(3S-cis)-3,6-dimethyl-1,4-dioxane-2,5-dione

Endpoint summary

Description of key information

Lactide (18:1 mixture of L-lactide and m-lactide) was tested in dogs in a 2-week dose range finding study and a subsequent 90d full study (similar to OECD 409). The primary toxic effect was irritation of the gastrointestinal tract at 100 mg/kg/d in the 90d study.

In addition, in an oral subchronic repeated dose toxicity study conducted with a suitable read-across partner, calcium lactate was used to assess the specific target organ toxicity of the target substance. Based on the results, no classification for specific target organ toxicity is warranted for L-lactide.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

not specified

Principles of method if other than guideline:

Doses for the 13-wk study were selected based on the results of a 2-week study. In the 13-wk study, four dogs per sex were assigned to each of four treatment groups (dosed with lactide at 0, 4, 20 and 100 mg/kg body weight/day). Each dog received daily oral doses of one to six gelatin capsules containing lactide. Doses were administered at approximately 1 hr after feed was withdrawn, and at approximately the same time each day for 13 weeks. Control dogs received the same number of empty capsules as dogs of the corresponding sex in the highest dose group. Doses were calculated based on the body weight of each dog during the previous week.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): lactide; 18:1 mixture of L-lactide (CAS no. 4511-42-6): m-lactide (CAS no. 13076-19-2)
- Lactide was manufactured and provided by Cargill, Inc USA
- bulk chemical stored at -20°C

For dose administration:
- removal from -20°C, 2 hours to reach ambient temperature
- lactide was loaded into 0.5 oz gelatin capsules
- each capsules contained between 0.1 g and 4 g of lactide
- no filling material was used to fill those capsules that contained less than 4g of chemical
- Capsules were prepared weekly, were stored on capsule boards, refrigerated in plastic bags containing desiccant
--> a prior stability study indicated that lactide was stable for at least 14 days when stored under these conditions
- Capsules were removed from refrigerator and allowed 2 hours to reach ambient temperature before dose administration

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc ( Madison, WI, USA)
- Age at study initiation: ranged from 5 to 10 months
- Fasting period before study: 1 hour before dosing
- Housing: individual housed in stainless-steel cages on racks and were exercised at least twice weekly throughout the quarantine and study period
- Diet (e.g. ad libitum): dogs were fed Certified Canine Chow 5007 (PMI Feeds, Inc) for approximately 2 hours each day
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26°C
- Humidity (%): 35 - 85%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: capsule

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Each dog received daily oral doses of one to six gelatin capsules containing lactide. Doses were administered at approximately 1 hr after feed was withdrawn, and at approximately the same time each day for 13 weeks.
Control dogs received the same number of empty capsules as dogs of the corresponding sex in the highest dose group. Doses were calculated based on the body weight of each dog during the previous week.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Single daily dose

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

4 mg/kg bw/day (actual dose received)

Remarks:

low dose

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

mid dose

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

high dose

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Observations:
- Dogs were observed twice daily for mortality or moribundity.
- Cageside observations were performed daily, approximately 1 hr after dosing.
- Once a week, each dog was removed from its cage and examined closely for detailed clinical signs of toxicity.
- Throughout each study:
dogs were weighed weekly, and food consumption was measured once a week over a 2-hour period.

Clinical pathology:
- blood and urine samples were obtained from each dog for clinical pathology and urinalysis determinations during quarantine, during week 5 and 9, and within 3 days prior to terminal sacrifice

Dogs were fasted overnight prior to blood collection for haematologic, clinical chemistry and coagulation analyses

Haematologic analyses included:
total leucocyte count, erythrocyte count, haemaglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count and differential leucocyte count. Reticulocyte counts and red blood cell morphology were evaluated.

Clinical chemistry analysis included:
By using a Roche Cobas Fara clinical chemistry analyser: blood urea nitrogen, creatinine, serum glucose, serum aspartate aminotransferase, serum alanine aminotransferase, alkaline phosphatase, glutamyl transferase, sodium, potassium, chloride, calcium, inorganic phosphate, total protein, albumin, albumin/total protein ratio, total bilirubin and cholesterol.

