Azelaic acid, calcium salt (1:1)

Endpoint summary

Description of key information

Data have been read across from the structural analogues, dilithium azelate, azelaic acid and calcium. Although effects were observed in the acute oral toxicity study with dilithium azelate, these effects were a result of the concentration of lithium present, and do not correlate with the expected toxicity effects of calcium azelate. This conclusion is supported by the LD50 values for azelaic acid (>5,000 mg/kg bw) and calcium (930 mg/kg bw, equivalent to 5258 mg/kg calcium azelate), which shows the calcium azelate would not meet the criteria for classification for acute oral toxicity.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

16 April to 11 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, guideline study, available as an unpublished report.

Justification for type of information:

Please see attached read across justification for full details of the read across approach.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany. Animals were 8 to 10 weeks old and nulliparous and non-pregnant.
- Acclimatisation: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions and animals were individually housed for the pilot study and group housed for the main study.
- Housing: Individual housing of animals in the pilot study and group housing of four animals per cage in the main study in labeled Makrolon cages (MIV type; height 18 cm.) containing sterilized sawdust as bedding material and paper as cage-enrichment. The rats had free access to pelleted rodent diet and tap water.
- Environmental conditions: Temperature and relative humidity were set to achieve limits of 18 to 24 deg C, and 40 to 70% humidity. Rate of air exchange was at least 10 changes per hour and there was a 12 hour light/12 hour dark cycle.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- Preparation: The preparations (w/w) were kept at room temperature protected from light and were dosed within 4 hours after adding the vehicle to the test substance. Homogeneity was obtained to visually acceptable levels.
- Dosing: Oral gavage, using plastic feeding tubes. The test item preparations were stirred on a magnetic stirrer during dosing. The concentration of the test substance in vehicle was varied to allow constant dosage volume in terms of mL/kg body weight.
- Fasting: Animals were deprived of food overnight prior to dosing and until 3-4 hours after administration of the test substance. Water was available.

Doses:

In the absence of any other toxicity information a "Pilot study" was conducted in which a single female rat was orally dosed with the test substance at 2000 mg/kg body weight. Based on the Pilot results, a fixed dose level of 2000 mg/kg body weight was then selected for the main study which was conducted in a stepwise approach on three animals. One animal at a time was treated at 2000 mg/kg body weight. When two animals were found dead, five animals were treated at a lower dose level of 300 mg/kg body weight.

No. of animals per sex per dose:

For the main study four female rats were treated at 2000 mg/kg body weight, and five female rats were subsequently treated at 300 mg/kg body weight.

Control animals:

no

Details on study design:

- Observations: Clinical observations were made on the day of dosing and then once daily until Day 15. Morbidity and viability checks were made twice daily and individual body weights were recorded on days 0, 8 and 15. At the end of the observation period, all animals were sacrificed by oxygen/carbon dioxide procedure and subjected to necropsy. Descriptions of all internal macroscopic abnormalities were recorded.

Statistics:

The Fixed Dose Procedure does not require any statistical analyses of the data.

Preliminary study:

In the preliminary study in one animal at 2000 mg/kg bw no mortality occurred. Hunched posture, piloerection and ptosis were noted between Days 1 and 12. The animal showed body weight loss between Days 1 and 8 and showed body weight gain again between Days 8 and 15. No findings were noted during macroscopic post mortem examination.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 mg/kg bw

Based on:

test mat.

Mortality:

At 2000 mg/kg, two animals were found dead, on Day 4 and 5 of the study.
At 300 mg/kg, no mortality occurred.

Clinical signs:

other: At 2000 mg/kg, hunched posture, piloerection and/or chromodacryorrhoea (snout) were noted for the animals between Days 1 and 4. At 300 mg/kg, no clinical signs were noted for any of the animals.

Gross pathology:

At 2000 mg/kg, abnormalities of the stomach (glandular mucosa: focus/foci, several, black-brown) were found in one of the animals found dead. Macroscopic post mortem examination of the other animal that died during the study and of the surviving animals at termination did not reveal any abnormalities. Autolysis was noted for both animals found dead. This was considered not toxicologically relevant.
At 300 mg/kg, no abnormalities were found at macroscopic post mortem examination of the animals.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The highest dose level of Dilithium azelate that did not produce mortality in Wistar rats was established as 300 mg/kg body weight and the estimated minimum lethal dose level was 2000 mg/kg body weight. Based on the mortality rate at 2000 mg/kg body weight, it was concluded that the oral LD50 value of Dilithium azelate was within the range of 300-2000 mg/kg body weight. Based on these results Dilithium azelate should be classified as Category 4.

