Diphenyl carbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Non mutagenic with and without metabolic activation (Ames test) in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100,  OECD 471,Timm (1989)

- Non mutagenic with and without metabolic activation (Ames test) in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, OECD 471, Loveday (1991)

- Non mutagenic with and without metabolic activation (HPRT test) with Chinese hamster V79 cells, OECD 476, Wollny (1996)

- Increased frequencies of structural chromosomal aberrations in V79 cells with and without metabolic activation (chromosome aberration assay) OECD 473, Czich (1996)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Plate incorporation assay

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene (S. typhimurium), Tryptophan gene (E. coli WP2)

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli strain (WP2)

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver microsomal fraction

Test concentrations with justification for top dose:

0, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Because of strong cytotoxicity at >= 500 µg/plate in a non-activation range-finding study, the concentrations were limited to 150 µg/plate.
exp. I and II without S9-mix: 0.5 - 150 µg/plate
exp. I and II with S9-mix: 15 - 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

as appropriate (see below)

Remarks:

Appropriate reference mutagens (sodium azid, 9-aminoacridine, 2-AA, ENNG, 2-NF) were used as positive controls

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

A test result was considered positive if for any strain a significant increase (2- to 3-fold in dependence on strain) over the negative control in the number of revertants per plate was observed which was concentration-dependent.

Statistics:

none

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli strain (WP2)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: >= 500 µg/plate without S9 mix, 5000 µg/plate with S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Without metabolic activation: diphenyl carbonate was toxic to all four Salmonella strains at the dose levels of 0.15 mg/plate and higher.  At 0.05 mg/plate, slight toxicity was seen in strains TA 98 and TA 1537.  E. coli showed no toxicity at any dose.  No bacterial strain demonstrated any mutagenicity in conjunction with DPC.  All test plate counts remained within acceptable historical background parameters.  
With metabolic activation: Extreme toxicity was seen in all four Salmonella typhimurium strains at 5 mg/plate.  In addition, TA1535 and TA1537 also showed increased toxicity to the test chemical at 1.5 mg/plate.  Slight toxicity was also observed at 1.5 and 0.5 mg/plate in the Salmonella strains.  E. coli was not affected by the test chemical at any dose level.  As with the non-activated assay, no mutagenicity was observed at any dose level.

Conclusions:

Diphenyl carbonate showed no mutagenic properties in the Ames Test with S. typhimurium TA 1535, TA 1537, TA 98, TA 100 or E. coli WP2.

Executive summary:

The mutagenic potential of the substance was investigated in a study which was conducted in accordance with OECD 471.

During the study S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 were exposed to the test material in DMSO. Due to strong cytotoxicity at 500 µg/plate in a non-activation range-finding study, the concentrations were limited to 150 µg/plate. Experiments I and II were performed without S9 mix in the test concentration range of 0.5 to 150 µg/plate; Experiments I and II were performed with S9 mix in the test concentration range of 15 to 5000 µg/plate.

Without metabolic activation, diphenyl carbonate was toxic to all four Salmonella strains at dose levels of 0.15 mg/plate and higher. At 0.05 mg/plate, slight toxicity was seen in strains TA 98 and TA 1537. E. coli showed no toxicity at any dose. No bacterial strain demonstrated any mutagenicity in conjunction with the test material. All test plate counts remained within acceptable historical background parameters. 

With metabolic activation, extreme toxicity was seen in all four Salmonella typhimurium strains at 5 mg/plate. In addition, TA1535 and TA1537 also showed increased toxicity to the test chemical at 1.5 mg/plate. Slight toxicity was also observed at 1.5 and 0.5 mg/plate in the Salmonella strains. E. coli was not affected by the test chemical at any dose level. As with the non-activated assay, no mutagenicity was observed at any dose level.

Under the conditions of this study, diphenyl carbonate did not cause a positive response in either the presence or absence of metabolic activation and is therefore non-mutagenic in the Ames test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Limitation: only 4 strains tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 strains tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

exp. I without S9 mix: 1; 3.3; 10; 33.3; 100, 333.3 µg/plate;  with S9 mix: 10; 33.3; 100; 333.3; 1000, 5000 µg/plate;
exp. II without S9 mix: 3.3; 10; 33.3; 100; 333.3, 1000 µg/plate; with S9 mix: 10; 33.3; 100; 333.3; 1000, 5000 µg/plate;

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: as appropriate

Remarks:

Appropriate reference mutagens (sodium azid, 4-NOPD, 2-AA) were used as  positive controls.