Coagulation analyses included:
Prothrombin time and activated partial prothrombin time, fibrinogen

Urine analyses included:
Urine specific gravity, urine microscopic sediment, urine pH, ketones, protein, glucose, bilirubin and occult blood

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

- Groups means and standard deviations for: body weights, food consumption, clinical pathology parameters, for terminal body weights and for absolute and relative organ weights
- Body weights, food consumption and clinical pathology parameters were evaluated by two-way repeated ANOVA, and if significant by Dunnett´s test
- Mean body weights, mean organ weights and organ/body, organ:brain ratios for each treated group were compared to those of the control group by a two-tailed Students t-test for each sex

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Emesis once in each of two female dogs (one in the 0 mg/kg and one in the 100 mg/kg group during week 9). Bloody diarrhoea was seen once during week 6 in one female dog of the 100 mg/kg group

Mortality:

mortality observed, treatment-related

Description (incidence):

Emesis once in each of two female dogs (one in the 0 mg/kg and one in the 100 mg/kg group during week 9). Bloody diarrhoea was seen once during week 6 in one female dog of the 100 mg/kg group

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

stomach foci in one male/female from the 100 mg/kg , and one female from the 4mg/kg dose group

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

moderately severe ulceration of the stomach mucosa seen in one female dog of the high dose group

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects observed at the highest tested dose 100 mg/kg/day

Dose descriptor:

LOAEL

Remarks:

(local)

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: gastrointestinal irritation

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: gastrointestinal irritation

Critical effects observed:

not specified

Table 1: Incidence of gross lesions in dogs treated for 13 weeks with oral doses of lactide
Dose group		0 mg/kg	4 mg/kg	20 mg/kg	100 mg/kg
Males
Stomach focus		0	0	0	1
Females
Stomach focus		0	1	0	1

Incidence= number of dogs in a given dose group with a given lesion. n=4 for all dose groups

Conclusions:

Lactide acts primarily, if not only, as an irritant after oral administration. 13 week NOAEL is 100 mg/kg bw/d.

Executive summary:

In a subchronic toxicity study lactide (18:1 mixture of l-lactide and m-lactide) was administered to 4 beagle dogs/sex/dose by capsule at dose levels of 4, 20, 100 mg/kg bw/day for 13 weeks.

The only apparent toxic effect at 100 mg/kg/day was gastrointestinal irritation. Therefore, the local LOAEL is 100 mg/kg/d. No systemic effects were reported at 100 mg/kg/d. Thus, the systemic NOAEL for orally administered lactide under the conditions in this study was considered to be 100 mg/kg/day.

This subchronic study in dog is acceptable and satisfies the principle requirement for a subchronic oral study similar to OECD 409 in dog.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

Principles of method if other than guideline:

Dose range finding study: In a two weeks study, two dogs per sex were assigned to each of six treatment groups (dosed with lactide at 0, 10, 100, 400, 1000 and 2500 mg/kg body weight/day). Each dog received daily oral doses of one to six gelatin capsules containing lactide. Doses were administered at approximately 1 hr after feed was withdrawn, and at approximately the same time each day for 14 days. Control dogs received the same number of empty capsules as dogs of the corresponding sex in the highest dose group. Doses were calculated based on the body weight of each dog during the previous week.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): lactide; 18:1 mixture of L-lactide (CAS no. 4511-42-6): m-lactide (CAS no. 13076-19-2)
- Lactide was manufactured and provided by Cargill, Inc USA
- bulk chemical stored at -20°C

For dose administration:
- removal from -20°C, 2 hours to reach ambient temperature
- lactide was loaded into 0.5 oz gelatin capsules
- each capsules contained between 0.1 g and 4 g of lactide
- no filling material was used to fill those capsules that contained less than 4g of chemical
- Capsules were prepared weekly, were stored on capsule boards, refrigerated in plastic bags containing desiccant
--> a prior stability study indicated that lactide was stable for at least 14 days when stored under these conditions
- Capsules were removed from refrigerator and allowed 2 hours to reach ambient temperature before dose administration

