Lanthanum trihydroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria (Ames): Harlan (2013): Negative with and without metabolic activation.

In vitro cytogenicity in mammalian cells (Chrom Ab): Harlan (2013): Negative with and without metabolic activation.

In vitro gene mutation in mammalian cells (CHO HPRT): Damment et al. (2005) Lanthanum tricarbonate: Negative with and without metabolic activation.

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

Study period:

26 September 2012 - 6 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: guidelines published by the Japanese Regulatory Authorities, including METI, MHLW and MAFF.

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OPPTS harmonised guidelines.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth.
- Properly maintained: yes. Stored at approximately -196 °C in a liquid nitrogen freezer. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Additional strain / cell type characteristics:

other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate (10 % liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test
Experiment 1: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. The test material formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment. Formulated concentrations were adjusted to allow for the purity of the test material. All formulations were used within four hours of preparation and were assumed to be stable for this period. Prior to use, the solvent was dried to remove water using molecular sieves.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

- EXPERIMENT 1
METHOD OF APPLICATION: in agar (direct plate incorporation)
0.1 mL aliquots of the bacterial cultures were dispensed into sets of test tubes, followed by 2 mL molten top agar (0.6 % agar, 0.5 % NaCl with 5 mL of 1.0 mM histidine and 1.0 mM biotin for Salmonella typhimurium or 1.0 mM tryptophan solution for E. coli), 0.1 mL of the appropriate test material solution or the vehicle or positive control substance and 0.5 mL S9-mix (for the plates with metabolic activation) or 0.5 mL phosphate buffer (for the plates without metabolic activation). The contents were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
0.1 mL of the appropriate bacterial culture was dispensed into a test tube followed by 0.5 mL of S9 mix or phosphate buffer and 0.1 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates.
The positive and untreated controls were dosed using the standard plate incorporation method described above.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


DETERMINATION OF CYTOTOXICITY
- Method: Examined for effects on the background lawn of bacterial growth.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- All tester strain cultures should be in the range of 0.9 to 9 x 10⁹ bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. However, slight reductions in revertant colony frequency were noted in Experiment 1 (plate incorporation method) at 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES
A preliminary toxicity test was performed with TA100 and WP2uvrA in both the absence and presence of S9-mix with ten different concentrations of the test material, ranging from 0.15 to 5000 µg/plate. The test material was toxic only at 5000 μg/plate in TA100 both in the presence and absence of metabolic activation.

DEFINITIVE STUDY
Results for the negative controls (spontaneous mutation rates) are presented in Table 2 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The mean number of revertant colonies for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 3 and Table 4 for Experiments 1 and 2, respectively.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. However, slight reductions in revertant colony frequency were noted in Experiment 1 (plate incorporation method) at 5000 μg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A test material precipitate (fine in appearance) was noted at 5000 μg/plate; this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 2: Spontaneous Mutation Rates (Concurrent Vehicle Controls)

Experiment	Mean number of colonies/plate
	Base-pair Substitution Type			Frameshift Type
	TA100	TA1535	WP2uvrA	TA98	TA1537
1	108	18	30	19	11
2	108	26	34	13	10

 

Table 3: Experiment 1

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
- - - - - - - -	Solvent 5 15 50 150 500 1500 5000	90 96 104 99 107 100 76 53*	20 20 20 19 17 14 11 9*	29 28 30 26 24 27 22 18*	21 22 18 24 22 15 19 10*	12 13 12 11 11 11 10 7*
+ + + + + + + +	Solvent 5 15 50 150 500 1500 5000	113 111 110 125 110 92 90 63*	15 13 13 12 12 13 9 10*	33 40 32 30 30 33 27 16*	21 21 24 20 25 28 24 14*	16 15 15 13 12 12 12 13*
Positive Controls
-	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean no. colonies/plate	479	367	584	163	598
+	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate	1883	315	374	161	387

*Precipitate

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

 

