Reaction mass of calcium dihydroxide and calcium peroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Hydrogen peroxide:

Key study: OECD Guideline 471. Hydrogen peroxide was found to be mutagenic in an in-vitro Ames test performed with S. typhimurium TA100, whereas it was negative in Ames tests carried out with other S. typhimurium strains and with E. coli WP2 strain.

Key study: OECD Guideline 473. Based on the 10 mammalian cell cytogenicity studies evaluated in the EU Risk Assessment Report, hydrogen peroxide has the potential to induce chromosomal aberrations in mammalian cells without metabolic activation. No concluding statement on genotoxicity of hydrogen peroxide to mammalian cells after metabolic activation can be given as the data available are very poor. The only available study (in which very high concentrations were tested) indicates positive results.

Key study: OECD Guideline 476. Based on the 11 mammalian cell mutagenicity studies evaluated in the EU Risk Assessment Report, hydrogen peroxide has the potential to induce mutations in mammalian cells without metabolic activation. No concluding statement on mutagenicity of hydrogen peroxide to mammalian cells after metabolic activation can be given as the data available are very poor. The only available study, however, indicates negative results.

Calcium hydroxide:

Key study: Calcium hydroxide (up to 300 µM) failed to induce chromosome aberrations even in the presence of exogenous metabolic activation suggesting the lack of clastogenic activity under test conditions (3 -30 h treatment with an incubation time of 27h).

Key study: It was concluded that calcium hydroxide did not cause DNA damage as assessed by the comet assay.

The test item (80 µg/mL) was exposed to cells (mouse lymphoma or human fibroblasts) in culture directly. The cell culture tubes were incubated at 37°C for 1 h.

Key study: It was concluded that calcium hydroxide did not induce DNA lesions in CHO cells in-vitro as assessed by the comet assay. The cells were exposed to the test item (100 µg/mL) for 3 h at 37 ºC.

Kety study: Calcium hydroxide did not induce DNA damage in human peripheral lymphocytes as assessed by the in-vitro single-cell gel (comet) assay. The cells were exposed to the test item (100 µg/mL) for 1h at 37 ºC.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was carried out in accordance with the principles layed down for the standard S. typhimurium plate-incorporation assay with metabolic activation by the S9 mix.

Principles of method if other than guideline:

Method: other: slightly modified from Ames, B.N. et al.: Mutation Research 31, 347-361 (1975)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2

Additional strain / cell type characteristics:

other: tryptophan-requiring

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Without S9 mix: 0.0033 to 0.67 mg per plate in S. typhimurium strains TA1535, TA1538 and TA98; from 0.001 to 0.33 mg per plate in strains TA1537 nd TA100; from 0.033 to 3.3 mg per plate in E. coli strain WP2
With S9 mix: 0.01 to 3.3 mg per plate in all five S. typhimurium strains and from 0.01 to 30 mg per plate in E. coli strain WP2

Vehicle / solvent:

Hydrogen peroxide was dissolved in 0.067 M potassium or sodium phosphate buffer, pH 7

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 mix: 2-nitrofluoren (TA98, TA1538), sodium azide (TA100, TA1535), 9-aminoacridine (TA1537), furylfuramide or N-methyl-N'-nitro-N-nitrosoguadinine (E. coli); With S9 mix: 2-Anthramine (all tested strains)

Details on test system and experimental conditions:

The S9 mix contained 10 % Aroclor 1254-induced S9 from male Sprague-Dawley rats. All platings were performed in duplicate and all tests were repeated. An additional test was performed with the test substance to see if the substance supported the growth of histidine-requiring strains of S. typhimurium in the absence of added histidine. In addition to the tested strains of S. typhimurium also strain SL4024 was used in this test.

Evaluation criteria:

Test results were considered valid only if the positive control compounds induced increases in mutant counts to at least twice background.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

Hydrogen peroxide was found to be mutagenic in an in-vitro Ames test performed with S. typhimurium TA100, whereas it was negative in Ames tests carried out with other S. typhimurium strains and with E. coli WP2 strain.

Executive summary:

The mutagenicity of hydrogen peroxide was tested in the Ames test with S. typhimurium strains TA98, 100, 1535, 1537 and 1538 and E. coli WP2 strain. The test substance was found to increase the number of revertant colonies of S. typhimurium TA100 significantly, both in the absence and the presence of S9 metabolic activation. The test was negative in all other strains tested in the study. It was concluded that hydrogen peroxide exhibits mutagenicity to S. typhimurium TA100 in the Ames test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well described, peer reviewed risk assessment report based on fully available data.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

not specified

Principles of method if other than guideline:

In the EU Risk Assessment Report on hydrogen peroxide, 10 different studies on in vitro mammalian cell cytogenicity were evaluated.

