Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test with Hydrogen peroxide-urea (1:1) positive results were obtained in TA98 and TA102 in the absence of metabolic activation. As Hydrogen peroxide-urea (1:1) breaks down to hydrogen peroxide and urea, data from these can be used as source data in a read-across approach. Hydrogen peroxide was a weak mutagen in bacterial and mammalian cell in vitro assays. Urea was not mutagenic in bacterial test systems. Mixed results were obtained with mammalian cells. Positive results were only obtained at high dose levels.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
Test substance: Carbamide peroxide, i.e hydrogen peroxide - urea (1:1)
CAS number: 124.43-6
Purity: not staterd
Target gene:
his operon
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
30% S9 from induced both rat and hamster liver
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
30% S9 from induced both rat and hamster liver
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
30% S9 from induced both rat and hamster liver
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
without
Test concentrations with justification for top dose:
according to guideline
Vehicle:
water
Negative controls:
yes
Positive controls:
yes
Positive control substance:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: positive without metabolic activation
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: positive without metabolic activation
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes

Strain: TA100

 

Dose

No Activation
(Negative)

No Activation
(Negative)

No Activation
(Weakly Positive)

30% RLI
(Negative)

30% HLI
(Negative)

 

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

 

ug/Plate

Mean

±SEM

Mean

±SEM

Mean

±SEM

Mean

±SEM

Mean

±SEM

 0         

131

3.8

177

7.8

164

.9

137

8.4

134

.6

10         

166

6.1

217

17.6

172

11.3

33         

145

8.7

181

4.5

211

5

100         

162

9

149 s

8.5

267

3.2

138

9.5

144

7

150         

105 s

19.8

248

10.4

200         

100 s

21.9

165 s

11.1

333         

66 s

5.8

149

3.5

141

5.7

1000         

t

141

2

152

4.9

3333         

147

6.1

162

6.3

10000         

142

3.2

165

3.7

Positive Control

433

12.1

554

2.1

383

9

796

10.4

393

9.8

 

Strain: TA102

 

Dose

No Activation
(Positive)

No Activation
(Weakly Positive)

30% RLI
(Negative)

30% HLI
(Negative)

 

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

 

ug/Plate

Mean

±SEM

Mean

±SEM

Mean

±SEM

Mean

±SEM

 0         

283

11.7

239

11.1

389

5.6

391

7.9

10         

281

12.4

33         

357

13.4

282

7.7

100         

409

3.5

408

18.7

379

6.4

393

9.5

150         

424

11.9

200         

522

9.4

433

9.3

333         

635

7.5

429

25.2

348

34.2

1000         

789

15.7

430

14.8

396

9.5

3333         

435

4.7

389

12.4

10000         

441

8.6

424

7.7

Positive Control

1008

168.7

546

2.3

1254

215.7

2412

36.5

 


 

 

Strain: TA1535

 

Dose

No Activation
(Negative)

 

Protocol

Preincubation

 

ug/Plate

Mean

±SEM

 0         

11

3.4

10         

14

1.5

33         

15

.7

100         

15

2.6

150         

11 s

3

200         

9 s

.6

Positive Control

263

4

 

Strain: TA1538

 

Dose

No Activation
(Negative)

 

Protocol

Preincubation

 

ug/Plate

Mean

±SEM

 0         

5

.3

10         

3

.9

33         

6

.9

100         

6

1.5

150         

6

1.5

200         

3 s

0

Positive Control

292

9.9

 

Strain: TA98

 

Dose

No Activation
(Equivocal)

No Activation
(Equivocal)

No Activation
(Positive)

No Activation
(Weakly Positive)

30% RLI
(Negative)

30% HLI
(Negative)

 

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

 

ug/Plate

Mean

±SEM

Mean

±SEM

Mean

±SEM

Mean

±SEM

Mean

±SEM

Mean

±SEM

 0         

15

2.3

13

.3

17

2.2

25

3.8

23

1.2

19

1.5

10         

18

.9

23

.9

20

3.4

30

4.5

33         

17

3.8

20

3.5

24

0

38

3.5

100         

26

1.2

42

4.4

35

2.6

50

6.7

20

1.5

23

1.5

150         

25

3

52

2.6

27

7.3

200         

13 s

1.2

37

6.7

4 s

1.2

333         

6 s

1.9

27

3.3

25

1.8

1000         

t

27

1.5

27

4

3333         

19

1.5

25

1.8

10000         

25

1.2

28

1.5

Positive Control

229

11

297

9.3

268

6.7

246

2

335

22.7

410

16

 

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; t = Toxic;

 

Conclusions:
Positive results were obtained in TA98 and TA102 in the absence of metabolic activation.
Executive summary:

Hydrogen peroxide-urea (1:1) (name in test report: carbamide peroxide) was tested in the AMES test using S. typhimurium strains TA98, TA100, TA102, TA1535, and TA1538 according to the OECD 471 test guideline both without and with metabolic activation (30% S9 supernatant from induced rat and hamster liver, respectively). Positive results were obtained in TA98 and TA102 in the absence of metabolic activation.

