Calcium acetylide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In aqueous solution, calcium carbide rapidly decomposes to calcium hydroxide and acetylene. Testing of calcium carbide in in vitro assays is technically not feasible and no studies for the endpoint genetic toxicity in vitro were available for the target substance calcium carbide. Instead, read-across to existing data on the reaction products calcium hydroxide/oxide and acetylene were to assess the genotoxic potential of calcium carbide.

Acetylene did not induce any mutagenic effects on S. typhymurium when tested under the conditions of this test, both with and without metabolic activation. However, due to technical difficulties for the generation of higher concentrations, concentration levels were only up to 31 μg/plate.

In addition, acetylene did not lead to a toxicologically significant increase in the mutation frequency with or without metabolic activation in a gene mutation assay in mammalian cells conducted according to OECD 490.

The other reaction product, calcium hydroxide, did not contribute to DNA damage in the COMET assay in different cellular models. In addition, calcium hydroxide was tested negative in a chromosomal aberration test up to 300 μM with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

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:

As calcium carbide instantly hydrolyses to Ca(OH)2 and C2H2 upon contact with water/moisture the assessment is based on the degradation products. For details please refer to the read across report in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Remarks:

Due to technical difficulties: it is difficult to get high test concentrations of the gas acetylene in liquid suspension.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Remarks:

Due to technical difficulties: it is difficult to get high test concentrations of the gas acetylene in liquid suspension.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Remarks:

Due to technical difficulties: it is difficult to get high test concentrations of the gas acetylene in liquid suspension.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Due to technical difficulties (getting high test concentrations of the gas acetylene in liquid suspension), acetylene was not tested for its toxic potential. The solubility of the gas in the test solvent limited the highest test concentration applied. The highest concentration applied was only 31 μg/plate. The raw data are presented in the attached pdf document.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results
negative

Acetylene did not induce any mutagenic effects on S.typhymurium strains TA97, TA98 and TA100, when tested under the conditions of this test, both with and without metabolic activation. However, due to technical difficulties for the generation of higher concentrations, concentration levels were only up to 31 μg/plate.

Executive summary:

The main objective of this study was to validate an appropriate method in order to test vapor-phase compounds in the Ames mutagenicity test. Several parameters were investigated and 12 vapor phase substances, including acetylene, were tested in the Ames test in order to validate the protocol. S. typhimurium strains TA97, TA98 and TA100 were exposed to acetylene, in acetone at concentrations of 0.30, 1.00, 3.00, 10.00 and 31.00 μg/plate in the presence and absence of mammalian metabolic activation (Aroclor-induced hamster and rat liver S9) in the pre-incubation method. The response was considered positive when at least a two-fold increase in spontaneous revertants over background at two increasing dose levels was detected. Acetylene could not be tested up to cytotoxic concentrations since no cytotoxicity screening was performed, due to difficulties of preparing the sample. Due to the difficulty of getting high test concentrations on the gas acetylene in liquid suspension, the highest concentration tested was only 31.00 μg/plate. The results revealed no mutagenicity response. The positive controls induced the appropriate responses in the corresponding strains. The authors of the study report suggested that additional experiments shall be performed with some of the vapors, including acetylene, in order to be possible to test higher concentration levels.

 

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 2

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:

As calcium carbide instantly hydrolyses to Ca(OH)2 and C2H2 upon contact with water/moisture the assessment is based on the degradation products. For details please refer to the read across report in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: cultured human dental pulp cells (D824)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No increases in chromosomal aberrations were seen in cells treated with calcium hydroxide, at all concentrations levels, with and without metabolic activation and at both exposure duration (3 h and 30 h). Calcium hydroxide did not increase the percentage of cells with polyploidy and endoreduplication.The results can be seen in more detail in the tables below.

Remarks on result:

other: all strains/cell types tested

Table 1: Chromosome aberrations in D824 cells induced by treatment with varying concentrations of Ca(OH)2 for 3 h, without metabolic activation

3 h exposure time without metabolic activation
				Type of structural aberrations (%)
	Concentration	Relative cell No(%)	No of metaphases scored	G	B	E	D	O	F	Aberrant metaphases (%)	Polyploidy & endoreduplication (%)
Control	0	100	500	0.8	0	0	0	0	0	0.8	2.8
Ca(OH)2	30	100	100	1	0	0	0	0	0	1.0	6.0
	100	110	200	1.5	0	0	0	0	0	1.5	4.5
	300	96	100	2	0	0	0	0	0	2.0	6.0