Table 4: Experiment 2

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
- - - - - - - -	Solvent 5 15 50 150 500 1500 5000	107 95 91 81 88 98 102 89*	26 25 21 19 15 22 23 27*	32 28 27 25 28 31 30 25*	17 16 20 14 16 13 16 17*	10 11 10 12 9 12 10 5
+ + + + + + + +	Solvent 5 15 50 150 500 1500 5000	102 103 102 94 96 99 93 92*	13 15 13 13 13 14 14 13*	35 36 36 31 38 34 33 29*	25 25 27 25 24 23 21 21*	13 11 12 13 14 15 12 14*
Positive Controls
-	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean no. colonies/plate	385	259	758	168	725
+	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate	527	161	242	111	190

*Precipitate

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

Conclusions:

Under the conditions of this study, the test material was considered to be non-mutagenic.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14. Furthermore, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at seven dose levels, both with and without metabolic activation. The dose levels assessed were 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. However, slight reductions in revertant colony frequency were noted in Experiment 1 (plate incorporation method) at 5000 μg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A test material precipitate (fine in appearance) was noted at this dose level, though it did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. 

The vehicle controls gave revertant colony counts within the normal range. The positive controls gave the expected increases in revertants, validating the sensitivity of the assay and the efficacy of the S9-mix.

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 September 2012 - 21 March 2013

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

- Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in the testing laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

- Cell Culture
Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented in-house with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 °C with 5 % CO₂ in humidified air.
The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate metabolising system (S9)

Test concentrations with justification for top dose:

Preliminary Toxicity Test:
0, 7.42, 14.84, 29.67, 59.34, 118.7, 237.4, 474.8, 949.5 an 1899 µg/mL in both the absence and presence of metabolic activation.

Experiment 1:
0, 6.25, 12.5, 25, 50, 100 and 200 µg/mL in both the absence and presence of metabolic activation.

Experiment 2
0, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL in the absence of metabolic activation.
0, 6.25, 12.5, 25, 50, 100 and 200 µg/mL in the presence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was suspendable in Eagle's minimal essential medium with HEPES buffer (MEM) at 18.99 mg/mL but the best suspension was in dimethyl sulphoxide at 189.93 mg/mL.

- Preparation of test material in vehicle
The test material was accurately weighed, suspended in DMSO and serial dilutions prepared.
There was no significant change in pH when the test item was dosed into DMSO and the osmolality did not increase by more than 50 mOsm.
The test material was formulated within two hours of it being applied to the test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

CULTURE CONDITIONS
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 - 9.15 mL MEM, 10 % (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.65 - 0.75 mL heparinised whole blood

WITH METABOLIC ACTIVATION (S9) TREATMENT
After approximately 48 hours incubation at approximately 37 °C, 5 % CO₂ in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test material was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20 % S9-mix (i.e. 2 % final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.
In Experiment 2, 1 mL of 10 % S9-mix (i.e. 1 % final concentration of S9 in standard co-factors) was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.
After 4 hours at approximately 37 °C, 5 % CO₂ in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 °C in 5 % CO₂ in humidified air.

WITHOUT METABOLIC ACTIVATION (S9) TREATMENT
In Experiment 1, after approximately 48 hours incubation at approximately 37 °C with 5 % CO₂ in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then re-suspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test material solution or positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 °C, 5 % CO₂ in humidified air the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test material dose solution positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 °C, 5 % CO₂ in humidified air for 24 hours.

Experiment 1
There were 2 exposure conditions conducted for Experiment 1:
i) 4 hour exposure to the test material without S9-mix followed by 20 hour culture in treatment-free media prior to cell harvest.
ii) 4 hour exposure to the test material with S9-mix (2 %) followed by 20 hour culture in treatment-free media prior to cell harvest.

Experiment 2
There were 2 exposure conditions conducted for Experiment 2:
i) 24 hour continuous exposure to the test material without S9-mix prior to cell harvest.
ii) 4 hour exposure to the test material with S9-mix (1 %) followed by 20 hour culture in treatment-free media prior to cell harvest.