Details on test system and experimental conditions:

Salmonella typhimurium reverse mutation assay
Appropriate reference mutagens (sodium azid, 4-NOPD, 2-AA) were used as  positive controls.

Evaluation criteria:

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A significant response is described as: increase in number of revertants by a factor of 2 (TA 100) or 3 (TA 1535, TA 1537, TA 98), respectively, and/or a dose-dependent increase in the number of revertants

Statistics:

no evaluated statistical method available at time of testing

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: without S9-mix: distinct reduction in the number of revertants occurred in all strains (except TA 1537) at 1000 µg/plate. With S9 mix: no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation in the overlay agar at 333.3 µg/plate and higher concentration.

A distinct reduction in the number of revertants occurred in all strains (except TA 1537) at 1000 µg/plate without S9 mix. With S9 mix no cytotoxicity became obvious.Appropriate reference mutagens (sodium azide, 4-NOPD, 2-AA) were used as positive controls and showed the expected results.

Conclusions:

Diphenyl carbonate showed no mutagenic effects in the Ames Test with S. typhimurium TA 1535, TA 5137, TA 98, and TA 100.

Executive summary:

The mutagenic potential of the substance was investigated in a study which was performed in accordance with OECD 471 though only four strains of bacteria were tested. S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were exposed to the test material at concentrations up to 5000 µg/plate in DMSO, both with and without Aroclor induced rat liver S9 metabolic activation.

A distinct reduction in the number of revertants occurred in all strains (except TA 1537) at 1000 µg/plate without S9 mix. With S9 mix no cytotoxicity became obvious. Appropriate reference mutagens (sodium azide, 4-NOPD, 2-AA) were used as positive controls and showed the expected results.

Under the conditions of this study, diphenyl carbonate did not cause a positive response in either the presence or absence of metabolic activation and is therefore non-mutagenic in the Ames test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1984

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver microsomal fraction

Test concentrations with justification for top dose:

Without S9 mix: 3, 5, 10, 30, 50, 70, 100 µg/mL
With S9 mix: 10, 30, 50, 100, 300, 500 µg/mL

Vehicle / solvent:

DMSO (not to exceed 1% final DMSO concentration in  nutrient medium).

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

as appropriate

Positive control substance:

other: see below

Details on test system and experimental conditions:

Positive controls:
without S9: ethylmethanesulfonate 600 µg/ml (4.8 mM)
with S9: cyclophosphamide 0.71 µg/ml (2.5 µM)
The chromosomes were prepared 18 and 28 hours after start of treatment with TS.  The treatment interval was 4 hours with metabolic activation and 18 and 28 hours without metabolic activation.  In each experimental group, two parallel cultures were set up.  Per culture 100 metaphases were scored for structural chromosome aberrations.

Statistics:

Statistical significance at the five per cent level (p<0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence of S9 mix, the mitotic indices were reduced after treatment with the highest evaluated concentration at each preparation interval, whereas in the presence of S9 mix no reduction was observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the TS in the culture medium was observed at concentrations of 300 ug/mL and above.  In the absence of S9 mix, the  mitotic indices were reduced after treatment with the highest evaluated concentration at each preparation interval, whereas in the presence of S9 mix no reduction was observed.  
In the  absence of metabolic activation 30 and 50 ug/mL and in the presence of  metabolic activation 100, 300, and 500 ug/mL of the test article induced  a statistically significant aberration frequency. The laboratory's  historical negative control range of aberrant cells is 0-4% and the  observed aberration frequencies after treatment with the TS are:

                       Exp.I                Exp.II
solvent  (-S9)                  0.0 %                 2.0 %
5 µg/ml  (-S9)                  0.5 %                 1.0 %
30 µg/ml (-S9)                 9.5 %                 9.5 %
50 µg/ml (-S9)                 7.5 %                 -
70 µg/ml (-S9)                  -                  4.5 %
solvent  (+S9)                  2.0 %                 0.0 %
50 µg/ml (+S9)            0.0 %                 -
100 µg/ml (+S9)                 -                  7.5 %
300 µg/ml (+S9)              17.0 %              13.0 %
500 µg/ml (+S9)             15.5 %              21.5 %

Reference mutagens showed the expected results (up to 24.5%  and 20% aberrant cells, respectively).

Conclusions:

In the in vitro chromosome aberration assay diphenyl carbonate led to increased frequencies of structural chromosomal aberrations in V79 cells.

