A mixture of: esters of C14-C15 branched alcohols with 3,5-di-t-butyl-4-hydroxyphenyl propionic acid; C15 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate; C13 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 17 1991 to June 18 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

other: Acute inhalation toxicity

Limit test:

yes

Test material

Test material information

Constituent 1

Test material form:

liquid

Specific details on test material used for the study:

Not specified

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
5 male and 5 female Sprague-Dawley rats were received from Charles River (UK) Limited, Margate, Kent, England on 9 May 1991. A t the time of arrival the batch o f animals were within the weight range 121-150 g. A further 5 male and 5 female rats were assigned t o the study in order to dose the additional group (Group 2). These additional animals were received on 9 May 1991, in the weight range 124-150 g.

Housing
The rats were housed in a semi-barrier maintained animal room with a target room temperature of 20°C and a relative humidity of -ca 55% +/- 10%. Temperature and humidity extremes recorded were 19 - 22°C and 34 - 45% respectively. Although these environmental extremes deviated from the protocol-stated target ranges, they were not considered to have affected the integrity of the study. A 12 h light-dark cycle was controlled by a time switch; light hours being 0700-1900 h. The animals were housed in Room N21 of the Block N Rodent Inhalation Toxicology Complex at the Elphinstone Research Centre.

Animal maintenance
Rats were housed 5 per cage according to sex in suspended stainless steel cages. All cages were suspended over trays containing absorbent paper. Each cage was supplied with a polypropylene water bottle (capacity 500 ml). with a rubber washer and melamine cap. Cages and cage tray papers were changed as necessary during the study period whilst water bottles were cleaned or changed as required. Each afternoon, when other work in the room was finished, floors were swept then washed with a disinfectant solution . Approximately once each week walls, benches and racking within the animal room were washed with a disinfectant solution. The disinfectant used was 1% Tego from Th Gofdschmidt and Company Limited, Middlesex, England.

Diet
The food and water used on the study were considered to contain no contaminants insufficient quantity to have had any influence on the outcome o f the study.

Food
Rat and Mouse (Modified) No. 1 Diet SQC Expanded, supplied by Special Diets Services Limited, Stepfield, Witham, Essex was available.

Water
The animals had access to domestic mains quality drinking water.

Food and water were available adlibitum except during the 4 h exposure period.

Animal identification
Each animal was given a unique earmark which identified it within the study and corresponded to that animal's study number.
Each animal was ascribed a cage card which identified that animal by project number, animal number, sex, treatment group and cage number.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

snout only

Vehicle:

air

Geometric standard deviation (GSD):

ca. 0.49 - ca. 3.3

Details on inhalation exposure:

The test atmospheres were generated by metering the test material via a peristaltic pump to a Schlick atomiser which was located at the top of the exposure chamber. The concentration within the exposure chamber was controlled by adjusting the rate o f feed of the test material and the air flow rate to the atomiser. Chamber air flow rates were monitored continuously and the values recorded at 30 min intervals.
Filtered, oil-free compressed air , for the production of the test atmosphere, was supplied by oi 1-free Hydrovane compressors. The aluminum exposure chamber (ADG Instruments Limited, Hitchin, Herts, England) was cylindrical in cross section and had a volume of approximately 41.5 liters . An extract duct from the chamber was connected by way of a high efficiency filter to a metered vacuum system. The exposure chamber was located inside an extract chamber for the protection of the operators and the environment.
The exposure system was truly dynamic, incorporating a single pass of the freshly generated material. The aerosol particles dispersed throughout the chamber and exited from the base through the trap to a filtered vacuum line. The inhalation exposures were conducted in a room (N19) adjacent to the animal holding room.
Prior to exposure, each animal was removed from it's cage and examined for general health status. The ear number was checked, the animal weighed and then loaded into a tapered, polycarbonate restraint tube which fitted onto the exposure chamber and sealed by means of a push fit through a rubber '0' ring. All the animals were exposed on a single tier to eliminate any exposure variation. Only the animals' snouts were exposed to the test atmosphere. The animals were not allowed access t o food or water during the 4 h exposure period.
During the exposure the animals were observed at regular intervals for signs of any adverse reactions to treatment. On completion of the 4 h exposure period, the animals were removed from the chamber, unloaded from the restraint tubes, returned t o their cages in the animal holding room and observed for clinical signs.
The Group 1 animals (5 males and 5 females) were exposed to 4.07 mg liter -1 while trying to achieve the target limit concentration of 5 mg liter -1 via the inhalation route by snout only exposure for a single continuous 4 h period. The exposure was conducted on 17 May
1991.
The Group 2 animals were dosed to ensure the target limit was achieved and were exposed to 7.53 mg liter -1. This exposure was conducted on 3 June 1991.
During the exposure period the temperature within the exposure chamber was measured by a mercury thermometer located at the animals' breathing zone whilst the relative humidity was monitored using wet/dry bulb mercury thermometers. The chamber temperature and
relative humidity were measured at 30 min intervals throughout the exposure period.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Chamber concentrations were measured at regular interval s during the exposure period. The gravimetric method used employed silica gel sorbent sampling tubes.

Duration of exposure:

4 h

Remarks on duration:

As per guideline

Concentrations:

Group 1 (5 males and 5 females): 4.07 mg/ litre -1
Group 2 (5 males and 5 females): 7.53 mg/ litre -1

No. of animals per sex per dose:

5 male and 5 female.