CELL HARVEST
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075 M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 °C to ensure complete fixation.

SLIDE PREPARATION
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. When the slides were dry, they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

QUALITATIVE SLIDE ASSESSMENT
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose level for mitotic index evaluation.

MITOTIC INDEX
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

SCORING OF CHROMOSOME DAMAGE
Where possible, the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50 % of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44 - 48 chromosomes, any gaps, breaks or rearrangements were noted. In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
Precipitate observations were made from the blood-free cultures at the end of the exposure period. In the 4(20) hour exposure group in the absence of S9, precipitate was noted at the end of exposure at and above 237.4 μg/mL. However, due to the suspension of the test material, precipitate persisted on the slides at and above 118.7 μg/mL, which became obscuring at and above 474.8 μg/mL. This difference is due to the fact that precipitate in the pellet was observed in all dose levels tested in the contemporaneous blood cultures at the end of the exposure period.
In the 4(20) hour exposure group in the presence of S9, precipitate was noted at the end of exposure at and above 59.34 μg/mL. Also, precipitate persisted on the slides at and above this dose level and became obscuring at and above 474.8 μg/mL. In the 24 hour exposure group in the absence of S9, precipitate was noted at the end of exposure at all dose levels tested. Again, precipitate persisted on the slides at and above 59.34 μg/mL which became obscuring at and above 237.4 μg/mL. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 949.5 μg/mL in the 4(20) hour exposure groups and up to 474.8 μg/mL in the 24 hour exposure group. The selection of the maximum dose level was based on precipitate rather than the onset of toxicity in all exposure groups tested because it had persisted on to the slides at the end of exposure.

CHROMOSOME ABERRATION TEST - EXPERIMENT 1
The qualitative assessment of the slides determined that there was no toxicity present and that there were metaphases suitable for scoring present at the maximum test material dose level tested in both exposure groups (200 μg/mL). Precipitate observations were made at the end of exposure and precipitate in the pellet was noted at all dose levels tested in both exposure groups. Also, the precipitate persisted after exposure on the slides at and above 100 and 200 μg/mL, in the absence and presence of S9, respectively.
The mitotic index summary data are given in Table 1. These data show there was no dose-related reduction in Mitotic Index either in the absence or presence of S9.
The maximum dose level selected for metaphase analysis was 100 and 200 μg/mL in the absence and presence of S9, respectively, due to the presence of precipitate on the slides.
A summary of the chromosome aberration data is given in Table 2. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either exposure group, which included at least one precipitating dose level.
A summary of the polyploid cell frequency data is also given in Table 2. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

CHROMOSOME ABERRATION TEST - EXPERIMENT 2
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 100 and 200 μg/mL in the absence and presence of S9, respectively. Precipitate observations were made at the end of exposure and precipitate in the pellet was noted at all dose levels tested in both exposure groups. Also, the precipitate persisted after exposure on the slides at and above 50 μg/mL.
The mitotic index data summary are given in Table 1. These data show there was no dose-related reduction in Mitotic Index either with or without S9.
The maximum dose level selected for metaphase analysis was, therefore, 50 and 100 μg/mL in both the presence and absence of S9 due to the presence of precipitate on the slides.
A summary of the chromosome aberration data is given in Table 3. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either exposure group, which included at least one precipitating dose level.
A summary of the polyploid cell frequency data is also given in Table 3. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Table 1 Summary of Mitotic Index Data from Experiments 1 and 2

Dose Level (µg/mL)	Experiment 1				Experiment 2
	4 Hour Treatment -S9		4 Hour Treatment +S9		24 Hour Treatment -S9		4 Hour Treatment +S9
	Mean	% of Control	Mean	% of Control	Mean	% of Control	Mean	% of Control
0 3.13 6.25 12.5 25 50 100 200	2.98 - - - 4.18 4.83 3.93* -	100 - - - 140 162 132 -	5.70 - - - - 5.33 6.98 4.80*	100 - - - - 93 122 84	6.15 - - - 6.80 4.60* 4.85* -	100 - - - 111 75 79 -	6.40 - - 6.40 5.78 5.70* - -	100 - - 100 90 89 - -
Mitomycin C 0.4	0.98	33	-	-	2.05	33	-	-
Cyclophosphamide 5	-	-	0.98	17	-	-	1.20	19

Precipitate was present for all dose levels evaluated.