Executive summary:

The genotoxicity of the test material was investigated in a study which was conducted in accordance with OECD 473.

During the study Chinese hamster lung fibroblasts (V79) were exposed to the test material in DMSO both with and without metabolic activation. Without S9 mix the concentrations tested were 3, 5, 10, 30, 50, 70 and 100 µg/mL; with S9 mix, the concentrations tested were 10, 30, 50, 100, 300 and 500 µg/mL.

Precipitation of the test material in the culture medium was observed at concentrations of 300 µg/mL and above. In the absence of S9 mix, the mitotic indices were reduced after treatment with the highest evaluated concentration at each preparation interval, whereas in the presence of S9 mix no reduction was observed. 

In the absence and in the presence of metabolic activation, 30 and 50 µg/mL and 100, 300 and 500 µg/mL of the test material, respectively, induced a statistically significant aberration frequency. The laboratory's historical negative control range of aberrant cells is 0 to 4 %. The observed aberration frequencies after treatment were: 9.5 and 7.5 % at 30 and 50 µg/mL respectively in Experiment I and 9 % at 30 µg/mL in Experiment II in the absence of metabolic activation; 17.0 and 15.5 % at 300 and 500 µg/mL respectively in Experiment I and 7.5, 13 and 21.5 % at 100, 300 and 500 µg/mL respectively in Experiment II in the presence of metabolic activation.

Under the conditions of the study, diphenyl carbonate led to increased frequencies of structural chromosomal aberrations in V79 cells.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver microxomal fraction

Test concentrations with justification for top dose:

Experiment I:
Without S9 mix: 3, 30, 60, 100 µg/mL
With S9 mix: 30, 300, 600, 1000, 2000 µg/mL
Experiment II:
Without S9 mix: 10, 30, 100, 120, 150 µg/mL
With S9 mix: 100, 300, 600, 1000, 2000 µg/mL

Vehicle / solvent:

DMSO. Final DMSO concentration in the medium did not  exceed 1%.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

as appropriate (see below)

Details on test system and experimental conditions:

Positive control substances
Without S9 mix: ethylmethanesulfonate (EMS) 0.6 mg/ml
With S9 mix: 7,12-dimethylbenzo(a)anthracene (DMBA) 3.85 µg/ml)

Evaluation criteria:

A test article is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response for one of the test points.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 60 µg/mL and above without and at 600 µg/mL and above with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Relevant toxic effects occurred at concentrations of 60 ug/mL and above without and at 600 ug/mL and above with metabolic activation.  Visible precipitation of the test article occurred at the two highest concentrations with and without metabolic activation.  The cloning efficiency of the cells was reduced at the maximal concentrations tested without S9 mix. 

No substantial increase in the number of mutant colonies occurred at any  of the evaluated concentrations neither in the presence nor absence of  metabolic activation. There was no indication of a dose dependent  increase in the number of colonies even below the threshold of biological  relevance.  The number of mutant colonies in any of the TS groups with or  without metabolic activation remained well within the range of historical  negative controls.

Conclusions:

Diphenyl carbonate did not induce mutagenic effects in the in vitro gene mutation assay (HPRT test) with Chinese hamster V79 cells.

Executive summary:

The mutagenic potential of the test material was investigated in a study which was conducted in accordance with OECD 476.

During the study Chinese hamster lung fibroblasts (V79) were exposed to the test material in DMSO both with and without S9 activation. Experiments I and II were performed without S9 mix in the test concentration range of 0.5 to 150 µg/plate; Experiments I and II were performed with S9 mix in the test concentration range of 30 to 2000 µg/plate.

Relevant toxic effects occurred at concentrations of 60 and 600 µg/mL and above without and with metabolic activation, respectively. Visible precipitation of the test material occurred at the two highest concentrations with and without metabolic activation. The cloning efficiency of the cells was reduced at the maximal concentrations tested without S9 mix. 

No substantial increase in the number of mutant colonies occurred at any of the evaluated concentrations either in the presence or absence of metabolic activation. There was no indication of a dose dependent increase in the number of colonies even below the threshold of biological relevance. The number of mutant colonies in any of the test material groups with or without metabolic activation remained well within the range of historical negative controls.

Under the conditions of the study, diphenyl carbonate did not induce mutagenic effects in a gene mutation assay (HPRT test) with Chinese hamster V79 cells.

