Alkaline earth metal and zinc hydroxy(substituted)acetate complexes and alkaline earth metal glyoxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (bacteria)

Key, Ames, OECD 471, GLP, negative (Schulz, 2018)

Cytogenicity (mammalian cells)

Read across: WoE, FF6, chromosomal aberration, OECD 473, GLP, negative (Herold, 1999)

Read across: WoE, ZnCl₂, chromosomal aberration, positive (Deknudt and Deminatti, 1978)

Read across: WoE, ZnAc, chromosomal aberration, positive (Thompson, 1989)

Gene mutation (mammalian cells)

Read across: WoE, ZnCl2, gene mutation in mammalian cells, negative (Anmacher and Paillet, 1980)

Read across: WoE, ZnAc, gene mutation in mammalian cells, positive (Thompson, 1989)

Read-across: WoE, sulfonate, QSAR, negative (OECD QSAR Toolbox v4.4)

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:

Oct 2018 - Noc 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal activation (Phenobarbital/β-naphthoflavone induced rat liver S9)

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment Ia:
Strains TA 98: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

For each strain and dose level, including the controls, three plates were used.
Experiment I and Ia (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension (cf. 3.4.3 Precultures),
2000 μL Overlay agar
Experiment II (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37°C for 60 minutes.
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension (cf. 3.4.3 Precultures),
After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube.
The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above 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.

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

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

Species / strain:

S. typhimurium TA 98

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:

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:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I and Ia) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in three independent experiments with and without liver microsomal activation. Each concentration, including the 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
Because of contamination, which led to irregular background growth, data evaluation was not possible in experiment I in strain TA 98 with and without S9 mix. Therefore, this part of experiment was repeated as a plate incorporation assay at the following concentrations (reported as experiment Ia):
Experiment Ia:
Strains TA 98: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate. No precipitation of the test item occurred in the overlay agar on the incubated agar plates.
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, except of strain TA 98 with and without S9 mix, in which reduced background growth was observed in experiment Ia at 5000 μg/plate.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate. No precipitation of the test item occurred in the overlay agar on the incubated agar plates.
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, except of strain TA 98 with and without S9 mix, in which reduced background growth was observed in experiment Ia at 5000 μg/plate.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the sample at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). A minor increase in revertant colony number, not reaching the threshold of twice the number of the corresponding solvent control, was observed in experiment Ia at 5000 μg/plate. Since no relevant increase in the number of revertant was observed in experiment II, which was performed as pre-incubation assay, this effect can be judged as biologically irrelevant.

Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

Conclusions:

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.

Executive summary:

The assay was performed according to OECD guideline 471 and EU Method B.13/14 under GLP-conditions in three independent experiments with and without liver microsomal activation. Each concentration, including the 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

Because of contamination, which led to irregular background growth, data evaluation was not possible in experiment I in strain TA 98 with and without S9 mix. Therefore, this part of experiment was repeated as a plate incorporation assay at the following concentrations (reported as experiment Ia):

Experiment Ia:

Strains TA 98: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate

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

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate. No precipitation of the test item occurred in the overlay agar on the incubated agar plates.

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, except of strain TA 98 with and without S9 mix, in which reduced background growth was observed in experiment Ia at 5000 μg/plate.

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

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). A minor increase in revertant colony number, not reaching the threshold of twice the number of the corresponding solvent control, was observed in experiment Ia at 5000 μg/plate.

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, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/ml

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

625 µg/ml

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
not determined

RANGE-FINDING/SCREENING STUDIES:
Following 4 hours treatment a slight depression in cell growth was observed at concentration exceeding 1481 µg/mL after 18 and 26 hours continuous treatment, however, all cultures treated with the test article showed a dose depending cytotoxic effect. Inhibition in cell growth of at least 50 % was observed at concentrations exceeding 1481 µg/mL (18 hours treatment) and 658 µg/mL (26 hours treatment).

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Marked increases in the frequencies of aberrant cells compared with untreated controls were seen in cultures treated with the positive control chemicals ethyl methanesulfonate and cyclophosphamide both in the 1st and 2nd experiment.

Chromosome aberration test (CA) in mammalian cells:

First Experiment:
Marked cytotoxicity - as expressed by a reduction in degree of confluency versus the controls - was observed at concentration of 5,000 µg/mL both with and without metabolic activation. The number of aberrant metaphases in cultures treated with the test article, except the top concentration with metabolic activation, did not exceed 3.5 %. At a concentration of 5,000 µg/mL in the presence of metabolic activation the number of aberrant metaphases was 13.0 %.

