Registration Dossier

Administrative data

Description of key information

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed in essential accordance with the OECD Guideline for Testing of Chemicals No. 408 with restrictions. No FOB and motor activity measurements were performed as they were not requested by the guideline at the time the study was performed.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
None relevant for the integrity and validity of the study: no FOB and motor activity measurements were performed as they were not requested by the guideline at the time the study was performed (there were no indications for neurotoxic effects); blood clot
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
other: C57BL/6NCrlBR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 5 weeks
- Weight at study initiation:
- Fasting period before study: no data
- Housing: individually in suspended, stainless steel cages with wire bottom
- Diet (e.g. ad libitum): Purina Rodent Chow 5002 (meal) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature: 65 to 71 °F
- Humidity (%): 41 to 78
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness
Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
The treated (and control) water for this study was prepared twice weekly and administered to the animals on the day of preparation. The drinking water solutions were made by adding preweighed amounts of 35 % hydrogen peroxide to distilled water. The solutions were mixed in carboys for at least 15 minutes prior to dispending the animals. Following administration, unused portions of treated water were stored refrigerated. All equipment for water solution preparation and administration was passivated with nitric acid before use.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the 100-ppm hydrogen peroxide stock solution and the 3000-ppm hydrogen peroxide stock solution used to dose mice at the 100 ppm and 3000 ppm hydrogen peroxide levels were taken together with a blank sample and analysed for concentration and homogeneity. A colourimetric analytical method designated Test Method APG No. 332 was applied which uses a ferrus thiocyanate reagent. The colour absorbance was measured with a Perkin Elmer Lambda 18 Spectrophotometer. Subsequently, on each of four date one distilled water blank, one control sample of 100 ppm and 3000 ppm hydrogen peroxide and five samples of 100 ppm and 3000 ppm dose solutions were analysed by the same method. Additionally, the 35 % hydrogen peroxide solution was analysed by an iodometric titration at 30 day intervals up to 120 days to test the stability of the solution under the storage conditions (4 °C, vented closed container).
Duration of treatment / exposure:
Approximately 90 days
Frequency of treatment:
Mice received drinking water ad libitum
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 or 3000 ppm
Basis:
nominal in water
No. of animals per sex per dose:
Number of animals per group: 15/sex per treatment group; 10/sex were killed after ceasing the exposure period, whereas 5/sex were submitted to a six week recovery period.
Control animals:
yes
Details on study design:
C57BL/6NCRlBR mice were chosen due to their particular sensitivity to hydrogen peroxide because of a deficient detoxification pathway. The strain can therefore be regarded as a very sensitive animal model for this particular substance.
Observations and examinations performed and frequency:
Clinical signs: daily
Mortality: twice daily
Body weight: weekly
Food consumption: weekly
Water consumption: twice weekly
Blood analysis, haematology, clinical chemistry analyses: blood samples were taken immediately before the scheduled necropsy
Ophthalmic examinations: the eyes of all animals were checked for lesions before the study and one week before the study termination and only animals showing no lesions were used in the study
Sacrifice and pathology:
Animals that died before the study termination underwent a complete necropsy upon dicovery of death. Animals sacrificed at their scheduled termination (days 91-93 of treatment period, days 133-134 of recovery period) were anaesthetised, bled for haematology and clinical chemistry determinations, sacrificed via exsanguination then necropsied. Animals were not fasted prior to sacrifice. The weights were determined of brain, liver, kidneys, spleen, testes, adrenals and heart. Samples from various tissues were saved in 10 % buffered formalin. All slides of organs and tissues in the control and high dose groups as well as tissues from mice that died out of schedule were investigated by an experienced pathologist and histological examinations were performed on all gross lesions, the tongue, esophagus, stomach, duodenum, ileum, jejunum, caecum, colon and rectum of all animals from all groups.
Statistics:
Body weights, food consumption, water consumption, absolute organ weights, organ:brain weight ratios, haematology and clinical chemistry data were analysed using the Ebar-Squared trend test. The test compared data from the high-dose group to control and computed a p-value to indicate whether the measured parameter was significantly different (p < 0.05 for statistically significant difference). Subsequent analyses compared data from the next highest dosage group to control, in the direction of the overall trend, and generated another p-value. These analyses continued in a stepwise manner for successively lower groups until the p-value was greater 0.05. When the trend test returned a value greater than 0.05, no subsequent comparisons of the lower dosage groups were performed.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
No treatment-related deaths occurred and no treatment-related clinical signs were noted at any time of the study. Male and females exhibited significant reductions in body weight at 3000 ppm. Food and water consumption were significantly reduced at 3000 ppm and notably reduced at 1000 and 300 ppm. Males receiving 3000 ppm displayed significant reductions in total protein and globulin levels in the blood, possibly caused by mucosal hyperplasia occurring in the duodenum of these animals. Necropsy revealed no treatment-related gross lesions. Tissue slides indicated an increase in the cross sectional diameter and wall thickness of the duodenum. Subsequent microscopic evaluations revealed mild mucosal hyperplasia in eight of nine males receiving 3000 ppm and in seven of ten males receiving 1000 ppm. Minimal mucosal hyperplasia was noted in one of ten males receiving 300 ppm. Minimal to mild mucosal hyperplasia was also seen in ten of ten females receiving 3000 ppm and in eight of ten females receiving 1000 ppm. No other areas of the gastrointestinal tract were affected. No evidence of cellular atypia or architectual disruptions nor any other indications of neoplastic changes were observed; therefore, the treatment-related mucosal hyperplasia noted was not considered as a neoplastic lesion.
Dose descriptor:
NOEL
Effect level:
100 ppm
Sex:
male/female
Basis for effect level:
other: 26 and 37 mg/kg bw/day for males and females, respectively; dose-related reductions in food and water consumption were seen at the next higher doses level of 300 ppm; additionally, duodenal mucosal hyperplasia was observed at 300 ppm
Critical effects observed:
not specified