GLP compliance:

not specified

Type of assay:

other: in vitro chromosomal aberration and in vitro micronucleus test

Species / strain / cell type:

mammalian cell line, other: CHO, CHL, CHC, V79, mouse skin cells and splenocytes, human embryonic fibroblasts, D98/AH2 (variant of HeLa) cells

Details on mammalian cell type (if applicable):

details see supporting studies

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

details see supporting study: Procter & Gamble 1985

Metabolic activation:

without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Overall range (without metabolic activation): 0.83 µM to 7.35 mM (28.1 ng/mL to 0.25 mg/mL)
Overall range (with metabolic activation): 0.33 M to 3.3 M (11.1 mg/mL to 111 mg/mL)
For details see supporting studies.

Vehicle / solvent:

no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

For study details please see supporting studies.

Key result

Species / strain:

mammalian cell line, other: CHO, CHL, CHC, V79, mouse skin cells and splenocytes, human embryonic fibroblasts, D98/AH2 (variant of HeLa) cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

in 8 of 10 studies

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

mammalian cell line, other: CHO, CHL, CHC, V79, mouse skin cells and splenocytes, human embryonic fibroblasts, D98/AH2 (variant of HeLa) cells

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

in 2 of 10 studies

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

From the 10 studies evaluated in the EU Risk Assessment Report, 8 gave positive results without metabolic activation, namely Sawada et al. 1988, Stich et al. 1978, Procter & Gamble 1985, Ishidate et al. 1984, Stich and Dunn 1986, Tsuda, 1981, Tachon 1990, and Oya et al. 1986. These studies were performed using different cell lines, different endpoints and different concentrations of hydrogen peroxide.
In two studies, which were performed using either mouse splenocytes or human cervical carcinoma cells (D98/AH2, a variant of HeLa), ambiguous results were obtained without metabolic activation: Dreosti et al. 1990, and Estervig and Wang 1984.

The only study performed with metabolic activation is the one of Procter & Gamble 1985 in which a positive result was obtained in CHO cells after metabolic acitivation with S9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

 

Table 1: Summary of mammalian cell cytogenetic assays performed with hydrogen peroxide in vitro.

Cell line	Endpoint	Test conditions	Results		Reference
			with activation	without activation
CHL	CA	1.6 mM	+	NT	Sawada et al. 1988
CHO	CA	no data	+	NT	Stich et al. 1978
CHO	CA	0.83-1.48 µM (- S9) 0.33-3.3 M (+ S9)	+	+	Procter & Gamble 1985
Chinese hamster fibroblasts	CA	7.35 mM	+	NT	Ishidate et al. 1984
CHO	CA,,	0-25 µmol	+	NT	Stich and Dunn 1986
CHO-K1, V79, CHC, BALB/c mouse back skin cells	CA	0-1.0 mM	+	NT	Tsuda 1981
Mouse (C57BL/6J) splenocytes	MNT	0-20 µM	*	NT	Dreosti et al. 1990
V79	MNT	10-20 µM	+	NT	Tachon 1990
D98/AH2 (variant of HeLa)	CA	0-88 µM	*	NT	Estervig and Wang 1984
human embryonic fibroblasts	CA	0.01-1.0 mM	+	NT	Oya et al. 1986

CA      Chromosomal aberrations

MNT   Micronucleus test

CT      Chromatid translocations

 

+         positive result

*         ambiguous result

NT      not tested

Conclusions:

Based on the 10 mammalian cell cytogenicity studies evaluated in the EU Risk Assessment Report, hydrogen peroxide has the potential to induce chromosomal aberrations in mammalian cells without metabolic activation. No concluding statement on genotoxicity of hydrogen peroxide to mammalian cells after metabolic activation can be given as the data available are very poor. The only available study (in which very high concentrations were tested) indicates positive results.

Executive summary:

The genotoxic activity of hydrogen peroxide in vitro was evaluated in an EU Risk Assessment Report in 2003. Ten different studies were assessed which used different mammalian cell lines (CHO, CHL, CHC, V79, mouse skin cells and splenocytes, human embryonic fibroblasts, D98/AH2 (variant of HeLa) cells) and different endpoints (chromosomal aberration, micronucleus test, chromatid translocations). The studies without metabolic activation altogether cover a concentration range from 0.83 µM to 7.35 mM (28.1 ng/mL to 0.25 mg/mL). Only one study is available that evaluates the effect after metabolic activation with S9; here, a concentration range from 330 mM to 3.3 M was tested. In the studies which were tested without metabolic activation, 8 of 10 studies gave positive results. Based on these data hydrogen peroxide has the potential to induce chromosomal aberrations in mammalian cells without metabolic activation. No concluding statement on genotoxicity of hydrogen peroxide to mammalian cells after metabolic activation can be given as the data available are very poor. The only available study (in which very high concentrations were tested) indicates positive results.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well described, peer reviewed risk assessment report based on fully available data.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

Principles of method if other than guideline:

In the EU Risk Assessment Report on hydrogen peroxide, 11 different studies on mammalian cell mutagenicity were evaluated.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mammalian cell line, other: CHO, V79, L5178Y, CV-1

Details on mammalian cell type (if applicable):

details see supporting studies

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

details see supporting studies

Metabolic activation:

without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Overall range (all evaluated studies): 0.2 µM to 10 mM
For details see supporting studies.