Table 2: Chromosome aberrations in D824 cells induced by treatment with varying concentrations of Ca(OH)2 for 30 h, without metabolic activation

30 h exposure time without metabolic activation
				Type of structural aberrations (%)
	Concentration (µM)	Relative cell No(%)	No of metaphases scored	G	B	E	D	O	F	Aberrant metaphases (%)	Polyploidy & endoreduplication (%)
Control	0	100	500	1	0	0	0	0	0	1.0	4.8
Ca(OH)2	30	73	100	0	0	0	0	0	0	0	1.5
	100	67	100	1	0	0	0	0	0	1.0	1.0
	300	66	100	1	0	0	0	0	0	1.0	2.0

Table 3: Chromosome aberrations in D824 cells induced by treatment with varying concentrations of Ca(OH)2 for 3 h, with metabolic activation

3 h exposure time with metabolic activation
					Type of structural aberrations (%)
	Concentration (µM)	PMS	Relative cell No(%)	No of metaphases scored	G	B	E	D	O	F	Aberrant metaphases (%)	Polyploidy & endoreduplication (%)
Control	0	0	100	100	0	0	0	0	0	0	0	2.0
Ca(OH)2	100	5	88	100	1	0	0	0	0	0	1.0	1.0
	300	5	85	100	1	0	0	0	0	0	1.0	0
Cyclophosphamide	50	0	61	100	1	0	0	0	0	0	1.0	1.0
	50	5	40	100	19	9	5	0	0	0	31.0	0

G: Gaps, B: Breaks, E: Exchanges, D: Dicentric chromosomes, O: Ring chromosomes, F: Fragmentations

Conclusions:

Interpretation of results
negative

Ca(OH)2 does not induce chromosomal aberrations in cultured human dental pulp cells (D824), after exposure of 3h with and without metabolic activation, and 30 h without metabolic activation.

Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration), cultured human dental pulp cells (D824) were exposed to several antiseptic agents used in root canal procedure, including calcium hydroxide. The cells were exposed to calcium hydroxide in glycerol at concentrations of 0, 30, 100, 300 µM with and without metabolic activation. Calcium hydroxide was tested up to cytotoxic concentrations. Solvent controls did not induce any positive response. Positive controls induced the appropriate response. There was no evidence or a concentration related positive response of chromosomal aberrations induced by exposure to calcium hydroxide.

 

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

As calcium carbide instantly hydrolyses to Ca(OH)2 and C2H2 upon contact with water/moisture the assessment is based on the degradation products. For details please refer to the read across report in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

No DNA breakage was detected after a treatment of peripheral lymphocytes by formocresol, paramonochlorophenol, or calcium hydroxide at 100 μg/mL.

Conclusions:

Interpretation of results:
negative

Exposure to calcium hydroxide may not be a factor that increases the level of DNA lesions in human peripheral lymphocytes as detected by single-cell gel (comet) assay.

Executive summary:

In a mammalian cell single-cell gel electrophoresis assay (comet assay), a total of 10 μL of calcium hydroxide at 100 μg/mL was added to human peripheral lymphocytes from 10 volunteers for 1 hour at 37 °C. The negative control group was treated with vehicle control (PBS) for 1 hour at 37 °C, as well. For the positive control group, lymphocytes were exposed to hydrogen peroxide at 100 μM during 5 minutes on ice.

No DNA breakage was detected after treatment of peripheral lymphocytes with calcium hydroxide at 100 μg/mL.

Exposure to calcium hydroxide may not be a factor that increases the level of DNA lesions in human peripheral lymphocytes as detected by single-cell gel (comet) assay.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

As calcium carbide instantly hydrolyses to Ca(OH)2 and C2H2 upon contact with water/moisture the assessment is based on the degradation products. For details please refer to the read across report in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

other: human fibroblasts cells

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

The results showed that all compounds tested did not cause DNA damage for the tail moment or tail intensity parameters. These findings suggest that formocresol, paramonochlorophenol, and calcium hydroxide do not promote DNA damage in mammalian cells and that the comet assay is a suitable tool to investigate genotoxicity.

Conclusions:

Interpretation of results:
negative

Calcium hydroxide did not cause DNA damage for the tail moment or tail intensity parameters. These findings suggest that calcium hydroxide do not promote DNA damage in mammalian cells and that the comet assay is a suitable tool to investigate genotoxicity.