Table 1: Results of analysis of stock solution (35 % hydrogen peroxide), dose solutions (100, 300, 1000 and 3000 ppm), and mean hydrogen peroxide consumption.

Stability of refrigerated 35 % test material

Range of % of target concentration in dose solutions

Hydrogen peroxide consumption (mg/kg/day) based on water consumption and nominal conc.

Time point of sampling

Total % hydrogen peroxide

% change from initial analysis

Group

Target concentration (ppm)

Initial range, % of target

Males

Females

Initial analysis

35.1

NA

1

0

NA

NA

NA

30-day analysis

35.0

-0.3

2

100

94.8-102

26 ± 6.0

37 ± 10.0

60-day analysis

34.7

-1.1

3

300

98.0-105

76 ± 17.1

103 ± 25.5

90-day analysis

34.7

-1.1

4

1000

102-105

239 ± 56.4

328 ± 81.4

120-day analysis

34.7

-1.1

5

3000

101-104

547 ± 95.3

785 ± 194.3

NA: not applicable

Table 2: Development of body weights, food and water consumption throughout the study

sex

males

females

group

0

1

2

3

4

0

1

2

3

4

ppm

0

100

300

1000

3000

0

100

300

1000

3000

Body weight [g/animal]

Day 0

19.7

19.7

19.6

19.6

19.7

16.6

16.6

16.7

16.7

16.7

Day 21

23.2

22.5

22.3

22.7

21.6

19.6

20.0

19.9

19.6

19.1

Day 42

24.9

24.5

24.1

24.5

23.6¯

22.1

22.5

22.3

21.7

21.7

Day 63

26.3

25.5

25.5

25.7

24.9¯

23.2

21.7

22.7

22.5

22.7

Day 91

28.1

27.1

27.3

27.2

26.7¯

25.1

24.9

25.2

24.8

24.1

Weight gain

9.2

8.4

8.7

9.2

7.3¯

8.5

10.3

10.6

10.4

8.9

Mean food consumption [g/animal/week]

Day 7

31

33

31

32

24¯

27

31

26

27

27

Day 35

39

41

35

38

36

56

45

40¯

36¯

39¯

Day 63

40

40

38

40

34¯

68

69

68

62

56¯

Day 91

43

41

43

41

36¯

47

48

46

35¯

39¯

Mean water consumption [g/animal/week]

Day 7

39

41

38

33

25¯

38

42

44

33

26¯

Day 35

52

52

43

47

33¯

90

75

56¯

61¯

52¯

Day 63

39

35

35

32

26¯

46

42

42

42

33¯

Day 91

40

37

41

36

35¯

47

50

48

48

43

-- Indicates a decrease in comparison with controls

Table 3: Results of clinical chemistry (blood samples)

parameter changed

control

100 ppm

300 ppm

1000 ppm

3000 ppm

Males

Total protein

g/dL

4.7

4.6

4.8

4.5

4.2¯

Globulin

g/dL

2.0

2.1

1.9

1.9

1.5¯

Females

Total protein

g/dL

4.9

4.8

4.6

4.7

4.5

Globulin

g/dL

2.3

2.2

2.1

2.1

2.0

-- Indicates a decrease in comparison with controls

Table 4: Incidence of histopathological findings

Parameter

Control

100 ppm

300 ppm

1000 ppm

3000 ppm

m

f

m

f

m

f

m

f

m

f

number of animals examined

9

10

10

10

10

10

10

10

9

10

duodenum

- mucosal hyperplasia

0

0

0

0

1

0

7

8

8

10

Conclusions:
No treatment-related effects were observed at 100 ppm dose level and the LOEL, based on decreased food and water consumption and the observation of duodenal mycosal hyperplasia, was 300 ppm.
Executive summary:

A 90-day oral, subchronic toxicity study with a 35 % aqueous solution of hydrogen peroxide dissolved in drinking water to produce concentrations ranging from 100 to 3000 ppm was performed with C57BL/6NCrlBR mice under GLP conditions and in essential accordance with OECD Guideline No. 408. C57BL/6NCRlBR mice were chosen due to their particular sensitivity to hydrogen peroxide because of a deficient detoxification pathway. The strain can therefore be regarded as a very sensitive animal model for this particular substance. Groups of 15 males and 15 females received different doses of hydrogen peroxide dissolved in their drinking water. After the 90 -day exposure duration, 10 animals/sex of each dose group were sacrificed, while the remaining five animals/sex were submitted to a six week recovery period. No treatment-related mortality or clinical signs were noted throughout the study. No other treatment-related effects were observed at the 100 ppm dose level. At 300 ppm, the consumption of food and water was reduced. Tissue slides indicated an increase in the cross sectional diameter and wall thickness of the duodenum. Subsequent microscopic evaluations revealed mild mucosal hyperplasia in eight of nine males and ten of ten females receiving 3000 ppm and in seven of ten males and eight of ten females receiving 1000 ppm. Minimal mucosal hyperplasia was noted in one of ten males but in none of the females receiving 300 ppm. No other areas of the gastrointestinal tract were affected. Microscopically, no evidence of cellular atypia or architectural disruptions nor any other indications of neoplastic changes were observed; therefore, the treatment-related mucosal hyperplasia noted in the study was not considered as neoplastic lesion.

Based on dose-related reductions in food and water consumption and the observation of duodenal mucosal hyperplasia the lowest observed effect level in the study was 300 ppm and the no observed effect level (NOEL) was 100 ppm (26 and 37 mg/kg/day for males and females, respectively). Clinical pathologic effects (decreased total protein and globulin blood levels) were limited to the 3000 ppm level. All effects noted during the treatment period were reversible; animals sacrificed following the recovery period were considered biologically normal.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
73.53 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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.
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Alpk:ApfSD (Wistar derived)
Sex:
male/female
Details on test animals 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
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
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).
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.
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.
Dose descriptor:
NOAEL
Effect level:
2.9 mg/m³ air
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
14.6 mg/m³ air
Sex:
male/female
Critical effects observed:
not specified

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 ppmA

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

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).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
8.2 mg/m³
Study duration:
subacute
Species:
rat

Additional information

In order to be systemically available, a chemical needs to be absorbed, either via the oral, inhalatory or dermal route. Dissolution of solids is generally assumed to be a prerequisite for absorption. As the reaction mass of calcium carbonate and calcium dihydroxide and calcium peroxide is a solid inorganic multi-constituent substance, this means that Ca2+, OH- and hydrogen peroxide are the species to be taken into account when assessing its toxicity.

 

Oral repeated dose toxicity

* Ca2 +: A comprehensive evaluation of all available human data on prolonged oral exposure to various calcium compounds is published in “Opinion of the Scientific Committee on Food on the Tolerable Upper Intake Level of Calcium” dd 4 April 2003. In this document, a tolerable upper intake level (UL) of 2500 mg of calcium per day for calcium intake from all sources is proposed for adults, which corresponds to a dose of approximately 36 mg calcium/kg bw/day taking into account an average body weight of 70 kg/person.

The Tolerable Upper Intake Level (UL) derived by the SCF is considered as sufficient and adequate for risk characterisation. In conclusion, the conduct of any further repeated-dose toxicity studies in animals would not contribute any new information and is therefore not considered to be required.

 

* H2O2: A key study is available that assesses the repeated oral exposure to hydrogen peroxide. In this oral toxicity study (Freeman 1997) 35 % hydrogen peroxide was applied to mice via the drinking water for 90 days. Reduced food and water consumption were seen at ≥ 300 ppm. Body weight was reduced in mice receiving 3000 ppm during most of treatment period in male animals. Further signs of treatment indicated the duodenum as target organ with local mucosal hyperplasia at ≥ 1000 ppm (corresponding to 239 and 328 mg/kg bw/d for males and females, respectively). Mucosal hyperplasia in the duodenum was not found in any dose group after recovery. The no observed adverse effect level (NOAEL) was 100 ppm (26 mg/kg bw/day in males and 37 mg/kg bw/day in females).

