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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: no deviation from OECD guideline (OECD 411). Not GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): DGA
- Substance type: clear, colorless
- Physical state: liquid
- Lot/batch No.: 9F10
- Expiration date of the lot/batch: 15 July 2001
- Stability under test conditions: stability information was not provided
- Storage condition of test material: room temp

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, strain Hsd:SD
- Age at study initiation: 6 weeks
- Weight at study initiation: 143-247 grams
- Fasting period before study:
- Housing:
* before the study: rats were groups-housed by sex
* during the study: animals were housed individually in stainless steel cages
- Diet (e.g. ad libitum): Teklad Certified LM-485 rodent diet, ad libitum, except overnight prior to scheduled blood collectiong
- Water (e.g. ad libitum): ad libitum
- Acclimation period: min 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70% (during 2 days of the study, relative humidity was outside this range. However, this is not considered to have had any adverse
effect on the outcome of this study)
- Photoperiod (hrs dark / hrs light): 12h light, 12h dark


Administration / exposure

Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: between 10 and 20% of the body surface
- Type of wrap if used: gauze pad, rubber dam and an elastic bandage
- Time intervals for shavings or clipplings: minimum of twice weekly


REMOVAL OF TEST SUBSTANCE
- Washing (if done): gently cleansed with gauze soaked in warm water and gently dried
- Time after start of exposure: 6h


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 ml/kg bw /d
- Concentration (if solution): 0 - 17- 87- 175 mg/kg bw/d
- Constant volume or concentration used: yes


VEHICLE = deionized water
- Amount(s) applied (volume or weight with unit): 0.5 ml/kg bw/d
- Lot/batch no. (if required): 071099, 201099, 011199, 091199, 171199, 221199, 031299, 081299, 151299, 171299, 281299


USE OF RESTRAINERS FOR PREVENTING INGESTION: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
nominal concentration (mg/kg bw/d): 0 - 50 - 250 - 500 respectively
actual concentration (mg/kg bw/d): 0 - 17 - 87 - 175 respectively
Duration of treatment / exposure:
approximately 6h
Frequency of treatment:
once daily, 90 consecutive days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
17
Basis:
analytical per unit body weight
Remarks:
Doses / Concentrations:
87
Basis:
analytical per unit body weight
Remarks:
Doses / Concentrations:
175
Basis:
analytical per unit body weight
No. of animals per sex per dose:
10 male and 10 female rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose level selected by the sponsor
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no satellite group
Positive control:
no data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: once daily


BODY WEIGHT: Yes
- Time schedule for examinations:
at the time of randomisation
prior to dose administration on day 1
weekly (after that)
on day 91 (fasted)


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (weekly)


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before treatment + prior to terminal sacrifice
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 91, prior to terminale sacrifice
- Anaesthetic used for blood collection: Yes, CO2
- Animals fasted: Yes , overnight
- How many animals: all surviving animals (= all animals, 80)
- Following parameters were examined.
* Hematology: differential white blood cell count, hematocrit, hemoglobin, mean corpuscular hemoglobin,
mean corpuscular hemoglobin concntration, mean corpuscular volume, platelet count, red blood cell count and morphology,
white blood cell count
* Coagulation: prothrombin time, acctivated partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:on day 91, prior to terminale sacrifice
- Animals fasted: Yes , overnight
- How many animals: all animals (80)
- Following parameters were examined:
* serum clinical chemistry: alanine aminotransferase, albumin, albumin/globulin ratio (calculated), aspartate aminotransferase, calcium,
chloride, cholesterol, creatinine, creatine phosphokinase, globulin (calculated), glucose, phosphorus, potassium, sodium, total bilirubin,
total protein, triglycerides, urea nitrogen


URINALYSIS: Yes
- Time schedule for collection of urine: on day 90, urine was collected overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined:
* volume, specific gravity, appearance/color, semi-quantitative estimation: pH, protein, glucose, ketone, urobilinoen, bilirubin,
blood, leukocytes, nitrites, microscopic examination of spun deposit

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on day 28 and day 90 during treatment
- Dose groups that were examined: all
- Battery of functions tested: observation of animals / sensory activity / grip strength / motor activity / other: loss of righting reflex,
spontaneous locomotor activity, right pupil examination, various reflex responses


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
external surface of the body, all orifices, cranial, thoracic and abdominal cavities together with their content