*Precipitate was present on the slide at evaluation for these dose levels.

 

Table 2 Summary of Chromosome Aberration Data for Experiment 1

Treatment Period (hours)	Dose Level (µg/mL)		No. and % Cells Showing Structural Aberrations† (%)	No. and % Cells Showing Gaps (%)	No. and % Cells Showing Numerical Aberrations‡ (%)
4 (-S9-mix)	0	Total %	2 1.0	2 1.0	0 0.0
	25	Total %	3 1.5	1 0.5	0 0.0
	50	Total %	1 0.5	0 0.0	0 0.0
	100	Total %	4 2.0	0 0.0	0 0.0
	MMC 0.4	Total %	50*** 50.0	6 6.0	0 0.0
4 (+S9-mix)	0	Total %	4 2.0	6 3.0	0 0.0
	50	Total %	1 0.5	2 1.0	0 0.0
	100	Total %	2 1.0	2 1.0	0 0.0
	200	Total %	5 2.5	3 1.5	0 0.0
	CP 5	Total %	38*** 25.3	6 4.0	0 0.0

Results are the mean values from two replicates.

MMC = Mitomycin C; CP = Cyclophosphamide.

†Includes chromatid and chromosome breaks, chromatid and chromosome exchanges.

‡Includes polyploids and other numerical chromosome aberrations.

***p = <0.001

 

Table 3 Summary of Chromosome Aberration Data for Experiment 2

Treatment Period (hours)	Dose Level (µg/mL)		No. and % Cells Showing Structural Aberrations†(%)	No. and % Cells Showing Gaps (%)	No. and % Cells Showing Numerical Aberrations‡(%)
24 (-S9-mix)	0	Total %	1 0.5	2 1.0	0 0.0
	25	Total %	0 0.0	0 0.0	0 0.0
	50	Total %	4 2.0	0 0.0	1 0.5
	100	Total %	1 0.5	2 1.0	0 0.0
	MMC 0.4	Total %	36*** 36.0	6 6.0	0 0.0
4 (+S9-mix)	0	Total %	0 0.0	0 0.0	0 0.0
	12.5	Total %	1 0.5	1 0.5	0 0.0
	25	Total %	1 0.5	1 0.5	0 0.0
	50	Total %	1 0.5	0 0.0	0 0.0
	CP 5	Total %	37*** 37.0	5 5.0	0 0.0

Results are the mean values from two replicates.

MMC = Mitomycin C; CP = Cyclophosphamide.

†Includes chromatid and chromosome breaks, chromatid and chromosome exchanges.

‡Includes polyploids and other numerical chromosome aberrations.

***p = <0.001

Conclusions:

Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.

Executive summary:

The potential of the test material to induce chromosomal aberrations was investigated in vitro in accordance with the standardised guidelines OECD 473 and EU Method B.10.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. The dose levels used in Experiment 1 in both the presence and absence of metabolic activation and Experiment 2 in the presence of metabolic activation were 6.25, 12.5, 25, 50, 100 and 200 µg/mL. In Experiment 2 in the absence of metabolic activation, the dose levels selected were 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL.

Four treatment conditions were used for the study:

In Experiment 1, cells were exposed for 4 hours in the presence of an induced rat liver homogenate metabolising system (S9 at a 2 % final concentration) with cell harvest after a 20 hour expression period and a 4 hour exposure in the absence of metabolic activation with a 20 hour expression period.

In Experiment 2, the 4 hours exposure period with addition of S9 was repeated (using a 1 % final S9 concentration) whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations in the exposure groups dosed in the presence or absence of S9, which included at least one precipitating dose level.

Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles. Study is read across to from structurally similar substance.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Remarks:

data published

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was derived from the livers of male Fischer 344 rats that had been pretreated with a combination of sodium phenobarbitone (i.p. at 40 mg/kg) and beta-naphthoflavone (i.p. at 100 mg/kg) for 3 consecutive days prior to killing.

Test concentrations with justification for top dose:

50, 250, 500, 1000, 1500, and 2000 µg/mL in the absence of S9 mix
25, 250, 500, 2500 and 5000 µg/mL in the presence of S9 mix

Vehicle / solvent:

- sterile distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: ethyl methane sulphonate (750 µg/mL); +S9: benzo[a]pyrene (25 µg/mL)

Details on test system and experimental conditions:

DURATION
- Exposure duration: 3 h ( in the absence and in the presence of S9)
- Selection time (if incubation with a selection agent): 7 days
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: 2 independant assays
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

- significant increase of mutation frequency
- dose-response relationship

Statistics:

The significance of any changes in mutation frequency compared with the control was determined using the statistical methods described in Statistical Evaluation of Mutagenicity Test Data (Kirkland, 1989).

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: -S9: reduction in survival at 2000 µg/mL (exp. I: 80 - 90%, exp. II: approx. 38% ) and at 1500 µg/mL (exp. I: approx. 35%; exp. II: approx. 44%); +S9: no or little toxicity at the highest dose of 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

There were no significant effects on mutation frequency in the presence of S9 mix. In the absence of S9 mix increases in mutation frequency were obtained at isolated concentrations in the first experiment. However, there was no obvious dose-response relationship and the effects were not reproducible in a second experiment, indicating that they had arisen by chance and do not indicate a mutagenic effect of Lanthanum carbonate.

Maximum revertant colonies, control data and data for cytotoxicity (if observed):

(* p< 0.05; n.d. no data given)

 

Without S9: experiment I:

Conc. (µg/mL)	No. of colonies	control (%)	Mutation frequency (x 10E-6)
2000	21.3	16.4	11.4
1500	83.7	64.4	0.9
1000	104.3	80.3	8.5
500	128.3	98.7	16.5
250	117.3	90.3	n.d.
50	124.7	95.9	n.d.
solvent control	130	100	2.5
positive control	n.d.	n.d.	574.4*

 

Without S9: experiment II:

Conc. (µg/mL)	No. of colonies	control (%)	Mutation frequency (x 10E-6)
2000	101.3	62.1	14.9
1500	92.3	56.6	14.1
1000	100.3	61.5	2.0
500	121.7	74.7	0.9
250	110.7	67.9	n.d.
50	147.0	90.2	n.d.
solvent control	163	100	15.5
positive control	n.d.	n.d.	540.9

 

With S9: experiment I:

Conc. (µg/mL)	No. of colonies	control (%)	Mutation frequency (x 10E-6)
5000	129.0	111.9	7.5
2500	144.8	125.6	6.8
500	91.5	79.3	9.2
250	97.3	84.4	16.0
25	123.7	107.2	n.d.
solvent control	115.3	100	10.6
positive control	n.d.	n.d.	89.6*

 

With S9: experiment II:

Conc. (µg/mL)	No. of colonies	control (%)	Mutation frequency (x 10E-6)
5000	104.7	96.3	1.5
2500	74.7	68.7	12.4
500	102.0	93.3	1.7
250	101.0	92.9	1.8
25	83.7	77.0	n.d.
solvent control	108.7	100	5.0
positive control	n.d.	n.d.	65.6*

 

Conclusions:

The test substance did not significantly increase the number of mutations at the hprt locus at any concentration, in either the absence or presence of metabolic activation.

Executive summary:

The genetic toxicity of the test substance was examined in accordance with OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test).

The effect of the test substance on the forward mutation of mammalian cells was examined using Chinese hamster Ovary (CHO) cells, without and with metabolic activation using an S9 liver fraction mix. The target gene was the hprt locus.