Key result

Species / strain:

mammalian cell line, other: CHO, V79, L5178Y, CV-1

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

in 7 of 11 studies

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

mammalian cell line, other: CHO, V79, L5178Y, CV-1

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

in 3 of 11 studies

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

From the 11 studies evaluated in the EU Risk Assessment Report, 7 gave positive results without metabolic activation, namely Hsie et al. 1993, Nassi-Calo et al 1989, Kruzewski and Szumiel 1993, Ziegler-Skylakakis and Andrae 1987, Moraes et al. 1990, Moraes et al. 1990, Procter & Gamble 1986, Wangenheim and Bolcsfoldi 1988. These studies were performed using different cell lines, different endpoints and different concentrations of hydrogen peroxide.
In three studies, which were all performed using V79 cells, negative results were obtained without metabolic activation: Bradley and Erickson 1981, Bradley et al. 1979, and Tsuda 1981. Additionally, the study of Speit 1986 with V79 cells gave ambiguous results.

The only study performed with metabolic activation is the one of Procter & Gamble 1986 in which a negative result was obtained after metabolic activation with S9.

Remarks on result:

other: strain/cell type: CHO, V79, L5178Y, CV-1

Remarks:

Migrated from field 'Test system'.

Table 1: Summary of mammalian cell gene mutation assays performed with hydrogen peroxide in vitro.

Cell line	Endpoint	Test conditions	Results		Reference
			without activation	with activation
CHO	HGPRT	0, 0.2, and 0.4 µM	+	NT	Hsie et al. 1993
V-79	HGPRT	10, 20, 30, and 40 µM	+	NT	Nassi-Calo et al 1989
V-79, CHO	HGPRT	10, 20, 40, 60, and 80 µM	*	NT	Speit 1986
V-79	HGPRT	27.2-585 µM	-	NT	Bradley and Erickson 1981
L5178Y-S(LY-S) L5178 (LYR)	HGPRT	0.3-5.0 µM	+	NT	Kruzewski and Szumiel 1993
V-79	6-TG resistance	353 µM	-	NT	Bradley et al. 1979
V-79	6-AG and ouabin resistance	0, 0.1, 0.2, 0.3, 0.5, and 1.0 mM	-	NT	Tsuda 1981
V-79	6-TG resistance	0.5-4.0 mM	+	NT	Ziegler-Skylakakis and Andrae 1987
CV-1	mutation of supF locus of the pZ189 plasmid	0.5-10 mM	+	NT	Moraes et al. 1990
L5178Y	TK-locus	0.0018-0.1 µg/mL	+	-	Procter & Gamble 1986
L5178Y	TK-locus forward mutation	18.6, 37.2, 79.5, 199.0, and 496 µM	+	NT	Wangenheim and Bolcsfoldi 1988

HGPRT:Hypoxanthine-guanine phosphoribosyltransferase

6-TG: 6-thioguanine

6-AG: 6-azaguanine

 

+      positive result

-       negative result

*       ambiguous result

NT   not tested

 

Conclusions:

Based on the 11 mammalian cell mutagenicity studies evaluated in the EU Risk Assessment Report, hydrogen peroxide has the potential to induce mutations in mammalian cells without metabolic activation. No concluding statement on mutagenicity of hydrogen peroxide to mammalian cells after metabolic activation can be given as the data available are very poor. The only available study, however, indicates negative results.

Executive summary:

The mutagenic activity of hydrogen peroxide in vitro was evaluated in an EU Risk Assessment Report in 2003. Eleven different studies were assessed which used different mammalian cell lines (CHO, V79, CV-1, and L5178Y) and different endpoints (HGPRT, thymidine kinase, 6-thioguanine resistance, 6-azaguanidine resistance, ouabain resistance, mutation of the supF locus of the pZ189 plasmid). The studies altogether cover a concentration range from 0.2 µM to 10 mM. From the 11 studies evaluated in the EU Risk Assessment Report, 7 gave positive results, 3 studies gave negative results and 1 study gave an ambiguous result (all without metabolic activation). In the only study that was performed with metabolic activation a negative result was obtained. Based on the aforementioned studies hydrogen peroxide has the potential to induce mutations in mammalian cells without metabolic activation. No concluding statement on mutagenicity of hydrogen peroxide to mammalian cells after metabolic activation can be given as the data available are very poor. The only available study, however, indicates negative results.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Hydrogen peroxide:

Key study: OECD Guideline 474 (drinking water, rats). GLP study. No statistically significant increases in the frequency of micronucleated PCEs were observed in the 6000 ppm dose group, neither was any decreased ratio polychromatic/normochromatic erythrocytes noted. Animals receiving cyclophosphamide responded as expected. In conclusion, hydrogen peroxide did not show any genotoxic effects at the tested concentrations.

