2-imino-1-methylimidazolidin-4-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1987

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study does not report the cytotoxicity observed in each test culture. Without a cytotoxicity measure for each test culture, it is impossible to determine whether the observed apparent positive response would be considered positive (biologically relevant) by current standards. That is, the concentration yielding the positive response might have caused excessive cytotoxicity - beyond that currently considered acceptable. Furthermore the concentrations are higher than those that are recommended by current test guidelines. For example OECD 473 in its current version recommends 10 mM or 2 mg/mL as highest test concentration. In the present study 5, 10 and 15 mg/L were chosen as concentrations. This means that also the lowest concentration of 5 mg/L is many times higher than the highest concentration recommended by OECD 473 (5 mg/L corresponds to 44 mM Creatinine, which is 4 times higher than recommended). Also at this concentration no significant effects were observed. At concentrations of 10 mg/L and above osmotic stress is also to be expected but not fully assessed within the report. Additionally, there was no information provided regarding test material details (purity, impurities), there is no reported positive control, there are no historical control data for the solvent “medium” and the test was not performed under GLP. Above and beyond all these aspects of inadequate reporting and interpretation, as well as concentration many times over the recommendation of todays Guidelines, which are located in the range of cytotoxicity and osmotic stress, Creatinine is an endogenous substance. Creatinine is a breakdown product of Creatine in muscle and is usually produced at a fairly constant rate by the body (depending on muscle mass). According to “Wissenschaftliche Tabellen, 7. Auflage, 1969, J.R. Geigy S.A., Basel, Schweiz” considerable amounts of Creatinine can be found in the different compartments and body fluids: Cord blood plasm or serum: 11.8 mg/L (6.4 – 17.2 mg/L) Blood plasm or serum, child (4-21 weeks): 9.5 mg/L (7.9 – 11.1 mg/L) Blood plasm or serum, child (1-6 years): 11.9 mg/L (7.5 -16.3 mg/L) Blood plasm or serum, adult:12.4 mg/L (6.6 – 18.2 mg/L) Liquor cerebrospinalis: 6-14 mg/L Gastric juice: 12-33 mg/L Urine, male (20-45 years): 1.8 (1.2 - 2.5 g/24h) Urine, female (20-45 years): 1.17 (0.01 - 1.33 g/24h) Sweat: 4.6 mg/L (2.1 - 8.4 mg/L) Mother’s milk: 11 mg/L (8 - 19 mg/L) Furthermore Creatinine is part of the diet and can be found in lean meat in constant content of 0.02 – 0.04 % (G.Mayer, H.Knapp, RÖMPP, Thieme Verlag, 2009).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

OECD TG 473 was adopted in 1983 and may have been available at the time of the test. It is unclear whether the test was conducted prior to or after 1983; for deviations see details on materials and methods below, and applicant's summary and conclusions.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese Hamster Lung Fibroblasts (CHL)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's MEM + 10% calf serum

Additional strain / cell type characteristics:

other: 25 chromosomes ( Chinese Hamsters normally have 22 chromosomes)

Metabolic activation:

without

Test concentrations with justification for top dose:

5, 10, and 15 mM

Vehicle / solvent:

Cell culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

cell culture medium

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. This experiment was only conducted without S9 mix.

DURATION:
The test agent was added and allowed to remain in the cultures until chromosome preparations were made 24 and 48 hours after the start of the treatment.

NUMBER OF CELLS EVALUATED:
44x10^3/ml cells were seeded in a 6cm plastic petri dish with 5ml culture medium and allowed to grow for 3 days. It is unclear how many cells were treated with test chemical. 100 metaphases per test concentration were analyzed.

DETERMINATION OF CYTOTOXICITY
- Method: In a preliminary experiment, test agent was added to a 3 day old monolayer culture and allowed to grow for 2 days. Medium was removed, cells were stained and an estimate of the test agent cell inhibition was determined microscopically based on the amount of cell staining. This approach was used for selecting test concentrations in the full experiment. There was no individual culture measure of cytotoxicity in the full experiment.

OTHER EXAMINATIONS:
- One hundred well spread metaphases were evaluated for structural chromosome and chromatid gaps, breaks and exchanges. These along with fragments were enumerated as a percentage of cells containing the particular event. Determination of polyploidy: only the incidence of obvious polyploidy cells per 100 metaphases was recorded.