Test substance concentrations of 50, 250, 500, 1000, 1500, and 2000 µg/mL in the absence of S9 mix and 25, 250, 500, 2500 and 5000 µg/mL in the presence of S9 mix were used.

Some evidence of cytotoxicity was observed at both 1500 and 2000 µg/plate in the absence of metabolic activation, but little evidence of toxicity was seen at the highest dose 5000 µg/plate, in the presence of metabolic activation.

The test substance did not significantly increase the number of mutations at the hprt locus at any test substance concentration, in either the absence or presence of metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance, lanthanum tricarbonate, is justified on the basis that toxicological effects will be driven by the metal cation species (La3+) which is analogous to the registered substance and the read-across substance.

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: -S9: reduction in survival at 2000 µg/mL (exp. I: 80 - 90%, exp. II: approx. 38% ) and at 1500 µg/mL (exp. I: approx. 35%; exp. II: approx. 44%); +S9: no or little toxicity at the highest dose of 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse gene mutation assay (Ames)

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14. The study was assigned a reliability score of 1 in accordance with the criteria detailed by Klimisch (1997). Furthermore, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at seven dose levels, both with and without metabolic activation. The dose levels assessed were 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. However, slight reductions in revertant colony frequency were noted in Experiment 1 (plate incorporation method) at 5000 μg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A test material precipitate (fine in appearance) was noted at this dose level, though it did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. 

The vehicle controls gave revertant colony counts within the normal range. The positive controls gave the expected increases in revertants, validating the sensitivity of the assay and the efficacy of the S9-mix.

The test material was considered to be non-mutagenic under the conditions of this test.

In vitro cytogeneicity assay (chromosomal aberration)

The potential of the test material to induce chromosomal aberrations was investigated in vitro in accordance with the standardised guidelines OECD 473 and EU Method B.10. The study was assigned a reliability score of 1 in accordance with the criteria detailed by Klimisch (1997).

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. The dose levels used in Experiment 1 in both the presence and absence of metabolic activation and Experiment 2 in the presence of metabolic activation were 6.25, 12.5, 25, 50, 100 and 200 µg/mL. In Experiment 2 in the absence of metabolic activation, the dose levels selected were 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL.

Four treatment conditions were used for the study:

In Experiment 1, cells were exposed for 4 hours in the presence of an induced rat liver homogenate metabolising system (S9 at a 2 % final concentration) with cell harvest after a 20 hour expression period and a 4 hour exposure in the absence of metabolic activation with a 20 hour expression period.

In Experiment 2, the 4 hours exposure period with addition of S9 was repeated (using a 1 % final S9 concentration) whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations in the exposure groups dosed in the presence or absence of S9, which included at least one precipitating dose level.

Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.

In vitro gene mutation assay (CHO HPRT) (read across to lanthanum tricarbonate)

The genetic toxicity of the test substance was examined in accordance with OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test).

The effect of the test substance on the forward mutation of mammalian cells was examined using Chinese hamster Ovary (CHO) cells, without and with metabolic activation using an S9 liver fraction mix. The target gene was the hprt locus.

Test substance concentrations of 50, 250, 500, 1000, 1500, and 2000 µg/mL in the absence of S9 mix and 25, 250, 500, 2500 and 5000 µg/mL in the presence of S9 mix were used.

Some evidence of cytotoxicity was observed at both 1500 and 2000 µg/plate in the absence of metabolic activation, but little evidence of toxicity was seen at the highest dose 5000 µg/plate, in the presence of metabolic activation.

The test substance did not significantly increase the number of mutations at the hprt locus at any test substance concentration, in either the absence or presence of metabolic activation.

Read-across to structurally similar substance, lanthanum tricarbonate, is justified on the basis that toxicological effects will be driven by the metal cation species (La3+) which is analogous to the registered substance and the read-across substance.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material does not require classification for genetic toxicity.