Chloramide

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

No satellite group. The wet weight of Epididymides and uterus were not taken.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Mich.
- Age at study initiation: no data
- Weight at study initiation: 23.9 to 24.6 g for males/ 19.1 to 19.5 g for females
- Fasting period before study: no
- Housing: 5 animals per cage
- Diet : ad libitum (Purina certified Chow 5002, Ralston-Purina Co., St Louis, Mo)
- Water: ad libitum (tap water)
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 40- 60
- Air changes (per hr): no data
- Photoperiod : 12 hrs dark / 12 hrs light

IN-LIFE DATES: no data

Route of administration:

oral: drinking water

Vehicle:

other: carbonate buffer

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Monochloramine stock solutions (about 3,500 ppm) were prepared by adding 50 ml of the stock chlorine solution to 500 ml of distilled water. This solution was added slowly to 400 ml of distilled water containing 5 ml of concentrated ammonium hydroxide and 2 ml of 6N hydrochloric acid. The actual dosing solutions were prepared by diluting the stock monochloramine solution with pH 9.4 carbonate buffer. Because stability studies indicated that monochloramine solutions undergo at 7 to 10 percent decomposition over a period of three days at room temperature, the actual drinking water solutions were prepared at a concentration 10 percent desired, i.e., 15, 22, 55, 110, and 220 mg/l.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the monochloramine concentration by the DPD method showed that the 220 mg/l solutions averaged 190 +/- 7 mg/l when the bottles were removed from the animal cages after three days, thus confirming the previously determined decomposition rates. Similar levels of loss were observed for the lower concentrations of monochloramine.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 12.5, 25, 50, 100 and 200 mg/l (0, 2.5, 5, 10, 20 and 40 mg/kg/d)
Basis:
nominal in water

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale: The planned dosage levels had been selected based on previous studies conducted by the authors' laboratory and at the National Toxicology Program laboratories and were projected from the concentrations of chemical added to the drinking water and normal drinking water consumption.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Body weughts were recorded at the initiation of the study, weekly during exposure, and at the time of necropsy.


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was determined three times a week.

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected via cardiac puncture prior to sacrifice.
- Anaesthetic used for blood collection: Yes (60 mg/kg of pentobarbital)
- Animals fasted: No data
- How many animals: 10 males and 10 females per dose level


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected via cardiac puncture prior to sacrifice.
- Animals fasted: No data
- How many animals: 10 males and 10 females per dose level


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

None

Statistics:

The standard statistical procedure used was a one-factor analysis of variance (ANOVA) for normally distributed measures. Males and females were considered separately in all statistical analyses, and the parameters analyzed were body weight, organ weights, organ-to-body weight ratios, water and food consumption, hematology, and clinical chemistry measurements. The differences between the control and treatment groupswere analyzed pairwise using an ANOVA contrast prodecure at an adjusted significance level to control the false positive rate (overall alpha = 0.05). Because of the high variability of some of the clinical chemistry measures, a nonparametric analysis of variance, i.e., the Kruskal-Wallis test, was also employed to determine differences among the dosage groups and to compare pairwise each dosage group to the control group. analyses of the gross and microscopic pahtology diagnoses are limited to descriptive statistics.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gains were significantly depressed at the three highest concentrations in males and the two highest concentrations in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

food consumption was decreased in both males and females, and the change was significant for the females at the two highest concentrations.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No animals died during the study. Toxic effects were not evident during routine cage-side examination.

BODY WEIGHT AND WEIGHT GAIN
The body weight gains were significantly depressed at the three highest concentrations in males and the two highest concentrations in females.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was decreased in both males and females, and the change was significant for the females at the two highest concentrations.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Significant differences were alsoobserved in the water consumption of males at the two highest concentrations and of females at all monochloramine concentrations.

HAEMATOLOGY
A variety of changes in hematology was observed, although no effect was determined to be consistently treatment or dosage-related. The most consistent effects in both sexes were increased WBCs. In males an increase in lymphocytes at 100 and 200 mg/l and in females a decrease in neutrophils at 100 mg/l and a significant decrease in MCV at all dosage levels were the most remarkable changes.

CLINICAL CHEMISTRY
Several minor changes in clinical chemistries were noted, with no consistent treatment-related pattern. ALK-P was decreased in males at all concentration (significant at 25, 50, and 100 mg/l), whereas AST was significantly increased in females at all dosage levels (significant at 50 and 200 mg/l). LDH was significantly decreased at 25 mg/l in males but was significantly increased at 200 mg/l.

ORGAN WEIGHTS
Compared with the controls, numerous significant reductions in absolute organ weights were evident in the male and female animals drinking 200 mg monochloramine/l, with lesser effects seen for the lower concentrations. For example, for both males and females, liver and heart weights were decreased at the top two concentrations, and spleen weights (relative and absolute) were decreased at the two high dosages for females. At the highest dosage, the kidney and lung weights were decreased for both males and females, and absolute testes and spleen weight, some organs - brain, kidney, lung, and testes (males) and brain and kidney (females) - were significantly increased at the higher dosages compared with the control values.

