3), and no changes during the functional observational battery performed during week 4 (with no changes in the mean locomotor activity or the mean fore limb or hind limb grip strength values) were observed.

 

The mean daily food consumption of test item-treated males and females was less than that of their respective controls. Additionally, test item-related findings included reduced body weight development at 1000 mg/kg/day, minor changes in the hematology parameters (typical of a mild inflammatory reaction) at 1000 mg/kg/day, mild activation of liver enzymes (considered to be a metabolic reaction of adaptive nature), elevated potassium in males at 300 mg/kg/day and both sexes at 1000 mg/kg/day, increased phosphorus levels in females at 1000 mg/kg/day, macroscopically evident crateriform retractions in the stomachs of rats at 1000 mg/kg/day, and liver enlargement in males at 300 mg/kg/day and 1000 mg/kg/day, as well as reduced mean absolute organ weights and/or ratios in the heart, thymus, adrenal and spleen at 1000 mg/kg/day, increased liver weights and/or ratios at 1000 mg/kg/day and 300 mg/kg/day. Microscopically, squamous cell hyperplasia and hyperkeratosis were noted in the stomach of females treated with 300 mg/kg/day and in both sexes treated with 1000 mg/kg/day, parakeratosis, submucosal inflammation, submucosal edema, and pustules recorded in male and females treated with 1000 mg/kg/day, whereas forestomach ulceration and erosion were recorded in females. These findings were considered to be adverse.

 

Based on the results of this study, 100 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL). Based upon the microscopical changes in the stomachs of rats treated with 300 mg/kg/day or 1000 mg/kg/day, the no-observed adverse-effect-level (NOAEL) was considered to be 100 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and Guideline conform study
System:
gastrointestinal tract
Organ:
stomach

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-03-07 and 2001-07-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
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 and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road Margate, Kent
- Age at study initiation: 8 weeks
- Weight at study initiation: 230 – 290 g (males), 160 – 217 g (females)
- Fasting period before study: Animals had no access to food or water during each 6-hour inhalation exposure.
- Housing: 5 animals of the same sex in suspended stainless steel cages fitted with mesh
front, back and floor with stainless steel sheet sides and suspended on racks
- Diet: ad libitum (pelleted SDS Rat and Mouse No 1 SQC modified maintenance diet)
- Water: ad libitum (tap water)
- Acclimation period: approximately 2 weeks (All animals (including spares) were acclimatized to the restraint procedure for 3 days prior to the start of treatment. The rats were restrained once per day for 20, 40 and 60 minutes respectively for a 3 day period.

DETAILS OF FOOD AND WATER QUALITY: There was no information available that any non-nutrient substance likely to influence the outcome of this study could reasonably be expected to be present in the diet of the drinking water, both of which were routinely subjected to regular chemical analyses.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
1.2 µm
Geometric standard deviation (GSD):
2.17
Remarks on MMAD:
The MMAD was determined for all of the tested concentrations. The following MMAD values were determined:
- Low dose group: 1.4 µm EAD (σg = 2.27)
- Mid dose group: 1.2 µm EAD (σg = 2.17)
- High dose group: 1.3 µm EAD (σg = 2.18)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each exposure system comprised a snout-only inhalation chamber, rat restraining tubes, a concentric jet atomiser, a glass elutriator and diluent air control system. Accessories included a diluent air supply line and an air extract line, which attached to the bottom of the exposure chamber.

- 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 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 “O” ring seals located in the exposure ports of the animal exposure sections.

- Source and rate of air: A compressor supplied air to the atomisers and diluent lines. The air was filtered to remove any residual particulate and was dried (dew point ~2°C).

- System of generating aerosols: The test item delivery system for each group comprised a polypropylene syringe located on a syringe driver (Precidor, Model 5003). The liquid test material was delivered to the inlet of the concentric jet atomiser via a PolyTetraFluoroEthylene (PTFE) needle.The atmosphere exiting each atomiser was delivered into a glass column with a volume of approximately 11 litres (elutriator) and further diluted with air that entered via a pair of inlet ports adjacent to the atomiser in the base of the unit. The test atmospheres exited the elutriator through a 22 mm diameter flexible pipe located at the centre top of the column and were delivered directly to the top of the exposure chambers.

