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Repeated dose toxicity: oral

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Hydrogen peroxide, 35% w/w

Test animals

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

Administration / exposure

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
Doses / concentrations
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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.