Second Experiment:
Cultures treated with the test article continuously in the absence of metabolic activation at concentrations exceeding 1250 µg/mL (18 hours sampling time) and 625 µg/mL (26 hours sampling time) showed marked reduction in degree of confluency and damage of cell integrity. For this reason at a concentration of 5000 µg/mL less than 200 metaphases were evaluated at 18 hours sampling time and the cultures were not scorable at the late sampling time. Following 4 hours treatment (with metabolic activation) a slight depression in confluency was observed at concentration of 5000 µg/mL.

In the absence of metabolic activation in one of the duplicate cultures and in both cultures in the presence of metabolic activation a slight inerease in the aberration rate was found at a concentration of 5000 µg/mL at the 18 hours sampling time. At the 26 hour sampling time the maximum aberration score determined in negative controls and cultures treated with the test article was 2 %.
At concentrations of 5000 and 2500 µg/mL the incidence of endoreduplications was recorded.

Conclusions:

It is concluded that the test item induces chromosomal aberrations in cultured chinese hamster V79 cells at a concentration of 5000 µg/ml when tested under the experimental conditions reported.

Executive summary:

The test item was examined for the ability to cause chromosomal damage in cultured chinese hamster V79 cells following in vitro treatment in the presence and absence of S9 metabolic activation. The test procedure followed OECD Guideline 473. The study was performed under GLP conditions.

Two independent assays for chromosomal aberrations were performed and two parallel cultures were set up for each experimental point.

Range finder experiment: marked cytotoxicity (as expressed by an inhibition of cell growth - was found at concentrations exceeding 1481 µg/ml (4 hours treatment). After continuous treatment reduction in cell growth of at least 50 % was observed at concentrations exceeding 1481µg/mL (18 hours treatment) and 658µg/mL (26 hours treatment) respectively. On the basis of these findings concentrations of 1250, 2500 and 5000 µg/mL wereevaluated in the 1st experiment, both in the in the absence ar presence of S9 mix. In this experiment cells were treated for 4 hours and harvested 18 hours after the start of treatment.

In the 2nd experiment the cells were continuously treated in the absence of S9 metabolism until cell sampling after 18 or 26 hours. In the presence of S9 metabolism, the treatment duration was 4 hours again and cells were harvested after a subsequent 14 or 22 hours recovery period. For the 2nd experiment concentrations of 625, 1250, 2500 and 5000µg/mL (without S9 mix) or 1250, 2500 and 5000 µg/mL (with S9 mix) were evaluated for early harvest time. For the late harvest time only the highest scorable concentration was analysed. 200 metaphases spread from 2 cultures per experimental point (100 per culture) were selected for chromosomal aberration analysis.

In the cytogenetic experiments a reduced degree of confluency indicated cytotocity at a concentration of 5000µg/mL after 4 hours treatment. After continuous treatment cytotoxicity was observed at concentrations exceeding 1250µg/mL (18 hours sampling time) and 625µg/mL (26 hours sampling time).

In the 1st experiment the test article induced an increased and statistically significant incidence of structural chromosomal aberrations at the concentration of 5000 µg/mL in the presence of metabolic activation. At the same concentration with and without metabolic activation a slight, however, statistically significant increase in aberration frequency was observed in the 2nd experiment.

The positive substances induced sufficient aberrations confirming the effectiveness of the test procedures.

It is concluded that the test item induces chromosomal aberrations in cultured chinese hamster V79 cells at a concentration of 5000 µg/ml when tested under the experimental conditions reported. Therefore, a mutagenic effect of the test article can not be excluded completely. As in an AMES-Test no evidence of a mutagenic effect to bacteria occurred, the mutagenic potential of the test article should be investigated by in vivo mutagenicity testing.

The source substance contains the major organic moieties of the target substance. Therefore, the results are also applicable for the target substance.

Reference 3

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

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

zinc acetate

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Dose-dependent positive responses to zinc acetate were obtained in the presence and absence of the S9 activation system, although the S9 reduced both the clastogenic response and the toxicity.

Conclusions:

Dose-dependent positive responses to zinc acetate were obtained in the presence and absence of the S9 activation system, although the S9 reduced both the clastogenic response and the toxicity. These results indicate that zinc is an effective clastogen when presented to a susceptible cell population in an appropriate form. However, in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.