Genetic toxicity in vivo

Description of key information

- Non mutagenic (micronucleus test) in mice, OECD 474, Herbold (1999)

- Equivocal (micronucleus test) in mice, OECD 474, Völkner (2000)

- Non mutagenic (UDS assay) in rats, OECD 486, San (2002)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Hsd/Win: NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

according to Guideline

Route of administration:

intraperitoneal

Vehicle:

TS was suspended in 0.5% aqueous Cremophor emulsion, using a microdismembrator for 5 minutes. The suspensions were stirred with a magnetic mixer during administration. CP was dissolved in  deionized water.
administered volume: 10 mg/kg bw

Details on exposure:

animals were treated intraperitoneally

Duration of treatment / exposure:

animals were treated twice, separated by 24 hours

Frequency of treatment:

diphenyl carbonate was administered twice, whereas the positive control cyclophosphamide was administered only once.

Post exposure period:

24 hours after the last dose the animals were sacrificed.

Dose / conc.:

75 mg/kg bw/day

Remarks:

2 applications separated by 24 hours

Dose / conc.:

150 mg/kg bw/day

Remarks:

2 applications separated by 24 hours

Dose / conc.:

300 mg/kg bw/day

Remarks:

2 applications separated by 24 hours

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide, dissolved in deionized water, single i.p. injection with 20 mg/kg bw

Tissues and cell types examined:

bone marrow smears
No. of cells scored for micronuclei: 2000 polychromatic erythrocytes (PCEs)/animal
PCE/normochromatic erythrocytes (NCE)-ratio determined for 2000 PCEs/animal

Details of tissue and slide preparation:

according to Guideline

Evaluation criteria:

A test was considered positive if there was a relevant and significant increase in the number of micronucleated polychromatic erythrocytes showing micronuclei in comparison to the negative control.

Statistics:

Wilcoxon's non-parametric rank sum test; p<0.001 for significance

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Symptoms of toxicity (e.g. apathy, loss of weight) after administration of >= 2x75 mg/kg bw; no substance-induced mortalities; PCE/NCE ratio reduced in highest dose group

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Symptoms of toxicity (apathy, roughened fur, loss of weight, spasm,  twitching, difficulty in breathing, slitted eyes and closed eyes) after  administration of >= 2x75 mg/kg; no substance-induced mortalities; 
the  ratio between polychromatic and normochromatic erythrocytes was reduced in the highest  dose group by > 30%, indicating cytotoxic effects in the bone marrow; 
No indication of a test substance dependent  clastogenic effect at any diphenyl carbonate dose.
Micronucleated PCEs/2000 PCEs (MNPCE in %)  (mean values of 5 animals in each group):
vehicle control: 3.6 (0.18%)
2 x 75 mg/kg: 2.6 (0.13%)
2 x 150 mg/kg: 2.8 (0.14%)
2 x 300 mg/kg: 5.6 (0.28%)
No statistically significant increase. The highest dose group with the mean of 0.28% MNPCE includes one animal  with an exceptional high number of 16 MNPCE. Since this value is clearly  different from the other findings in this group (1-5 MNPCE), it is  interpreted as outlier. Without this outlier the MNPCE-value for this  group would be 3.0 (0.15%).

The appropriate reference mutagen (cyclophosphamide, single i.p. application of 20 mg/kg bw) was used as positive control and showed the expected results (23.4 MNPCE/ 2000 PCEs). Also vehicle controls showed the expected results.

Conclusions:

In the mouse bone marrow micronucleus test (MNT) with two intraperitoneal injection of diphenyl carbonate in concentrations of 75, 150, and 300 mg/kg bw no indications of a clastogenic effect were found. Relevant systemic exposure was demonstrated by symptoms of toxicity starting at 75 mg/kg bw and an altered PCE/NCE ratio.

Executive summary:

An in vivo micronucleus test was conducted in accordance with OECD 474 and under GLP conditions.

During the study male Hsd/Win: NMRI mice were dosed twice in an intraperitoneal fashion with the test material at 75, 150 and 300 mg/kg bw suspended in 0.5 % aqueous Cremophor emulsion. The doses were administered 24 hours apart. Test material was administered at a dose volume of 10 mg/kg bw. Twenty four hours after the last dose the animals were sacrificed.

Symptoms of toxicity were observed after administration of all dose levels, though no test material-induced mortalities occurred. The ratio between polychromatic and normochromatic erythrocytes was reduced in the highest dose group by > 30 %, indicating cytotoxic effects in the bone marrow. These symptoms demonstrate relevant systemic exposure. No indication of a test material-dependent clastogenic effect was observed at any dose.