- Air flow rate: 60 L/minute

- Method of particle size determination: The particle size distribution of the test item droplets was determined gravimetrically. The distribution was measured using a cascade impactor (Marple Model 296 Personal Cascade Impactor, Andersen Instruments Inc., Smyrna, USA) operated at an airflow of 2 L/minute.


TEST ATMOSPHERE
- Brief description of analytical method used: During the exposure (6 hours), samples were taken after approximately 1, 3 and 5 hours to determine the chamber concentration of the test item. The samples were collected by drawing chamber atmosphere through a glass microfibre filter mounted in an open faced filter holder. Each sample was removed from a spare animal exposure port through a modified blanking plug. The atmosphere samples were taken by drawing a previously selected volume of chamber air through the filter at a calibrated flow of 5 litres/minute using a laboratory pump. The air volume of each sample collected was measured using and in-line wet type gas meter.
- Samples taken from breathing zone: yes


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations during the study were confirmed by analysis using HPLC. The analysed chamber concentrations of Troysol LAC were 75% (0.0038 mg/L), 113% (0.0113 mg/L) and 92% (0.036 mg/L) of the targets for nominal concentrations of 0.005 mg/L, 0.01 mg/L and 0.04 mg/L respectively. Deviations from these mean values was generally within 20% of these mean values, indicating that satisfactory control of the system was exercised. At the Low and Intermediate exposure concentrations (Groups 2 and 3), the ratios of analysed to nominal concentration (12% and 27% respectively) are lower than is normally seen for droplet generation systems. This is considered to be due in part to the low feed rate of the liquid test article, which does not produce efficient nebulisation and in part to absorption by some of the materials in the exposure system. At High exposure concentration (Group 4), the calculated delivery efficiency (36%) was within the range normally seen for a liquid droplet exposure.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Duration of exposure per day: 6 hours a day
Dosing regime: 5 days/week
Dose / conc.:
0.037 mg/L air (analytical)
Dose / conc.:
0.011 mg/L air (analytical)
Dose / conc.:
0.004 mg/L air (analytical)
No. of animals per sex per dose:
15
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Dose levels were selected on the results obtained from a preliminary range finding study carried out at Huntingdon Life Sciences (Study number TCC 013/003204).
- Rationale for selecting satellite groups: Recovery from any effects was evaluated by using recovery animals.
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- any observation, considered to be of possible importance, made at any time during the study;
- any observation, considered to be of possible importance, made during transfer to restraining tubes (prior to exposure), during exposure, on return to holding cages (after exposure) and as late as possible in the working day;
- a careful external examination made at weekly intervals when special attention was given to the detection of audible respiratory sounds;
- during the recovery period the animals were examined once daily during RecoveryWeek 1 (Study Week 5) and once during Recovery Week 2 (Study Week 6)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (beginning one week before the start of exposure); body weights were recorded before exposure on the day and also at necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: weekly basis, commencing 1 week before the start of exposures

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily basis, commencing 1 week before the start of exposures

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 4 of dosing
- Anaesthetic used for blood collection: Yes (Isoflurane anaesthesia)
- Animals fasted: Yes (overnight; water was returned for at least one hour before blood sampling)
- How many animals: All main study animals from each group in week 4 of dosing
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 4 of dosing
- Animals fasted: Yes (overnight; water was returned for at least one hour before blood sampling)
- How many animals: All main study animals from each group in week 4 of dosing
- Parameters checked in table No.2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: organ weights (adrenals, heart, kidneys, liver, lungs, spleen and testes)

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all superficial tissues were examined visually and by palpation; brain, pituitary gland and cranial nerves; all subcutaneous tissues were examined; thoracic viscera, thymus, lymph nodes and heart; abdominal viscera; urinary bladder; gastrointestinal tract as whole, the stomach and caecum; lungs; liver, kidneys; gonads; adrenals; uterus; intra-abdominal lymph nodes; reporductive organs)
HISTOPATHOLOGY: Yes (abnormalities, adrenals, larynx, liver, lungs, bronchi, spleen, epididymides, nasal turbinates, testes, trachea (including bifurcation), trachea (including bifurcation), kidneys)
Statistics:
Summary statistics (eg means and standard deviations) presented in this report were calculated from computer stored individual raw data. The summary statistics and the individual data were stored in the computer to a defined number of decimal places, different for each parameter. For presentation purposes, however, they were usually rounded to fewer significant figures. Therefore, in general, it was not possible to reproduce the presented mean and standard deviations exactly using the presented individual data.