GROSS PATHOLOGY
No compound-reated gross or microscopic lesions were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
No target tissues were identified in monochloramine-treated animals, and all lesions observed were interpreted as either agonal changes or incidental background findings.

Dose descriptor:

NOAEL

Effect level:

8.5 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Decreased organ weights

Critical effects observed:

not specified

Conclusions:

Under the conditions of this test, no treatment-related pathology was observed.

Executive summary:

Separate groups of male and female B6C3F1 mice were administered monochloramine in drinking water for 90 consecutive days. Monochloramine was supplied at 12.5, 25, 50, 100, and 200 mg/L. Criteria evaluated included mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, gross pathology, and histopathology. Overall, the correlation of the biochemical, hematological, and organ weight data, in the absence of histopathology and observable clinical signs of toxicity, suggests that the monochloramine induced effects via an indirect mechanism, e.g., nutritional deficiencies, rather than a direct toxicological effect on specific organs or tissues.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5.79 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

Quality of whole database:

The key studies are similar to OECD test guideline (408) and are of medium quality (Klimish score = 2)

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

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

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

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.

Remarks:

Doses / Concentrations:
0.025
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0.079
Basis:
nominal conc.

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

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.

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.

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

Critical effects observed:

not specified

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD guideline 412 and GLP compliant

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Remarks:

other: range finding test

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study, according to GLP.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this study was to determine dose-levels of substance to be administered in the definitve study OECD guideleine 412.

GLP compliance:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Analytical verification of doses or concentrations:

yes

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

0.287 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD guideline 412 and GLP compliant

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral Route:

Monochloramine is the second most widely used disinfectant for drinking water purification in the United States. Humans may be exposed to monochloramine through the consumption of drinking water and foods and through other means, including showering, bathing, and swimming in water containing this disinfectant. Therefore, there has been an interest in determining the potential health effects of monochloramine. Indeed, several Monochloramine dose-repeated studies via drinking water with rats or mice have been performed.

Daniel et al. (1990) conducted a 90-day drinking water study with monochloramine at 0, 25, 50, 100, and 200 mg/L in Sprague-Dawley rats (10/sex/dose group). Based on body weight and water consumption data, the chloramine intake has been estimated to be 0, 1.8, 3.4, 5.8 and 9.0 mg/kg-day for male rats and 0, 2.6, 4.3, 7.7, and 12.1 mg/kg-day for female rats. Mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weight, gross pathology, and histopathology were examined. Water consumption was significantly reduced (p < 0.05) in all treated groups in a concentration-related manner. Food consumption was significantly reduced in high-dose male rats. At the 200 mg/l dose level, the average weight gain for male and female treated rats was 51 % of controls; water consumption at this dose level was 31 -34 % of controls. Significant changes (p < 0.05) in hematologic and clinical chemistry parameters included decreased hematocrit at 100 mg/l, decreased red blood cell counts at 100 and 200 mg/l in males, and decreased serum calcium levels in all treated male groups. These findings were not dose-related, were within the normal ranges, and were not considered biologically significant. There were significant reductions (p < 0.05) in organ weights (absolute, relative or both). For example, spleen and liver weight reductions occurred in both males and females at the highest dose but, histopathologic examinations did not reveal any treatment-related changes in these tissues. A NOAEL = 100 mg/l (equivalent concentration to 7.71 and 5.79 mg/kg bw/ day female and male rats, respectively) was identified. However, the authors considered that further investigations were needed to distinguish systemic toxic effects and weight gain depression from taste aversion.

In a second 90 -day study (Daniel et al., 1991), male and female B6C3F1 mice (10/sex/dose group) were treated with monochloramine in the drinking water. The dose levels of the disinfectant in water were 0 (controls), 12.5, 25, 50, 100 and 200 mg/l and provided doses (calculated from water consumption and body weight data) of 0, 2.5, 5.0, 8.6, 11.1 and 15.6 mg/kg/day for male mice; and 0, 2.8, 5.3, 9.2, 12.9, and 15.8 mg/kg/day for female mice. Mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, gross pathology and histopathology. A significant, dose-related decrease in water consumption (p < 0.05) occurred in females at all dose levels and in males at the two highest dose levels. Food consumption was decrease in females at the two highest dose levels. Final body weight and body weight gain were decreased (p < 0.05) in both sexes at the two highest concentration levels. No overt clinical signs of toxicity and gross or histopathologic changes could be detected. Changes in hematologic and clinical chemistry parameters were not concentration-related and were attributed to the decrease in water and nutrient consumption and altered electrolyte balance. Changes in organ weights included a significant (p< 0.05) decrease in liver, heart, and lung weight in males and in liver, heart, and spleen weight in females at concentrations of 100 and 200 mg/l. Significant decreased organ weights (including liver, heart, lung and spleen) occurred at 100 mg/l and 200 mg/l, including minor (less than 10 %) depression of body weight relative to controls. These effects were considered as a consequence of decreased water consumption associated with taste aversion and not chemically induced. The authors concluded that, based on the decreased organ weights, weight gain, food and water consumption, 50 mg/l (8.6 mg/kg bw/ day in males; 9.2 mg/kg bw/day in females) was the NOAEL. The authors suspect that monochloramine induces effects via an indirect mechanism (e.g., nutritional deficiencies), rather than a direct toxicological effect on specific organs or tissues.

