Reaction mass of calcium carbonate and calcium dihydroxide and calcium peroxide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out in accordance with the respective OECD Guidelines and under GLP conditions. No deviations were reported that were likely to negatively influence the outcomes of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen peroxide

EC Number:

231-765-0

EC Name:

Hydrogen peroxide

Cas Number:

7722-84-1

Molecular formula:

H2O2

IUPAC Name:

hydrogen peroxide

Details on test material:

Hydrogen peroxide, 50% w/w

Test animals

Species:

rat

Strain:

other: Alpk:ApfSD (Wistar derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rodent Breeding Unit, Alderley Park, Macclesfield, UK
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: for groups 1-4, 242.5 +/- 9.6 g (males), 190.2 +/- 12.3 g (females); four groups five and six, 300.3 +/- 9.1 g (males), 233.8 +/- 11.0 g (females)
- Fasting period before study:
- Housing: five rats, sexes separately, in stainless steel cages
- Diet (e.g. ad libitum): CT1 supplied by Special Diet Services Ltd, Witham, UK ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: not applicable

Details on inhalation exposure:

Test atmospheres were generated using a glass concentric-jet atomiser to generate fine aerosol directly into a 3-necked quick-fit round bttomed flask, heated by placing it in a waterbath at 80 °C. The test substance was pumped to the atomiser using a peristaltic pump, typically operating at a pump speed giving a flow rate of test material of approximately 1 mL/min. Clean, dry air was passed through the atomiser at nominal flow rates of 2, 10 or 15 L/minute for groups 2, 3 and 4 respectively, and (together with heated generation air at 25 L/minute) carried the atmosphere to the lng term exposure chamber. Diluting air was added directly to the exposure chambers at a flow rate of 500-600 L/min. Air flows were monitored continuously using variable area flowmeters.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test atmospheres were sampled by passing the atmosphere, at a fixed flow rate for a known time period, through a known volume of de-ionised water in a midget impinger. The resulting solutions were analysed by flow injection analysis using a LC Module 1 (Waters) separations module at a flow rate of 5 L/minute, a dilute Cobalt-bicarbonate reagent mobile phase, and a 486 series UV detector (Waters) at 260 nm. The limit of detection of the method was assessed to be approximately 0.1 mg/mL corresponding to an atmosphere concentration of 0.1 ppm.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours daily, 5 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
2.03, 10.3, 23.3, 58.1/27.3 ppm (2.88, 14.6, 33, 82.4/38.7 mg/m3)
Basis:
analytical conc.

No. of animals per sex per dose:

five males, five females

Control animals:

yes

Details on study design:

Another group (group 4 of the main test) of animals was exposed to 60 ppm at day 1, 4, 5 and 6. Thereafter, the exposure level was reduced to 30 ppm at day 11 and 12. The treatment was terminated on day 13 and animals were sacrificed due to toxicity. The group treated with a target concentration of 25 ppm was introduced later in the test after termination of the test group 4 (60/30 ppm).

Examinations

Observations and examinations performed and frequency:

Clinical observations: prior to the start of the study, frequently during exposure and at the end of the 6 hour exposure duration, daily before exposure
Body weight: before study initiation, weekly during exposure study
Food consumption: continuously throughout the study
Clinical pathology (haematology, blood clinical chemistry): at termination of study

Sacrifice and pathology:

Animals were killed by an overdose of halothane Ph. Eur. vapour followed by exsanguination. Weights of adrenal glands, kidneys, liver, lungs and testes were measured. All animals were subjected to full examination post mortem including external and careful internal examination of all organs and structures. Slides were prepared from various organs and tissues. All submitted tissues from control and high exposure animals together with the lungs, liver, kidney, trachea, nasal passages and abnormal tissues from the low and mid exposure groups were routinely processed, embedded in paraffin wax, sectioned at 5 micrometre and stained with haematoxylin and eosin. Examination by light microscopy was performed for these tissues.

Statistics:

Body weights were considered by analysis of covariance on initial body weight, separately for males and females. Haematology and blood clinical chemistry were consdired by analysis of variance. Male and female data were analysed together. Organ weights were considered by analysis of variance and of covariance on final body weight, separately for males and females. Analyses were carried separately for main study and additional group 5 and 6 animals. Unbiased estimates of differences from control were provided by the difference between each treatment group least-squares mean and the control group least-squares mean. Differences from control were tested statistically by comparing each treatment group least-squares mean with the contol group least squares mean using a two-sided Student's t-test, based on the error mean square in the analysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical signs were seen in animals exposed to 10.3 ppm and greater and in general the number and severity of these clinical signs increased with repeated exposure at low doses, whereas the onset of clinical signs was earlier at higher doses but also a certain degree of recovery from symptoms was seen at higher doses. Signs included reddening of the nose, stains around the snout, stains around the mouth, signs of salivation, signs of respiratory tract irritation, irregular breathing, signs of urinary incontinence, piloerection, chomodacryorrhoea, hunched posture, increased response to touch, thin appearance. Some evidence of recovery from these symptoms was seen during periods of non-exposure. Body weights gradually decreased in males exposed to 23.3 ppm and in males and females exposed to 58.1/27.3 ppm. Food consumption was affected in males exposed to 23.3 ppm and in males and females exposed to 58.1/27.3 ppm. Minor effects on haematology were seen at exposure levels of 23.3 ppm, which were considered as not biologically and toxicologically significant. In both sexes there was a minimal decrease in albumin and total protein levels at 23.3 ppm exposure. Kidney weight was increased in females exposed to 23.3 ppm and lung/body weight ratio in males and kidney/body weight ratios in females exposed to 23.3 ppm was increased. Treatment-related findings were seen in the nasal and oral cavities of rats at the necropsy following termination of the study. Staining of the nares was seen at 10 ppm and above and mouth staining was at 25 ppm. In both instances, no dose-response could be found. Increased incidences of findings in exposed animals over controls during the microscopic examinations were seen in the nasal cavity, larynx and lung including necrosis, inflammation and perivascular neutrophil infiltration.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

