Calcium carbonate

Endpoint summary

Description of key information

Repeat dose toxicity - oral: A 14 day range finder study, a 28 day and a 90-day repeat dose oral toxicity study in rats are available that have evaluated the repeated dose toxicity of uncoated nano calcium carbonate. A 28 day study in the mouse is also available, as well as a number of human studies on bulk calcium carbonate. None of the studies described any severe or adverse toxicological effects following oral administration of the test material.

Repeat dose toxicity - dermal: No studies on the repeated dose dermal toxicity of bulk calcium carbonate are available.

Repeat dose toxicity - inhalation: A 90 day inhalation toxicity study in the rat is has been performed using uncoated nano calcium carbonate in which no systemic toxicity was observed. Local effects were observed in the lung, these were irritation and increased lung weights. The NOAEC was 0.212 mg/L. The results of these studies are read across to the bulk calcium carbonate.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Bulk calcium carbonate and uncoated nano calcium carbonate are chemically identical. The only difference between them is the particle size, which may result in minor differences in physico-chemical properties. However, in biological systems and in the environment, both bulk and uncoated nano calcium carbonate are expected to have identical (eco)toxicological and environmental fate properties.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Uncoated nano calcium carbonate (Source): Refer to IUCLID section 1.2 for information on purity and impurities.
Bulk calcium carbonate (Target): Refer to IUCLID section 1.2 for information on purity and impurities.
The majority of new studies have been performed using uncoated nano calcium carbonate as the test material, hence it has been designated as the ‘Source’ in the read-across justification. However, for some endpoints data on bulk calcium carbonate are available and used to read-across to uncoated nano calcium carbonate so for these endpoints, marked with a * in the read-across justification document, bulk calcium carbonate is the ‘Source’.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification attached in Section 13 of the dataset
4. DATA MATRIX
Please refer to the justification attached in Section 13 of the dataset

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Specific details on test material used for the study:

Bulk calcium carbonate

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically significant effects observed at any dose level.

Critical effects observed:

no

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Bulk calcium carbonate and uncoated nano calcium carbonate are chemically identical. The only difference between them is the particle size, which may result in minor differences in physico-chemical properties. However, in biological systems and in the environment, both bulk and uncoated nano calcium carbonate are expected to have identical (eco)toxicological and environmental fate properties.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Uncoated nano calcium carbonate (Source): Refer to IUCLID section 1.2 for information on purity and impurities.
Bulk calcium carbonate (Target): Refer to IUCLID section 1.2 for information on purity and impurities.
The majority of new studies have been performed using uncoated nano calcium carbonate as the test material, hence it has been designated as the ‘Source’ in the read-across justification. However, for some endpoints data on bulk calcium carbonate are available and used to read-across to uncoated nano calcium carbonate so for these endpoints, marked with a * in the read-across justification document, bulk calcium carbonate is the ‘Source’.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification attached in Section 13 of the dataset
4. DATA MATRIX
Please refer to the justification attached in Section 13 of the dataset

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Specific details on test material used for the study:

Bulk calcium carbonate

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of clinical signs; mortality; body weight; effects on food consumption; ophthalmoscopic examination; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1, GLP sub-acute toxicity study combined with a reproductive toxicity screening test performed according to OECD 422.
Klimisch 2, GLP 90-day study done to a protocol similar to the OECD 408.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Bulk calcium carbonate and uncoated nano calcium carbonate are chemically identical. The only difference between them is the particle size, which may result in minor differences in physico-chemical properties. However, in biological systems and in the environment, both bulk and uncoated nano calcium carbonate are expected to have identical (eco)toxicological and environmental fate properties.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Uncoated nano calcium carbonate (Source): Refer to IUCLID section 1.2 for information on purity and impurities.
Bulk calcium carbonate (Target): Refer to IUCLID section 1.2 for information on purity and impurities.
The majority of new studies have been performed using uncoated nano calcium carbonate as the test material, hence it has been designated as the ‘Source’ in the read-across justification. However, for some endpoints data on bulk calcium carbonate are available and used to read-across to uncoated nano calcium carbonate so for these endpoints, marked with a * in the read-across justification document, bulk calcium carbonate is the ‘Source’.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification attached in Section 13 of the dataset
4. DATA MATRIX
Please refer to the justification attached in Section 13 of the dataset

