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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
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Publicly available literature, non GLP
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
not applicable
Principles of method if other than guideline:
No guideline indicated as study is a subchronic inhalation study. The conduct was similar to OECD guideline 413 “Subchronic Inhalation Toxicity: 90-day Study
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Chlorine
EC Number:
231-959-5
EC Name:
Chlorine
Cas Number:
7782-50-5
Molecular formula:
Cl2
IUPAC Name:
Active chlorine generated from chloride of ambient water by electrolysis
Details on test material:
chlorine gasSupplied by Matheson (Morrow, GA) as a 3 % Cl2 in nitrogen mixture

Test animals

Species:
monkey
Strain:
other: Rhesus monkey (Macaca mulatta)
Sex:
male/female
Details on test animals or test system and environmental conditions:
4 monkeys per sex and groupAge: not statedbody weightmales: 2.5 kgfemales: 2.4 kg

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: none, dilution with air
Remarks on MMAD:
MMAD / GSD: Not applicable, chlorine is a gas
Details on inhalation exposure:
No vehicle (dilution with clean air only)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Mean measured chlorine concentration0.1±0.03 ppm0.5±0.1 ppm2.3±0.4 ppm
Duration of treatment / exposure:
one year
Frequency of treatment:
5days per week, 6h per day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:0, 0.1, 0.5 and 2.5 ppmBasis:nominal conc.
Remarks:
Doses / Concentrations:0.1±0.03 ppm, 0.5±0.1 ppm and 2.3±0.4 ppm Basis:analytical conc.
No. of animals per sex per dose:
4 monkeys per sex and group
Control animals:
yes, concurrent no treatment
Details on study design:
Three groups of four male and four female Rhesus monkeys each were exposed to chlorine for a period of 6 hours per day for 52 weeks (exposure on week days only). Monkeys were exposed to nominal concentrations of 0, 0.1, 0.5 and 2.5 ppm Cl2corresponding to analytical concentrations of 0.1±0.03, 0.5±0.1 and 2.3±0.4 ppm Cl2.Clinical signs and mortality were recorded twice daily and the animals underwent a detailed physical examination monthly during the treatment period of the study. Body weights and electrocardiograms were obtained monthly. Ophthalmological examinations were performed prior to initiation of exposures and at the termination of the study.Pulmonary function evaluations, which included pulmonary diffusing capacity of carbon monoxide and distribution of ventilation, were conducted three times prior to the start and monthly thereafter. Haematology, serum chemistry, and urinalysis tests were conducted on all monkeys prior to exposure initiation and at monthly intervals thereafter. At the end of the treatment period, all animals were fasted overnight, anaesthetised and sacrificed by exsanguination. Macroscopic and microscopic examinations were conducted.Nominal and analytical concentrations were regularly determined during the study. No MMAD and GSD were determined in this study and particle size measurements were not possible to perform fore chlorine is a gas.
Positive control:
no positive control