Significance tests were conducted for main and recovery data at the 5% and 1% levels and the results are indicated in the report using the following asterisk notation:

Main groups:

Williams’ test (Groups 2, 3 and 4 versus Group 1) * p≤ 0.05 **p≤ 0.01

Recovery groups:

Student’s ‘t’>test (Groups 2, 3 and 4 versus Group 1) + p≤ 0.05 ++p≤ 0.01
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to "details on results".
Mortality:
no mortality observed
Description (incidence):
Please refer to "details on results".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to "details on results".
Urinalysis findings:
not examined
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):
Please refer to "details on results".
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
MORTALITY:
No deaths occurred during the study.

CLINICAL OBSERVATIONS:
There were no treatment-related clinical signs noted throughout the study.
Following daily exposures, clinical signs considered to be related to the method of restraint were observed (brown staining on head/body and red staining round the eyes/snout). These are considered to be unrelated to treatment.

BODY WEIGHT:
The body weight gain of the males treated with 0.04 mg/L was slightly lower than that of the control animals throughout the treatment period and remained lower during the recovery period.

FOOD CONSUMPTION:
Group cumulative food intake was statistically significantly lower than the controls amongst high dose (0.04 mg/L) males over the 4-week exposure period, and remained lower during the 2-week recovery period. There were no other treatment-related effects on food consumption.

WATER CONSUMPTION:
There were no signs of a reaction to the treatment.

HAEMATOLOGY:
There were no treatment-related effects.

BIOCHEMISTRY:
There were no treatment-related effects.
Occasional differences from control values for certain parameters attained statistical significance, but, since these were slight or not consistent across the sexes, and in the absence of any corroborating histopathological evidence, are considered to be of no toxicological significance.

ORGAN WEIGHTS:
There were considered to be no effects of treatment on the organ weights recorded. Occasional slight inter-group differences in organ weights were not corroborated with histopathological evidence and are thus considered to be of no toxicological significance.

MACROSCOPIC PATHOLOGY:
The macroscopic examination of the animals after the 4-week treatment period and after the 2-week recovery period revealed no treatment-related effects.

MICROSCOPIC PATHOLOGY:
- Larynx:
Necrosis of the ventral cartilage was reported in all treated groups with the severity increasing with the dose. Epithelial hyperplasia in ventral and arytenoid areas was reported in a proportion of animals from all treated groups. The numbers of affected animals and the severity of lesions were less in the lower dosage groups. The high dose Group 4 also showed epithelial hyperplasia in the lateral and/or ventrolateral areas with occasional keratinisation in lateral areas in a few animals. Squamous metaplasia in the ventrolateral area was reported in some animals in Groups 3 and 4.

Recovery group: Necrosis of the ventral cartilage was reported in animals from all groups with the incidence and severity related to the dose of the test compound. A few Group 4 animals and one Group 3 female showed epithelial hyperplasia in the ventral area over the ventral cartilage.

- Lungs:

Alveolar duct thickening was reported in animals in Group 4 and in a proportion of animals in
Group 3. Goblet cell hyperplasia was present in some Group 4 animals but also a few females from Groups 2 and 3, and one male Control.

- Nasal turbinates:
Eosinophilic inclusions in the olfactory epithelium were reported in some animals in all groups but were generally more frequent and/or more severe in the treated groups and this tendency increased with the dosage.