Key study: OECD Guideline 474 (intraperitoneal, mice). GLP study. In all groups treated with hydrogen peroxide, the mean values of MPE were similar to those of their respective vehicle controls. A slight statistically significant increase in the MPE number in the low-dose group after 24 hours was considered as biologically insignificant. A statistically significant decrease in the PE/NE ratio in most treated groups after 24 and 48 hours showed that hydrogen peroxide effectively affected the bone marrow cells. It was concluded from the findings that under the experimental conditions hydrogen peroxide did not induce cytogenetic damage in bone marrow cells of mice when administered by the intraperitoneal route.

Key study: OECD Guideline 486. GLP study. In vivo treatment with 25 or 50 mg/kg hydrogen peroxide did not produce a group mean NNG value greater than zero (-2.1- -2.7 respectively) nor were any more than 0.7 % cells found in repair at either dose or time point. It was concluded that hydrogen peroxide failed to induce unscheduled DNA synthesis following treatment in vivo, detected under the experimental conditions employed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed under GLP conditions and in accordance with the OECD Guideline for Testing of Chemicals No. 474.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: C57BL/6NCr1BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 24.92 +/- 1.48 g, females: 20.81 +/- 1.48 g
- Assigned to test groups randomly: yes, preselected based on body weight and health status, assigned randomly by computerised stratification
- Fasting period before study: no data
- Housing: individually in stainless steel wire mesh suspended cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow No. 5002 ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2
- Humidity (%): 50 +/- 10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness


IN-LIFE DATES: From: 19 September 1994 To: 25 October 1994

Route of administration:

oral: drinking water

Vehicle:

Hydrogen peroxide was dissolved in distilled water.

Details on exposure:

The drinking water solutions were prepared by adding hydrogen peroxide (35 % solution) to distilled water and thoroughly mixing. Control water was similarly mixed. Drinking water solutions were prepared weekly in autoclavable polypropylene carboys equipped with spigots. After preparation, the test water solutions were refrigerated until use.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily ad libitum

Post exposure period:

None

Remarks:

Doses / Concentrations:
0, 200, 1000, 3000 or 6000 ppm (male: 0, 42.4, 164, 415 or 536 mg/kg bw/day) (female: 0, 48.5, 198, 485 or 774 mg/kg bw/day)
Basis:
nominal in water

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Five males and five females were taken from the control group in the second week of treatment. These animals were treated with 20 mg/kg cyclophosphamide by a single intraperitoneal injection on day 13.

Tissues and cell types examined:

On day 14 of the exposure phase, a gross examination was performed on all animals including the general appearance, all orifices, cranial, thoracic and abdominal cavities, and contents. At necropsy, all gross lesions and the entire gastrointestinal tract including the esophagus were save from all mice except those serving as positive control for the micronucleus assay. The esophagus was transected near the forestomach, the duodenum was transected approximately 2 cm from the gastric pyloric sphincter and the stomach was incised along the greater curvature with the incision extending down the 2 cm segment of attached duodenum. Gastric contents were gently removed. The opened stomach and duodenum segment were pinned flat for fixation by immersion in 10 % neutral buffered formalin. A section was trimmed from the gastric pyloric antrum along the lesser curvature and continuing across the sphincter and including proximal duodenum. Tissues were processed to haematoxylin and eosin stained slides. Each section was microscopically examined from all animals in the control and all test groups.

Details of tissue and slide preparation:

On test day 14, bone marrow smears were prepared at necropsy from the five male and five female mice treated with cyclophosphamide, from 10 male and 10 female mice each from the 0 (negative control), 200, 100, 3000 and 6000 ppm dose groups and from those mice not assigned to any treatment group. Slides from negative control, 6000 ppm hydrogen peroxide dose group and positive indicator group were evaluated for micronuclei. Two thousand polychromatic erythrocytes from each evaluated animal were scored for micronuclei.

Evaluation criteria:

A statistical increase in the frequency of micronucleated polychromatic erythrocytes as found by one-way analysis of variance (ANOVA) (one-tailed with significance level set to 5 %).

Statistics:

Mean body weight, body weight gain, water consumption and food consumption data were statistically analysed by one-way analysis of variance (ANOVA). Exposure group values were compared to controls by Dunnett's tests when the ratio of variance indicated a signficant amont-to-within group variation. The incidence of clinical observations did not warrant statistical evaluation. For the micronucleus assay, a Shapiro-Wilkes test indicated that the data for percent micronucleated polychromatic erythrocytes (MNPCEs) and proportion of PCEs among 1000 erythrocytes were normally distributed. Therefore, parametric statistical analyses were performed on the data. Data for the proportion of MNPCEs (frequency) and proportion of PCEs among 1000 erythrocytes (PCE frequency) were transformed prior to analysis using the arcsine square-root function. Data were analysed using a one-way analysis of variance (ANOVA). The dose groups were compared using each animal as an experimental unit. All analyses were one-tailed, with significance set at the 5 % level. Positive indicator data were analysed separately from the analyses for effects of the test material.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Mouse PCE and MNPCE frequency

Hydrogen peroxide concentration (ppm)	Number and sex	Mean % PCE	Mean % MNPCE
0	10 M	62.2	0.42
	10 F	67.4	0.18
6000	10 M	67.4	0.49
	10 F	66.9	0.28
Positive control°	5 M	65.9	1.68*
	4 F°°	54.6*	1.50*

° CPA, 20 mg/kg; °° one animal removed from study due to technical error; * p < 0.05

Conclusions:

Hydrogen peroxide did not show any genotoxic effects at the tested concentrations.