2.9 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: all endpoints

2

Dose descriptor:

LOAEL

Effect level:

14.6 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: all endpoints

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Results of the clinical chemistry haematology

Parameter changed	Control group 1 (0 ppm)	2 ppm	10 ppm	Control group 2 (0 ppm)^A	25 ppm^A
Males
Mean cell volume (fl)	56.1	56.2	56.0	55.3	55.3*
Mean cell haemoglobin (pg)	19.4	19.6	19.4	19.0	18.8*
Albumin (g/L)	33.6	32.8	33.1	32.7	31.3*
Total protein (g/L)	66.	64.9	65.1	63.3	57.8**
Females
Red blood cell count (10e12/L)	7.56	7.72	7.42	7.77	7.43*
Albumin (g/L)	33.2	33.0	32.8	32.6	31.3*
Total protein (g/L)	61.1	61.3	60.2	61.4	57.0*
Glucose (mmol/L)	12.4	13.7	12.9	12.8	9.6**

A) 25 ppm group is compared to control group 2 since animals were treated simultaneously. *p <0.05, **p <0.01 (student's t-test, two-sided)

Table 2: Microscopic findings

Target organs	Control (0 ppm)	2 ppm	10 ppm	25 ppm
Nasal cavity	No finding	No finding	Necrosis and inflammation (sqamous epithelium, anterior region of nasal cavity) 3/5 males, 2/5 females	Rhinitis 1/5 males Necrosis and inflammation (sqamous epithelium, anterior regions of nasal cavity) 4/5 males, 4/5 females
Larynx	No finding	Inflammation 1/5 females	No finding	Mononuclear cell infiltration 2/5 females Epithelia erosion 1/5 males
Lung	No finding	Increase in perivascular neurophil infiltration 1/5 males Haemorrhage 2/5 males, 1/5 females	Increase in perivascular neurophil infiltration 1/5 males Haemorrhage 2/5 males	Increase in perivascular neurophil infiltration 1/5 males, 2/5 females

Applicant's summary and conclusion

Conclusions:

Whole body exposure to hydrogen peroxide vapour for 6 hours per day, 5 days per week for a period of 28 days at concentrations of 2.03, 10.3 or 23.3 ppm resulted in signs of general toxicity in males exposed to 23.3 ppm and were consistent with the material being a respiratory tract irritant. Treatment-related microscopic changes were seen in the nasal cavity in animals exposed to 10.3 ppm or above. The no observed effect level (NOEL) for the study was considered to be 2.03 ppm hydrogen peroxide.

Executive summary:

A repeated dose inhalation toxicity study was performed with male and female Alpk:APfSD (Wistar-derived) rats exposed to hydrogen peroxide vapours for 6 hours per day, 5 days per week for a period of 28 days at concentrations of 2.03, 10.3 or 23.3 ppm. The study was carried out under GLP conditions and in accordance with OECD Guideline No. 412. Treatment of a group exposed initially to 58.1 ppm and subsequently to 27.3 ppm was terminated before schedule due to the toxicity of the test material. Clinical observations were consistent with the material being a respiratory tract irritant (reddened noses, stains around the nose, abnormal respiratory noise) and in general the time to onset, incidence and severity of clinical signs increased with exposure concentration and repeated exposure. Males exposed to 23.3 ppm hydrogen peroxide showed lower food consumption and body weight gain compared to controls. Minimal changes in albumin and total protein blood levels were found in males and females exposed to 23.3 ppm. Histopathological, treatment-related changes were seen in the anterior-most regions of the nasal cavity lined with squamous epithelium, where minimal to slight necrosis (with associated inflammation) and rhinitis were seen in animals exposed to 10.3 and 23.3 ppm hydrogen peroxide. Inflammation and epithelial erosion in the larynx and increased perivascular neutrophil infiltration in the lungs were considered unlikely to be related to treatment in the absence of a clear dose response relationship. The no observed effect level (NOEL) for the study was considered to be 2.03 ppm hydrogen peroxide (corresponding to 2.9 mg/m3).