 

* OH-: When administered via the oral route, the hydroxide ions will be neutralised in the GI tract. No further assessment is therefore deemed required.

The NOAEL of the reaction mass of calcium peroxide, calcium hydroxide and calcium carbonate was calculated from the NOAEL available for hydrogen peroxide (26 mg/kg bw/d) by taking into account the composition of the reaction mass:

According to the applicable chemical reaction, the amount of hydrogen peroxicde formed is equimolar to the amount of calcium peroxide present in the reaction mass. As the concentration of calcium peroxide in the reaction mass is ca. 75%, 100 mg of the reaction mass contains 75 mg of calcium peroxide, which corresponds to 1.04 mmol of calcium peroxide. Therefore, 1.04 mmol (= 35.36 mg) of hydrogen peroxide is formed upon dissolution of 100 mg of calcium peroxide. As a consequence, the NOAEL for the reaction mass of calcium peroxide, calcium hydroxide and calcium carbonate was calculated to be 100*26/35.36 = 73.53 mg/kg bw/d.

 

Inhalatory repeated dose toxicity

* Ca2+and OH-: An adopted Recommendation from the Scientific Committee on Occupational Exposure Limits (SCOEL) for calcium oxide (CaO) and calcium dihydroxide (Ca(OH)2) is in place, setting a short-term exposure limit (STEL) of 4 mg/m³ respirable dust, and a 8 -h TWA of 1 mg/m³ respirable dust which is considered protective against long-term exposure to calcium oxide and calcium dihydroxide. In particular, two ethically approved human volunteer studies taken into account by SCOEL indicate chemosensory feel and local irritation of mucous membranes in the respiratory tract being the primary effect upon inhalation exposure of alkaline mineral materials due to a pH shift.

Following the criteria of Annex XI, point 1.1.2 of Regulation No 1907/2006, the SCOEL recommendation is considered as adequate for purpose of classification and labelling and for the risk assessment.

 

* H2O2: A key study is available that assesses the repeated inhalatory exposure to hydrogen peroxide. In this 28 -days inhalation study in the rat (Kilgour 2002), local effects appeared in the nose with necrosis and inflammation at ≥10 ppm followed by respiratory irritation and reduced body weight gain in higher exposure concentrations. The no observed adverse effect level (NOAEL) was 2.9 mg/m³ (2.03 ppm).

The NOAEC of the reaction mass of calcium peroxide, calcium hydroxide and calcium carbonate was calculated from the NOAEC available for hydrogen peroxide (2.9 mg/m3) by taking into account the composition of the reaction mass:

 

According to the applicable chemical reaction, the amount of hydrogen peroxide formed is equimolar to the amount of calcium peroxide present in the reaction mass. As the concentration of calcium peroxide in the reaction mass is ca. 75%, 100 mg of the reaction mass contains 75 mg of calcium peroxide, which corresponds to 1.04 mmol of calcium peroxide. Therefore, 1.04 mmol (= 35.36 mg) of hydrogen peroxide is formed upon dissolution of 100 mg of calcium peroxide. As a consequence, the NOAEC for the reaction mass of calcium peroxide, calcium hydroxide and calcium carbonate was calculated to be 100*2.9/35.36 = 8.20 mg/m3.

 

Dermal repeated dose toxicity

In the REACH registration dossiers for hydrogen peroxide and calcium hydroxide, elaborate documentation is available that confirms that both these substances have a low bioavailability through the skin. As a consequence, assessment of the repeated dose toxicity for the dermal exposure route is not considered to be required as systemic effects following dermal exposure are not expected due to the low absorption rate of the substance. Furthermore, calcium hydroxide is neutralized in the blood while hydrogen peroxide is degraded in the blood, and therefore, the substance is not expected to be systemically available.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study available for read-across substance hydrogen peroxide. Effect NOAEL is re-calculated towards the reaction mass of calcium carbonate and calcium hydroxide and calcium peroxide.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Key study available for read-across substance hydrogen peroxide. Effect NOAEC is re-calculated towards the reaction mass of calcium carbonate and calcium hydroxide and calcium peroxide.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: duodenum

Justification for classification or non-classification

In accordance to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for repeated dose toxicity based on the available test results for the read-across substances hydrogen peroxide and calcium hydroxide. The dose at which the transient effects of mucosal hyperplasia in the duodenum was observed (239 mg/kg bw/d, which corresponds to 100*239/35.36 = 675 mg/kg bw/d of the reaction mass of calcium carbonate and calcium dihydroxide and calcium peroxid) is outside the classification criteria.

Furthermore, for the inhalatory route a STOT-RE is not required as repeated dose testing of both read-across substances (calcium dihydroxide and hydrogen peroxide) did not show any systemic effects. Both substances only exhibited local irritancy.