Chloramide

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study, according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chloramide

EC Number:

234-217-9

EC Name:

Chloramide

Cas Number:

10599-90-3

Molecular formula:

ClH2N

IUPAC Name:

chloranamine

Test material form:

other: aqueous solution

Details on test material:

- solution of Monochloramine at 1 %
- Due to the instability of the Monochloramine, the solution is prepared extemporaneously.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

25 males and 25 females.
Duration of acclimatisation: 10 days before commencement of treatment.
Age of the main animals at start of treatment: 53 to 59 days.
Weight range of the study animals at the start of treatment: Males: 192 to 226 g / Females: 141 to 171 g

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Details on inhalation exposure:

Route: Inhalation - snout only exposure.
Training for dosing: The animals on study were acclimated to the method of restraint, over a 3 day period preceding the first test substance exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The formulated samples were analysed using a method transferred from the Sponsor and involved the colorimetric measurement of Total Residual Chlorine (TRC) and Free Residual Chlorine (FRC). Monochloramine was calculated from the difference between the two measurements

Duration of treatment / exposure:

28 days

Frequency of treatment:

Duration of daily exposure: 6 hours, 5 days each week.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.025
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.079
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.25
Basis:
nominal conc.

No. of animals per sex per dose:

25 males and 25 females.
5 males and 5 females per dose

Control animals:

yes

Positive control:

none

Examinations

Observations and examinations performed and frequency:

SERIAL OBSERVATION:
1) Clinical observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
Signs associated with exposures
Detailed observations were recorded daily on exposure days at the following times in relation to exposure:
- Pre-exposure observation
- As each animal was returned to its home cage
- As late as possible in the working day
Detailed observations were recorded daily on non-exposure days at the following times in relation to dose administration:
- Early in the working day (equivalent to pre-exposure observation)
- As late as possible in the working day
Clinical signs
A detailed weekly physical examination was performed on each animal to monitor general health.

2) Body weight:
The weight of each animal was recorded twice during Week -1, on the day that exposures commenced (Day 1), twice weekly throughout the study and before necropsy.

3) Food consumption:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

4) Haematology, peripheral blood:
Blood samples were collected after overnight withdrawal of food and prior to exposure during Week 4.
- Haematocrit (Hct)
- Haemoglobin concentration (Hb)
- Erythrocyte count (RBC)
- Absolute reticulocyte count (Retic)
- Mean cell haemoglobin (MCH)
- Mean cell haemoglobin concentration (MCHC)
- Mean cell volume (MCV)
- Red cell distribution width (RDW)
- Total leucocyte count (WBC)
- Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt)
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyser and appropriate reagent in respect of:
- Prothrombin time (PT) - using IL PT-Fibrinogen reagent.
- Activated partial thromboplastin time (APTT) - using IL APTT reagent.

5) Haematology, bone marrow:
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment period.

6) Blood chemistry:
Blood samples were collected after overnight withdrawal of food and prior to exposure during Week 4.
- Alkaline phosphatase (ALP)
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Gamma-glutamyl transpeptidase (gGT)
- Total bilirubin (Bili)
- Urea
- Creatinine (Creat)
- Glucose (Gluc)
- Total cholesterol (Chol)
- Triglycerides (Trig)
- Sodium (Na)
- Potassium (K)
- Chloride (Cl)
- Calcium (Ca)
- Inorganic phosphorus (Phos)
- Total protein (Total Prot)
- Albumin (Alb)
- Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

Sacrifice and pathology:

TERMINAL PROCEDURES:

All main study animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.
Schedule Animals were killed following 4 weeks of exposures.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed in the table 1 "in "any other information on material and methods"
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

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):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

1) Clinical observations
Clinical signs associated with exposures for females exposed to 0.287 μg/L included abnormal gait (elevated, swaying, unsteady), these signs were observed on occasion on return to the home cage and remained evident until the end of day observations. There were no clinical signs on the days without exposures (Days 6-7, 13-14, 20-21, 27-28). Clinical signs of red staining to the nose, eyes and head were observed for a proportion of animals from all groups, the incidence of these signs was increased in test groups. Wet fur was observed on return to the home cage on occasion for all groups. These signs were considered to be due to the method of administration.

2) Body weight
There were no test article related effects on body weight.

3) Food consumption
There were no test article related effects on food consumption.

4) Haematology, peripheral blood
There were no test article related effects.
Mean white blood cell counts were lower for all males exposed to monochloramine and females exposed to 0.287 μg/L, principally due to neutrophils and lymphocytes. This finding lacked an exposure relationship for males and all individual values were within the background data range, therefore this was considered not to be test article related. All other differences from control were generally small, inconsistent between the sexes or considered to be due to high intra-group variation and therefore were not of toxicological importance.

