Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 15, 1980 to August 8, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was conducted under Good Laboratory Practice standards. The study was performed prior to the adoption (12 May 1981) of OECD test guideline 410 "Repeated Dose Dermal Toxicity: 21/28-day study"; however, the data are well documented and scientifically acceptable.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male and female rabbits were administered the test material on shaved intact or abraded skin. Following application of the test material, the application site was occluded for 6 hours , at which time the test material was wiped off. Animals were administered the test material 5 days per week for 3 weeks. Clinical signs, mortality, body weights, haematology, clinical chemistry, organ weights, gross pathology and histopathology were evaluated for all animals.
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): Diethyltoluamine (diethyltoluenediamine, DETA)
- Physical state: Brown liquid
- Analytical purity: 95.78%
- Composition of test material, percentage of components: 80:20 ratio of 2,4- and 2,6-diethyltoluenediamine

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Small Stock, Inc., Pea Ridge, Arkansas
- Age at study initiation: approximately 14 weeks old
- Weight at study initiation: males (2.89-2.91 kg), females (2.81-2.82 kg)
- Housing: individually housed in stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 to 23.3 °C
- Humidity (%): 35 to 55%
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: July 15, 1980 To: August 8, 1980

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Type of wrap if used: The test material was covered with a square of qauze (two single layers thick) and secured with surgical hypoallergenic adhesive tape. The gauze was covered with a larger square of clear plastic secured with adhesive tape.
- Time intervals for shavings or clipplings:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The test material was wiped off with gauze.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The volume of test material applied was adjusted as required twice weekly based on the most recent body weight. The mean total amount administered per animal was 0.046 +/- 0.0025 ml (males) and 0.045 +/- 0.0026 ml (females) for the 1.0 mg/kg group, 0.466 +/- 0.028 ml (males) and 0.453 +/- 0.291 ml (females) for the 10 mg/kg group, and 4.687 +/- 0.300 ml (males) and 4.501 +/- 0.266 in (females) for the 100 mg/kg group (all volumes are mean +/- S.D.). No solution was applied to the control rabbits.
- Constant volume or concentration used: no

VEHICLE
-No vehicle was used. The test material was applied, undiluted.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Three weeks (21 days)
Frequency of treatment:
Daily (five days/week)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1.0 mg/kg
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
10 mg/kg
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal per unit area
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
The backs of the rabbits were shaved free of hair using electric clippers with an angra head. The last five rabbits of each sex on each level were abraded on the shaved area using a nylon bristle brush. Rabbits were shaved and abraded weekly, at least the day before application of the next dose or earlier.

At the time of dosing, plastic collars were placed on the animals. The volume of material to be applied was determined from the animals' most recent body weight and was applied undiluted to the shaved area on the back. Dosage volumes were measured and applied using a 5 microliter Hamilton syringe for the 1 mg/kg group, a 50 microliter Hamilton syringe for 10 mg/kg group, and a 500 microliter Hamilton syringe for 100 mg/kg group.

The material was then covered with a square of gauze large enough to cover the liquid, two single layers thick, which was secured with surgical hypoallergenic adhesive tape. The gauze was covered with a larger square of clear plastic secured with adhesive tape. The animals were returned to their cages after application of the test material.

After six hours of exposure, the plastic, gauze and tape were removed and the treated area was wiped with gauze to remove any remaining test material. The collar was removed and the animal was returned to the cage.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
The animals were observed twice daily on weekdays when material was applied and removed for the appearance of toxicological and pharmacological signs; animals were observed once daily on weekends. Dermal irritation readings according to the method of Draize et al. (1965) were made daily immediately prior to dosing, including the first day of treatment. Body weights were determined at the beginning of the test and every three to four days thereafter. Surveillance was made to preclude loss of animals due to autolysis, misplacement or other management problems.

Reference: Draize et al. (1944) Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharm. Exp. Ther., Vol. 82, pp. 337-390.
Sacrifice and pathology:
All test and control animals were subjected to gross necropsy. The Liver, kidneys, heart, gonads, thyroid (with parathyroid), adrenals, lungs, brain and pituitary were weighed. Tissues were collected and fixed in 10% buffered formalin for histopathology examination.
Other examinations:
Animals were already assigned randomly to test or control groups. The first five animal numbers of each sex in each test and control group were then selected for clinical laboratory testing both before initiating the study and on the day of last treatment.

Hematology tests included determinations of hematocrit and hemoglobin concentration, red and white blood cell counts, differential white cell counts and platelet counts.

Blood chemistry tests included determinations of calcium, sodium, potassium, glucose, blood urea nitrogen, total bilirubin, total cholesterol, albumin, globulin and total protein concentrations and of serum lactic dehydrogenase, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase and serum alkaline phosphatase activities.
Statistics:
The Waller-Duncan k-ratio t-test was used to compare results of treated rabbits to those of concurrent controls (Duncan 1975). Differences between the results in a particular group before and after treatment were tested using Student's t-test.