Under the conditions of this study, the test material was non-mutagenic in the mouse bone marrow micronucleus test.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Hsd/Win: NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

according to Guideline

Route of administration:

intraperitoneal

Vehicle:

diphenyl carbonate was formulated in corn oil, the positive control cyclophosphamid was formulated in deionized water

Details on exposure:

Administration volume: 10 ml/kg bw

Duration of treatment / exposure:

24 and 48 hours after single dosing the bone marrow cells were collected for micronuclei analysis. 

Frequency of treatment:

once

Post exposure period:

see above

Dose / conc.:

30 mg/kg bw/day

Remarks:

24 hour preparation interval

Dose / conc.:

100 mg/kg bw/day

Remarks:

24 hour preparation interval

Dose / conc.:

300 mg/kg bw/day

Remarks:

24 hour preparation interval

Dose / conc.:

300 mg/kg bw/day

Remarks:

48 hour preparation interval

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide, 40 mg/kg bw

Tissues and cell types examined:

bone marrow smears
No. of cells scored for micronuclei: 2000 polychromatic erythrocytes (PCEs)/animal
PCE/normochromatic erythrocytes (NCE)-ratio determined for 2000 PCEs/animal

Details of tissue and slide preparation:

according to Guideline

Evaluation criteria:

A test was considered positive if there was a either a dose related or significant increase in the number of micronucleated polychromatic erythrocytes showing micronuclei for at least one of the test points.

Statistics:

non-parametric Mann-Whitney test; p < 0.05 for significance

Sex:

male/female

Genotoxicity:

ambiguous

Remarks:

in each of the highest dose groups one animal showed an exceptional high number (15 or 10) MNPCE, which are interpreted as outliers

Toxicity:

yes

Remarks:

PCE/NCE ratio reduced in the highest dose group

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Symptoms of toxicity were present in the pilot experiments at doses of >=  250 mg/kg bw, however, not recorded in the main experiments; no  substance-induced mortalities in the main experiment; the ratio between  polychromatic and normochromatic erythrocytes was reduced in the highest  dose group (48 h preparation interval) by > 40%, indicating cytotoxic effects in the bone marrow; 

Micronucleated PCEs (in %):

vehicle control: 0.065 (range 0-2)

30 mg/kg (24 h): 0.030 (range 0-2)

100 mg/kg (24 h): 0.060 (range 0-3)

300 mg/kg (24 h): 0.190 (range 0-15)

300 mg/kg (48h): 0.190 (range 0-10)

Statistical significance (p < 0.05) at 300 mg/kg (24 and 48 h). However, in each of the highest dose groups one animal showed an exceptional high number of 15 (24h) or 10 (48h) MNPCE. Since these values are clearly different from the other findings in this groups (0-6, 24h; 0-7, 48h), they are interpreted as outliers. In conclusion, the results were considered as to be equivocal.

Conclusions:

Under the conditions of this study, the results were considered as to be equivocal.

Executive summary:

The genotoxicity of the test material was investigated in a study which was conducted in accordance with OECD 474 and under GLP conditions.

Several pilot tests were performed with i.p. administration at doses of 250 to 500 mg/kg bw with 2 male and 2 female mice each. Administration of ≥ 400 mg/kg bw led to the death of females. Throughout all doses males and females showed clinical signs (apathy, eyelid closure) and some females showed temporary tremors. Therefore 300 mg/kg bw was chosen as the MTD for both sexes.

During the study male and female Hsd/Win: NMRI mice were dosed with the test material formulated in corn oil at 30, 100, 300 mg/kg bw. The test material was administrated at 10 mL/kg via the intraperitoneal route. Twenty four (30, 100, 300 mg/kg bw) and 48 hours (300 mg/kg bw) after single dosing, the bone marrow cells were collected for micronuclei analysis.

No symptoms of toxicity were present in the main experiments and no test material-induced mortalities were seen; the ratio between polychromatic and normochromatic erythrocytes was reduced in the highest dose group (48 h preparation interval) by > 40 %, indicating cytotoxic effects in the bone marrow.

Micronucleated PCEs (in %) observed in the vehicle control, 30, 100 and 300 mg/kg dose groups were 0.065 (range 0-2), 0.030 (range 0-2), 0.060 (range 0-3), 0.190 (24 h; range 0-15) and 0.190 (48 h; range 0-10).