Executive summary:

The acute oral toxicity of Dilithium azelate to female Wistar rats was determined in a GLP-compliant, fixed-dose method study following OECD guideline 420 (Latour 2015). A preliminary study indicated no mortality of a single rat at 2000 mg/kg bw Dilithium azelate, therefore 2000 mg/kg bw was selected for the main study which was conducted in a stepwise approach on three further animals. Dilithium azelate was dosed by oral gavage and the rats were observed for mortality, systemic toxicity and bodyweight gain.When two animals were found dead at 2000 mg/kg bw, five animals were treated at a lower dose level of 300 mg/kg bw for 15 days. No deaths or significant signs of toxicity were observed at 300 mg/kg bw Dilithium azelate. The oral LD50 value is within the range 300 mg/kg bw - 2000 mg/kg bw Dilthium azelate. Based on these results Dilithium azelate should be classified as Category 4. The study is considered to be relevant and reliable for use for this endpoint.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Please see attached read across justification for full details of the read across approach.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Review paper on tolerable upper intake levels for vitamins and minerals, quoting previous study (Sarabia et al 1999) for acute oral toxicity. A daily tolerable upper intake level was derived based on the evidence of different interventional studies of long duration in adults.

GLP compliance:

no

Test type:

other: No details reported

Limit test:

no

Specific details on test material used for the study:

Tested as Biocal (TM), calcium gluconate stabilised with glycine

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Weights: 400-450 g male, 300-350 g female
Cages: 315 x 445 x 240 mm stainless steel with grated flooring and collecting tray
Feeding: Free access to water and normalised diet (Nutrimentos (TM) Diet No. 3)

Route of administration:

oral: unspecified

Vehicle:

not specified

Details on oral exposure:

Dosing: Gastric catheter with syringe

Doses:

10, 11, 12, 13, 14, 15 g Biocal/kg bw

No. of animals per sex per dose:

6 groups of 10 male and 10 female rats

Control animals:

not specified

Details on study design:

Feeding: Animals were starved of solid food for 10 hours prior and 1 hour post dosing
Photoperiod: 12 hours light to 12 hours dark

Statistics:

Statistics: One-way analysis of variance with student-Newman-Keuls method to test difference between means (p < 0.05 considered statistically significant). LD50 according to Litchfield and Wilcoxon.
Results: Data are presented as mean ± standard deviation.

Preliminary study:

Not specified

Sex:

female

Dose descriptor:

LD50

Effect level:

13.5 other: g/kg

Based on:

test mat.

95% CL:

12.8 - 14.3

Sex:

male

Dose descriptor:

LD50

Effect level:

13 other: g/kg

Based on:

test mat.

95% CL:

12.2 - 13.9

Mortality:

Not specified

Clinical signs:

other: Not specified

Gross pathology:

Not specified

Other findings:

Not specified

Conclusions:

Review paper on tolerable upper intake levels for vitamins and minerals, quoting previous study (Sarabia et al 1999) for acute oral toxicity. The acute oral toxicity of calcium (dosed as Biocal (TM) calcium gluconate stabilised with glycine) gave an LD50 of 13.5 g/kg for female rats and 13 g/kg for male rats.

Executive summary:

Limited details are available for the acute toxicity study but the data have been included in a review by the European Commission's Scientific Committee on Food so the results are considered to be reliable. The data are taken from a review paper on tolerable upper intake levels for vitamins and minerals, quoting previous study (Sarabia et al 1999) for acute oral toxicity. The acute oral toxicity of calcium (dosed as Biocal (TM) calcium gluconate stabilised with glycine) gave an LD50 of 13.5 g/kg for female rats and 13 g/kg for male rats.

Reference 3

Endpoint:

acute toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Justification for type of information:

Please see attached read across justification for full details of the read across approach.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Preliminary screening for acute oral toxicity involved dosing two rats at 5.0 g/kg bw. The substance was considered to be non-toxic and no further testing was conducted if no mortality occurred. Otherwise, further testing was undertaken to determine an oral LD50. Classification for acute toxicity followed the criteria in Black et al (1972).

GLP compliance:

no

Test type:

other: Not specified

Limit test:

yes

Specific details on test material used for the study:

No details reported

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Animals: Limit test - Sprague-Dawley rats, otherwise either Sprague-Dawley rats or ICR mice
Feeding: Animals were fasted for at least 16 hours prior to dosing
Weight: Animals were weighed at dosing to determine dosing volume, start of test animals weighed 200-300 g (rats) or 25-40 g (mice)

Route of administration:

oral: gavage

Vehicle:

other: Either corn oil or propylene glycol

Details on oral exposure:

Dosing: Animals were dosed using syringes and special oral dosing needles

Doses:

Spacing: Single dose of 5 g/kg bw for limit test, otherwise geometically spaced doses

No. of animals per sex per dose:

Limit test: 2 rats, otherwise five rats or mice per concentration

Control animals:

not specified

Details on study design:

Duration: 14 days
Observations: Any deaths during 14 days following exposure were included in final moratlity results

Statistics:

LD50 and 95% confidence limits: Moving average interpolation method of Weil (1952)

Preliminary study:

No mortality was observed in the preliminary study, with an LD50 >5000 mg/kg bw, so the main study was not conducted.

Sex:

male

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

No mortality was observed in the preliminary study, with an LD50 >5000 mg/kg bw, so the main study was not conducted.

