TROYSOL LAC

Repeated dose toxicity: inhalation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid

Test animals

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 or test system 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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.037 mg/L air (analytical)

Doses / concentrations 2

Dose / conc.:

0.011 mg/L air (analytical)

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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.

Target system / organ toxicity

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

Applicant's summary and conclusion

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).