There was statistical significance (p < 0.05) at 300 mg/kg (24 and 48 h). However, in each of the highest dose groups, one animal showed an exceptionally high number of 15 (24 h) or 10 (48 h) MNPCE. Since these values are clearly different from the other findings in these groups (0-6 at 24 h; 0-7 at 48 h), they are interpreted as outliers.

Under the conditions of this study, the results were considered as to be equivocal.

Reference 3

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

according to Guideline

Route of administration:

oral: gavage

Vehicle:

vehicle for diphenyl carbonate: 0.5 % carboxymethylcellulose in water
vehicle for postive control substance: deionized water

Details on exposure:

administration volume: 20 ml/kg bw (DMN: 10 ml/kg bw)

Duration of treatment / exposure:

2-4 hours and 12-16 hours

Frequency of treatment:

once

Post exposure period:

not applicable

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males per group
No. of rats/group used for hepatocyte cultures: 3 

Control animals:

yes, concurrent vehicle

Positive control(s):

dimethylnitrosamine 35 mg/kg bw 

Tissues and cell types examined:

rat liver hepatocytes
No. of hepatocytes scored/animal: 150

Details of tissue and slide preparation:

according to Guideline

Evaluation criteria:

Criteria for positive result: A dose-related increase with at least one  dose significantly elevated (at least 5 grains) above the negative  control. 

Statistics:

none

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at 1000 mg/kg bw ataxia and tremors were observed > 4 hours after dosing in all animals

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the main experiment at 1000 mg/kg bw ataxia and tremors were observed > 4 hours after dosing in all animals.

Average net grains: 
-2.0/-2.7 NG at 250 mg/kg bw (2-4/12-16 h)
-1.8/-3.0 NG at 500 mg/kg bw
-2.8/-1.4 NG at 1000 mg/kg bw

Conclusions:

Diphenyl carbonate was tested in the Unscheduled DNA Synthesis (UDS) Test with mammalian liver cells in vivo (OECD 486). Male rats (5 per group) were exposed to 250, 500, and 1000 mg/kg bw. Diphenyl carbonate was concluded to be negative in this test.

Executive summary:

The Unscheduled DNA Synthesis (UDS) Test was conducted with mammalian liver cells in vivo in accordance with OECD 486 and under GLP conditiond. A pilot gavage study was conducted with dose levels of 1, 10, 100, 1000, 1500 and 2000 mg/kg bw. At 1500 and 2000 mg/kg bw 2/5 animals died within two days of dose administration. At 1000 mg/kg bw the two treated animals were lethargic and showed tremors. 1000 mg/kg bw was determined as the MTD.

In the main test, male rats (5 per group) were exposed to the test material at 250, 500 and 1000 mg/kg bw in 0.5 % carboxymethylcellulose in water by gavage at a dose volume of 20 mL/kg bw. Rats were exposed for 2-4 hours and 12-16 hours.

At 1000 mg/kg bw, ataxia and tremors were observed > 4 hours after dosing in all animals. The average net grains were determined at both the 2-4 hour and 12-16 hour intervals. The results at both of these intervals were as follows: -2.0 and -2.7 NG at 250 mg/kg bw, -1.8 and -3.0 NG at 500 mg/kg bw, and -2.8 and -1.4 NG at 1000 mg/kg bw.

Under the conditions of this study, diphenyl carbonate was concluded to be negative in the UDS assay.

Additional information

In Vitro

There are four key studies available to address this endpoint. All were conducted in accordance with standardised guidelines under GLP conditions. All were awarded a reliability score of 1 in accordance with the criteria for assessing data quality as set forth by Klimisch et al. (1997).

 

The first study (Timm, 1989) was performed in accordance with OECD 471 though only four strains of bacteria were tested. S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were exposed to the test material at concentrations up to 5000 µg/plate in DMSO, both with and without Aroclor induced rat liver S9 metabolic activation.

A distinct reduction in the number of revertants occurred in all strains (except TA 1537) at 1000 µg/plate without S9 mix. With S9 mix no cytotoxicity became obvious. Appropriate reference mutagens (sodium azide, 4-NOPD, 2-AA) were used as positive controls and showed the expected results.

Under the conditions of this study, diphenyl carbonate did not cause a positive response in either the presence or absence of metabolic activation and is therefore non-mutagenic in the Ames test.