Control animals:

not specified

Details on study design:

Atmospheric Characterisation

Chamber Concentration:
Chamber concentrations were measured at regular interval s during the exposure period. The gravimetric method used employed silica gel sorbent sampling tubes. The input side of the tube was positioned and temporarily sealed in a port in the exposure chamber at the animals' breathing zone. Chamber air was drawn through the sorbent tube at a measured rate of 1.0 litre.min -1 using a vacuum pump. The air flow during each sample was controlled by a flow meter and timed for a suitable period.
Each sorbent tube was weighed before and after sampling in order t o calculate by difference the weight o f material collected. The total chamber concentration was estimated by further calculation using the sample air volume. The background moisture content was determined t o be 0.27 and 0.11 mg.litre -1 for Groups 1 and 2 respectively and these values were subtracted from the above derived chamber concentration.

Nominal Chamber Concentration
The nominal chamber concentration was estimated using the following equation:

Nominal concentration = Weight of material used (mg) / Total air flow through chamber (litres)

Particle size distribution:
The particle size distribution o f the dispersed material inside the exposure chamber was estimated twice during each exposure period using a Marple (Model 296) Cascade Impactor (Anderson Samplers Inc., Atlanta, Georgia, USA). This device consisted of an in line sampler and a series of impaction stages capable of fractionating the aerosol into the size range 0.25->10 µm. The device was positioned on the chamber at the animal's breathing zone chamber air sampled, using a vacuum pump, at a constant rate o f 2.0 1itres.rnina1 for a recorded time period.
The implication substrates (for each stage) and back up sorbent tube were weighed before and after sampling and the weight increase on each substrate determined to be the mass of particles in the size range o f that impactor stage. From the results obtained, the total weight of particles collected on the impaction stages and back up sorbent tube were summed and the percent particle mass in each size range calculated. The data are presented as showing the percent of particle mass smaller that the stated aerodynamic particle diameter, and the respirable mass fraction determined from the particle size distribution.

Observation on the animals

Clinical signs
A17 the rats were observed for clinical signs at frequent intervals throughout the exposure period, for the first 1 h post dosing and thereafter at least once (normally twice) daily during the subsequent 14 day observation period. The onset, intensity and duration of any signs observed were recorded.

Body Weights
All the rats were weighed immediately. before dosing and on Days 2, 3, 4, 7, 10 and 14 post exposure.

Terminal studies

Necropsy examination:
A t the end of the 14 day observation period a11 the animals were sacrificed by an intraperitoneal overdose of pentobarbitone sodium (Expiral, Abbot) and subjected to a macroscopic post mortem examination.
Each rat was examined externally prior to opening the abdominal and thoracic cavities. Any gross lesions observed were recorded in descriptive terms, including location(s) , size (mm) , colour and number. The respiratory tract was subjected to detailed macroscopic examination for signs of irritancy or local toxicity. All organs were examined in situ.

Lung : Body weight ratio:
The lungs o each animal were removed and weighed to allow calculation of lung : body weight ratios.
On completion of the necropsy, the animals' carcasses were disposed of; no tissues were retained.

Results and discussion

Preliminary study:

Not specified

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 7.53 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

There were no mortalities during the course of the study.

Clinical signs:

other: An unkempt appearance was noted for all Group 1 animals immediately post dose and one animal (179, Group 2) appeared subdued and showed hunched posture over the first 24 h post dose. Otherwise no other abnormalities were detected following treatment.

Body weight:

There was no effect on body weight profile following exposure to ANOX BF

Gross pathology:

All group 1 animals were seen to have no abnormalities.
Slightly mottled lungs i n all but 2 Group 2 animals and pale/discoloured lungs were noted in one Group 2 male (No. 12) and 1 Group 2 female (No 20). These were deemed to be in accordance with normal background findings i n acute rat studies at IRI and not attributable to treatment.

Other findings:

Lung : body weight ratios were generally within normal limits for all animals, although Group 2 males tended towards the bottom of the expected range.

Any other information on results incl. tables

The chamber air flow rate was monitored continuously and recorded at 30 min intervals throughout the exposure period. The values recorded were a constant 12.0 litre.min^-1, for both groups.

Chamber Atmos~heric Conditions
Concentration
The gravimetric concentration of the test aerosols in the exposure chamber was derived from the mean of several measurements made during the exposure periods. The adjusted overall mean values obtained were 4.07 mg litre^-1 for Group 1 and 7.53 mg. litre^-1 for Group 2.

Particle Size Distribution
Estimation of the particle size distribution revealed that the percentage of particles <3.5 µm for Group 1 was 94.0 and for Group 2 was 93.9.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

ANOX BF did not produce any evidence of toxicity in Sprague-Dawley rats following exposure for 4 hours to an atmosphere containing 7.53 mg. liter -1 (measured gravimetrically).

Executive summary:

This study was undertaken to investigate the acute inhalation toxicity (limit test) of ANOX BF  in two groups of rats (5male and 5 female) following a single 4 hour snout only exposure to the test article.

The study was conducted in compliance with OECD (403) guidelines for acute toxicity testing and in accordance with internationally recognised standards of GLP.

Group 1 animals were exposed to a measured gravimetric ^{conce-1ntration} of 4.07 mg. litre ^-1 (87.85 mg. litre^-1 nominal). Group 2 animals were exposed to a measured gravimetric concentration of 7.53. litre^-1 (70.14 mg. litre^-1 nominal). The particle size determination indicated 94.0% and 93.9% of the test aerosol particles to be <3.5 µm for groups 1 and 2 respectively.

There were no mortalities, clinical signs or affects to body weight following exposure to the test article. No abnormalities detected at post mortem observations were considered to be indicative of a reaction to treatment with the test article.

The lung : body weight ratio values were generally all within normal limits.

In conclusion, ANOX BF did not produce any evidence of toxicity in Sprague-Dawley rats following exposure for 4 hours to an atmosphere containing 7.53 mg. litre^-1 (measured gravimetically).