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc ( Madison, WI, USA)
- Age at study initiation: ranged from 5 to 10 months
- Fasting period before study: 1 hour before dosing
- Housing: individual housed in stainless-steel cages on racks and were exercised at least twice weekly throughout the quarantine and study period
- Diet (e.g. ad libitum): dogs were fed Certified Canine Chow 5007 (PMI Feeds, Inc) for approximately 2 hours each day
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26°C
- Humidity (%): 35 - 85%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: capsule

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Each dog received daily oral doses of one to six gelatin capsules containing lactide. Doses were administered at approximately 1 hr after feed was withdrawn, and at approximately the same time each day for 14 days.
Control dogs received the same number of empty capsules as dogs of the corresponding sex in the highest dose group. Doses were calculated based on the body weight of each dog during the previous week.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

14 days

Frequency of treatment:

Single daily dose

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Dose / conc.:

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

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

2

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Observations:
- Dogs were observed twice daily for mortality or moribundity.
- Cageside observations were performed daily, approximately 1 hr after dosing.
- Once a week, each dog was removed from its cage and examined closely for detailed clinical signs of toxicity.
- Throughout each study:
dogs were weighed weekly, and food consumption was measured once a week over a 2-hour period.

Clinical pathology:
- blood and urine samples were obtained from each dog for clinical pathology and urinalysis determinations during quarantine and on the day of terminal sacrifice.

Dogs were fasted overnight prior to blood collection for haematologic, clinical chemistry and coagulation analyses

Haematologic analyses included:
total leucocyte count, erythrocyte count, haemaglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count and differential leucocyte count. Reticulocyte counts and red blood cell morphology were evaluated.

Clinical chemistry analysis included:
By using a Roche Cobas Fara clinical chemistry analyser: blood urea nitrogen, creatinine, serum glucose, serum aspartate aminotransferase, serum alanine aminotransferase, alkaline phosphatase, glutamyl transferase, sodium, potassium, chloride, calcium, inorganic phosphate, total protein, albumin, albumin/total protein ratio, total bilirubin and cholesterol.

Coagulation analyses included:
Prothrombin time and activated partial prothrombin time, fibrinogen

Urine analyses included:
Urine specific gravity, urine microscopic sediment, urine pH, ketones, protein, glucose, bilirubin and occult blood

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

- Groups means and standard deviations for: body weights and food consumption.
- Body weights and food consumption were evaluated by two-way repeated ANOVA, and if significant by Dunnett´s test.
- Mean body weights were compared to those of the control group by a two-tailed Students t-test for each sex.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Emesis occured in the two highest dose groups of both sexes. Diarrhoea was also observed, occuring in one male dog in the 100 mg/kg group and inboth female dogs in the 2500 mg/kg group. One female dog that experienced a high incidence of emesis was also observed to be hypoactive on days 4-14 of the study

BODY WEIGHT AND WEIGHT GAIN
Male dogs in the 1000 mg/kg group and dogs of both sexes in the 2500 mg/kg group showed a net loss between day 1 and terminal sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was slightly depressed for male dogs in the 1000 mg/kg group during the second week and for male dogs in the 2500 mg/kg group during both weeks of the study

HAEMATOLOGY
no effect

CLINICAL CHEMISTRY
no effect

URINALYSIS
no effect

ORGAN WEIGHTS
A decrease in absolute and relative thymus weight (> 60% reduction) was observed in one male dog in the 1000 and in all dogs of the both sexes in the 2500 mg/kg dose group. A decrease in absolute and relative spleen weights were observed in one male dog in the 1000 and in both male dogs in the 2500 mg/kg day dose group.

GROSS PATHOLOGY
Gross lesions indicative of irritation were present in one female and both male dogs in the 400 mg/kg group and in all four dogs in the 2500 mg/kg group, but were abesent in the 1000 mg/kg group.These lesions included dark discoloration of the stomach and focal stomach lesions.