OTHER:
- Colcemid was added to all cultures 2 hours before harvest to arrest cells in metaphase
- Cells were harvested by trypsinization, fixed, cell pellets dropped onto slides, metaphase spreads were stained and scored microscopically.

Evaluation criteria:

Negative: < 5 %
Inconclusive: 5 to <10 %
Positive: ≥ 10 %

Statistics:

While statistics were not used to evaluate whether a response was positive or negative two different types of potency values were calculated for any chemical giving a positive response. D20 value was calculated, which is the concentration required to induce any aberration in 20 % of metaphases. TR value was calculated, which indicates the incidence of aberrant cells with chromatid exchange per unit concentration.

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

The result was determined positive; however, because there is no cytotoxicity measure for the individual test cultures, it is impossible to confirm that the response is biologically relevant and would be considered positive by today’s standards.

Cytotoxicity / choice of top concentrations:

not determined

Remarks:

In a preliminary experiment, the laboratory used a method to provide an estimate of cytotoxicity to establish the test concentrations. There is no estimate in the full experiment of individual test culture cytotoxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Creatinine at the 24 hour exposure gave 43 % of the cells containing aberrations at the 10 mM concentration. It should be noted that this includes 10 % cells with gaps (which are not normally included when scoring aberrations) and after 48 hours of exposure, gave 10 % of the cells with aberrations at the 10mM concentration.

Remarks on result:

other: strain/cell type: Chinese Hamster Lung Fibroblasts (CHL)

Remarks:

Migrated from field 'Test system'.

Creatinine (CAS No 60-27-5)
Solvent: Medium
Time (h)	Concentration (mg/mL)	Number of metaphases	Polyploidy (%)	Judge	Cells with structural chromosome aberrations (%)
					CTG	CTB	CTE	FRG	CSB	CSE	Total	Judge
24	None	100	0		0	0	0	0	0	0	0
24	5	100	1	–	1	1	2	0	0	0	4	–
24	10	100	1	–	10	32	15	0	0	0	43	+
24	15	–	–	TOX	–	–	–	–	–	–	–	TOX
48	5	100	3	–	1	0	1	0	0	0	2	–
48	10	100	17	+	1	6	7	0	0	0	10	+
48	15	–	–	TOX	–	–	–	–	–	–	–	TOX

 

Conclusions:

Interpretation of results (migrated information):
ambiguous without metabolic activation Because there is no concurrent measurement of test culture cytotoxicity, it’s impossible to reach a conclusion of creatinine’s clastogenicity. Also, the test was only conducted without S9, which provides no information as to whether S9 would alter results

Overall, it is not possible to make a firm conclusion as to whether creatinine is or is not clastogenic. This is based on the fact that there is no concurrent individual cytotoxicity measure which would provide information as to whether the test concentration used would be considered appropriate by today’s standards. It is now recognized that excess cytotoxicity can give positive results that are not biologically relevant.

Executive summary:

In a mammalian cell chromosome aberration assay, Chinese hamster lung fibroblast cultures (CHL cells) were exposed to creatinine at concentrations of 0, 5, 10, and 15 mg/mL without exogenous metabolic activation. While test concentrations were selected based on a preliminary study to access relative cell growth inhibition in the monolayer cultures, there were no cytotoxicity measures associated with the specific test cultures in the full experiment. Exposure was for 24 and 48 hours. Two hours before harvest, colcemid was added to block cells in metaphase, cells were harvested, fixed, slides prepared and stained. 100 well spread metaphases were evaluated and the percentage of cells containing chromosome or chromatid gaps, breaks and rearrangements enumerated. The 15 mg/mL cultures were not evaluated due to severe toxicity (likely no metaphases available to score). The 10 mg/mL cultures, both the 24 and 48 hour exposures, were reported by the test laboratory to be positive.

However this study does not report the cytotoxicity observed in each test culture. Without a cytotoxicity measure for each test culture, it is impossible to determine whether the observed apparent positive response would be considered positive (biologically relevant) by current standards. That is, the concentration yielding the positive response might have caused excessive cytotoxicity - beyond that currently considered acceptable.