Recovery group: Eosinophilic inclusions in the olfactory epithelium were reported in all groups with the incidence and severity apparently correlating with the dose of the test compound.
Key result
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
0.004 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
0.037 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.004 mg/L air (analytical)
System:
respiratory system: upper respiratory tract
Organ:
larynx
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Exposure of rats to the test item at 0.037 mg/L caused signs indicative of respiratory tract irritation. There was also a reduced bodyweight gain, the cause of which is not known but, in the absence of other changes, is considered probably secondary to the respiratory tract effects. At lower exposure levels (0.0113 or 0.0038 mg/L), signs indicative of respiratory tract irritation were present. Therefore the LOAEL for local effects was set at 0.0038 mg/L. (or 3.8 mg/m3). The test substance did not induce any clear sytemic effects, therefore the NOEAL systemic was set at 0.037 mg/L (>37 mg/m3).
Executive summary:

Three groups of rats (each of 15 males and 15 females) of the Crl:CD®(SD)IGS BR strain were exposed to the test item, 6 hours a day, 5 days a week for 4 consecutive weeks using a snout only exposure system. A fourth group, acting as control, was exposed to air only. Five males and 5 females from each group were monitored over a further 2-week withdrawal period to evaluate recovery.

The study mean analysed concentrations of the test item were close to the targets of 0.005, 0.01 and 0.04 mg/L with analysed levels of 0.0038, 0.0113 and 0.037 mg/L for the Low, Intermediate and High dose respectively.

 

The animals tolerated exposure to the test item well, with only a slightly lower bodyweight gain and food consumption being recorded for males of the high dose group. There was no evidence of any systemic toxicity noted at pathological examinations.

 

There was evidence of local, dosage-related and treatment-related changes to the respiratory tract, following 4 weeks of exposures as shown by histopathological changes to the larynx (including necrosis of the ventral cartilage and epithelial hyperplasia in all treated groups, and squamous metaplasia in intermediate and high dose groups), lungs (alveolar duct thickening in the intermediate and high dose groups and bronchiolar goblet cell hyperplasia predominantly in the high dosage group), and nasal turbinates (eosinophilic inclusions in the olfactory epithelium). Although there was evidence of recovery from these changes in so much that the lung changes showed full resolutions, there were still pathological changes noted in the larynx and nasal turbinates. These changes are considered to represent signs of local irritation by the test substance.

 

Exposure of rats to the test item at 0.037 mg/L caused signs indicative of respiratory tract irritation. There was also a reduced bodyweight gain, the cause of which is not known but, in the absence of other changes, is considered probably secondary to the respiratory tract effects. At lower exposure levels (0.0113 or 0.0038 mg/L), signs indicative of respiratory tract irritation were present. Therefore the LOAEL for local effects was set at 0.0038 mg/L. (or 3.8 mg/m3). The test substance did not induce any clear sytemic effects, therefore the NOEAL systemic was set at 0.037 mg/L (>37 mg/m3).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
37 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and Guideline conform study

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-03-07 and 2001-07-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
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 and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road Margate, Kent
- Age at study initiation: 8 weeks
- Weight at study initiation: 230 – 290 g (males), 160 – 217 g (females)
- Fasting period before study: Animals had no access to food or water during each 6-hour inhalation exposure.
- Housing: 5 animals of the same sex in suspended stainless steel cages fitted with mesh
front, back and floor with stainless steel sheet sides and suspended on racks
- Diet: ad libitum (pelleted SDS Rat and Mouse No 1 SQC modified maintenance diet)
- Water: ad libitum (tap water)
- Acclimation period: approximately 2 weeks (All animals (including spares) were acclimatized to the restraint procedure for 3 days prior to the start of treatment. The rats were restrained once per day for 20, 40 and 60 minutes respectively for a 3 day period.

DETAILS OF FOOD AND WATER QUALITY: There was no information available that any non-nutrient substance likely to influence the outcome of this study could reasonably be expected to be present in the diet of the drinking water, both of which were routinely subjected to regular chemical analyses.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
1.2 µm
Geometric standard deviation (GSD):
2.17
Remarks on MMAD:
The MMAD was determined for all of the tested concentrations. The following MMAD values were determined:
- Low dose group: 1.4 µm EAD (σg = 2.27)
- Mid dose group: 1.2 µm EAD (σg = 2.17)
- High dose group: 1.3 µm EAD (σg = 2.18)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each exposure system comprised a snout-only inhalation chamber, rat restraining tubes, a concentric jet atomiser, a glass elutriator and diluent air control system. Accessories included a diluent air supply line and an air extract line, which attached to the bottom of the exposure chamber.