Examinations

Observations and examinations performed and frequency:
The monkeys were observed twice daily for clinical signs of toxicity and underwent a detailed physical examination once each month following sedation with ketamine. Electrocardiograms were obtained four times prior to the start of the study and monthly while the studyMortality: Twice daily during the treatment period of the study.Body weight : The monkeys were weighed monthly.Ophthalmoscopic examination: Prior to initiation of exposures and at the termination of the study the monkeys received an ophthalmoscopic examination.Haematology: Haematologic tests were conducted on all monkeys prior to exposure initiation and at monthly intervals thereafter. The determination included haematocrit, haemoglobin concentration, erythrocyte count, leukocyte count (total and differential), reticulocyte count, mean corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration.Clinical Chemisty: Clinical measurements were conducted on all monkeys prior to study start and monthly thereafter. They included alanine aminotransferase, aspartate aminotransferase, γ-glutamyltransferase, blood urea nitrogen, calcium, phosphorus, total bilibrubin, and total protein. Additional measurements included alkaline phosphatase and glucose. The serum electrolytes, sodium, potassium and chloride, were analysed.Urinalysis: Urinalysis was conducted prior to the study start and monthly thereafter. The determination included colour, microscopic examination of sediment, specific gravity, volume, and pH, protein, glucose, occult blood, nitrites, bilibrubin, ketones and urobilinogen.
Sacrifice and pathology:
Organ Weights The weights of heart, lung plus trachea, liver, gonads, kidneys, spleen, and brain were recorded for all monkeys.Gross and histopathology The following tissues were examined from monkeys in all study groups: adrenal (2), aorta (abdominal), bone marrow (sternum, smear and section), brain (cerebellum, cerebrum (2 levels),medulla), cervix, epididymis, oesophagus, eye/optic nerve (2), gallbladder, heart, kidney (2), lachrymal gland, large intestine (cecum, colon, rectum), larynx, liver, lung (all five lobes), lymph nodes (cervical, bronchial, mesenteric), mammary gland (male and female), nasal tissues (4 sections: at the first palantine ridge and just posterior to the third, fifth, and seventh palatine ridges), neuroganglia (lumbar and sacral, dorsal), ovary (2), oviduct (2), pancreas, parathyroid (2), peripheral nerve (sciatic, anterior tibial), pituitary, prostate, salvary gland (submaxillary), skeletal muscle (gastrocnemius), skin, small intestine (duodenum, jejunum, ileum), spinal cord (4 levels), spleen, stomach, (cardia, fundus, pylorus), testis (2), thymus, thyroid (2), tongue, trachea (2 levels), urinary bladder, and uterus. A grading system of trace, mild, moderate, and severe was used to define the lesions.
Other examinations:
Clinical observational neurologic examination were conducted prior to and at the termination of the study.Pulmonary function evaluations, which included pulmonary diffusing capacity of carbon monoxide (DLCO) distribution of ventilation (N2 washout), where conducted three times prior to the start of the study and monthly thereafter.Blood gas and pH evaluations were conducted during the pre-exposure period and at 3, 6, 9, and 12 months of the study.
Statistics:
Body weights, haematologic, serum chemistry, blood gas data, and absolute and relative (to body weight) organ weights were analysed by nonparametric analysis, using the Kruskal-Wallis one-way analysis of variance, followed where appropriate, with the Mann-Whitney U test. All statistical analyses compared the treatment groups with the control group by sex. Pulmonary physiology data were analysed using a reported-measures design analysis of variance (Winner, 1971) with treatment and time as factors, each animal being observed during each time period (three pre-initiation intervals and monthly for the duration of the study). All statistical tests were conducted using p<0.05 as the level of significance.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical signsAfter approx. 6 weeks of exposure, the 2.3 ppm group exhibited overt signs of ocular irritation (tearing, reddened eyes, rubbing at eyes) during daily exposures. Signs of ocular irritation were not observed at the lower exposure concentrations.MortalityNot reported.Body weight and body weight gainThere were no significant differences in body weights between the control group and any of the exposure groups at any interval during the study.Food consumption and compound intakeNot investigated.Water consumptionNot determinedOphthalmoscopic examinationThe monkeys exposed to 2.3 ppm Cl2 exhibited conjunctival irritation with some exudation during the ocular examination at the end of the study. The conjunctival irritation may have been further exacerbated by self-inflicted rubbing-trauma.There was no gross evidence of chronic changes in the conjunctiva nor were corneas affected. Blood analysisHaematologyThere were no exposure-related differences in haematology parameters evaluated.Clinical chemistryThere were no exposure-related differences in the clinical chemistry parameters evaluated.UrinalysisThere were no exposure-related differences in the urinalysis parameters evaluated.Sacrifice and pathologyAbsolute and relative organ weightsThe two statistically significant differences observed for organ weights (increased liver/body weight and absolute heart weight for the 0.5 ppm group females) were not concentration-related and therefore considered to be due to normal biological variability and the small sample population.Gross pathology and histopathologyMacroscopy:There were no exposure-related macroscopic changes in tissues of either sex in any exposure group.Microscopy:The only treatment-related histopathological change observed was focal epithelial hyperplasia which was characterised by increased cell numbers and loss of cilia and goblet cells in affected areas of the respiratory epithelium of the nose and trachea.• Nasal passages (please refer to table below): In affected areas small zones of respiratory epithelium exhibited hypercellularitiy with loss of goblet cells and cilia, with no apparent increase in epithelial thickness. In some areas the nuclei of epithelial cells in regions of hypercellularity showed altered polarity, suggesting that the lesion was an early stage of squamous metaplasia. However, frank squame cell formation was minimal, and this lesion will be referred to subsequently as epithelial hyperplasia. This lesion was most commonly present on the angular margins of the turbinates and was less frequently observed on the lateral wall or septum adjacent to these margins. Respiratory epithelial hyperplasia was in some cases associated with a mild suppurative inflammatory response. Respiratory epithelial hyperplasia was observed in the nasal passages of both sexes in the 2.3 ppm exposure group. It was also observed, but only in its very mildest form, in the 0.5 ppm group, females of the 0.1 ppm group, and in one male control monkey. It was evident in treated animals that these lesions increased in incidence and severity with increasing exposure concentration.• Trachea: the epithelial lesions resembled those seen in the nasal passages but were less severe and involved in only a small circumferential section of the ventral and ventolateral trachea. Again, the lesion was comprised of epithelial hyperplasia with minimal evidence of early non-keratinising squamous metaplasia.• Parasites (nematode: Anatrichosoma; mite: probably Pneumonyssus sp) and/or ova were observed in 11 of 32 animals and histological changes compatible with the presence of the parasites were observed in an additional 16 monkeys. The lesions caused by parasites were focal, and there was no evidence to suggest that they interfered with the recognition of possible exposure-related effects in the lung.• Granulomatous rhinitis was generally confined to the nasal mucosa anterior to the turbinates. However, in on of the monkeys exposed to chlorine (a 0.1 ppm group male) the granulomatous rhinitis and associated squamous metaplasia extended to the level of the turbinates and thus overlapped the area in which exposure-related lesions were present.OtherNo exposure-related differences were observed in the evaluated neurologic or electrocardiographic parameters. There was a statistically significant trend for increasing pulmonary diffusing capacity and distribution of ventilation values for both sexes over the course of 15 month (including three pre-initiation evaluations) during which pulmonary function evaluations were conducted. There were, however, no significant differences between control and exposed groups with regard to either pulmonary diffusing capacity or distribution of ventilation at any interval during the study (please refer to table below).