The study is considered to be fully reliable and suitable for assessment.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
without
Genotoxicity:
positive
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: positive in standard plate assay: the result is adopted for the target substance
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: weakly positive in the standard plate assay; result is adopted for the target substance
Key result
Species / strain:
S. typhimurium TA 102
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: positive in the standard plate and weakly in the preincubation assay; result adopted for the target substance
Key result
Species / strain:
S. typhimurium TA 1537
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: positive in the standard plate and weakly in the preincubation and liquid incubation assay; adopted for the arget substance
Key result
Species / strain:
S. typhimurium TA 1538
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: positive in the preincubation assay; adopted for the target substance
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Species / strain:
other: CHO cells; CH DON-6 cells, CH lung V79 cells, BALB/c mouse back-skin cells, Syrian hamster lung cells, human fibroblasts, human lymphocytes, mouse ascites tumour cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
in 10 of 14
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: results are for the source substance; may be adopted for the target substance
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: results are for the source substance; may be adopted for the target substance
Key result
Species / strain:
other: human lymphocytes, rat bone-marrow cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: results are for the source substance; may be adopted for the target substance
Key result
Species / strain:
other: Syrian hamster
Remarks:
V79 and CHC cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
1 study; micronuclei
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: results are for the source substance; may be adopted for the target substance
Key result
Species / strain:
other: mouse
Remarks:
splenocytes
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: results are for the source substance; may be adopted for the target substance
Key result
Species / strain:
other: variant of HeLa cells, and embryonic fibroplasts
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
2 studies; chomosome aberration
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: results are for the source substance; may be adopted for the target substance
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
in 1 of 6 studies
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: HPRT mutation seen with the source substance; can be adopted for the target substance
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
in 5 of 6 studies
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: HPRT mutation seen with the source substance; can be adopted for the target substance
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
2 studies
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: HGPRT mutation seen with the source substance; can be adopted for the target substance
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
6 studies
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: Mutations seen with the source substance include HGPRT; resistance to 6-thioguanine, or 8-azaguanine and ouobain; the result can be adopted for the target substance
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
3 studies
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: HGPRT; TK locus; result for the source substance can be adopted for the target substance
Key result
Species / strain:
other: African Green monkey
Remarks:
kideney cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: result for the source substance; can be adopted for the target substance
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: result for the source substance; can be adopted for the target substanceresult for the source substance; can be adopted for the target substance
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: result for the source substance; can be adopted for the target substance
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: result for the source substance; can be adopted for the target substance
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: result for the source substance; can be adopted for the target substance
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: result for the source substance; can be adopted for the target substance
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
several limitations of the study lead to this conclusion, differs from tthe authors' interpretation
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: mutation frequency was 2.4-fold increased at only 530 mmol/l (31.7 mg/ml). Result for the source substance; can be adopted for the target substance

Urea

Concentration

[mol/l]

Total growth

[suspension growth x cloning efficiency]

Mutation frequency

[mutations per
10E06 surviving cells]

Mutation index

[mutation frequency of treated culture / mutation frequency of control cultures]

0

 

80

 

0

 

99

 

1.320 x 10 E-01

107

102

1.1

2.640 x 10 E-01

73

150**

1..7

3.970 x 10 E-01

56

135

1.5

5.300 x 10 E-01

24

214***

2.4

6.620 x 10 E-01

8

256***

2.9

 