- 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 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 “O” ring seals located in the exposure ports of the animal exposure sections.

- Source and rate of air: A compressor supplied air to the atomisers and diluent lines. The air was filtered to remove any residual particulate and was dried (dew point ~2°C).

- System of generating aerosols: The test item delivery system for each group comprised a polypropylene syringe located on a syringe driver (Precidor, Model 5003). The liquid test material was delivered to the inlet of the concentric jet atomiser via a PolyTetraFluoroEthylene (PTFE) needle.The atmosphere exiting each atomiser was delivered into a glass column with a volume of approximately 11 litres (elutriator) and further diluted with air that entered via a pair of inlet ports adjacent to the atomiser in the base of the unit. The test atmospheres exited the elutriator through a 22 mm diameter flexible pipe located at the centre top of the column and were delivered directly to the top of the exposure chambers.

- Air flow rate: 60 L/minute

- Method of particle size determination: The particle size distribution of the test item droplets was determined gravimetrically. The distribution was measured using a cascade impactor (Marple Model 296 Personal Cascade Impactor, Andersen Instruments Inc., Smyrna, USA) operated at an airflow of 2 L/minute.


TEST ATMOSPHERE
- Brief description of analytical method used: During the exposure (6 hours), samples were taken after approximately 1, 3 and 5 hours to determine the chamber concentration of the test item. The samples were collected by drawing chamber atmosphere through a glass microfibre filter mounted in an open faced filter holder. Each sample was removed from a spare animal exposure port through a modified blanking plug. The atmosphere samples were taken by drawing a previously selected volume of chamber air through the filter at a calibrated flow of 5 litres/minute using a laboratory pump. The air volume of each sample collected was measured using and in-line wet type gas meter.
- Samples taken from breathing zone: yes


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations during the study were confirmed by analysis using HPLC. The analysed chamber concentrations of Troysol LAC were 75% (0.0038 mg/L), 113% (0.0113 mg/L) and 92% (0.036 mg/L) of the targets for nominal concentrations of 0.005 mg/L, 0.01 mg/L and 0.04 mg/L respectively. Deviations from these mean values was generally within 20% of these mean values, indicating that satisfactory control of the system was exercised. At the Low and Intermediate exposure concentrations (Groups 2 and 3), the ratios of analysed to nominal concentration (12% and 27% respectively) are lower than is normally seen for droplet generation systems. This is considered to be due in part to the low feed rate of the liquid test article, which does not produce efficient nebulisation and in part to absorption by some of the materials in the exposure system. At High exposure concentration (Group 4), the calculated delivery efficiency (36%) was within the range normally seen for a liquid droplet exposure.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Duration of exposure per day: 6 hours a day
Dosing regime: 5 days/week
Dose / conc.:
0.037 mg/L air (analytical)
Dose / conc.:
0.011 mg/L air (analytical)
Dose / conc.:
0.004 mg/L air (analytical)
No. of animals per sex per dose:
15
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Dose levels were selected on the results obtained from a preliminary range finding study carried out at Huntingdon Life Sciences (Study number TCC 013/003204).
- Rationale for selecting satellite groups: Recovery from any effects was evaluated by using recovery animals.
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- any observation, considered to be of possible importance, made at any time during the study;
- any observation, considered to be of possible importance, made during transfer to restraining tubes (prior to exposure), during exposure, on return to holding cages (after exposure) and as late as possible in the working day;
- a careful external examination made at weekly intervals when special attention was given to the detection of audible respiratory sounds;
- during the recovery period the animals were examined once daily during RecoveryWeek 1 (Study Week 5) and once during Recovery Week 2 (Study Week 6)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (beginning one week before the start of exposure); body weights were recorded before exposure on the day and also at necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: weekly basis, commencing 1 week before the start of exposures