 

The second key study (Loveday, 1991) was conducted in accordance with OECD 471. S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 were exposed to the test material in DMSO. Due to strong cytotoxicity at 500 µg/plate in a non-activation range-finding study, the concentrations were limited to 150 µg/plate. Experiments I and II were performed without S9 mix in the test concentration range of 0.5 to 150 µg/plate; Experiments I and II were performed with S9 mix in the test concentration range of 15 to 5000 µg/plate.

Without metabolic activation, diphenyl carbonate was toxic to all four Salmonella strains at dose levels of 0.15 mg/plate and higher. At 0.05 mg/plate, slight toxicity was seen in strains TA 98 and TA 1537. E. coli showed no toxicity at any dose. No bacterial strain demonstrated any mutagenicity in conjunction with the test material. All test plate counts remained within acceptable historical background parameters. 

With metabolic activation, extreme toxicity was seen in all four Salmonella typhimurium strains at 5 mg/plate. In addition, TA1535 and TA1537 also showed increased toxicity to the test chemical at 1.5 mg/plate. Slight toxicity was also observed at 1.5 and 0.5 mg/plate in the Salmonella strains. E. coli was not affected by the test chemical at any dose level. As with the non-activated assay, no mutagenicity was observed at any dose level.

Under the conditions of this study, diphenyl carbonate did not cause a positive response in either the presence or absence of metabolic activation and is therefore non-mutagenic in the Ames test.

 

The third key study (Wollny, 1996) was conducted in accordance with OECD 476. Chinese hamster lung fibroblasts (V79) were exposed to the test material in DMSO both with and without S9 activation. Experiments I and II were performed without S9 mix in the test concentration range of 0.5 to 150 µg/plate; Experiments I and II were performed with S9 mix in the test concentration range of 30 to 2000 µg/plate.

Relevant toxic effects occurred at concentrations of 60 and 600 µg/mL and above without and with metabolic activation, respectively. Visible precipitation of the test material occurred at the two highest concentrations with and without metabolic activation. The cloning efficiency of the cells was reduced at the maximal concentrations tested without S9 mix. 

No substantial increase in the number of mutant colonies occurred at any of the evaluated concentrations either in the presence or absence of metabolic activation. There was no indication of a dose dependent increase in the number of colonies even below the threshold of biological relevance. The number of mutant colonies in any of the test material groups with or without metabolic activation remained well within the range of historical negative controls.

Under the conditions of the study, diphenyl carbonate did not induce mutagenic effects in a gene mutation assay (HPRT test) with Chinese hamster V79 cells.

 

The fourth key study (Czich, 1996) was conducted in accordance with OECD 473. Chinese hamster lung fibroblasts (V79) were exposed to the test material in DMSO both with and without metabolic activation. Without S9 mix the concentrations tested were 3, 5, 10, 30, 50, 70 and 100 µg/mL; with S9 mix, the concentrations tested were 10, 30, 50, 100, 300 and 500 µg/mL.

Precipitation of the test material in the culture medium was observed at concentrations of 300 µg/mL and above. In the absence of S9 mix, the mitotic indices were reduced after treatment with the highest evaluated concentration at each preparation interval, whereas in the presence of S9 mix no reduction was observed. 

In the absence and in the presence of metabolic activation, 30 and 50 µg/mL and 100, 300 and 500 µg/mL of the test material, respectively, induced a statistically significant aberration frequency. The laboratory's historical negative control range of aberrant cells is 0 to 4 %. The observed aberration frequencies after treatment were: 9.5 and 7.5 % at 30 and 50 µg/mL respectively in Experiment I and 9 % at 30 µg/mL in Experiment II in the absence of metabolic activation; 17.0 and 15.5 % at 300 and 500 µg/mL respectively in Experiment I and 7.5, 13 and 21.5 % at 100, 300 and 500 µg/mL respectively in Experiment II in the presence of metabolic activation.

Under the conditions of the study, diphenyl carbonate led to increased frequencies of structural chromosomal aberrations in V79 cells.

 

In Vivo

There are three key studies to address this endpoint. All studies were conducted in accordance with standardised guidelines under GLP conditions. All three studies have been awarded a reliability score of 1 in accordance with the criteria for assessing data quality as set forth by Klimisch et al. (1997).

 

The first key study (Herbold, 1999) was conducted in accordance with OECD 474. Male Hsd/Win: NMRI mice were dosed twice in an intraperitoneal fashion with the test material at 75, 150 and 300 mg/kg bw suspended in 0.5 % aqueous Cremophor emulsion. The doses were administered 24 hours apart. Test material was administered at a dose volume of 10 mg/kg bw. Twenty four hours after the last dose the animals were sacrificed.