Furthermore the concentrations are higher than those that are recommended by current test guidelines. For example OECD 473 in its current version recommends 10 mM or 2 mg/mL as highest test concentration. In the present study 5, 10 and 15 mg/L were chosen as concentrations. This means that also the lowest concentration of 5 mg/L is many times higher than the highest concentration recommended by OECD 473 (5 mg/L corresponds to 44 mM Creatinine, which is 4 times higher than recommended). Also at this concentration no significant effects were observed.

At concentrations of 10 mg/L and above osmotic stress is also to be expected but not fully assessed within the report. Additionally, there was no information provided regarding test material details (purity, impurities), there is no reported positive control, there are no historical control data for the solvent “medium” and the test was not performed under GLP.

 

Above and beyond all these aspects of inadequate reporting and interpretation, as well as concentration many times over the recommendation of todays Guidelines, which are located in the range of cytotoxicity and osmotic stress, Creatinine is an endogenous substance.

Creatinine is a breakdown product of Creatine in muscle and is usually produced at a fairly constant rate by the body (depending on muscle mass). According to “Wissenschaftliche Tabellen, 7. Auflage, 1969, J.R. Geigy S.A., Basel, Schweiz” considerable amounts of Creatinine can be found in the different compartments and body fluids:

Cord blood plasm or serum: 11.8 mg/L (6.4 – 17.2 mg/L)

Blood plasm or serum, child (4-21 weeks): 9.5 mg/L (7.9 – 11.1 mg/L)

Blood plasm or serum, child (1-6 years): 11.9 mg/L (7.5 -16.3 mg/L)

Blood plasm or serum, adult:12.4 mg/L (6.6 – 18.2 mg/L)

Liquor cerebrospinalis: 6-14 mg/L

Gastric juice: 12-33 mg/L

Urine, male (20-45 years): 1.8 (1.2- 2.5 g/24h)

Urine, female (20-45 years): 1.17 (0.01-1.33 g/24h)

Sweat: 4.6 mg/L (2.1-8.4 mg/L)

Mother’s milk: 11 mg/L (8-19 mg/L)

Furthermore Creatinine is part of the diet and can be found in lean meat in constant content of 0.02 – 0.04 % (G.Mayer, H.Knapp, RÖMPP, Thieme Verlag, 2009).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08.05. - 09.07.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Gene involved in histidine synthesis.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item Creatinine was dissolved in deionised water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

During the described mutagenicity test and under the experimental conditions reported, Creatinine did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Creatinine is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

A study was performed to investigate the potential of Creatinine to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using th eSalmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation according to the OECD Guideline 471 ("Bacterial Reverse Mutation Test"). Each concentration, including the positive and negative controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                               33; 100; 333; 1000; 2500; and 5000 µg/plate

No precipitation of the test item occured up to the highest invested dose. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

 

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Creatinine at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Creatinine is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04.09. - 06.09.2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: DIN 38415 part 4

Deviations:

yes

Remarks:

only two strains of Salmonella typhimurium were used.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

gene involved in histidine synthesis

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

n. a.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

n. a.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal S9 Mix

Test concentrations with justification for top dose:

0.05, 0.15, 0.5, 1.5, 5 mg/plate

Vehicle / solvent:

none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-nitro-1,2-phenylendiamin

Details on test system and experimental conditions:

Testmedium: Mininmal-Glucose-Agar, Top-Agar
Incubation temperature: 37 +/- 1 °C
Test duration: 48 h
Number of replications: 4 replicates per each strain, 4 replicates for the negative control and 4 replicates for each positive control
Titer determination: TA98 > 1.000.000.000 Bact./mL; TA100 1.700.000.000 Bact./mL

Evaluation criteria:

A test substance is regarded as mutagenic if it induces in at least one of the two used strains, with our without S9 mix, an increase of the mutant number at least twice of the negative controll (Induction rate I >= 2) which shows an dose-response relationship.
Furthermore in each case in strain TA 100 an increase (Induction difference ID) of at least 100 mutants/plate in the mean and an increase of at least 30 mutants/plate in the mean for TA98 has to be obtained.