HISTOPATHOLOGY: NON-NEOPLASTIC
Lesions in the stomach, oesophagus and kidneys of dogs of both sexes were observed. Minimal or mild haemorrhage involving the mucosa or submucosa of the stomach was observed in one dog of each sex in the 400 mg/kg group and in one female and both male dogs in the 2500 mg/kg group. Stomach ulceration was seen in one male and both females dogs in the 2500 mg/kg group. Oesophageal mild ulceration was present in one 1000 mg/kg female dog and oesophagal erosion was present in one 2500 mg/kg female dog. Mild to moderate renal tubular regeneration was present in both male and both female dogs from the high dose group. Atrophy of the spleen and thymus was evident in male dogs from the 1000 mg/kg group and in dogs of both sexes from the 2500 mg/kg group.

Dose descriptor:

LOAEL

Remarks:

(systemic)

Effect level:

2 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: renal toxicity (Mild to moderate renal tubular regeneration)

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: renal toxicity (Mild to moderate renal tubular regeneration)

Dose descriptor:

LOAEL

Remarks:

(local)

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: irritation of the alimentary tract

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: irritation of the alimentary tract

Critical effects observed:

not specified

Table 1: Overview effects

		dose in mg/kg/d (2wk)
		0	10	100	400	1000	2500
Clinical signs	emesis*	-	-	-	-	4/4	4/4
Body weights	Reductions in body weight gain*	-	-	-	-	2/4	4/4
Organ weights	Reductions in absolute and relative thymus weights*	-	-	-	-	1m/4	4/4
	reductions in absolute and relative spleen weights*	-	-	-	-	1m/4	2m/4
Gross and microscopic lesions	Minimal or mild haemorrhage involving the mucosa and/or submucosa of the stomach*	-	-	-	1f1m/4	-	1f2m/4
	Stomach erosion of minimal severity*	-	-	-	-	-	1m/4
	Stomach ulceration*	-	-	-	-	-	1m2f/4
	Mild oesophageal ulceration/oesophageal erosion*	-	-	-	-	1f/4	1f/4
	Congestion of the small intestinal mucosa*	-	-	-	1m/4	-	4/4
	Mild to moderate renal tubular regeneration	-	-	-	-	-	4/4
Clinical pathology	Blood: total leucocyte count, erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, and dierential leucocyte count.	-	-	-	-	-	-
Clinical chemistry	Blood: blood urea; nitrogen, creatinine, serum glucose, serum aspartate ;aminotransferase, serum alanine aminotransferase, alkaline phosphatase, g-glutamyl transferase, sodium, potassium, chloride, calcium, inorganic phosphate, total protein, albumin, albumin/total protein ratio, total bilirubin and cholesterol. Prothrombin time and activated partial prothrombin time	-	-	-	-	-	-
	Urine: specifc gravity; urine microscopic sediment; pH, ketones, protein, glucose, bilirubin, and occult blood	-	-	-	-	-	-
*) effects considered to be related to irritation of the alimentary tract; m: male; f: female

Conclusions:

The primary toxic effect of lactide in dogs under the experimental conditions employed was irritation of the alimentary tract starting at 400 mg/kg/d. In addition, lactide appeared to exhibit renal toxicity. Regeneration of the renal tubular epithelium, frequently seen as a reparative or adaptive change following tubular epithelial necrosis, was observed in dogs of both sexes at the 2500 mg/kg/d.

Executive summary:

In a subacute oral toxicity (dose range finding) study, lactide (18:1 mixture of l-lactide and m-lactide) was administered to 2 beagle dogs/sex/dose by capsule at dose levels of 10, 100, 400, 1.000 and 2.500 mg/kg bw/day for 2 weeks. The primary toxic effect of lactide in dogs was irritation of the alimentary tract. As irritating effects occurred down to a daily dose of 400 mg/kg bw, a (local) LOAEL can be determined with 400 mg/kg bw/d. At a daily dose of 100 mg/kg bw/d no lesions were noted by gross and histopathology. Thus, the (local) NOAEL can be determined with 100 mg/kg bw/day.