Executive summary:

A chromosome aberration assay with zinc acetate was conducted in Chinese Hamster Ovary cells. Zinc acetate induced chromosome aberraions. in the presence and absence of metabolic activation. These results indicate that zinc is an effective clastogen when presented to a susceptible cell population in an appropriate form.

However,in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxicin vivounder standard feeding conditions.

Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.

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:

weight of evidence

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The results presented here indicate zinc is an effective mutagen when presented to a mouse lymphoma cells in an appropriate form. However, controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.

Executive summary:

A mouse lymphoma TK+/- assay was perormed with zinc acetate. The results indicate zinc is an effective mutagen when presented to a mouse lymphoma cells in an appropriate form. However, controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.

Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.

Reference 5

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

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Conclusions:

Zinc chloride was not genotoxic in L5178Y mouse lymphoma cells.

Executive summary:

Zinc chloride was not genotoxic in L5178Y mouse lymphoma cells.

Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.

Reference 6

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

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Species / strain:

lymphocytes: human

Remarks:

zinc chloride

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

not examined

Conclusions:

The results suggest that high levels of zinc (3 x 10E-3 M) are cytotoxic and that lower concentration (3 x 10E-5 M) can cause severe chromosome aberrations (dicentrics). It must be pointed out, however, that the concentration of zinc used in the present experiments are extremely high representing up to 1000 times the respective concentrations reported in the blood of people professionally contaminated by heavy metals and which have been shown to be 22.4 µg% for zinc.

Executive summary:

Chromosome aberrations in human lymphocytes induced by zinc chloride were analysed. The results suggest that high levels of zinc (3 x 10E-3 M) are cytotoxic and that lower concentration (3 x 10E-5) can cause severe chromosome aberrations (dicentrics). It must be pointed out, however, that the concentration of zinc used in the present experiments are extremely high representing up to 1000 times the respective concentrations reported in the blood of people professionally contaminated by heavy metals and which have been shown to be 22.4 µg% for zinc.

Due to the fact that the Zn ion is a constituent of the target substance, the result is relevant for the target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Cytogenicity (mouse)

Read across: WoE, FF6, micronucleus test, OECD 474, GLP, negative (Müller, 2001)

Read across: WoE, ZnCl₂, chromosomal aberration, negative (Deknudt and Gerber, 1979)

Link to relevant study records

Reference

Reference 1

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:

weight of evidence

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Sex:

male/female

Genotoxicity:

negative

Remarks:

2000 mg/kg b.w.

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg body weight
A preliminary study on acute toxicity was performed with a small group of animals under identical conditions to these in the mutagenicity study with respect to animals, vehicle, route, frequency and volume of administration to assess the approximate MTD (maximum tolerated dose).
Two animals per sex were treated with a single oral dose of 2000 mg/kg body weight. This dose corresponds to the limit dose in accordance with the OECD Guideline. After administration the animals were monitored at approximately 1, 6, 24 and 48 hours for any symptoms of acute toxicity.
None of the animals died.

RESULTS OF DEFINITIVE STUDY
Mean values of micronucleated polychromatic erythrocytes (MPCEs) of 0.20 % (males) and 0.22 % (females) were found for the vehicle control group. The range ofthe historical negative controls in the testing facility (since 1997) was 0.11 to 0.24 % in males and 0.12 to 0.27 % in females.
In treated groups mean MPCE values were in the range from 0.19 to 0.26 % in males and from 0.18 to 0.27 % in females.
The statistical analysis of the MPCE counts did not show any statistically significant difference in comparison to the vehicle control.
Treatment with the positive control chemical (Cyclophosphamide, 40 mg/kg body weight) induced statistically significant increases in the incidence of MPCEs with a group mean value of 2.79 % in males and 2.49 % in females.

Conclusions:

In none of the experimental groups treated with the test item an increase in MPCEs was observed. However, the treatments induced statistically significant decreases of the proportion of polychromatic erythrocytes among total erythrocytes in all cases in female animals and for the last sacrifice time in the male animals. But these values are in the range of the historical control data in the testing facility. Therefore it is deemed these changes are incidental and not caused by the administration of the test item.
Based on the results of the study reported here it is concluded that the test item does not induce micronuclei in polychromatic erythrocytes of NMRI-mice under the described experimental conditions. The test item is therefore considered to be non-mutagenic in the mouse bone marrow micronucleus test.

