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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Every detail is documented and reported; The protocol has been designed to meet the minimum requirements of the OECD Guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
not influencing outcome of study
Principles of method if other than guideline:
The study was conducted in accordance with the agreed protocol (P2074d) and 2 amendments, with the following exceptions:
* The temperature and relative humidity of the holding room and exposure room were routinely maintained at 19-25°C and 40-70% respectively. The holding room humidity was outside this range on 8 occasions, the lowest value was 34%. The exposure room temperature was below the limit on 18 occasions, the lowest value recorded was 15°C.
* The temperature and relative humidity of the exposure chambers were routinely maintained at 19-25°C and 45-70% respectively. Occasional deviations outside these ranges were noted, the temperature ranging between 15 and 18.5°C and the humidity between 28 and 90%.
* All animals were examined at week 4, rather than just groups 1 and 4 as indicated in the protocol amendment.
* Potassium was measured in all animals at week 4. This was not a protocol requirement.
* In addition to the tissues required by protocol the gonads were preserved in fixative.
These deviations were thought not to have adversely affected the integrity or outcome of the study.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test material was a colourless liquid with a characteristic odour, identified as 1,4 butane diamine, and was supplied by the sponsor.
Two bottles of the test article each containing 2 kg were delivered to HLE on the 22 June 1983 and 30 January 1984. The test article was stored at room temperature in the dark. No information was available to suggest that the conditions of storage would adversely affect the test article.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Sufficient random-bred Sprague Dawley rats of the Crl:DC(SD)BR strain, to provide 25 healthy animals of each sex, were obtained form Charles River (UK) Ltd., Manston Road, Margate, Kent. The animals were delivered on 12 January 1984.
The animals were obtained as weanlings of about 30 days of age and on receipt all animals were examined for signs of ill health.
5 Male and 5 female animals were killed on arrival and their lungs examined histopathologically. The results of the health screen indicated that the batch of animals was suitable for experimental purposes.
The animals were acclimatised to the experimental room for 11 days, towards the end of which they were examined by a veterinary surgeon, who confirmed their suitability for experimental use. At the start of treatment the animals weighed between 128 and 201 g.

All exposure chambers and the animal holding room were sanitised before use.

The animals were caged in groups of 5, by sex, in stainless steel wire mesch cages (All Type Tools Ltd., Woolwich, London) suspended over cardboard lined trays. The cardboard liners were replaced as often as was necessary to maintain hygiene. Exposure to the test atmosphere took place in a single room dedicated to the study. With the exception of these times, the animals were held in an adjacent room. After exposure periods, the animals were replaced in their holding cages.
The rooms were fully air conditioned providing at least 12 air changes per hour and routinely maintained at a temperature of 19-25°C and relative humidity of 40-70%. Fluorescent lighting was controlled automatically to give a cycle of 12 hurs light (0600 to 1800 hours) and 12 hours dark.

With the exception of the exposure periods, before laboratory investigations and necropsy, the animals were allowed free access to diet (SQC Rat and Mouse Maintenance Diet No.1 Expanded, Special Diets Services Ltd., Witham, Essex) throughout the study. The supplier provided a certificate of analysis of each batch supplied, detailing the composition and levels of specified contaminants (heavy metals, aflatoxins and insecticides).
The animals had free access to drinking water available from glass bottles attached to the cages except during the exposure period. The water is analysed periodically by both HLE and the Yorkshire Water Authority for specified contaminants including heavy metals, chlorinated hydrocarbons and polycyclic aromatic hydrocarbons. The results of all these analyses are maintained on file at HLE.
the diet and water were considered not to contain any contaminants at a level which might affect the integrity of outcome of the study.

The animals were allocated to treatment groups before the start of treatment, using a stratified procedure based on body weight. The positions of the cages within the battery were also randomised.
After allocation to treatment group, each animal was permanently numbered by ear tattoo as follows:

Group 1: Control; colour code buff; males 1-5, females 21-25
Group 2: Low; colour code green; males 6-10; females 26-30
Group 3; Intermediate; colour code blue; males 11-15; females 31-35
Group 4: High; colour code pink; males 16-20; females 36-40.

Each cage of animals was identified with a card (coloured according to the treatment group), bearing the following information: cage number, HLE project number, HLE dispensary number, atmosphere concentration, animal numbers and sex, route of administration, start date of study and Home Office Licensee.


Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
The atmosphere was generated in nose only inhalation chambers. The test article was fed continuously into the chamber with filtered air at a constant flow rate controlled between 8 and 16 litres/min. The flow rate selected was dependent on the type of generator used. The air flow rate to the chambers was monitored continuously and recorded at hourly intervals. The air flow rate was adjusted to ensure that the oxygen concentration of the atmosphere was >= 19%. The oxygen concentration was recorded at hourly intervals. Temperature and humidity of the atmosphere within the exposure chamber was maintained at 19-15°C and 45-70% respectively, monitored continuoulsy and recorded at hourly intervals.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The nominal concentrations of test article were calculated daily from the mass of test article transferred form the generators and the mean exposure chamber flow rates. The real concentrations were measured using a laser light scattering monitor (Simslin obtained from Rotheroe and Mitchell Ltd. Greenford, Middlesex) at hourly intervals during each exposure period, form a point representative of that occupied by the external nares of the experimental animals. Índividual concentrations did not vary by more than approx. 30% of the mean value.
The particle size distribution for each chamber was measured daily using a cascade impaction sampler.
Duration of treatment / exposure:
2 x 2 hours each day
Frequency of treatment:
5 days a week for 4 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.011, 0.05 and 0.25 mg/l
Basis:
other: measured concentrations
No. of animals per sex per dose:
5 males & 5 females per dose
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
The animals were exposed for 2x2 hour periods each day, 5 days/week for 4 weeks. As the necropsis could not be performed on one day the animals were exposed until the day before necropsy. An approximate 2 hour rest period (animals returned to their cages) separated the 2 exposure periods each day.









Examinations

Observations and examinations performed and frequency:
All animals were handled and examined before and after each exposure for signs of ill health, reactions to treatment and overt toxicity. an individual record of clinical changes was maintained for each animal.
The body weight of each animal was recorded immediately before the first exposure period and then at weekly intervals throughout the study.
The food consumed by each cage of animals was determined over 7 day periods at weekly intervals throughout the study.
As a visual assessment of the water consumption did not detect any consistent change in consumption, formal measurements were not instituted.

The eyes of all animals were examined once before the start of the exposure phase and again during week 4 of the study. On both occasions the eyes were examined using a Keeler direct ophthalmoscope following instillation of a mydriatic agent (1% tropicamide, Mydriacyl, Acon Labs. (UK) Ltd. Watford, Herts.).

All blood samples were taken by puncture of the retro-orbital plexus under light ether anaesthesia (diethyl ether, analar grade, BDH, Poole, Dorset), following an overnight fast (about 16 hours). The animals were not exposed to the test article on the day of blood sampling.
The following analyses were performed on blood samples withdrawn into EDTA anticoagulant:
* red blood cell count (RBC) and derived indices:
- mean cell haemoglobin (MCH)
- packed cell volume (PCV)
- mean cell haemoglobin concentration (MCHC)
* haemoglobin (Hb)
* mean cell volume (MCV)
* total (WBC) and differential white blood cell count
* platelet count
The following analyses were performed on blood samples withdrawn into lithium heparin anticoagulant:
* glutamate oxaloacetate transaminase (GOT)
* glutatmate pyruvate transaminase (GPT)
* alkaline phosphatase (Alk.P.)
* glucose
* calcium
* blood urea nitrogen (BUN)
* sodium
* inorganic phosphorous
* chloride
* potassium
* bilirubin
* creatinine
* total protein
* protein electorphoresis
* albumin
* albumin/globulin ratio (A/G ratio)
Sacrifice and pathology:
The following procedures were applied to all animals killed at the end of the study:
* The animals were killed by an intraperitoneal injection of sodium pentobarbitone (Euthatal, May and Baker, Dagenham, Essex) following an overnight period without food. The animals were examined externally including all orifices. The necropsies were conducted over 3 working days. As far as possible equal number of male and female animals from each treatment group were killed on each day.
* The following organs, dissected free from fat and contiguous tissue, were weighed before fixation: adrenals, liver, kidneys, lungs (with a fixed length of trachea), ovaries, testes. After excision and weighing the lungs were fixed by infusion of 10% neutral buffered formalin.
The following tissues were preserved in 10% neutral buffered formalin: adrenals, larynx, heart, nasal turbinates (3 levels), kidneys, spleen, liver, trachea, lungs (removed intact with mainstem bronchi), gonads (taken but not examined), gross lesions. Samples of the tissues from groups 1 and 4 animals and gross lesions from all animals were embedded in paraffin wax B.P., sectioned at a nominal thickness of 5 µm and stained with haematoxylin and eosin. Sections of the nasal turbinates for all animals from groups 2 and 3 were also processed after the identification of treatment-related lesions in group 4 animals. Sections of the specified tissues were examined using light microscopy by the study pathologist.
Statistics:
Data were processed to give group mean values with one standard deviation, where appropriate.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Threatment-related effects were seen in the anterior nasal cavity of group 4 rats.The effects consisted of low grade hyperplasia of the epithelial lining of the lateral wall and turbinates, and atrophy of the most anterior portions of the olfactory epithelium. These findings are indicative of low grade irritation to the anterior nasal cavity. Occasional rats showed minor extensions of olfactory epithelial atrophy into the mid-dorsal nasal cavity. In general the posterior nasal cavity, conductin airways and lung parenchyma resembled that of control rats.
Extension of the histopathological investigation to to 2 lower dose groups indentified minimal changes in the respiratory epithelium of the anterior nasal cavity of some rats in group 3, but no changes were seen in group 2.
There were no unusual findings in the other organs and tissues examined to suggest any systemic toxicity as a result of test article administration.