At 1.000 and 2.500 mg/kg/d effects on body weight, and absolute and relative organ weights were reported in thymus and spleen. These effects were considered to be related to the irritation of the alimentary tract. In contrast, a mild to moderate renal tubular regeneration was reported in all animals of the 2.500 mg/kg/d dose. Regeneration of the renal tububar epithelium is frequently seen as a reparative or adaptive change following tubular epithelial necrosis, and is suggestive of prior damage to this tissue. Although the mechanism of this effect is unknown, it cannot be excluded it is based on lactide toxicity. Based on the possible renal toxicity the (systemic) LOAEL is 2.500 mg/kg bw/day. The (systemic) NOAEL for orally administered lactide to dogs under the conditions of this study is 1.000 mg/kg/day.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Details on results:

All observed effects could be attributed to calcium overload/imbalance. No lactate toxicity was observed.

Dose descriptor:

NOAEL

Effect level:

50 000 mg/L drinking water

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

Conclusions:

5% calcium lactate in drinking water or diet does not result in adverse effects attributable to lactate.

Executive summary:

In a subchronic toxicity study (similar to OECD 408), Calcium lactate was administered to Fischer 344/DuCrj rats.

In Experiment I, Calcium lactate was mixed at 5, 2.5, 1.25, 0.6, and 0.3 % in the drinking water and the rats were given this solution ad libitum for 13 weeks. As a result, the inhibition of body weight gain in the 5 % group fell within 10 % of that in the control group. Some examination values showed variations in the hematological and hematobiochemical studies, but no controversial findings were obtained in the pathohistological search. Since the highest solubility of Calcium lactate is 5%, experiments II and III were carried out by giving blended diet in order to study the toxicity at higher doses. In experiment II, Calcium lactate was mixed at concentrations of 30, 20, 10, and 5 % in the B-blend powder diet and then the rats were given this diet ad libitum for 20 weeks. In experiment III, the rats were given the CRF-1 or the B-blend powder diet ad libitum for 8 weeks. As a result, in experiment II, nephrocalcinosis was observed in all the groups including the control group. The degree of the lesion was in reverse correlation with the administered concentrations of calcium and the lesion was seen more intensely in female rats. In experiment III, nephrocalcinosis resulting from the administration of the B-blend diet was already observed in the 4th week. Nephrocalcinosis as observed in experiments II and III was attributable to the small Ca/P value in the B-blend diet.

From the above results, the optimal dose for a long-term toxicity/carcinogenicity study has been determined to be 5 and 2.5 % based on the values obtained from experiment I.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

dog

Quality of whole database:

comparable to Guideline study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

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

Lactide is rapidly hydrolysed to lactic acid in water and in-vivo (gastric acid). Lactic acid is a ubiquitous and essential molecule of life. Lactate is non-toxic, any (local) effects are due to pH effects only.

In addition, in an oral toxicity (dose range finding) study, lactide (18:1 mixture of l-lactide and m-lactide) was administered to beagle dogs by capsule at dose levels of 10, 100, 400, 1.000 and 2500 mg/kg bw/day for 2 weeks, and 0, 4, 20 and 100 mg/kg/d for 13 weeks. The primary toxic effect of lactide in dogs was irritation of the alimentary tract. As irritating effects occurred down to a daily dose of 400 mg/kg bw (for 2 wks), the sub-chronic study was run with a maximum dose of 100 mg/kg bw/d.

At 1000 and 2500 mg/kg/d effects on body weight, and absolute and relative organ weights were reported in the 14-day dose range finding study for thymus and spleen. These effects were considered secondary to the irritation of the alimentary tract. In addition, a mild to moderate renal tubular regeneration was reported in all animals of the 2500 mg/kg/d dose. Regeneration of the renal tubular epithelium is frequently seen as a reparative or adaptive change following tubular epithelial necrosis, and is suggestive of prior damage to this tissue. Although the mechanism of this effect is unknown, lactide toxicity cannot be excluded. Based on the possible renal toxicity the (systemic) LOAEL is 2500 mg/kg bw/day. That's well above the Limit Dose of 1000 mg/kg/d for a sub-acute toxicity study.