Executive summary:

In a mammalian cell single-cell gel electrophoresis assay (comet assay), mouse lymphoma L5178Y cells and human fibroblasts were treated with calcium hydroxide

Exposure to calcium hydroxide did not cause DNA damage for the tail moment or tail intensity parameters. These findings suggest that calcium hydroxide does not promote DNA damage in mammalian cells and that the comet assay is a suitable tool to investigate genotoxicity.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

As calcium carbide instantly hydrolyses to Ca(OH)2 and C2H2 upon contact with water/moisture the assessment is based on the degradation products. For details please refer to the read across report in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

In the current study, the potential DNA damage associated with exposure to a number of antimicrobial endodontic compounds was assessed by the single cell gel (comet) assay in vitro. Formocresol, paramonochlorophenol, and calcium hydroxide, as well as chlorhexidine in all concentrations tested did not contribute to the DNA damage.

Conclusions:

Interpretation of results:
negative

Calcium hydroxide did not contribute to DNA damage in the COMET assay.

Executive summary:

In a mammalian cell single-cell gel electrophoresis assay (comet assay), Chinese hamster ovary (CHO) cells were exposed to calcium hydroxide at a final concentration ranging from 0.01% to 1%.

Calcium hydroxide did not contribute to DNA damage in the comet assay.

Based on the results of this study, there is no evidence for a genotoxic potential of calcium hydroxide.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 6

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:

As calcium carbide instantly hydrolyses to Ca(OH)2 and C2H2 upon contact with water/moisture the assessment is based on the degradation products. For details please refer to the read across report in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Based on the preliminary toxicity test, the maximum dose level in the Mutagenicity Test was set at 50% acetylene in air.

GENE MUTATION IN MAMMALIAN CELLS:
- Results from cytotoxicity measurements: As seen in the preliminary toxicity test, there was no evidence of toxicity following exposure to the test material in the presence of metabolic activation, as indicated by the %RSG and RTG values in both experiments (see Table 1 & 2 in box " Any other information on results incl. tables").
- Mutant frequency: The test material did not induce any statistically significant or dose-related increases in the mutant frequency x 10^-6 per viable cells in both experiments with and without metabolic activation. The GEF was not exceeded at any dose level and the mutant frequency values observed were in the acceptable range for vehicle controls (see Table 1 & 2 in box " Any other information on results incl. tables").

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle control mutant frequency values were within the acceptable range of historical control data. The positive controls produced marked incresaes in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

Table 1: Summary of Results from Experiment 1

Treatment (% Gas)		4-hours -S9				Treatment (% Gas)		4 -hours +S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	147.69		0		100	1.00	80.06
0.31		99	1.03	160.81		0.31		105	1.32	56.17
0.63		87	0.98	188.04		0.63		112	1.22	94.01
1.25		89	0.98	193.16		1.25		117	1.46	69.99
2.5		91	0.88	208.90		2.5		111	1.35	69.97
5		83	0.88	133.80		5		104	1.26	123.92
12.5		94	1.00	131.52		12.5		105	1.54	90.22
25		94	1.02	183.09		25		93	1.25	103.85
50		92	1.03	128.84		50		101	1.29	125.10
Linear trend				NS		Linear trend				**
EMS						CP
400 µg/mL		65	0.52	1308.82		2 µg/mL		59	0.29	2319.71

Table 2: Summary of Results from Experiment 2

Treatment (% Gas)		24-hours -S-9				Treatment		4-hours +S-9
						(% Gas)
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	68.36		0		100	1.00	88.17
0.31		119	1.10	91.53		0.31		91	1.10	80.11
0.63		130	1.42	72.61		0.63		102	1.25	77.86
1.25		117	1.03	70.17		1.25		106	1.14	61.45
2.5		127	1.09	72.00		2.5		100	1.05	79.45
5		115	1.06	75.15		5		103	1.17	76.50
12.5		108	1.01	98.11		12.5		93	1.04	92.51
25		85	0.87	99.30		25		103	1.27	107.67
50		83	0.82	104.44		50		117	1.37	78.42
Linear trend				*		Linear trend				NS
EMS						CP
150 µg/mL		59	0.29	1289.77		2 µg/mL		62	0.27	645.07

MF§                5‑TFT resistant mutants/10^6 viable cells 2 days after treatment

%RSG            Relative Suspension Growth

RTG                Relative Total Growth

$$                   Evidence of heterogeneity (poor correlation between A and B viability plates)

NS                  Not significant

*                     Significant at p< 0.05

**                    nSignificant at p< 0.01

Conclusions:

In conclusion, the test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5187Y cells and is therefore considered to be non-mutagenic.