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
2.3 ppm
Sex:
male/female
Basis for effect level:
other: corresponding to 6.9 mg/m3 (20.9 mg/kg bw/d assuming a body weight of 2.5 kg and a respiratory volume of 0.021 m3/min. similar to humans)
Dose descriptor:
NOAEL
Effect level:
0.5 ppm
Sex:
male/female
Basis for effect level:
other: corresponding to 1.5 mg/m3 (4.5 mg/kg bw/d assuming a body weight of 2.5 kg and a respiratory volume of 0.021 m3/min. similar to humans)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Incidence of histopathological lesions observed in monkeys exposed to chlorine for 1 yeara

 

Nasal mucosal lesions

 

Exposure concentration
[ppm]

Trace

Mild

Tracheal mucosal lesionsb
(Mild)

 

Males

 

0

1

0

0

0.1

0

0

0

0.5

3

0

0

2.3

2

2

0

 

Females

 

0

0

0

0

0.1

3

0

0

0.5

3

0

0

2.3

2

2

2

 

 

 

 

a     four monkeys/sex/group were examined.

b     Epithelial hyperplasia with loss of cilia and goblet cells

 Pulmonary function values at selected intervals for monkeys exposed to chlorine for 1 yeara

 

Pulmonary diffusing capacity [mL CO/min/mm Hg]

Distribution of ventilation [No. breaths to 1 % N2]

Study interval

0 ppm

0.1 ppm

0.5 ppm

2.3 ppm

0 ppm

0.1 ppm

0.5 ppm

2.3 ppm

 