** p ≤0.01; *** p ≤0.001

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Hydrogen peroxide – urea (1:1) readily breaks down to hydrogen and urea in aqueous solution. On this basis data on hydrogen peroxide and urea, respectively, may be used for read across. Neither hydrogen peroxide nor urea was determined to be genotoxic in vivo. This result is adopted for hydrogen peroxide – urea (1:1).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Sex:
not specified
Genotoxicity:
positive
Remarks:
the only study with positive results
Toxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: Mouse; increased chromatid aberrations (local) following i.p. injection. Local effect; response presumed to depend on the presence or absence of RBCs.
Key result
Sex:
not specified
Genotoxicity:
negative
Remarks:
in 8 studies
Toxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: species tested: rat, mouse, Drosophila
Key result
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: micronuclei in bone marrow polychromatic RBCs
Key result
Sex:
not specified
Genotoxicity:
negative
Remarks:
rat; 1 study
Toxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: hepatocyte UDS assay
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Sex:
male
Genotoxicity:
positive
Toxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: result for the source substance; can be adopted for the target substance
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test:
The induction of DNA damage (SCE) following long-term exposure to hydrogen peroxide -urea (1:1) was examined in-vivo using Chinese hamsters.
- Short description of test conditions:
20 Chines hamsters per dose group received 0 or 70 mg hydrogen peroxide -urea (1:1) /kg bw for 15 weeks (5 days/week) by oral gavage. The positive control group received cyclophosphamide. Another two animal groups received Rembrandt tooth whitener at doses of 500 and 2000 mg/kg bw (content of hydrogen peroxide -urea (1:1) not specified).
- Parameters analysed / observed:
The SCE frequency of bone marrow cells was determined. Further, gastroduodenal tissues were examined for histopathological changes.
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay
Test material information:
Composition 1
Specific details on test material used for the study:
CAS: 124-43-6
Purity: not specified
Species:
hamster, Chinese
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
route: oral gavage
exposure duration: 15 weeks
frequency: 5 per week
Duration of treatment / exposure:
15 weeks
Frequency of treatment:
5/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle (water)
Dose / conc.:
70 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
Tissues and cell types examined:
SCE frequency of bone marrow cells .
Histopathology of gastro duodenal tissue
Genotoxicity:
negative
Vehicle controls valid:
yes
Positive controls valid:
yes
Conclusions:
No genotoxicity seen in vivo following long-term adminsitration to Chinese hamsters.
Executive summary:

Reportedly, no DNA damage (SCE) was seen in Chinese hamsters dosed with 70 mg hydrogen-peroxide - urea (1:1)/kg bw and day for 15 weeks. Further, no changes were seen in in the gastro-duodenal tissue (Li et al., 1993).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

Only few studies on hydrogen peroxide – urea (1:1) itself (other name: Carbamide peroxide) were located. However, as Carbamide peroxide breaks down to one molecule of each hydrogen peroxide and urea, data from these can be used as source data in a read-across approach.

Hydrogen peroxide-urea (1:1) itself was tested in the Ames test using S. typhimurium strains TA98, TA100, TA102, TA1535, and TA1538 according to the OECD 471 test guideline both without and with metabolic activation (30% S9 supernatant from induced rat and hamster liver, respectively). Positive results were obtained in TA98 and TA102 in the absence of metabolic activation (NTP, 1999).

 

Hydrogen peroxide (H2O2) was a mutagen in a variety of in vitro bacterial test systems, but a high percentage of ambiguous or negative results were also obtained which is attributable to modifying factors and also indicates that hydrogen peroxide is a weak mutagen (SCCP, 2007). Hydrogen peroxide induced chromosome aberration in vitro in mammalian cells from several species (rat, mouse, Chinese and Syrian hamster, human cells). Positive results were obtained in 10 of 14 studies, 2 studies each gave ambiguous or negative results. The lowest effective dose in the various test systems ranged from 0.34 to 510 µg/mL (SCCP, 2007; ECB, 2003). Regarding mutations in mammalian cells in vitro, SCCP evaluated six mutagenicity studies with mammalian cells (Chinese hamster V79 lung fibroblasts). HPRT mutation was seen in only one study (lowest effective dose 17 µg/mL) whereas no mutation at this locus was seen in the other five studies (highest ineffective doses ranged from 3.4 to 20 µg/mL). Thus it appears that the responses depend on whether hydrogen peroxide does reach the DNA and on cellular repair abilities (SCCP, 2007). The percentage of positive results was higher in ECB’s evaluation. Of the 12 mammalian cell gene mutation assays (in vitro) that ECB evaluated, hydrogen peroxide caused mutations of the HGPRT or TK locus or resistance in 9 of 12 studies without metabolic activation (with metabolic activation not tested). The species from which cells lines were derived included Chinese hamster, mouse, and the African Green monkey (ECB, 2003).

 

Urea was not genotoxic in bacterial cell assays in vitro (Mortelmans,et al., 1986). This is also evidenced in EPA’s Toxicological Review of Urea (2011). Mixed results were obtained in assay with mammalian cells (EPA, 2011). Positive results were most often weak and only seen at high urea test concentrations. For example, Ishidate (1981, 1997) reported only a weak induction of chromosome aberration in Chinese hamster lung fibroblasts at urea concentrations up to 13 mg/mL. Likewise, urea reportedly caused mutations at the TK locus were tested using mouse lymphoma L5178Y cells in vitro. However, the test concentrations of 0, 8, 16, 24, 32, and 40 mg/mL exceeded the maximal recommended test concentration of 5 mg/ml (OECD guideline 476) by far. Excessive cytotoxicity was seen at the highest dose level; a significant increase of CA formation was only seen at 32 mg/mL. As this this 6.4-fold above the recommended dose, and because there was no dose-related effect at lower dose levels, it is considered that a false-positive effect was reported (Wangenheim and Bolcfoldi, 1988).