Executive summary:

A GLP compliant study according to OECD Guideline 474 was performed. Groups of 5 male and 5 female NMRI-mice were exposed to the test item at the limit dose of 2000 mg/kg body weight. The test item was administered orally using a metal catheter. Deionised water (10 mI/kg body weight) served as vehicle control and Cyclophosphamide (CPA) at a dose of 40 mg/kg body weight - also administered orally - was used as positive control.

A dose of 2000 mg/kg body weight was chosen as the maximum dose in accardance with the guideline because no of the animals died at this dose in the pre-experiment. Bone marrow smears were prepared at 12 hours (dose group), at 24 hours (vehicle contral, positive control, dose graup) and at 48 hours (dose group) after dosing.

Two thousand polychromatic erythrocytes per animal were analysed for the presence of micronuclei. To investigate bone marrow toxicity the proportion of polychromatic erythrocytes among total erythrocytes was evaluated on the basis of 200 erythrocytes.

The frequency of micronucleated polychromatic erythrocytes (MPCEs) in the vehicle control group was within the physiological range. Treatment with CPA induced statistically significant inereases in the incidence of MPCEs.

In none of the experimental groups treated with the test item an increase in MPCEs was observed. However, the treatments induced statistically significant decreases of the proportion of polychromatic erythrocytes among total erythrocytes in all cases in female animals and for the last sacrifice time in the male animals. But these values are in the range of the historical control data in the testing facility. Therefore it is deemed these changes are incidental and not caused by the administration of the test item.

Based on the results of the study reported here it is concluded that the test item does not induce micronuclei in polychromatic erythrocytes of NMRI-mice under the described experimental conditions. The test item is therefore considered to be non-mutagenic in the mouse bone marrow micronucleus test.

The source substance contains the major organic moieties of the target substance. Therefore, the results are also applicable for the target substance.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other: in mice with normal diet

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other: in mice with calcium deficient diet

Additional information on results:

Treatment with heavy metals as well as the low-calcium diet significantly reduced the body weight of the mice, and the effect was more pronounced when treatments with heavy metals and low calcium diet were combined. In general, the intoxicated animals on a low calcium diet had lost weight, were weak, seemed anaemic and had brittle femurs. Serum calcium was reduced in animals on a low-calcium diet, and this effect was accentuated by intoxication with heavy metals whereas this intoxication as such did not significantly influence calcium levels in animals on a normal diet. The number of dicentrics as well as the number of cells carrying structural aberrations was not significantly increased in mice kept on a normal diet and treated with zinc.

The number of dicentrics as well as the number of cells carrying structural aberrations was significantly increased in mice kept on a calcium-deficient diet and treated with zinc.

Treatment and diet	Body weight (g)	Serum calcium (mg/100ml)	Cells with structural aberrations	Type and ChromatidGaps
				Chromatid aberrations		Chromatid aberrations
				gaps	Breaks	Gaps	Fragments	Dicentrics
Control+ Ca	29.90±0.12	10.24±0.06	1.80±0.60	1.20±0.49			0.6±0.35
Control - Ca	21.80±0.27 !!	9.45±0.15 !!	2.00±0.63	1.80±0.60			0.4±0.28
Zinc+ Ca	17.90±0.23 **	9.76±0.29 *!	2.80±0.75	1.80±0.60	0.2±0.2		0.4±0.28	0.4±0.28
Zinc - Ca	12.05±0.25 **!!	8.76±0.24	5.00±1.00 **	3.20±0.80		0.4±0.28	0.6±0.35	1.2±0.49
^a All values represent means -+ standard errors (Poisson errors for counting data). ^Statistically significant differences from the respective controls without heavy metals are indicated as * and ** for the p <= 0.05 and p <= 0.01 levels. ^Differences from the respective treated group with calcium in the diet are shown as ! and !! for the p <= 0.05 and p <= 0.01 levels. ^b Exact probability 0.06

Conclusions:

The present experiments confirm that zinc can cause severe chromosomal anomalies in animals kept on a low calcium diet. The number of dicentrics as well as the number of cells carrying structural aberrations was not significantly increased in mice kept on a normal diet and treated with zinc.

Executive summary:

Mice kept on a normal (1.1% calcium) or low-calcium (0.03%) diet were exposed for one month to zinc chloride (0.5% Zn). The concentrations, given in a poor calcium diet, represent a LD 50 (30 days). After the mice were killed bone-marrow cells were assayed for chromosomal aberrations, and serum calcium was determined. The number of dicentrics as well as the number of cells carrying structural aberrations was significantly increased in mice kept on a calcium-deficient diet and treated with zinc. The number of dicentrics as well as the number of cells carrying structural aberrations was not significantly increased in mice kept on a normal diet and treated with zinc.

Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in bacteria

in vitro

OECD 471 (Schulz 2018)

The assay was performed according to OECD guideline 471 and EU Method B.13/14 under GLP-conditions in three independent experiments with and without liver microsomal activation. Each concentration, including the 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

Because of contamination, which led to irregular background growth, data evaluation was not possible in experiment I in strain TA 98 with and without S9 mix. Therefore, this part of experiment was repeated as a plate incorporation assay at the following concentrations (reported as experiment Ia):

Experiment Ia:

Strains TA 98: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate

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

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate. No precipitation of the test item occurred in the overlay agar on the incubated agar plates.

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, except of strain TA 98 with and without S9 mix, in which reduced background growth was observed in experiment Ia at 5000 μg/plate.

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

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test sample at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). A minor increase in revertant colony number, not reaching the threshold of twice the number of the corresponding solvent control, was observed in experiment Ia at 5000 μg/plate.

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, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Cytogenicity

in vitro

OECD 473 (Herold 1999)

The test item was examined for the ability to cause chromosomal damage in cultured chinese hamster V79 cells following in vitro treatment in the presence and absence of S9 metabolic activation. The test procedure followed OECD Guideline 473. The study was performed under GLP conditions.

Two independent assays for chromosomal aberrations were performed and two parallel cultures were set up for each experimental point.

Range finder experiment: marked cytotoxicity (as expressed by an inhibition of cell growth - was found at concentrations exceeding 1481µg/ml (4 hours treatment). After continuous treatment reduction in cell growth of at least 50 % was observed at concentrations exceeding 1481µg/mL (18 hours treatment) and 658µg/mL (26 hours treatment) respectively. On the basis of these findings concentrations of 1250, 2500 and 5000µg/mL wereevaluated in the 1st experiment, both in the in the absence ar presence of S9 mix. In this experiment cells were treated for 4 hours and harvested 18 hours after the start of treatment.

In the 2nd experiment the cells were continuously treated in the absence of S9 metabolism until cell sampling after 18 or 26 hours. In the presence of S9 metabolism, the treatment duration was 4 hours again and cells were harvested after a subsequent 14 or 22 hours recovery period. For the 2nd experiment concentrations of 625, 1250, 2500 and 5000 µg/mL (without S9 mix) or 1250, 2500 and 5000 µg/mL (with S9 mix) were evaluated for early harvest time. For the late harvest time only the highest scorable concentration was analysed. 200 metaphases spread from 2 cultures per experimental point (100 per culture) were selected for chromosomal aberration analysis.

In the cytogenetic experiments a reduced degree of confluency indicated cytotocity at a concentration of 5000 µg/mL after 4 hours treatment. After continuous treatment cytotoxicity was observed at concentrations exceeding 1250µg/mL (18 hours sampling time) and 625 µg/mL (26 hours sampling time).

In the 1st experiment the test article induced an increased and statistically significant incidence of structural chromosomal aberrations at the concentration of 5000 µg/mL in the presence of metabolic activation. At the same concentration with and without metabolic activation a slight, however, statistically significant increase in aberration frequency was observed in the 2nd experiment.

The positive substances induced sufficient aberrations confirming the effectiveness of the test procedures.

It is concluded that the test item induces chromosomal aberrations in cultured chinese hamster V79 cells at a concentration of 5000 µg/ml when tested under the experimental conditions reported. Therefore, a mutagenic effect of the test article can not be excluded completely. As in an AMES-Test no evidence of a mutagenic effect to bacteria occurred, the mutagenic potential of the test article should be investigated by in vivo mutagenicity testing.

The source substance contains the major organic moieties of the target substance. Therefore, the results are also applicable for the target substance.

Zinc chloride (Deknudt and Deminatti 1978)

Chromosome aberrations in human lymphocytes induced by zinc chloride were analysed. The results suggest that high levels of zinc (3 x 10E-3 M) are cytotoxic and that lower concentration (3 x 10E-5) can cause severe chromosome aberrations (dicentrics). It must be pointed out, however, that the concentration of zinc used in the present experiments are extremely high representing up to 1000 times the respective concentrations reported in the blood of people professionally contaminated by heavy metals and which have been shown to be 22.4 µg% for zinc.