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

NOAEC

Effect level:

0.26 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: slight changes in bronchoalveolar lavage parameters

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Bulk calcium carbonate and uncoated nano calcium carbonate are chemically identical. The only difference between them is the particle size, which may result in minor differences in physico-chemical properties. However, in biological systems and in the environment, both bulk and uncoated nano calcium carbonate are expected to have identical (eco)toxicological and environmental fate properties.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Uncoated nano calcium carbonate (Source): Refer to IUCLID section 1.2 for information on purity and impurities.
Bulk calcium carbonate (Target): Refer to IUCLID section 1.2 for information on purity and impurities.
The majority of new studies have been performed using uncoated nano calcium carbonate as the test material, hence it has been designated as the ‘Source’ in the read-across justification. However, for some endpoints data on bulk calcium carbonate are available and used to read-across to uncoated nano calcium carbonate so for these endpoints, marked with a * in the read-across justification document, bulk calcium carbonate is the ‘Source’.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification attached in Section 13 of the dataset
4. DATA MATRIX
Please refer to the justification attached in Section 13 of the dataset

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

NOAEC

Remarks:

Local

Effect level:

>= 0.212 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

other: bronchoalveolar lavage (BAL) parameters

Key result

Dose descriptor:

NOEC

Remarks:

systemic

Effect level:

0.399 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic effects noted at highest exposure level

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

399 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1, GLP sub-chronic 90-day study performed according to OECD 413.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Bulk calcium carbonate and uncoated nano calcium carbonate are chemically identical. The only difference between them is the particle size, which may result in minor differences in physico-chemical properties. However, in biological systems and in the environment, both bulk and uncoated nano calcium carbonate are expected to have identical (eco)toxicological and environmental fate properties.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Uncoated nano calcium carbonate (Source): Refer to IUCLID section 1.2 for information on purity and impurities.
Bulk calcium carbonate (Target): Refer to IUCLID section 1.2 for information on purity and impurities.
The majority of new studies have been performed using uncoated nano calcium carbonate as the test material, hence it has been designated as the ‘Source’ in the read-across justification. However, for some endpoints data on bulk calcium carbonate are available and used to read-across to uncoated nano calcium carbonate so for these endpoints, marked with a * in the read-across justification document, bulk calcium carbonate is the ‘Source’.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification attached in Section 13 of the dataset
4. DATA MATRIX
Please refer to the justification attached in Section 13 of the dataset

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

NOAEC

Effect level:

0.26 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: slight changes in bronchoalveolar lavage parameters

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Bulk calcium carbonate and uncoated nano calcium carbonate are chemically identical. The only difference between them is the particle size, which may result in minor differences in physico-chemical properties. However, in biological systems and in the environment, both bulk and uncoated nano calcium carbonate are expected to have identical (eco)toxicological and environmental fate properties.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Uncoated nano calcium carbonate (Source): Refer to IUCLID section 1.2 for information on purity and impurities.
Bulk calcium carbonate (Target): Refer to IUCLID section 1.2 for information on purity and impurities.
The majority of new studies have been performed using uncoated nano calcium carbonate as the test material, hence it has been designated as the ‘Source’ in the read-across justification. However, for some endpoints data on bulk calcium carbonate are available and used to read-across to uncoated nano calcium carbonate so for these endpoints, marked with a * in the read-across justification document, bulk calcium carbonate is the ‘Source’.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification attached in Section 13 of the dataset
4. DATA MATRIX
Please refer to the justification attached in Section 13 of the dataset

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

NOAEC

Remarks:

Local

Effect level:

>= 0.212 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

other: bronchoalveolar lavage (BAL) parameters

Key result

Dose descriptor:

NOEC

Remarks:

systemic

Effect level:

0.399 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic effects noted at highest exposure level

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

212 mg/m³

Study duration:

subchronic

Species:

rabbit

Quality of whole database:

Klimisch 1, GLP sub-chronic 90-day study performed according to OECD 413.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeat dose toxicity - oral:

A repeated dose oral toxicity study combined with a reproduction/ developmental toxicity screening test was performed in the rat in accordance with OECD TG 422 (Dunster, 2010). Uncoated nano calcium carbonate was administered by gavage to three groups, each of ten male and ten female Wistar rats, for up to forty-eight consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 0, 100, 300 and 1000 mg/kg bodyweight/day. Clinical signs, behavioural assessments, bodyweight change, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Although administration of the test material resulted in treatment-related effects at all dose levels, these effects were considered not to represent an adverse effect of treatment. Hence, the NOAEL for systemic toxicity was taken as 1000 mg/kg bodyweight/day.

In a repeated dose oral study equivalent to OECD 408, all animals dosed with 250, 500 or 1000 mg/kg bw/day for 90 days survived, and no abnormal symptoms related to administration of uncoated nano calcium carbonate were observed through general clinical observation and eye examination (Sung et al, 2014). Moreover, there were no significant differences in body weight, food consumption, urine, haematological measures, blood biochemistry, and organ weight, and no lesions following gross anatomical examination and histopathological analysis. Thus, the NOAEL for uncoated nano calcium carbonate in this study was >1000 mg/kg bw/day.

The results of these studies are read across to bulk calcium carbonate.

The daily oral administration of uncoated nano calcium carbonate to mice over a period of 28 days at dose levels of 0, 13, 130 and 1300 mg/kg bw/day, did not produce any obvious symptoms of toxicity or mortality even at the highest dose administered (Huang et al, 2009). The NOAEL was therefore reported to be 1300 mg/kg bw/day.

Calcium carbonate occurs in the natural environment and humans are widely exposed to naturally occurring calcium carbonate, e.g. via drinking water on a day to day basis. Calcium carbonate is also a food additive approved by the Council Directive 95/2/EC on food additives (the substance has the acronym E 170). Ingested calcium and carbonate ions are actively regulated by the body. The European Commission Scientific Committee on Food has produced a report on the Tolerable Upper Intake Level of Calcium (2003). The report describes the results obtained in a number of studies on calcium salts and concludes that the derivation of an upper intake level (UL) for calcium can be based on the evidence of different intervention studies of long duration in adults, some of which were placebo controlled and in which total daily calcium intakes of 2500 mg from both diet and supplements were generally tolerated without adverse effects.

The document prepared by the Scientific Committee on Food describes a number of potential adverse effects following calcium supplementation. These might include the following:

1). Kidney function: Some studies have indicated that perimenopausal women with total calcium intakes between 2 and 3 g/day may show a tendency for compromised glomerular function as indicated by increases in serum creatinine. However, no such effect was observed in another study with women receiving comparable calcium amounts. Therefore, it is not clear whether this finding can be attributed to the ingestion of calcium.

2). Milk-alkali syndrome: Manifestation of the milk-alkali syndrome through the combined intake of calcium both from food and especially from supplements and of absorbable alkalinising substances is facilitated by renal insufficiency, alkalosis and dehydration due to vomiting and anorexia and/or the use of thiazide diuretics, which increase renal tubular calcium reabsorption. All reported cases of milk-alkali syndrome in association with the prolonged or acute ingestion of calcium supplements used calcium carbonate as the nutrient source. In these reports the supplemental calcium intakes were reported as between 1.0 and 23 g/day. These patients also differ in their medical history, use and duration of use of drugs and alkali consumption, and their diets. Their dietary calcium intakes are often not known.

The use of calcium carbonate supplements in doses up to 2000 mg/day, and thereby achieving total daily calcium intakes up to more than 3000 mg/day, for preventive purposes in presumably healthy subjects, has not provoked the development of the milk-alkali syndrome, whereas the administration of large amounts (11.2 g calcium/day) of calcium carbonate in addition to large amounts of milk (1.8 g calcium/day) over 7 days to 20 gastric/duodenal ulcer patients resulted in reversible hypercalcaemia (2.8 mmol/L) in nine patients and renal insufficiency in all.