Males

Males

Pre-initiation

2.0±0.3

2.4±0.2

2.0±0.7

2.0±0.4

34±4

24±6

36±6

36±8

4 months

2.0±0.4

2.1±0.2

1.7±0.4

1.8±0.3

48±19

44±6

42±4

48±16

8 months

2.1±0.9

2.2±0.5

2.2±0.9

2.7±0.5

33±5

32±9

46±13

35±9

12 months

2.8±0.6

2.9±0.6

3.6±0.8

2.8±0.7

42±6

45±18

43±8

52±26

 

Females

Females

Pre-initiation

1.8±0.5

2.0±0.6

1.8±0.1

1.8±0.4

39±14

32±3

33±8

28±7

4 months

1.6±0.4

2.0±0.5

1.7±0.5

1.9±0.5

55±8

50±12

42±9

45±21

8 months

1.8±0.7

2.6±1.1

2.1±0.5

1.6±0.8

42±14

30±3

37±11

30±4

12 months

2.1±0.7

2.8±0.5

2.6±0.4

2.9±1.1

56±25

40±18

46±14

51±15

aValues are the means ±SD from four monkeys/sex/group. There were no statistically significant differences observed between the control and the exposure groups at any of the 12 monthly intervals for either pulmonary function parameter.

Applicant's summary and conclusion

Conclusions:
Treatment-induced histopathological changes were found in the respiratory epithelium of the nasal passages and trachea and were limited to focal, concentration-related epithelial hyperplasia with loss of cilia and decreased numbers of goblet cells in affected areas. These changes in nose and trachea were focal and mild in monkeys exposed to 2.3 ppm Cl2 and were not found in all animals in these exposure groups. Tracheal lesions were confined to the 2.3 ppm group. The lesions observed at 2.3 ppm were not present in all animals. At the lower Cl2 concentrations, similar though less prominent respiratory epithelial lesions were observed. The latter changes were very minimal and were confined to the nasal passages of some treated monkeys and one control animal.The results of this study indicate that 2.3 ppm chlorine acts as an upper respiratory irritant in monkeys, while 0.5 and 0.1 ppm induce changes of questionable clinical significance.
Executive summary:

The parasite Anatrichosoma spp. was present in many of the animals and induced moderate to severe, granulomatous rhinitis. However, the lesions associated with this nematode were generally confined to the nasal vestibule in the region lined by squamous epithelium. Furthermore, inflammatory responses to the nematode appeared to have no major effect on the shape and diameter of the vestibular lumen, and thus probably had little effect on nasal airflow patterns or consequently on regional deposition of Cl2 in the nose.

In this study, treatment-related responses were confined to ocular and respiratory tract irritation. Histopathological examinations revealed that treatment-induced lesions were limited to the respiratory epithelium of the nose and trachea. These lesions were characterised by mild, focal, epithelial hyperplasia in the absence of epithelial thickening with an associated loss of cilia and goblet cells in the affected areas.
Nasal and tracheal lesions were induced by exposure to 2.3 ppm chlorine, while less distinct but similar changes were also present in the nasal passages of some animals in the 0.5 and 0.1 ppm groups in the absence of tracheal lesions. These findings indicate a concentration-related response relationship for Cl2-induced airway toxicity.

No histological lesions were observed in this study at sites other than the nasal cavity and trachea. There was also no indication that exposure to chlorine concentrations up to 2.3 ppm for 1 year produced neither electrocardiogram abnormalities nor haematological changes.

Blood gases, distribution of ventilation, diffusing capacity, and histological evaluations were conducted and there were no exposure-related effects found in blood gas or pulmonary function parameters. These findings were further verified by the lack of histological lesions. LO(A)EL: 2.3 ppm corresponding to 6.9 mg/m3 (20.9 mg/kg bw/d assuming a body weight of 2.5 kg and a respiratory volume of 0.021 m3/min. similar to humans) NO(A)EL: 0.5 ppm corresponding to 1.5 mg/m3 (4.5 mg/kg bw/d assuming a body weight of 2.5 kg and a respiratory volume of 0.021 m3/min. similar to humans)

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