Monochloramine was administered in the drinking-water at concentrations of 0, 1, 10 and 100 mg/L to male Sprague-Dawley rats (Abdel-Rahman et al., 1984) and haematological parameters were examined after 3 and 10 months of treatment. Only four animals per group were sampled at each time-point. A statistically significant decrease in haematocrit was observed after 3 months of treatment with 10 and 100 mg/L. While a similar trend was apparent at 10 months, the results were not statistically significant.

Bercz et al. (1982) studied the ability of chloramine to induce haematological effects and alterations in clinical chemistry measures including serum thyroxine (T4) concentrations in adult African green monkeys (five males and seven females). Chloramine was administered in the drinking-water at a concentration of 100 mg/L for a period of 6 weeks (total body dose, 10 mg/kg bw per day). No significant effects were observed.

Poon et al. (1997) specifically examined the effects of 200 ppm [mg/L] (equivalent to 21.6 mg/kg per day) monochloramine in drinking-water in male Sprague-Dawley rats treated for 13 weeks. The study included a group of control rats that were given the same volume of water as that consumed by the chloramine-treated animals. The animals were monitored for changes in levels of T4, hepatic drug metabolizing enzymes, haematological parameters and measures of immune response. The authors concluded that the minor changes seen in these parameters were largely associated with decreased water and food consumption.

Moore et al., (1980) examined the effects of a 30 day exposure of monochloramine in drinking water in doses ranging from 2.5 to 200 ppm on the blood characteristics of male A/J mice. Experimental results indicate that doses of 100 ppm or 200 ppm monochloramine failed to initiate any clinically significant compensatory response on the part of the red blood cells of the A/J strain. With the exception of HCT, all indicators of prehemolytic or hemolytic stress (i.e., osmotic fragility, heinz body counts, METHB formation, GSH levels, RBC count, HB levels, RETIC counts, MCV, MCHC, and MCH) failed to show any statistically significant treatment-related changes. In the case of HCT, which is a classic identifying characteristic of anemia if it is significantly lowered, values actually increased significantly (p= 0.013) in the A/J strain, averaged over the 3 highest monochloramine treatments. The changes in HCT may have been partially a result of reduced intake of drinking water at the high monochloramine treatment levels. Thus, these results indicate that monochloramine ingested daily orally via drinking water not appear to produce evidence of hemolysis even at the high dose levels.

Inhalation route:

The inhalation route of administration was chosen to simulate the conditions of potential human exposure.

Two studies are reported for this administration route.

The objective of the first study was to determine dose-levels of monochloramine to be administered in a repeated-dose toxicity study 28 days.

In the second study, the objective of this study was to assess the toxic potential of monochloramine, in a 4 weeks inhalation study toHan Wistar rats according to OECD Guideline 412 and EC Methods 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 53.5μg/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 the main 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.

Therefore experimental results of the main test indicate that doses of 0.0294, 0.0767 or 0.287 μg/L of monochloramine failed to initiate any clinically or pathologically significant effect and thus any systemic effect.

Furthermore, according to the result of the preliminary test, the NOAEL of 0.287 µg/L was assigned to the local effect and was used for the human health risk assessment.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

Experimental results of the main test indicate that doses of monochloramine tested failed to initiate any clinically or pathologically significant effect and thus any systemic effect.

As no adverse effect was observed in the definitive test, it is not possible to determine a robust NOAEL for the systemic effect. Indeed, as the default NOAEL determined in this repeated dose toxicity study 28 days was based on the highest dose tested, it is not considered as robust enough to be used for the risk assessment (systemic toxicity).

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

As experimental results of the main test indicate that doses of 0.0294, 0.0767 or 0.287 μg/L of monochloramine failed to initiate any clinically or pathologically significant effect and thus any systemic effect and as the result of the preliminary test (histopathological changes in the respiratory system and slight to marked erosion/ulceration/necrosis (nasal turbinates, larynx, trachea, tracheal bifurcation and lung)) suggested a acute irritation, the NOAEL of 0.287 µg/L was assigned to the local effect and was used for the human health risk assessment.

Justification for classification or non-classification

According to the outcome of the preliminary assay of the repeated dose toxicity study 28 days, it is suggested to classify the monochloramine as H372; STOT RE cat 1. Indeed, in this preliminary assay the monochloramine induced adverse and irreversible effects on the respiration tract after two days of exposure at 6hours/day at the concentrations of 18.8 and 53.5 µg/L. These adverse effects, corresponding to histopathological changes on the respiration tract were mainly due to a local effect and suggested an acute irritation of the respiration system. These result of the preliminary test show the evidence that the monochloramine would have adverse effects after 28 days of repeated exposure for concentration below the guidance value of 600 µg/L of the CLP regulation (or 200 µg/liter 6h/L after 90 days of exposure) and justify the classification as H372, Causes damage to organs (respiration tract) through prolonged or repeated exposure by inhalation route.