Executive summary:

In a mammalian cell gene mutation assay at the TK +/- locus, L5187Y cells cultured in vitro were exposed to acetylene (in air) at concentrations of 0, 0.31, 0.63, 1.25, 2.5, 5, 12.5, 25 and 50% in the presence and absence of mammalian metabolic activation. In experiment 1, the test material did not induce any statistically significant or dose-related increases in the mutant frequency in the absence of metabolic activation after the 4 hour treatment period. In the presence of metabolic activation, a modest dose-related (linear trend) statistically significant response was observed after 4 hours of treatment, which did not reach statistical significance and did not exceed the GEF on an individual level. This response could not be reproduced in experiment 2 and was therefore considered to be of no toxicological significance.

In experiment 2, a very modest dose-related (linear trend) statistically significant response was observed after 24 hours in the absence of metabolic activation. Statistically significant increases in mutant frequency were not observed on any individual dose level and the GEF was also not exceeded at any dose level. Mutant frequency values were in the acceptable range for vehicle controls. Therefore, the response was considered to be of no toxicological significance. The positive controls did induce the appropriate response. Based on the results, the test item can be considered as non-mutagenic under the conditions of the test.

The study is classified as acceptable, as it was performed according to Test Guideline OECD 490 for in vitro mutagenicity (mammalian forward gene mutation).

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In Syrian golden hamsters the cheek pouches were painted daily for 5 days in 5 animals with 50 μL of a 4% lime solution. No increase was observed in micronucleated cells in the cheek pouches when compared to a similar number of controls.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

As calcium carbide instantly hydrolyses to Ca(OH)2 and C2H2 upon contact with water/moisture the assessment is based on the degradation products. For details please refer to the read across report in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

No increase was observed in micronucleated cells in the cheek pouches when compared to a similar number of controls

Conclusions:

Interpretation of results: negative
In Syrian golden hamsters the cheek pouches were painted daily for 5 days in 5 animals with 50 μl of a 4% lime solution.No increase was observed in micronucleated cells in the cheek pouches when compared to a similar number of controls

Executive summary:

In a mammalian cell micronucleus test, the cheek pouches of 5 Syrian golden hamsters were dermally exposed daily for 5 days with 50 μl of a 4% lime solution.

There was no evidence for an increase in micronucleated cells in the cheek pouches when compared to a similar number of controls.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In aqueous solution, calcium carbide rapidly decomposes into calcium hydroxide and acetylene. Calcium hydroxide dissociates into calcium and hydroxyl ions. As hydroxyl ions are readily buffered in biological tissue, only calcium ions and gaseous acetylene need to be assessed for potential genotoxic effects. Calcium is the most abundant mineral in the human body and part of the normal diet (approx 700 mg/day; SCF 2003). Thus, calcium is not expected to have genotoxic potential. This conclusion is supported by experimental data on calcium oxide and calcium hydroxide. Nishimura et al., 2008 tested calcium hydroxide negative in a chromosomal aberration test up to 300 μM with and without metabolic activation. Calcium hydroxide has been studied in the Comet assay in vitro, showing no DNA damage. Da Silva et al., 2007 tested calcium hydroxide in human lymphocytes (100 μg/mL); Ribeiro et al 2004 and 2008 tested calcium in human fibroblasts, mouse lymphoma cells (80 μg/mL) and CHO-cells (100 μg/mL). The tests are summarized in MAK (2013). Nair et al., 1992 treated Syrian golden hamsters with lime solution. The cheek pouches were painted daily for 5 days in 5 animals with 50 μL of a 4% lime solution. No increase of micronucleated cells in the cheek pouches was observed when compared to a similar number of controls.

The second decomposition product, acetylene, has been used for over 100 years as an anesthetic and industrial chemical, and few complications of using this gas have surfaced (US EPA, HPV review 2003). This assumption is supported by a negative Ames test (Hughes et al., 1984), and a negative in vitro gene mutation test in mammalian cells conducted according to OECD 490 (Flanders, 2011), and by the US EPA HPV review (2003) and the evaluation of carcinogenicity (Reichert 1984, IARC86, IARC87, IARC99).

Justification for classification or non-classification

Based on the known composition of the test substance and available results on the possible genotoxic properties of the test substance and its decomposition products it can be anticipated that the test substance is not genotoxic. Therefore, no classification according to the CLP criteria set out in Regulation (EC) No. 1272/2008 is warranted.