Executive summary:

The genetic toxicity of hydrogen peroxide was tested in a valid mammalian erythrocyte micronucleus assay using male and female C57BL/6NCr1BR mice in accordance with OECD Guideline No. 474 and GLP. The animals received the test substance ad libitum via drinking water at nominal concentrations of 0, 200, 1000, 3000 or 6000 ppm (male: 0, 42.4, 164, 415 or 536 mg/kg bw/day) (female: 0, 48.5, 198, 485 or 774 mg/kg bw/day) for 14 days. Positive control animals received a single intraperitoneal injectional of 20 mg/kg cyclophosphamide on day 13 of the study. No specific gross findings were attributable to exposure to hydrogen peroxide. Microscopical findings of degenerative and regenerative alterations in the mucosa of the stomach and/or the duodenum were present in the 3000 and 6000 ppm groups and considered to be substance related. No statistically significant increases in the frequency of micronucleated PCEs were observed in the 6000 ppm dose group, neither was any decreased ratio polychromatic/normochromatic erythrocytes noted. Animals receiving cyclophosphamide responded as expected. In conclusion, hydrogen peroxide did not show any genotoxic effects at the tested concentrations.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

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 under GLP conditions and in accordance with the OECD Guideline for Testing of Chemicals No. 474, 1994.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Swiss OF1/ICO:OF1 (IOPS Caw)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Iffa Crédo, L'Arbresle, France
- Age at study initiation: approximately 6 weeks
- Weight at study initiation:
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: five per sex in polycarbonate cages
- Diet (e.g. ad libitum): AO4 C pelleted diet (U.A.R., Villemoisson-sur-Orge, France) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness

Route of administration:

intraperitoneal

Vehicle:

Water was used as the vehicle.

Details on exposure:

The test substance was administered once by intraperitoneal route using a dose volume of 25 mL/kg, which allowed to test higher doses with less concentrated solutions. The quantity of the test substance administered to each animal was adjusted according to the body weight recorded at the time of dosing. The vehicle control animals received the vehicle alone, under the same conditions. The positive control animals received cyclophosphamide, by oral routed, at a volume of 10 mL/kg.

Duration of treatment / exposure:

Once by intraperitoneal injection

Frequency of treatment:

Once

Post exposure period:

24 and 48 hours

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000 mg/kg body weight
Basis:
nominal in water
first cytogenetic test

Remarks:

Doses / Concentrations:
0, 250, 500, 1000 mg/kg body weight
Basis:
nominal in water
second cytogenetic test

No. of animals per sex per dose:

five males, five females

Control animals:

yes

Positive control(s):

Cyclophosphamide, administered by oral route in 10 mL/kg at a dose of 50 mg/kg body weight.

Tissues and cell types examined:

For each animal, the micronuclei were counted in 20000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Details of tissue and slide preparation:

At the time of sacrifice, all the animals were killed after CO2 inhalation in excess. The femurs of the mice were removed and the bone marrow eluted out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with May-Grünwald-Giemsa. All the slides were coded for scoring.

Evaluation criteria:

A positive response was assumed if a statistically significant increase in the number of micronucleated polychromatic erythrocytes (MPE) when compared to the vehicle group occurred, which doubled the number of MPE of the historical control data, i.e. a number greater than 3.6/1000 PE. The results were considered as negative if the above criteria was not fully met.

Statistics:

The mean number of MPE and the PE/NE ratio from the treated groups were compared to simultaneous vehicle groups. The inter-group comparison was performed using: for MPE the X-square test, for PE/NE ratio the Student's t-test in which p = 0.05 was used as the lowest level of significance.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results of the second cytogenetic study are given in Table 1. The mean values of MPE of all groups treated with hydrogen peroxide were similar to those of their respective controls. A slight, statistically significant increase in the MPE number of the low-dose group after observed after 24 hours was considered as biologically insignificant, because the MPE value was within the range of historical controls, no dose-effect relationship was noted, and the increase was essentially attributed to one animal which had 13 MPE/2000 PE. The PE/NE ratio was statistically significant lower at the three doses at 24 hours and at 250 and 1000 mg/kg after 48 hours, showing that hydrogen peroxide effectively affected the bone marrow cells. The clinical signs and mortality noted in the first cytogenicity test are given in Table 2. Due to the marked mortality in the 2000 mg/kg dose group, a second cytogenetic study was carried out. In this second test, no mortality and no clinical signs were observed in the 250 and 500 mg/kg dose groups of both sexes and in the female 1000 mg/kg dose group. After treatment with 1000 mg/kg, one of sixteen males died, which was replaced by ne of the supplementary group. Hypoactivity and piloerection was noted in the other treated males at 1000 mg/kg. No macroscopic abnormalities (abdominal cavity) were seen at necropsy at doses of 250, 500 and 1000 mg/kg except for a discolourated spleen in one male at 1000 mg/kg (Table 3).
The mean values of micronucleated polychromatic erythrocytes were within the historical range in the two vehicle groups.
Cyclophosphamide induced a highly significant increase (p < 0.001) in the number of MPE. In addition, the PE/NE ratio decreased significantly (p < 0.05) showing the toxic effects of the positive control substance to bone marrow cells.