Symptoms of toxicity were observed after administration of all dose levels, though no test material-induced mortalities occurred. The ratio between polychromatic and normochromatic erythrocytes was reduced in the highest dose group by > 30 %, indicating cytotoxic effects in the bone marrow. These symptoms demonstrate relevant systemic exposure. No indication of a test material-dependent clastogenic effect was observed at any dose.

Under the conditions of this study, the test material was non-mutagenic in the mouse bone marrow micronucleus test.

 

The second key study (Völkner, 2000) was also conducted in accordance with OECD 474. Several pilot tests were performed with i.p. administration at doses of 250 to 500 mg/kg bw with 2 male and 2 female mice each. Administration of ≥ 400 mg/kg bw led to the death of females. Throughout all doses males and females showed clinical signs (apathy, eyelid closure) and some females showed temporary tremors. Therefore 300 mg/kg bw was chosen as the MTD for both sexes.

Male and female Hsd/Win: NMRI mice were dosed with the test material formulated in corn oil at 30, 100, 300 mg/kg bw. The test material was administrated at 10 mL/kg via the intraperitoneal route. Twenty four (30, 100, 300 mg/kg bw) and 48 hours (300 mg/kg bw) after single dosing, the bone marrow cells were collected for micronuclei analysis.

No symptoms of toxicity were present in the main experiments and no test material-induced mortalities were seen; the ratio between polychromatic and normochromatic erythrocytes was reduced in the highest dose group (48 h preparation interval) by > 40 %, indicating cytotoxic effects in the bone marrow.

Micronucleated PCEs (in %) observed in the vehicle control, 30, 100 and 300 mg/kg dose groups were 0.065 (range 0-2), 0.030 (range 0-2), 0.060 (range 0-3), 0.190 (24 h; range 0-15) and 0.190 (48 h; range 0-10).

There was statistical significance (p < 0.05) at 300 mg/kg (24 and 48 h). However, in each of the highest dose groups, one animal showed an exceptionally high number of 15 (24 h) or 10 (48 h) MNPCE. Since these values are clearly different from the other findings in these groups (0-6 at 24 h; 0-7 at 48 h), they are interpreted as outliers.

Under the conditions of this study, the results were considered as to be equivocal.

 

In third key study (San, 2002) diphenyl carbonate was tested in the Unscheduled DNA Synthesis (UDS) Test with mammalian liver cells in vivo in accordance with OECD 486. A pilot gavage study was conducted with dose levels of 1, 10, 100, 1000, 1500 and 2000 mg/kg bw. At 1500 and 2000 mg/kg bw 2/5 animals died within two days of dose administration. At 1000 mg/kg bw the two treated animals were lethargic and showed tremors. 1000 mg/kg bw was determined as the MTD.

In the main test, male rats (5 per group) were exposed to the test material at 250, 500 and 1000 mg/kg bw in 0.5 % carboxymethylcellulose in water by gavage at a dose volume of 20 mL/kg bw. Rats were exposed for 2-4 hours and 12-16 hours.

At 1000 mg/kg bw, ataxia and tremors were observed > 4 hours after dosing in all animals. The average net grains were determined at both the 2-4 hour and 12-16 hour intervals. The results at both of these intervals were as follows: -2.0 and -2.7 NG at 250 mg/kg bw, -1.8 and -3.0 NG at 500 mg/kg bw, and -2.8 and -1.4 NG at 1000 mg/kg bw.

Under the conditions of this study, diphenyl carbonate was concluded to be negative in the UDS assay.

 

Discussion

Diphenyl carbonate showed no mutagenic properties in two independently performed bacterial reverse mutation assays with multiple strains of Salmonella typhimurium and E. coli WP2, nor did it show mutagenic properties in a mammalian cell gene mutation assay using Chinese hamster V79 cells.

In the in vitro chromosome aberration assay diphenyl carbonate led to increased frequencies of structural chromosomal aberrations in V79 cells both in the absence and in the presence of metabolic activation. This positive result could not be confirmed in vivo by two mouse bone marrow micronucleus assays performed with evidence of target cell exposure. An in vivo/in vitro UDS assay on rat liver with oral doses up to the MTD gave negative results.

It is therefore concluded that the genotoxic properties observed in vitro are not expressed in vivo.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to mutagenicity.