Due to the fact that the Zn ion is a constituent of the target substance, the result is relevant for the target substance.

However,in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions. This is confirmed by a negative in vivo micronucleus assay with zinc chloride (Deknudt and Gerber, 1979, see below).

Zinc acetate (Thompson 1989)

A chromosome aberration assay with zinc acetate was conducted in Chinese Hamster Ovary cells. Zinc acetate induced chromosome aberraions. in the presence and absence of metabolic activation. These results indicate that zinc is an effective clastogen when presented to a susceptible cell population in an appropriate form.

Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.

However, in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions. This is confirmed by a negative in vivo micronucleus assay with zinc chloride (Deknudt and Gerber, 1979, see below).

in vivo

OECD 474 (Müller 2001)

A GLP compliant study according to OECD Guideline 474 was performed. Groups of 5 male and 5 female NMRI-mice were exposed to the test item at the limit dose of 2000 mg/kg body weight. The test item was administered orally using a metal catheter. Deionised water (10 mI/kg body weight) served as vehicle control and Cyclophosphamide (CPA) at a dose of 40 mg/kg body weight - also administered orally - was used as positive control.

A dose of 2000 mg/kg body weight was chosen as the maximum dose in accardance with the guideline because no of the animals died at this dose in the pre-experiment. Bone marrow smears were prepared at 12 hours (dose group), at 24 hours (vehicle contral, positive control, dose graup) and at 48 hours (dose group) after dosing.

Two thousand polychromatic erythrocytes per animal were analysed for the presence of micronuclei. To investigate bone marrow toxicity the proportion of polychromatic erythrocytes among total erythrocytes was evaluated on the basis of 200 erythrocytes.

The frequency of micronucleated polychromatic erythrocytes (MPCEs) in the vehicle control group was within the physiological range. Treatment with CPA induced statistically significant inereases in the incidence of MPCEs.

In none of the experimental groups treated with the test item an increase in MPCEs was observed. However, the treatments induced statistically significant decreases of the proportion of polychromatic erythrocytes among total erythrocytes in all cases in female animals and for the last sacrifice time in the male animals. But these values are in the range of the historical control data in the testing facility. Therefore it is deemed these changes are incidental and not caused by the administration of the test item.

Based on the results of the study reported here it is concluded that the test item does not induce micronuclei in polychromatic erythrocytes of NMRI-mice under the described experimental conditions. The test item is therefore considered to be non-mutagenic in the mouse bone marrow micronucleus test.

The source substance contains the major organic moieties of the target substance. Therefore, the results are also applicable for the target substance.

Zinc chloride (Deknudt and Gerber 1979)

Mice kept on a normal (1.1% calcium) or low-calcium (0.03%) diet were exposed for one month to zinc chloride (0.5% Zn). The concentrations, given in a poor calcium diet, represent a LD 50 (30 days). After the mice were killed bone-marrow cells were assayed for chromosomal aberrations, and serum calcium was determined. The number of dicentrics as well as the number of cells carrying structural aberrations was significantly increased in mice kept on a calcium-deficient diet and treated with zinc. The number of dicentrics as well as the number of cells carrying structural aberrations was not significantly increased in mice kept on a normal diet and treated with zinc.

Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.

Gene mutation in mammalian cells

in vitro

Zinc chloride (Anmacher and Paillet 1980)

Zinc chloride was not genotoxic in L5178Y mouse lymphoma cells.

Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.

Zinc acetate (Thompson 1989)

A mouse lymphoma TK+/- assay was perormed with zinc acetate. The results indicate zinc is an effective mutagen when presented to a mouse lymphoma cells in an appropriate form. Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance. However, controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.

Sulfonate (OECD QSAR Toolbox v4.4)

Genetic toxicity to mammalian cells was predicted using read-across analysis with the OECD QSAR toolbox v4.4. Two closely analogue substances with negative results were found after categorisation according to structural similarity. It was categorized for structural similarity as no alert for gene mutation was identified in the target substance. This prediction is outside the applicability domain, because of the higher logP of the target substance. However, as the target substance has a higher logP and a higher MW, this prediction can be assumed to be worst-case. Thus, the target substance is consiered to be negative for gene mutation in mammalian cells.

The source substance is the major organic moiety of the target substance. Therefore, this result shows that no genetic toxicity can be attributed to the organic moiety of the target substance.

Justification for classification or non-classification

Based on these results the substance is not classified according to Regulation (EC) No 1272/2008.