In conclusion, on the basis of the available evidence, a calcium dose which by itself might cause milk-alkali syndrome cannot be identified. The primary cause of milk-alkali syndrome appears to be when significantly large doses of calcium are ingested and is more prevalent in those people suffering from renal insufficiency.

3). Kidney stones: Observational studies on the relationship between total calcium intake and kidney stone incidence and interventional studies with calcium supplements do not allow definition of a calcium intake on a population basis which promotes kidney stone formation. On dietary calcium intakes in the range of the recommended dietary intake the risk of nephrolithiasis is determined by other dietary components and by genetic factors.

4). Interaction with minerals: Single-dose experiments demonstrate interference of both dietary and supplemental calcium with the absorption of other minerals. However, this effect is not demonstrable in long-term observational and interventional studies at dietary calcium intakes in the range of recommended intakes and at supplemental calcium of up to 2000 mg/day in adults and up to 1200 mg/day in one study with infants.

In conclusion, calcium carbonate ingestion in humans up to the tolerable upper intake level of 2500 mg calcium per day (equivalent to a dose of calcium carbonate of 104 mg/kg bw/day for a 60 kg person) does not cause any adverse effects. Effects are only seen when this dose is significantly exceeded. Furthermore, as calcium carbonate is used as a food additive with an ADI not allocated, it can be concluded that intake of calcium carbonate should not pose a need for concern regarding its toxicity via the oral route.

 

Repeat dose toxicity - dermal:

Currently, no studies are available that have evaluated the repeated dose dermal toxicity of calcium carbonate in either the bulk or nano form. Calcium carbonate is an inorganic ionic solid. It is expected to partition strongly to water rather than organic media. While it is not possible to measure or accurately predict an octanol/water partition coefficient for inorganic ionic substances, such a value would be expected to be very low. Electrolytes are also known not to penetrate the skin in any significant quantity. Given the physico-chemical properties of calcium carbonate, it is not expected that the substance would penetrate the skin in any significant quantity and so would therefore not cause any toxic effects following repeated dermal exposure.

 

Repeat dose toxicity - inhalation:

A study investigating the sub-chronic (13 week) toxicity of inhaled uncoated nano calcium carbonate in rats is available (van Triel, 2015) and may be read across to bulk calcium carbonate. Five main groups of 10 male and 10 female rats each were exposed by nose-only inhalation exposure to 0 (control), 0.026 (± 0.002), 0.123 (± 0.006), 0.212 (± 0.013) or 0.399 (± 0.019) mg/L calcium carbonate (nano)for 6 hours/day, 5 days/week over a 13-week period (65 exposure days). Animals of the main groups were sacrificed on the day after the last exposure. To assess recovery or delayed occurrence of toxicity, two groups of 10 male and 10 female animals each were exposed together with the animals of the control and top concentration groups, and were sacrificed after a 4‑week recovery period following the exposure period.

The exposure conditions were close to their respective targets. The aerodynamic particle size distribution of the test atmospheres was highly comparable across the groups with an average mass median aerodynamic diameter (MMAD) in the range of 1.29 – 1.35 µm and a geometric standard deviation (gsd) of 1.52 – 1.54. The relative contribution of nanoparticles (< 100 nm) in the various test atmospheres was determined to be very low. Scanning electron microscopy of aerosol samples confirmed that the particles were primarily present in agglomerates, which varied in size (ranging 60 nm – 30 µm, with trace amounts of primary particles) and shape, with little to no difference between the groups.

The exposure to the test material was well tolerated by the animals. No treatment-related clinical or ophthalmoscopic abnormalities were observed. A transient decrease in growth (females) or food consumption (males), observed in the top concentration group shortly after the initiation of exposure, was no longer observed after a few weeks. Haematology results, clinical chemistry analysis and necropsy findings did not show any treatment-related changes. No indications for systemic toxicity of inhaled calcium carbonate (nano) were observed in this study. 