Table 1: Results of the second cytogenicity study.

Group a)	Dose (mg/kg)	MPE/1000 PE b)		PE/NE ration c)		Time of sacrifice d) (hours)
		Mean	SD	Mean	SD
Vehicle	-	0.9	0.9	0.6	0.1	24
Test substance	250	2.2**	1.6	0.5*	0.1
	500	1.2	1.0	0.5*	0.1
	1000	1.6	1.2	0.5*	0.1
Cyclophosphamide	50	37.6***	8.2	0.5*	0.1
Vehicle	-	1.7	0.8	0.7	0.2	48
Test substance	250	1.9	0.8	0.5*	0.2
	500	1.1	0.5	0.6	0.1
	1000	1.2	0.8	0.5*	0.1

a) 10 animals (5 males, 5 females) per group; b) based on scoring of 2000 polychromatic erythrocytes; c) based on a total of 1000 erythrocytes (PE + NE); d) time following treatment (intraperitoneal injection, dissolved in water); MPE: micronucleated polychromatic erythrocytes; PE: polychromatic erythrocytes; NE: normochromatic erythrocytes; SD: standard deviation; * p < 0.05, ** p < 0.01, *** p < 0.001 (X-square test for MPE, Student's t-test for PE/NE ratio)

Table 2: Clinical signs and mortality noted in the frist cytogenicity study.

Dose (mg/kg)	Time point	Clinical signs	Males (animals/total number of animals)	Females (animals/total number of animals)
500	2 h – 48 h	None	--	--
1000	2 h	Piloerection	2/10	0/10
	24 h – 48 h	None	--	--
2000	2 h	Convulsions before death	5/13	7/13
	2 h	Convulsions, hypoactivity	3/8*	--
	2 h	Convulsions, piloerection, hypoactivity	3/8*	--
	2 h	Piloerection, hypoactivity	2/8*	1/6*
	2 h	Hypoactivity	--	5/6*
	24 h	None	--	3/6*
	24 h	Mortality	1/8*	--
	24 h	Piloerection, hypoactivity	5/7**	--
	24 h	Hypoactivity	2/7**	3/6*

* Animals remaining after first intraperitoneal injection; ** Animals remaining after second intraperitoneal injection

Table 3: Clinical signs and mortality noted in second cytogenic study.

Solution at 4 % hydrogen peroxide
Dose (mg/kg)	Time point	Clinical signs	Males (animals/total number of animals)	Females (animals/total number of animals)
1000	2 h	Hypoactivity	9/16	--
	2 h	Piloerection	3/16	--
	2 h	Hypoactivity, piloerection	3/16	--
	2 h	Coma	1/16	--
	24 h	Mortality	1/16	--
	24 h	Piloerection	15/16	--

Conclusions:

Hydrogen peroxide did not reveal any genotoxic potential under the experimental conditions of this test.

Executive summary:

The potential of hydrogen peroxide to induce cytogenetic damage to the bone marrow cells of Swiss OF1 mice was tested in a micronucleus assay in accordance with OECD Guideline No. 474 and under GLP conditions. Preliminary toxicity tests were performed to define the doses to be used in the cytogenetic test. Six groups of 5 male and 5 female mice received the test substance by a single intraperitoneal injection at doses of 250, 500 and 1000 mg/kg. Two groups of 5 males and 5 females received the vehicle (water) alone. One group of 5 males and 5 females was treated with the positive control substance cyclophosphamide administered by a single oral dose of 50 mg/kg. For each animal, bone marrow cell smears were prepared and the micronuclei were counted in 2000 polychromatic erythrocytes. The polychromatic (PE) to normochromatic (NE) erythrocyte ratio was established by scoring 1000 erythrocytes (PE + NE). In the two vehicle control groups, the mean values of micronucleated polychromatic erythrocytes (MPE) were in the range of historical controls. Cyclophosphamide induced a highly significant increase in the number of MPE and significantly decreased the PE/NE ratio, indicating the cytotoxicity of the control substance. In all groups treated with hydrogen peroxide, the mean values of MPE were similar to those of their respective vehicle controls. A slight statistically significant increase in the MPE number in the low-dose group after 24 hours was considered as biologically insignificant. A statistically significant decrease in the PE/NE ratio in most treated groups after 24 and 48 hours showed that hydrogen peroxide effectively affected the bone marrow cells. It was concluded from the findings that under the experimental conditions hydrogen peroxide did not induce cytogenetic damage in bone marrow cells of mice when administered by the intraperitoneal route.