Effect levels

Dose descriptor:
NOEL
Effect level:
11 mg/m³ air
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Necropsy

The majority of animals were unremarkable. There were no lesions of any unusual nature or incidence to suggest any gross toxic effect.

Histopathology

The respiratory tract of control animals was generally unremarkable apart from occasional foci of foamy histiocyte accumulation or pneumonitis. Histopathology findings in the other tissues examined were generally infrequent or of a minor nature such as foci of leucocyte accumulation in the liver.

There wer treatment-related effects in the anterior nasal cavity of group 4 rats.These consisted of low grade hyperplasia of the epithelial lining of the lateral wall and turbinates, and atrophy of the most anterior portions of the olfactory epithelium. These findings are indicative of low grade irritation of the anterior nasal cavity.

Occasional rats also showed minor extensions of olfactory epithelial atrophy into the mid-dorsal nasal cavity. In general, however, the posterior nasal cavity, conducting airways and lung parenchyma resembled that of control rats. Minimal changes were also found in the respiratory epithelium of the anterior nasal cavity of some rats in group 3, but no changes were seen in group 2 rats.

There were no unusual findings in the other organs and tissues examined to suggest any systemic toxicity as a result of test article adminstration.

Applicant's summary and conclusion

Conclusions:
NOEL inhalation: 11 mg/m3 (0.01 mg/l)
Executive summary:

This study was performed to evaluate the toxicity of 1,4 butane diamine when repeatedly administered to the rat by inhalation for 2x2 hours each day, 5 days/week for 4 weeks.

Groups of Sprague-Dawly rats, comprising 5 animals of each sex were exposed at target dose levels of 0, 0.01, 0.05, 0.25 mg/l. These were groups 1, 2, 3 and 4 respectively.

A dose level of 0.011 mg/l (=11 mg/m3) was identified as the NOEL (No Observed Effect Level)

There were no deaths during the course of this study.

There were no signs that could be unequivocally attributed to treatment.

The body weight gains of treated animals were similar to those of the controls

The food consumption of the treated animals was similar to those of the controls.

No treatment-related ophthalmic defects were found.

There were no changed in the haematological parameters that could be attributed to treatment.

There were no lesions of any unusual nature or incidence to suggest any gross toxic effect.

The organ weights of treated animals were similar to those of the controls.

Histopathologically, a treatment-related low grade irritation of the anterior nasal cavity of high and intermediate dose group animals only was noted.

There were no organ or tissue changes to suggest any systemic toxicity.

The exposure chamber temperature recorded for the control and treated groups ranged between 15 and 20°C, and the humidity between 28 and 90%.

The exposure chamber flow rate for the control and treated groups ranged between 8 and 16 litres/min.

The exposure chamber oxygen concentration for the control and treated groups ranged between 21 and 22%.

The overall mean measured concentrations were 0.011, 0.05, and 0.27 mg/l for groups 2, 3 and 4 respectively.

The mean mass median aerodynamic diameters for the treated groups were 1,92, 2,57 and 3.00 µm for groups 2, 3, and 4 respectively. All values were below the upper respirable limit of 8 µm.