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily basis, commencing 1 week before the start of exposures

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 4 of dosing
- Anaesthetic used for blood collection: Yes (Isoflurane anaesthesia)
- Animals fasted: Yes (overnight; water was returned for at least one hour before blood sampling)
- How many animals: All main study animals from each group in week 4 of dosing
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 4 of dosing
- Animals fasted: Yes (overnight; water was returned for at least one hour before blood sampling)
- How many animals: All main study animals from each group in week 4 of dosing
- Parameters checked in table No.2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: organ weights (adrenals, heart, kidneys, liver, lungs, spleen and testes)

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all superficial tissues were examined visually and by palpation; brain, pituitary gland and cranial nerves; all subcutaneous tissues were examined; thoracic viscera, thymus, lymph nodes and heart; abdominal viscera; urinary bladder; gastrointestinal tract as whole, the stomach and caecum; lungs; liver, kidneys; gonads; adrenals; uterus; intra-abdominal lymph nodes; reporductive organs)
HISTOPATHOLOGY: Yes (abnormalities, adrenals, larynx, liver, lungs, bronchi, spleen, epididymides, nasal turbinates, testes, trachea (including bifurcation), trachea (including bifurcation), kidneys)
Statistics:
Summary statistics (eg means and standard deviations) presented in this report were calculated from computer stored individual raw data. The summary statistics and the individual data were stored in the computer to a defined number of decimal places, different for each parameter. For presentation purposes, however, they were usually rounded to fewer significant figures. Therefore, in general, it was not possible to reproduce the presented mean and standard deviations exactly using the presented individual data.

Significance tests were conducted for main and recovery data at the 5% and 1% levels and the results are indicated in the report using the following asterisk notation:

Main groups:

Williams’ test (Groups 2, 3 and 4 versus Group 1) * p≤ 0.05 **p≤ 0.01

Recovery groups:

Student’s ‘t’>test (Groups 2, 3 and 4 versus Group 1) + p≤ 0.05 ++p≤ 0.01
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to "details on results".
Mortality:
no mortality observed
Description (incidence):
Please refer to "details on results".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to "details on results".
Urinalysis findings:
not examined
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):
Please refer to "details on results".
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results".
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
MORTALITY:
No deaths occurred during the study.

CLINICAL OBSERVATIONS:
There were no treatment-related clinical signs noted throughout the study.
Following daily exposures, clinical signs considered to be related to the method of restraint were observed (brown staining on head/body and red staining round the eyes/snout). These are considered to be unrelated to treatment.

BODY WEIGHT:
The body weight gain of the males treated with 0.04 mg/L was slightly lower than that of the control animals throughout the treatment period and remained lower during the recovery period.

FOOD CONSUMPTION:
Group cumulative food intake was statistically significantly lower than the controls amongst high dose (0.04 mg/L) males over the 4-week exposure period, and remained lower during the 2-week recovery period. There were no other treatment-related effects on food consumption.

WATER CONSUMPTION:
There were no signs of a reaction to the treatment.

HAEMATOLOGY:
There were no treatment-related effects.

BIOCHEMISTRY:
There were no treatment-related effects.
Occasional differences from control values for certain parameters attained statistical significance, but, since these were slight or not consistent across the sexes, and in the absence of any corroborating histopathological evidence, are considered to be of no toxicological significance.

ORGAN WEIGHTS:
There were considered to be no effects of treatment on the organ weights recorded. Occasional slight inter-group differences in organ weights were not corroborated with histopathological evidence and are thus considered to be of no toxicological significance.

MACROSCOPIC PATHOLOGY:
The macroscopic examination of the animals after the 4-week treatment period and after the 2-week recovery period revealed no treatment-related effects.

MICROSCOPIC PATHOLOGY:
- Larynx:
Necrosis of the ventral cartilage was reported in all treated groups with the severity increasing with the dose. Epithelial hyperplasia in ventral and arytenoid areas was reported in a proportion of animals from all treated groups. The numbers of affected animals and the severity of lesions were less in the lower dosage groups. The high dose Group 4 also showed epithelial hyperplasia in the lateral and/or ventrolateral areas with occasional keratinisation in lateral areas in a few animals. Squamous metaplasia in the ventrolateral area was reported in some animals in Groups 3 and 4.