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The method used in the study complied with UKEMS Test Guidelines, which is in accordance with the OECD Guideline for testing of Chemicals No. 486. The study was performed under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

Additionally to the in vivo UDS test an in vitro test with hydrogen peroxide in different concentration on liver cells received from negative control animals was performed

Principles of method if other than guideline:

In vivo rat liver UDS assay according to Kelly et al. (1993), in: Supplementary Mutagenicity Tests UKEMS Recommended Procedures. Eds. D.öJ. Kirkland and M. Fox, Cambridge University Press.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd. (UK), Margate, UK
- Age at study initiation: 42-52 days
- Weight at study initiation: 194-263 g
- Assigned to test groups randomly: yes (due to deaths some additional rats were dose which had not been included in the randomisation process)
- Fasting period before study:
- Housing: not more than 3 in polypropylene cages with wire mesh lids
- Diet (e.g. ad libitum): laboratory chow diet (Special Diet Services Ltd., RM1.[E].SQC) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 to 10


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 48-59
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours darkness

Route of administration:

intravenous

Vehicle:

Water for injection (Phoenix Pharmaceuticals Ltd., Gloucester, UK, batch number 515004)

Details on exposure:

Dosing preparations were made by diluting hydrogen peroxide in water for injection. Dilutions were made using water for injection. The test article preparations were protected from light, stood on ice prior to use and were used within approximately 4 hours of initial formulation. Animals were administered the appropriate concentration dosing solution at a dose rate of 0.2 mL/min, to a total administered dose volume of approximately 25 mL/kg.

Duration of treatment / exposure:

25-33 minutes (at a dose rate of 0.2 mL/min)

Frequency of treatment:

Once by intravenous administration

Post exposure period:

2-4 or 12-14 hours

Remarks:

Doses / Concentrations:
0, 25 or 50 mg/kg
Basis:
nominal in water

No. of animals per sex per dose:

five to six

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetamidfluorene (2-AAF) was freshly suspended in corn oil using a Silverson homogeniser at 7.5 mg/mL to serve as the positive control for the 12-14 hour experiment. Dimethylnitrosamine (DMN) dissolved in purified water at 1.0 mg/mL was used as the positive control for the 2-4 hour experiment. Both positive controls were administered by oral gavage.

Tissues and cell types examined:

Liver, hepatocytes

Details of tissue and slide preparation:

Animals were anaesthetised. The abdominal surface was rinsed with 70 % ethanol, a V-shaped incision was made from the centre of the lower abdomen o the rib cage, and the skin and muscle removed to reveal the abdominal cavity. The inferior vena cava was then clamped superior to the kidney and a cotton tie was placed loosely around the hepatic portal vein. The vein was then cannulated using an appropriate catheter, the inner needle removed, and the ligature tightened. Cannulation of the superior vena cava was carried out via the right atrium, following cutting of the diaphragm and removal of the rib cage. The hepatic portal cannula was connected to a supply of calcium free Buffer 1 which had been gassed for at least 5-10 minutes with 5 % CO2 in air (v/v), and pumped at approximately 40 mL/min. The vena cava cannula was connected to a waste line and the liver washed free of blood, using approximately 400 mL Buffer 2 which was continually gassed with 5 % CO2 in air (v/v), at 40 mL/min until the reservoir volume had dropped from 400 mL to approximately 200 mL. The liver was then cut free and transferred to a sterile plastic petri dish with approximately 10 mL of the prewarmed (37 °C) Buffer 2. The liver capsule was removed and the hepatocytes carefully teased out using a combing method. The separated hepatocytes were gently washed through 150 micrometre nylon mesh with Williams E medium-Complete (WE-C) to a volume of 100 mL. Of this suspension, approximately 50 mL was taken and centrifuged at approximately 40 x g for 2-3 minutes. The resulting pellet was resuspended in approximately 40 mL WE-C. The centrifugation and resuspension procedure was repeated twice more and cells taken, diluted with an equal volume of 0.4 % (w/v) trypan blue and the proportion of viable cells (those with unstained nuclei) determined using a haemocytometer. The culture was then diluted to provide 1.5 x 10e5 viable cells/mL.
Medium was removed from the cells and the monolayers washed with 2 mL Williams E medium-Incomplete (WE-I) which was then replaced with 2 mL WE-I containing 10 microCi/mL [3H] thymidine. After 4 hours incubation at 37 °C in a 5 % CO2 in air (v/v) atmosphere, the medium was removed and the cells washed with three changes of WE-I containing 0.25 mM thymidine. Cultures were then incubated overnight with 3 mL of the same medium. To prepare for autoradiography, coverslips were washed with 2 mL phosphate buffered saline and the cells fixed with three changes of 2 mL glacial acetic acid:ethanol (1:3 v/v). The coverslips were then washed four times with purified water, allowed to dry and mounted onto previously labelled microscope slides, cells side up, with DPX.

