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

Repeated dose toxicity: oral

Administrative data

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

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. pp. 335-342

Materials and methods

Test guideline

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 Doses were administered at approximately 1 hr after feed was withdrawn, and at approximately the same time each day for 13 weeks.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

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 - 85%
- 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 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

mid dose

Doses / concentrations 4

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

Examinations

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

Results and discussion

Results of examinations

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

Effect levels

1

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

2

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

3

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.