Recovery group: Necrosis of the ventral cartilage was reported in animals from all groups with the incidence and severity related to the dose of the test compound. A few Group 4 animals and one Group 3 female showed epithelial hyperplasia in the ventral area over the ventral cartilage.

- Lungs:

Alveolar duct thickening was reported in animals in Group 4 and in a proportion of animals in
Group 3. Goblet cell hyperplasia was present in some Group 4 animals but also a few females from Groups 2 and 3, and one male Control.

- Nasal turbinates:
Eosinophilic inclusions in the olfactory epithelium were reported in some animals in all groups but were generally more frequent and/or more severe in the treated groups and this tendency increased with the dosage.

Recovery group: Eosinophilic inclusions in the olfactory epithelium were reported in all groups with the incidence and severity apparently correlating with the dose of the test compound.
Key result
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
0.004 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
0.037 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.004 mg/L air (analytical)
System:
respiratory system: upper respiratory tract
Organ:
larynx
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Exposure of rats to the test item at 0.037 mg/L caused signs indicative of respiratory tract irritation. There was also a reduced bodyweight gain, the cause of which is not known but, in the absence of other changes, is considered probably secondary to the respiratory tract effects. At lower exposure levels (0.0113 or 0.0038 mg/L), signs indicative of respiratory tract irritation were present. Therefore the LOAEL for local effects was set at 0.0038 mg/L. (or 3.8 mg/m3). The test substance did not induce any clear sytemic effects, therefore the NOEAL systemic was set at 0.037 mg/L (>37 mg/m3).
Executive summary:

Three groups of rats (each of 15 males and 15 females) of the Crl:CD®(SD)IGS BR strain were exposed to the test item, 6 hours a day, 5 days a week for 4 consecutive weeks using a snout only exposure system. A fourth group, acting as control, was exposed to air only. Five males and 5 females from each group were monitored over a further 2-week withdrawal period to evaluate recovery.

The study mean analysed concentrations of the test item were close to the targets of 0.005, 0.01 and 0.04 mg/L with analysed levels of 0.0038, 0.0113 and 0.037 mg/L for the Low, Intermediate and High dose respectively.

 

The animals tolerated exposure to the test item well, with only a slightly lower bodyweight gain and food consumption being recorded for males of the high dose group. There was no evidence of any systemic toxicity noted at pathological examinations.

 

There was evidence of local, dosage-related and treatment-related changes to the respiratory tract, following 4 weeks of exposures as shown by histopathological changes to the larynx (including necrosis of the ventral cartilage and epithelial hyperplasia in all treated groups, and squamous metaplasia in intermediate and high dose groups), lungs (alveolar duct thickening in the intermediate and high dose groups and bronchiolar goblet cell hyperplasia predominantly in the high dosage group), and nasal turbinates (eosinophilic inclusions in the olfactory epithelium). Although there was evidence of recovery from these changes in so much that the lung changes showed full resolutions, there were still pathological changes noted in the larynx and nasal turbinates. These changes are considered to represent signs of local irritation by the test substance.

 

Exposure of rats to the test item at 0.037 mg/L caused signs indicative of respiratory tract irritation. There was also a reduced bodyweight gain, the cause of which is not known but, in the absence of other changes, is considered probably secondary to the respiratory tract effects. At lower exposure levels (0.0113 or 0.0038 mg/L), signs indicative of respiratory tract irritation were present. Therefore the LOAEL for local effects was set at 0.0038 mg/L. (or 3.8 mg/m3). The test substance did not induce any clear sytemic effects, therefore the NOEAL systemic was set at 0.037 mg/L (>37 mg/m3).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
3.8 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and Guideline conform study

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated Dose Toxicity - Oral

In this sub-acute toxicity study, the test item was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest and the treatment periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, as well as liver, stomach, thymus of both sexes, adrenal glands of females of the mid- and low dose group, and all gross lesions from all animals.