Evaluation criteria:

The study would be considered valid if 1) the negative control animals had 0 net grains/nucleus (NNG) counts or less (i.e. a negative value, within historical range), 2) the positive control treatments had NNG values of five or more, with 50 % or more cells having NNG counts of five or greater.
The test article would be considered as positive in the assay if, at any dose and at either time point, 1) the test article yielded group mean NNG values greater than 0 NNG and 20 % or more of cells responding (mean NNG values similar to or greater than 5), 2) an increase was seen in both NNG and the percentage of cells in repair.
If the test article failed to induce UDS at any dose tested after both 2-4 and 12-14 hours exposure, it would be considered as clearly negative in this system.

Statistics:

No statistics reported.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The validity criteria of the study were fulfilled: the mean net grain count for vehicle-control animals was less than zero and the positive control substances induced increases in group mean net grain counts of five or more (9.4 and 10.4 respectively), and 50 % or more cells (84.7 % and 83.7 % respecively) had net grain counts of five or more.

Table 1: Group mean net grain count values at sacrifice after 12 -14 hours.

Dose (mg/kg)	Net nuclear grain count		Net grain count of cells in repair		Percent of cells in repair (NG ≥ 5)
	Mean	SD	Mean	SD	Mean	SD
0 (water)	-2.7	1.1	6.3	0.0	0.3	0.6
25	-2.6	0.4	5.7	0.9	0.7	0.6
50	-2.7	0.6	0	-	-	-
75 (2-AAF)	10.4	0.5	11.8	0.3	83.7	4.7

2 -AAF: 2 -acetamidfluorene

Table 2: Group mean net grain count values at sacrifice after 2 -4 hours.

Dose (mg/kg)	Net nuclear grain count		Net grain count of cells in repair		Percent of cells in repair (NG ≥ 5)
	Mean	SD	Mean	SD	Mean	SD
0 (water)	-2.6	0.4	0	-	-	-
25	-2.1	0.2	0	-	-	-
50	-2.6	0.5	0	-	-	-
10 (DMN)	9.4	1.2	10.4	0.8	84.7	8.0

DMN: dimethylnitrosamine

Conclusions:

Hydrogen peroxide had no in vivo genotoxic activity under the described test conditions.

Executive summary:

The genotoxicity potential of hydrogen peroxide was tested in an Unscheduled DNA Synthesis assay in rat liver according to OECD Guideline No. 486 and under GLP conditions. Male Wistar rats were administered intravenously the test solutions at concentrations of 1 or 2 mg/mL at a dose rate of 0.2 mL/min to achieve final doses of 25 mg/kg or 50 mg/kg. Animals were sacrificed either after 2 -4 hours or 12 -14 hours. In the 2 -4 hour experiment, dimethylnitrosamine (DMN) was used as the positive control substance and in the 12 -14 hour experiment, the positive control substances was 2 -acetamidfluorene (2 -AAF). Negative control animals were administered water only. After sacrifice, hepatocytes from the liver were sampled and cultures of hepatocytes were treated with [3H] thymidine. Slides were prepared from each animals with fixed hepatocytes and dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically and the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm was determined (the net grain count, NNG). Negative vehicle controls gave a group mean NNG value of less than zero with 0 -0.3 % cells in repair. Group mean NNG values were increased by 2 -AAF and DMN treatment to at least 9.4 and more than 80 % of cells were found to be in repair. In vivo treatment with 25 or 50 mg/kg hydrogen peroxide did not produce a group mean NNG value greater than zero (-2.1- -2.7 respectively) nor were any more than 0.7 % cells found in repair at either dose or time point. It was concluded that hydrogen peroxide failed to induce unscheduled DNA synthesis following treatment in vivo, detected under the experimental conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Calcium peroxide when in contact with water, on the one hand hydrolyses into hydrogen peroxide, and on the other hand results in a loss of active oxygen. Moreover, dissolving calcium peroxide in water, results in an increase of the pH. Under acidic conditions, the water solubility of the reaction mass will be very high because the hydroxide reacts with the acid.

CaO₂  + H₂O → CaO + H₂O₂

2CaO₂→ 2CaO + O₂

2CaO₂+ 2H₂O → 2Ca²⁺+ 4OH^- + O₂

 

The toxicity of the reaction mass is due to the hydrolysis product hydrogen peroxide (H2O2) and calcium hydroxide (Ca(OH)2). Under physiological conditions, calcium hydroxide ultimately dissociates into calcium cations (Ca2 +) and hydroxyl anions (OH-). Calcium, is an essential and abundantly available mineral nutrient. Hydroxyl anion is neutralised in body fluids.

Justification for classification or non-classification

Based on available data, the substance is not classified for mutagenicity according to CLP Regulation (EC) no. 1272/2008.