5) Blood chemistry
Mean phosphorus concentration was low for males exposed to 0.0767 or 0.287 μg/L and all females exposed to monochloramine, when compared with control (as low as 0.89X and 0.77X control, for males and females, respectively (exposure related in females only)). All other differences from control were generally small, inconsistent between the sexes, lacked exposure-relationship or were considered to be due to high intra-group variation and therefore were not of toxicological importance.

6) Organ weights
Mean heart weight was low for all females exposed to monochloramine (unadjusted and adjusted), compared with control (as low as 0.80X), however this finding lacked an exposure relationship.

7) Macropathology
The macroscopic examination performed after 4 weeks of exposures revealed no test substance related lesions. The incidence and distribution of all findings were consistent with the common background seen here.

8) Histopathology
No changes related to treatment with monochloramine were seen. All histological findings were considered incidental and unrelated to the test article.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

0.287 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

1) Formulation analysis:

The formulation concentrations were within 10% of nominal inclusion apart from Week/Day 3.2 for Group 4 which was 116% of nominal inclusion. The pH measurements for all formulations ranged between pH 10.15 and pH 11.13.

2) Atmosphere analysis and estimation of achieved dose:

Group	Vapour concentration (µg/L)
	Target	Achieved
2	0.025	0.0294
3	0.079	0.0767
3	0.25	0.287

The achieved daily mean vapour concentrations were 118, 97 and 115% of the target concentration. The pH measurements from the concentration samples ranged between pH 6.04 and pH 8.09.

Applicant's summary and conclusion

Conclusions:

Administration of monochloramine vapour, by the inhaled route to Han Wistar rats, 6 hours daily, for 4 weeks at achieved vapour concentrations of 0.0294, 0.0767 or 0.287 μg/L was well tolerated.
Test article-related findings were limited to clinical observations, which were transient in nature and therefore considered non-adverse. There were no other clinical or pathological reactions to exposure to monochloramine. Based on the results of this study the No Observed Adverse Effect Level (NOAEL) is considered to be greater than 0.287 μg/L, the No Observed Effect Level (NOEL) was considered to be 0.0767 μg/L.

Executive summary:

The objective of this study was to assess the toxic potential of monochloramine, in a 4 weeks inhalation study to Han Wistar rats according to OECD Guideline 412 and EC Method B8. The inhalation route of administration was chosen to simulate the conditions of potential human exposure.

A previous preliminary test was performed in order to determine the test dose to be performed in the definitive test. In this preliminary study of the repeated dose toxicity study 28 days, firstly Han Wistar rats were exposed to monochloramine vapour at 18.8 or 53.5 μg/L for 6 hours a day. Exposure of monochloramine vapour at 18.8 or 53.5 μg/L for 2 days was not tolerated and animals were euthanized on Day 3. Histopathological changes were observed in the respiratory system and were suggestive of acute irritation. Secondly, Han Wistar rats were exposed five days for 6 hours a day at 0.233, 0.649 or 1.88 μg/L, followed by a two day off exposure period. This exposure resulted in test article related breathing signs. The incidence of these signs at 0.649 and 1.88 μg/L and the likely progression indicate that these vapour concentrations would not be suitable for longer term administration. The findings at 0.233 μg/L were of lesser severity, it is therefore anticipated that target vapour concentrations up to this threshold would be tolerated in a longer term study.

In this study, 3 groups of 5 males and 5 females Han Wistar rats were exposed to monochloramine vapour, by the inhaled route 6 hours daily, for 4 weeks at the target nominal vapour concentrations of 0.025, 0.079 or 0.25 μg/L. During the study, clinical condition, body weight, food consumption, haematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken. This administration of monochloramine vapour at the achieved vapour concentrations of 0.0294, 0.0767 or 0.287 μg/L was well tolerated. There were no test-article related effects on body weight, food consumption or haematology parameters. There were also no macroscopic or microscopic changes. There were no treatment-related unscheduled deaths during the study and no relevant clinical signs were observed. Test article-related findings were limited to clinical observations, which were transient in nature and therefore considered non-adverse. There were no other clinical or pathological reactions to exposure to monochloramine. Based on the results of this study the No Observed Adverse Effect Level (NOAEL) is considered to be greater than 0.287 μg/L, the No Observed Effect Level (NOEL) was considered to be 0.0767 μg/L.