Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Published study

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., Giles, H.D., Heath, J.E., Hogan, D.B., Modderman, J.P. & Conn, R.E.
Year:
1999
Bibliographic source:
Food and Chemical Toxicology, 37. pp. 335-342

Materials and methods

Principles of method if other than guideline:
In the 2-wk 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). Doses for the 13-wk study were selected based on the results of the 2-wk 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 Doses were administered at approximately 1 hr after feed was withdrawn, and at approximately the same time each day for 14 days or 13 wk. 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:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
Pure-bred beagle dogs were purchased from Harlan Sprague Dawley, Inc. (Madison, WI, USA). Dogs were quarantined for a minimum of 2 wk after arrival, during which time a physical examination was conducted on each animal. The age of the dogs ranged from 5 to 10 months on day 1 of dosing. 24 dogs (12/sex) were used in the 2-wk study, and 32 dogs (16/sex) were used in the 13-wk study. Individual animals were identified by ear tattoo. Dogs were fed Certified Canine Chow #5007 (PMI Feeds, Inc.) for approximately 2 hr each day during the quarantine and study periods, and tap water was available ad lib. The dogs were individu- ally housed in stainless-steel cages on racks and were exercised at least twice weekly throughout the quarantine and study periods. Environmental conditions in the animal rooms were maintained at approximately 20±268C (68±788F) and 35±85% relative humidity, with 12 hr of fl̄uorescent light per day.

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 or 13 wk. 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
13 weeks
Frequency of treatment:
Single daily dose
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 10, 100, 400, 1000, and 2500 mg/kg bw/d for 14 days.
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 4, 20, 100 mg/kg bw/d for 13 weeks.
Basis:
actual ingested
No. of animals per sex per dose:
12 (14 days)
16 (13 weeks)
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
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-hr period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
In the 2-wk study, group means and standard deviations were calculated for body weight and food consumption data. However, owing to the small number of animals/sex in each group of the 2-wk study, statistical analysis was not performed on clinical pathology and organ weight data. In the 13- wk study, group means and standard deviations were calculated for body weights, food consumption, clinical pathology parameters, and for terminal body and 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. Analysis included change over time. Mean body weights, mean organ weights, and organ/body and organ/brain weight ratios for each treated group 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:
no effects observed
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
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:
no effects observed
Details on results:
Effects were primarily seen at the two highest dosages in the 14-day exposure regimen. All observed effects were attributable to the irritant nature of lactide - no direct systemic toxicity was involved.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
13 weeks
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
14 days
Effect level:
400 mg/kg bw/day (actual dose received)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Lactide acts primarily, if not only, as an irritant after oral administration. 14 day NOAEL is 400 mg/kg bw/d. 13 week NOAEL is 100 mg/kg bw/d.
Executive summary:

The primary toxic effect of lactide in dogs under the experimental conditions employed in these two studies was irritation of the alimentary tract. This was evidenced clinically by emesis, which occurred in male and female dogs in the two highest dose groups in the 2-wk study, and by diarrhoea, which occurred sporadically in female dogs in the 2500 mg/kg group in the 2-wk study. The decreased food consumption and accompanying weight loss (or deficits in body weight gain) that were observed in high-dose animals of both sexes in the 2-wk study were probably the result of irritation of the alimentary tract, and the hypoactivity observed in one female dosed at 2500 mg/kg was considered to be a secondary effect caused by irritation of the gut. The diarrhoea that occurred in one male dog in the 100 mg/kg dose group in the 2-wk study was not considered to be related to administration of lactide because the diarrhoea (a) did not occur in a dose- related manner, (b) occurred only once in this dog, and (c) was not accompanied by gross or histopathologic lesions that would indicate irritation of the gastrointestinal tract in this dog. Because the emesis and diarrhoea observed in the 13-wk study occurred with a sporadic and non-dose-related pattern, they were not considered to be related to lactide toxicity. The gross and histopathologic lesions observed in both these studies, including discoloration of the oesophagus and stomach, foci in the stomach, and haemorrhage, ulcer and erosion of the oesophagus and stomach, confirmed the irritant effect of the lactide on the gastrointestinal tract that had been observed previously (Cargill, Inc., 1993a). In addition, lactide appeared to exhibit renal toxicity in the 2-wk study, although the mechanism of this effect is unknown. 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 dose in the 2-wk study, and was suggestive of prior damage to this tissue. However, necrosis of the epithelium was not noted in any of the dogs in this study. Nevertheless, because regener- ation was evident in all dogs in the highest dose group, but not in any other dose group, the effct was considered to be related to lactide treatment. There was no evidence of kidney toxicity in any of the dogs in the 13-wk study. Lymphoid atrophy, especially of the thymus and spleen, is a common observation in toxicology studies where animals lose weight, experience a decrease in food intake, or are otherwise stressed. Because irritation of the alimentary tract is likely to produce discomfort and stress, the thymic and splenic atrophy and the concomitant decreases in absolute and relative weights of these organs observed in the 1000 and 2500 mg/kg dose groups in the 2-wk study were considered to be secondary, stress- induced effects rather than primary toxic effects of lactide. The data described above agree with the results of previous studies in rats and rabbits that indicated a very low acute toxicity for lactide (Cargill, Inc., 1993a,b). The dark discoloration of the stomach mucosa seen in the dogs in these two studies was similar to that described in rats treated with single oral doses of lactide at 5000 mg/kg, and was consistent with the known irritant effects of the compound. Based on these results, the primary toxic effect of lactide after subchronic oral dosing in dogs was irritation of the gastrointestinal tract. The effects observed in this study extend our understanding of the non-specific response of a test species to a highly acidic substance administered in a localized high dose. Previous toxicological investigations with orally administered lactide provided information on the acute response of rodents (Cargill, Inc., 1993a), and this study provides data both on the response of another species and on adminis- tration of the compound in capsules. Lactide is known to be unstable in aqueous solution and breaks down quickly to form the highly acidic compound lactoyl lactic acid (the open-ring lactic acid dimer). In animals administered lactide in gelatin capsules, the compound is released in a bolus dose following dissolution of the gelatin capsule in the stomach, and is cleaved to form various lactate derivatives including lactoyl lactic acid and monomeric lactic acid, creating a strongly acidic environment. It is significant to note that irritation was the only toxicologic effect seen in dogs in the subchronic study, and that there was no evidence of other systemic toxicity at any dose after 13 wk of dosing. The irritation observed in this study was similar to that described for other acidic substances administered orally, and it is probable that the irritation is due to acidity of the cleavage products rather than to any inherent toxicity of the lactide itself. Lactate substances have been extensively tested and accepted as safe food ingredients (FASEB, 1978). Indeed, the absence of systemic toxic effects in this study is consistent with the safety of lactic acid in its various exposures as an endogenous product of mammalian metabolism, as a naturally occurring constituent of food, and as an added food ingredient. Because the only apparent toxic effect at the 100 mg/kg/day dose in the 13-wk study was gastrointestinal irritation, the NOAEL for orally administered lactide under the dosing conditions employed in that study was considered to be 100 mg/kg/day.