No systemic adverse effects were reported at the highest dose tested in the 90 d study (100 mg/kg/d). Therefore, the (systemic) NOAEL for orally administered lactide in a 90d study in dog is considered to be 100 mg/kg/day. The primary toxic effect after oral dosing was irritation of the gastrointestinal tract at 100 mg/kg/d.

In addition, in an oral subchronic repeated dose toxicity study conducted with a suitable read-across partner, calcium lactate was used to assess the specific target organ toxicity of the target substance. High levels of exposure to calcium lactate in the diet (up to 30% in feed) did not lead to any lactate-associated effects after sub-chronic exposure; all effects observed were due to the excessive intake of calcium. It could be concluded that any deleterious effects of lactic acid are caused by the effect of lactic acid on local pH, not on any biological effect of lactate. High dermal exposure to lactate, in cosmetic creams, did not lead to any effects, with the exception of slight irritation at the highest doses.

In addition, lactic acid is a natural metabolite found in humans and animals as it is endogenously produced from metabolic pathways such as glycogen breakdown, glycolysis and amino acid metabolism. Normal human blood contains 8-17 mg lactic acid/100 mL plasma, and the concentration of lactate in normal human skin is three times or more of that in the blood due to glycolytic enzymes, which actively convert glucose to lactic acid in the epidermis. Lactic acid has been detected in blood but also in several other body fluids and tissues. Concentrations of lactic acid increase significantly during intense exercise. At rest, blood concentrations have been reported of 1-1.5 mMol/L (90.1-135.12 mg/L), which can increase up to 10 mMol/L (900.8 mg/L) during exercise. External human exposure to lactic acid can occur via its natural presence in food, for example in fruit, vegetables, sour milk products, and fermented products such as sauerkraut, yogurt and beer. Based on the available information on concentrations of lactic acid in some of these products, an estimate of the daily consumption of lactic acid due to its natural presence in food was made using the ‘FAO/WHO standard European diet’. A (minimum) daily intake of 1.175 g/person/day was calculated using the available information. Another source of external exposure is its use as food additive; as such it is authorized in Europe (E270) and the United States (generally recognized as safe = GRAS). A daily intake of 1.65-2.76 g/person/day was estimated using the “Per Capita times 10” method, based on the amount of lactic acid placed on the market (EU and USA) as a food additive by Purac. Due to its role as a common and natural food ingredient, and ubiquitous metabolic product/substrate in mammals with proven low toxicity, it can safely be concluded that lactic acid does not contribute to any systemic effects. Lactic acid is a major and essential species in mammalian primary metabolism, and a ubiquitous ingredient in all kinds of food. There is no way of lowering exposure below minimum required levels or normal (or even abnormal) internal levels.  Furthermore, lactic acid bacteria, which produce lactic acid as their major fermented product, are abundantly found in humans, including breast milk and vaginal cavity, and are considered as probiotic bacteria. Infants can be exposed to lactic acid bacteria during breastfeeding. Lactic acid bacteria are known to be beneficial in preserving healthy vaginal functions such as maintenance of acidic vaginal pH and the prevention of infections.

The results obtained from sub-chronic toxicity studies (IUCLID section 7.5.1) and from a chronic toxicity study (IUCLID section 7.7) showed that there is no lactic acid specific adverse systemic effect after repeated exposure.

Altogether, given the existing data and taking a weight-of-evidence approach, there is no systemic toxicity concern of lactic acid/L-lactide and no classification is warranted.

Justification for classification or non-classification

L-lactide is to be considered non-toxic; the only effect are due to local irritation in the gastrointestinal tract as a consequence of the formation of lactate derivatives including lactoyl lactic acid and monomeric lactic acid. Therefore, based on the available data and according to CLP Regulation 1272/2008, L-lactide does not warrant classification for repeated dose toxicity.