1,4-Dioxane-2,5-dione, 3,6-dimethyl-, (3R,6R)-

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Toxicity of lactide in dogs after 2 and 13 weeks of daily oral dosing

Author:

Hebert C.D. et al.

Year:

1999

Bibliographic source:

Food and Chemical Toxicology 37, 335-342

Materials and methods

Test guideline

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 Doses were administered at approximately 1 hr after feed was withdrawn, and at approximately the same time each day for 14 days.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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

224-832-0

EC Name:

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

Cas Number:

4511-42-6

Molecular formula:

C6H8O4

IUPAC Name:

3,6-dimethyl-1,4-dioxane-2,5-dione

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

Test animals

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 (%): 35-8 %
- Photoperiod (hrs dark/hrs light): 12/12

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 6

Dose / conc.:

2 500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

2

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Observations:
- Dogs were observed twice daily for mortality or moribundity.
- Cageside observations were performed daily, approximately 1 h 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 Student's t-test for each sex.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

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 in both 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.

Effect levels

1

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)

2

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)

3

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

4

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Overview of effects

		dose in mg/kg/d (2 wk)
		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 moderaterenaltubularregeneration	-	-	-	-	-	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

Applicant's summary and conclusion

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, 1000 and 2500 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/day a (local) LOAEL can be determined with 400 mg/kg bw/day. At a daily dose of 100 mg/kg bw/day no lesions were noted by gross and histopathology. Thus, the (local) NOAEL can be determined with 100 mg/kg bw/day.

At 1000 and 2500 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 2500 mg/kg bw/day. The (systemic) NOAEL for orally administered lactide to dogs under the conditions of this study is 1000 mg/kg/day. L-lactide is the enantiomer of D-lactide and therefore suitable as read-across partner.