Clinical signs:

other: Not specified

Gross pathology:

Not specified

Other findings:

Not specified

Conclusions:

No mortality was observed in the preliminary study, with an LD50 >5000 mg/kg bw, so the main study was not conducted.

Executive summary:

Preliminary screening for acute oral toxicity involved dosing two rats at 5.0 g/kg bw. The substance was considered to be non-toxic and no further testing was conducted if no mortality occurred. Otherwise, further testing was undertaken to determine an oral LD50. Classification for acute toxicity followed the criteria in Black et al (1972). No mortality was observed in the preliminary study, with an LD50 >5000 mg/kg bw, so the main study was not conducted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

Data read across from proprietary and published studies on structural analogues: dilithium azelate, azelaic acid and calcium.

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data are available for acute oral toxicity for calcium azelate. Read across data for acute oral toxicity are available for dilithium azelate and azelaic acid but not for other simple metal salts. As acute oral toxicity could potentially derive from either or both the metal cation and the organic anion, data have been read across from a range of source substances, as part of a weight of evidence approach. Data are read across from dilithium azelate, as a structural analogue for calcium azelate as a whole, as well as from azelaic acid, being the same as the organic component of calcium azelate, and calcium gluconate, as a structural analogue for the calcium ion.

The data for dilithium azelate are taken from a GLP-compliant guideline study following OECD guideline 420, while the data on calcium and azelaic acid are taken from published data. The data on calcium are taken from a published study cited by the European Commission's Scientific Committee on Food, so the results are considered to be reliable. The data on azelaic acid are taken from a published summary of testing undertaken by the United States Air Force. Due to the limited details available on the published studies, these are included as weight of evidence.

There are some differences in the results between dilithium azelate and azelaic acid, and thus also calcium azelate, due to the nature of the substances; dilithium azelate is classified for acute oral toxicity category 4 (LD50: 300-2000 mg/kg), while azelaic acid is not (LD50 15,8000 mg/kg). The acute oral toxicity effects observed for dilithium azelate are related to the concentration of lithium present in the substance, as can be predicted based on theoretical calculations. Regarding the lithium ion, the lowest of several oral LD50 values for partially soluble lithium carbonate quoted in the ECHA Dissemination Portal file is 525 mg/kg based on a report in RTECS (and a very similar value of 526 mg/kg was also reported for lithium chloride). On the basis of atomic and molecular weight considerations (atomic weight of lithium being 6.94 g/mol, the molecular weight of Li₂CO₃ is 73.89 g/mol), 525 mg of Li₂CO₃ is equivalent to 98.63 mg lithium. Extrapolating this to dilithium azelate (MW 200.09 g/mol), 98.63 mg lithium is equivalent to 1,422 mg/kg of the salt, which is within the upper classification limit of acute toxicity (300-2000 mg/kg). Although reading across the result for dilithium azelate would present an effect which is not relevant for calcium azelate, the data have been included as part of a weight of evidence approach, along with data on calcium and azelaic acid.

Data are presented for calcium as it is the inorganic component of calcium azelate. The data on calcium gluconate stabilised with glycerine was presented as part of a review document by the Scientific Committee on Food (2003) for the derivation of a daily tolerable upper intake level for calcium. As the data are taken from a regulatory review, all relevant and reliable data on calcium should have been considered and the result has been selected as representative of calcium. The result, LD50 of 930 mg Ca/kg, equivalent to 5258 mg/kg calcium azelate, is comparable to other data for calcium, such as that in the ECHA dissemination portal (LD50 >2000 mg/kg calcium dihydroxide, equivalent to 1101 mg Ca/kg).

The data on azelaic acid have been taken from a regulatory document, summarising a preliminary screening study for acute oral toxicity which involved dosing two rats at 5,000 mg/kg bw. The substance was considered to be non-toxic following the criteria in Black et al (1972), with an LD50 >5000 mg/kg bw, and no further testing was conducted as no mortality occurred. The results are in line with those presented on the ECHA dissemination portal for azelaic acid, which give an LD50 of 15,800 mg/kg bw.

Although effects were observed in the acute oral toxicity study with dilithium azelate, these effects were a result of the concentration of lithium present, and do not correlate with the expected toxicity effects of calcium azelate. This conclusion is supported by the LD50 values for azelaic acid (>5,000 mg/kg bw) and calcium (930 mg/kg bw, equivalent to 5258 mg/kg calcium azelate), which shows the calcium azelate would not meet the criteria for classification for acute oral toxicity.

Justification for classification or non-classification

Calcium azelate is not expected to meet the criteria for classification for acute oral toxicity based on data read across from structural analogues: dilithium azelate, azelaic acid and calcium. The toxicity effects observed in the study with dilithium azelate were a result of the concentration of lithium present, and do not correlate with the expected toxicity effects of calcium azelate, while the LD50 values for azelaic acid (>5,000 mg/kg bw) and calcium (930 mg Ca/kg bw, equivalent to 5258 mg calcium azelate/kg) support that calcium azelate would not meet the criteria for classification for acute oral toxicity.