Statistics:

no data

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Parameter	Value
Induction rate I	TA98: at all concentrations used < 2  TA100: at all concentrations used < 2
Dose response relationship	Inexistent
Induction difference ID	TA98: at all concentrations used < 2  TA100: at all concentrations used < 2

Conclusions:

Interpretation of results (migrated information):
negative

Creatinine has no mutagenic potential under the conditions of this study.

Executive summary:

The test item Creatinine was investigated for its potential to induce gene mutations in a test according to DIN 38415 part 4 using two strains of Salmonella typhimurium (T98 and T100).

No substantial increases in revertant colony numbers of any of the two tester strains were observed following treatment with Creatinine at any concentration level, eighter in the presence or absence of metabolic activation (S9 -mix).

Therefore Creatinine is not considered to be mutagenic in bacterial cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Creatinine was tested negative for genotoxic effects in two Ames-Tests according to OECD 471 and DIN 38415 part 4, respectively. Concentrations up to the limit dose were used. No cytotoxic effects were observed.

Also one positive test on chromosome aberration is available for Creatinine. However this study does not report the cytotoxicity observed in each test culture. Without a cytotoxicity measure for each test culture, it is impossible to determine whether the observed apparent positive response would be considered positive (biologically relevant) by current standards. That is, the concentration yielding the positive response might have caused excessive cytotoxicity - beyond that currently considered acceptable.

Furthermore the concentrations are higher than those that are recommended by current test guidelines. For example OECD 473 in its current version recommends 10 mM or 2 mg/mL as highest test concentration. In the present study 5, 10 and 15 mg/L were chosen as concentrations. This means that also the lowest concentration of 5 mg/L is many times higher than the highest concentration recommended by OECD 473 (5 mg/L corresponds to 44 mM Creatinine, which is 4 times higher than recommended). Also at this concentration no significant effects were observed.

At concentrations of 10 mg/L and above osmotic stress is also to be expected but not fully assessed within the report. Additionally, there was no information provided regarding test material details (purity, impurities), there is no reported positive control, there are no historical control data for the solvent “medium” and the test was not performed under GLP.

 

Above and beyond all these aspects of inadequate reporting and interpretation, as well as concentration many times over the recommendation of todays Guidelines, which are located in the range of cytotoxicity and osmotic stress, Creatinine is an endogenous substance.

Creatinine is a breakdown product of Creatine in muscle and is usually produced at a fairly constant rate by the body (depending on muscle mass). According to “Wissenschaftliche Tabellen, 7. Auflage, 1969, J.R. Geigy S.A., Basel, Schweiz” considerable amounts of Creatinine can be found in the different compartments and body fluids:

Cord blood plasm or serum: 11.8 mg/L (6.4 – 17.2 mg/L)

Blood plasm or serum, child (4-21 weeks): 9.5 mg/L (7.9 – 11.1 mg/L)

Blood plasm or serum, child (1-6 years): 11.9 mg/L (7.5 -16.3 mg/L)

Blood plasm or serum, adult:12.4 mg/L (6.6 – 18.2 mg/L)

Liquor cerebrospinalis: 6 - 14 mg/L

Gastric juice: 12 - 33 mg/L

Urine, male (20-45 years): 1.8 (1.2- 2.5 g/24h)

Urine, female (20-45 years): 1.17 (0.01 -1.33 g/24h)

Sweat: 4.6 mg/L (2.1 - 8.4 mg/L)

Mother’s milk: 11 mg/L (8 - 19 mg/L)

Furthermore Creatinine is part of the diet and can be found in lean meat in constant content of 0.02 – 0.04% (G.Mayer, H.Knapp, RÖMPP, Thieme Verlag, 2009).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Creatinine was tested negative for genotoxic effects in two Ames-Tests according to OECD 471 and DIN 38415 part 4, respectively. Concentrations up to the limit dose were used. No cytotoxic effects were observed.

Also one positive test on chromosme aberration is available for Creatinine. However this study does not report the cytotoxicity observed in each test culture. Furthermore there was no information provided regarding test material details, there is no reported positive control, there are no historical control data for the solvent used and the test was not performed under GLP. Above and beyond all these aspects Creatinine is an endogenous substance and is also part of the diet (see under "Discussion").

Therefore Creatinine does not have to be classified.