HISTOPATHOLOGY: Yes
gross abnormalities, adrenals, aorta, whole brain, cecum, colon, duodenum, epididymides, esophagus, exorbital lachrymal gland,
eyes w/optic nerve, femur, fat (mesentery), heart, ileum, jejunum, kidneys, liver, lungs with mainstem bronchus, mammary gland(s), mesenteric
lymph nodes, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (mandibular lymph nodes), sciatic nerve, seminal vesicle(s),
skin (with subcutis from a site other than the treated site), spinal cord at three levels - cervical, midthoracic, lumbar - spleen, sternum with bone
marrow, stomach, testes, thigh musculature (skeletal muscle), thymus, thyroids/parathyroids, tongue, trachea, treated site (dorsal thoracic region
with subcutis), urinary bladder, uterus, vagina

Statistics:
evaluation of equality of means: one-way analysis of variance usiing the F distribution to assess statistical significance
is differences between the means are statistically significant, Dunnett's test was used to determine the degree of significance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
* no animals died during the study
* no clinical signs of toxicity observed during the study
* clinical signs of dermal irritation were noted.
-Erythema and edema of varying degrees was observed in both male and female 87 and 175 mg/kg bw/d groups.
-Very slight erythema first appeared on day 6, 7 or 8 of 87 - 175 mg/kg bw/d groups.
-Very slight edema first appeared on day 7 in females recieving 175 mg/kg bw/d and progressed to severe edema by the end of the study.
-Very slight edema was seen on days 28, 38 or 33 respectively in females (87mg/kg bw/d) and males (87 or 175 mg/kg bw/d). This progresses to moderate to severe during the following 90 days of treatment. There was slightly more eythema and edema in females (87 mg/kg bw/d) compared to males recieving the same dose.
- additional signs noted in the male/female 87 and 175 mg/kg bw/d dose groups were all related to irritation at the application site and included scab formation, sloughing, and black areas on the dosing site.


BODY WEIGHT AND WEIGHT GAIN
* no test article-related differences in group mean bw or body weight gains throughout the study


FOOD CONSUMPTION
* no test article-related differences in group mean food consumption throughout the study


FOOD EFFICIENCY
* no data


WATER CONSUMPTION
* no data


OPHTHALMOSCOPIC EXAMINATION
* no test article-related differences in ophthalmology examination, conducted during the final week of treatment


HAEMATOLOGY
* Females, 87 mg/kg bw/d: statistically significant increase in absolute and relative neutrophil counts
* no test article-related differences in erythrocyte morphology for males or females
* no test article-related differences in hematology for males


CLINICAL CHEMISTRY
* males, 175 mg/kg bw/d + females, 87 and 175 mg/kg bw/d: statistically significant increases in globulin + decreases in albumin/globulin ratios
* all other stat. significant differences were withing normal historical ranges : were not considered to be test article-related


URINALYSIS
* no test article-related changes in any of the urinalyses parameters observed in M or F rates at the end of the treatment period


NEUROBEHAVIOUR
* no test article-related neurotoxicity observed on day 28 or day 90.


ORGAN WEIGHTS
* no test article-related differences in absolute organ weights, relative organ to body weight ratios, or relative organ to brain weight-ratios
following 90 d of treatment.


GROSS PATHOLOGY
* scab formation of varying degrees was observed at the treatment site of males and females receiving 87 or 175 mg/kg bw/d (see table 9, p. 148)
* varous gross lesions on the skin at the treatment site were test article-related in male and females receiving 87 or 175 mg/kg bw/d
(namely respecitvely in 8/10 males and 10/10 females in 87 mg/kg bw/d dosing group; and 9/10 males and 9/10 females in 175 mg/kg bw/d).

HISTOPATHOLOGY: NON-NEOPLASTIC
test article-related microscopic changes were limited to the site of exposure and included ulceration, epidermal hyperplasia, fibrosis and
inflammation. there was some variation in the severity of these changes, however: most of the males and females in 87 - 175 mg/kg bw/d groups were affected with one or more of these changes. No evidence of a similar effect was seen in the control group and the lowest dose group.



Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
dermal effects
Effect level:
>= 17 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Dose descriptor:
NOAEL
Remarks:
systemic effets
Effect level:
>= 175 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: general systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Application of DGA to an intact cutaneous site for approximately six hours, once daily for 90 consecutive days to male and female Sprague-Dawley rats, results in ulceration, epidermal hyperplasia, fibrosis and/or inflamation at doses of 87 and 175 mg/kg bw/d. These changes represent local irritation following topical administration.
Based on the results of the study, the dermal NOAEL was at least 17 mg/kg bw/d while the systemic NOAEL was at least 175 mg/kg bw/d.