Exposure to the test material resulted in local changes in the lower airways. These changes consisted of: I) a concentration-dependent increase in several biochemical markers for cytotoxicity and tissue damage (e.g. ALP, GGT, LDH, total protein) in bronchoalveolar lavage (BAL) fluid of animals of the mid, high and top concentration main groups; II) slight changes in differential white blood cell numbers in BAL fluid of animals of the high and top concentration main groups, characterized by an increase in the number of neutrophils and – for females of the top concentration group only – a slight increase in the number of lymphocytes; and III) an increased lung weight in males and females of the top concentration main group. These findings were not accompanied by any microscopic changes in the lungs; histopathology did not reveal any treatment-related changes in the respiratory tract (or in any other tissues)[1]. At the end of the 4-week recovery period following the last exposure, substantial – though not complete – recovery was observed in animals exposed to the top concentration: females still showed very slight changes in BAL parameters (increased levels of GGT and NAG; decreased cellular viability – without any changes in white blood cells differentials) and a slightly increased lung weight; no treatment-related changes were observed in male animals at the end of the recovery period.

Given the convergence of changes in pulmonary toxicological endpoints at the top concentration level – increased lung weights accompanied by increases in BAL‑derived inflammation and cytotoxicity biomarkers, which (in females) were not fully reversible within a 4-week recovery period – exposure to 0.399 mg/L uncoated nano calcium carbonate was considered to have resulted in an adverse response in the lower airways. Exposure to 0.212mg/L uncoated nano calcium carbonate resulted in very limited alterations in BAL parameters only. These findings were not substantiated by any concomitant changes in lung weight or treatment-related histopathology. Therefore, the findings at the high concentration level were considered to be of no toxicological relevance and were judged as non-adverse.

Under the conditions of the current study, inhalation exposure to 0.399 mg/L uncoated nano calcium carbonate resulted in treatment-related changes in the lower airways, characterized by an increased lung weight accompanied by slight increases in BAL‑derived inflammation and cytotoxicity biomarkers. These changes were largely, but not fully, reversible within a 4‑week recovery period after the last exposure. Based on these observations, the NOAEC for local effects of sub-chronic inhalation exposure to uncoated nano calcium carbonate was placed at 0.212 mg/L. Since exposure to the test material did not induce any systemic toxicity, the NOEC for systemic effects was 0.399 mg/L.

[1] BAL measurements are usually a rather sensitive toxicological read-out parameter, and it is not uncommon to observe treatment-related changes at concentrations below a level at which histopathology is induced.

Justification for classification or non-classification

No systemic toxicological findings could be detected in rats after repeated administration of uncoated nano calcium carbonate by the oral route for a period of 90 days. The results of this study are read across to bulk calcium carbonate. Several potential adverse effects have been reported following calcium supplementation e.g. effects on kidney function, milk-alkali syndrome, kidney stones and interactions with minerals. However, these effects are more prevalent in those people suffering from renal insufficiency and following the ingestion of high doses of calcium (well above the recommended classification limits for STOT RE as defined in the Guidance on the Application of Regulation (EC) No 1272/2008). Therefore, a classification as STOT RE is not justified and no classification is proposed.

No systemic toxicity was observed in a 90-day inhalation study where rats were exposed to uncoated calcium carbonate at a maximum concentration of 0.399 mg/L, stated as the NOEC. The local effects observed at this level were limited to increased lung weights accompanied by increases in BAL‑derived inflammation and cytotoxicity biomarkers, which were reversible in males but were not fully reversible in females within a 4-week recovery period. Hence the NOAEC for local effects was established as 0.212 mg/L and the LOAEC at 0.399 mg/L. The results of this study are read across to bulk calcium carbonate. On the basis that the LOAEC is above the guidance value of 0.2 mg/L as the upper limit for classification of dusts as STOT RE2 and that the effects seen were minor and mostly reversible, a classification as STOT RE is not justified and no classification is proposed.