No mortality, no changes during daily observations or weekly detailed observations (weeks 1-3), and no changes during the functional observational battery performed during week 4 (with no changes in the mean locomotor activity or the mean fore limb or hind limb grip strength values) were observed.

 

The mean daily food consumption of test item-treated males and females was less than that of their respective controls. Additionally, test item-related findings included reduced body weight development at 1000 mg/kg/day, minor changes in the hematology parameters (typical of a mild inflammatory reaction) at 1000 mg/kg/day, mild activation of liver enzymes (considered to be a metabolic reaction of adaptive nature), elevated potassium in males at 300 mg/kg/day and both sexes at 1000 mg/kg/day, increased phosphorus levels in females at 1000 mg/kg/day, macroscopically evident crateriform retractions in the stomachs of rats at 1000 mg/kg/day, and liver enlargement in males at 300 mg/kg/day and 1000 mg/kg/day, as well as reduced mean absolute organ weights and/or ratios in the heart, thymus, adrenal and spleen at 1000 mg/kg/day, increased liver weights and/or ratios at 1000 mg/kg/day and 300 mg/kg/day. Microscopically, squamous cell hyperplasia and hyperkeratosis were noted in the stomach of females treated with 300 mg/kg/day and in both sexes treated with 1000 mg/kg/day, parakeratosis, submucosal inflammation, submucosal edema, and pustules recorded in male and females treated with 1000 mg/kg/day, whereas forestomach ulceration and erosion were recorded in females. These findings were considered to be adverse.

 

Based on the results of this study, 100 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL). Based upon the microscopical changes in the stomachs of rats treated with 300 mg/kg/day or 1000 mg/kg/day, the no-observed adverse-effect-level (NOAEL) was considered to be 100 mg/kg/day.

Repeated Dose Toxicity - Inhalation

Three groups of rats (each of 15 males and 15 females) of the Crl:CD®(SD)IGS BR strain were exposed to the test item, 6 hours a day, 5 days a week for 4 consecutive weeks using a snout only exposure system. A fourth group, acting as control, was exposed to air only. Five males and 5 females from each group were monitored over a further 2-week withdrawal period to evaluate recovery.

The study mean analysed concentrations of the test item were close to the targets of 0.005, 0.01 and 0.04 mg/L with analysed levels of 0.0038, 0.0113 and 0.037 mg/L for the Low, Intermediate and High dose respectively.

 

The animals tolerated exposure to the test item well, with only a slightly lower bodyweight gain and food consumption being recorded for males of the high dose group. There was no evidence of any systemic toxicity noted at pathological examinations.

 

There was evidence of local, dosage-related and treatment-related changes to the respiratory tract, following 4 weeks of exposures as shown by histopathological changes to the larynx (including necrosis of the ventral cartilage and epithelial hyperplasia in all treated groups, and squamous metaplasia in intermediate and high dose groups), lungs (alveolar duct thickening in the intermediate and high dose groups and bronchiolar goblet cell hyperplasia predominantly in the high dosage group), and nasal turbinates (eosinophilic inclusions in the olfactory epithelium). Although there was evidence of recovery from these changes in so much that the lung changes showed full resolutions, there were still pathological changes noted in the larynx and nasal turbinates. These changes are considered to represent signs of local irritation by the test substance.

 

Exposure of rats to the test item at 0.037 mg/L caused signs indicative of respiratory tract irritation. There was also a reduced bodyweight gain, the cause of which is not known but, in the absence of other changes, is considered probably secondary to the respiratory tract effects. At lower exposure levels (0.0113 or 0.0038 mg/L), signs indicative of respiratory tract irritation were present. Therefore the LOAEL for local effects was set at 0.0038 mg/L. (or 3.8 mg/m3). The test substance did not induce any clear sytemic effects, therefore the NOEAL systemic was set at 0.037 mg/L (>37 mg/m3).

Justification for classification or non-classification

Based on the available data, the test substance is not classified for repeated dose toxicity according to REACH Regulation (EC) No 1272/2008.