 

Overall conclusions regarding genotoxicity in vitro:

-       Hydrogen peroxide was a weak mutagen in bacterial and mammalian cell in vitro assays.

-       Urea was not mutagenic in bacterial test systems. Mixed results were obtained with mammalian cells. Positive results were obtained at high dose levels, and effects were either false-positive or marginal.

-       These result can be adopted for hydrogen peroxide – urea (1:1).

Genetic toxicity in vivo

As already stated earlier, hydrogen peroxide – urea (1:1) readily breaks down to hydrogen and urea in aqueous solution. The molar ratio is 1:1, i.e. hydrogen peroxide accounts for approx. 36% of the initial mass, and urea for 64%. On this basis data on hydrogen peroxide and urea, respectively, may be used for read across in instances where no or insufficient data are available for hydrogen peroxide – urea (1:1).

 

Hydrogen peroxide was a weak mutagen in vitro but was negative in vivo. ECB evaluated 9 in vivo mutagenicity studies conducted in the mouse, rat, and Drosophila melanogaster. The measured endpoints included cytogenicity (micronuclei and chromosome aberrations), DNA repair (UDS), DNA damage (quantity of 8-OH-2’- deoxyguanosine) and mutations (codon 61 of c-Ha-ras gene), and epidermal hyperplasia and dermal cellularity changes (as a pre-screen for carcinogenicity) in mammals, and the SLRL test in Drosophila melanogaster. Negative results were obtained in all studies. It was therefore concluded that hydrogen peroxide lacks genotoxicity in vivo (ECB, 2003).

 SCCP endorsed in their opinion on tooth whiteners (2007) the conclusions of the European Chemicals Bureau (2003) which are cited below (quoted references contained in the EU RAR on Hydrogen Peroxide, 2003).

"Hydrogen peroxide is a mutagen and genotoxicant in a variety of in vitro test systems. The responses observed were modified by the presence of degrading enzymes (catalase), the extent of formation of hydroxyl radicals by Fenton reaction, and the cells repair abilities.

Hydrogen peroxide has been studied for possible in vivo genotoxicity. Studies employing modern methodologies have explored DNA repair in liver cells of rats administered hydrogen peroxide by intravenous infusion for 30 minutes (CEFIC, 1997), as well as micronucleus formation in mice in the context of a 2-week drinking water exposure (Du Pont, 1995), or after a single intraperitoneal injection (CEFIC, 1995), all with a negative outcome. Intravenous administration of hydrogen peroxide in the in vivo-in vitro unscheduled DNA synthesis study ensured that the substance had a fair chance to reach the target (liver) cells, although the duration of exposure was limited (CEFIC, 1997). In the micronucleus study by oral drinking water exposure (Du Pont, 1995), the systemic fate of hydrogen peroxide was uncertain, and there was no decrease in the ratio of polychromatic/normochromatic erythrocytes in the bone marrow. In the other micronucleus study (CEFIC, 1995), a single intraperitoneal injection of a large dose of hydrogen peroxide somehow affected the bone marrow (because the PE/NE decreased), but the absence of micronucleus formation must be viewed with caution because of the presumably very short lifetime of hydrogen peroxide. With a view to exploring target tissue in vivo genotoxicity and mutagenicity as a pre-screen for carcinogenicity, hydrogen peroxide 0.2-3.2% solutions in ethanol were applied to the skin of Sencar mice twice weekly for 4 weeks (Society for Plastic Industry, 1997). There was no indication of induced DNA damage (increased 8-OH- dG), c-Ha-ras mutations, epidermal hyperplasia and dermal cellularity changes. Thus, at low concentrations, and with a low application frequency, hydrogen peroxide did not induce local mutagenicity in this tissue model.

In conclusion, the available studies are not in support of a significant genotoxicity/mutagenicity for hydrogen peroxide under in vivo conditions."

 

Regarding urea, mixed results were obtained with mammalian cells in vitro at high dose levels, and also in vivo. EPA could not rule out genetic toxicity in vivo, especially since carcinogenicity studies were too short and provided only “inadequate information to assess the carcinogenetic potential of urea” (EPA, 2011). Urea is generated in appreciable amounts in vivo in the course of protein and amino acid metabolism, and the available information is not considered to provide sufficient evidence that urea is genotoxic in vivo or has a carcinogenic potential.

 

As to hydrogen peroxide – urea (1:1) itself, only abstracts are available that provide additional information that no DNA damage (SCE) was seen in Chinese hamsters dosed with 70 mg hydrogen-peroxide - urea (1:1)/kg bw and day for 15 weeks. Further, no changes were seen in in the gastro-duodenal tissue (Li et al., 1993).

 

Overall, it is concluded that neither hydrogen peroxide nor urea are genotoxic in vivo. This result is adopted for hydrogen peroxide – urea (1:1).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on these data the substance is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.