Reference: Duncan, D.V. (1975) t-Tests and intervals for comparisons suggested by the data, Biometrics Vol. 31, pp. 339-359.

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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS
No behavioral abnormalities were observed in any of the rabbits during the study. Occasional coughing, sneezing and/or ocular discharge were observed in some of the rabbits, including controls, and were considered incidental.

BODY WEIGHT
On the first day of treatment, it was observed that a male rabbit to be treated with 100 mg/kg had not eaten for several days. On that same day, a possible replacement was designated. The replacement animal was shaved and treated; a blood sample was collected prior to treatment. On day 3 of treatment, the rabbit that had not eaten for several days appeared thin and had not resumed eating. The results of the clinical studies on the blood sample for the replacement animal were normal, and it was used as a replacement on day 3 of treatment. Since the cessation of food consumption by replaced animal occurred prior to treatment, its condition could not have been related to treatment. No mortality occurred in treated rabbits.

No significant differences were apparent among mean body weights of either sex throughout the study. However, two female rabbits receiving 100 mg/kg and one receiving 10 mg/kg failed to gain weight throughout the study. All of these showed initial weight gain followed by loss. Of the two rabbits receiving 100 mg/kg, one failed to regain her pretest body weight, while the other regained to 0.02 kg higher than her pretest body weight. The female receiving 10 mg/kg exhibited only approximately half of the total average weight gain of her group. No differences in rate of weight gain were apparent among male rabbits-treated at this dosage.

DERMAL IRRITATION
No evidence of skin irritation was observed in control rabbits of either sex. Slight erythema accompanied occasionally by slight edema was observed sporadically in some rabbits in the 1.0 mg/kg group. Rabbits in the 10 mg/kg group exhibited erythema and some edema, and the incidence of these was consistent by the third week of treatment. One female rabbit in the 10 mg/kg group exhibited a scab in the treated area of the skin in the third week of treatment. Rabbits in the 100 mg/kg group exhibited erythema accompanied by very slight to moderate edema through most of the treatment period. The severity of the edema increased during the study. Open wounds were observed in the skin of two males and two females of this group and small scabs were observed on one male and two females with no prior evidence of skin injury.

HEMATOLOGY
Occasional small differences in means of results were observed among groups before treatment; however, the results for all the groups were within the range of normal biological variation. In the results after treatment, occasional differences in hematology values were observed among groups of male rabbits, but these were not outside the range of normal biological variation. In female rabbits, a dose-related decrease in monocyte counts was observed; the counts were significantly different from control females for those receiving 10 and 100 mg/kg DETDA but not for those receiving 1.0 mg/kg. However, the counts in these two groups were within the range of counts (0-11) observed in rabbits of the same sex and approximate age. No other differences of biologic significance were observed in female rabbits.

CLINICAL CHEMISTRY
In rabbits before treatment, no differences in results between groups were observed that were outside the range of normal biological variation. In rabbits after treatment, dose-related decreases in serum calcium and sodium concentrations were observed in both male and female rabbits. The calcium concentrations were statistically significantly different from controls in females receiving 10 or 100 mg/kg DETDA and in males receiving 100 mg/kg. The sodium concentrations were statistically significantly different from controls in all treated groups of females and in males receiving 10 mg/kg DETDA but not those receiving 1 or 100 mg/kg. Decreased serum potassium concentrations were observed in male, but not female, rabbits; however, the results were not significantly different from controls. The cause of the decreases in serum electrolytes was not apparent. Although the decreases were statistically significant and dose-related, none of the electrolytes decreased to levels likely to impair normal physiologic processes. No other differences in chemistry results in male or female rabbits were observed that were outside the range of normal biological variation.

ORGAN WEIGHTS
No statistically significant differences were observed.

GROSS PATHOLOGY
All abnormalities observed at gross necropsy were considered incidental lesions or secondary to euthanasia.

HISTOPATHOLOGY
Chronic dermatitis was observed in treated skin of control and treated rabbits of either sex. The incidence and severity were greater in treated rabbits than in controls; however, the incidence of dermatitis increased with increasing dosage of DETDA. The severity of the lesions increased with dosage in female rabbits but was essentially constant in treated male rabbits. Because dermatitis was observed only occasionally in untreated skin, it was concluded that the dermatitis on treated skin was a direct result of application of DETDA.

Cysts were observed in thyroid glands of rabbits of either sex. The incidence was dose-related in males but not in females. These lesions are common histopathologic findings in rabbits of this age and strain, however, and the cysts observed were probably not a consequence of treatment. The remaining lesions observed occurred randomly in treated and control groups and were considered incidental.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: clinical signs/mortality
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: body weight
Dose descriptor:
NOAEL
Effect level:
>= 1 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: dermal irritation
Dose descriptor:
LOAEL
Effect level:
>= 10 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: dermal irritation
Dose descriptor:
NOEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: haematology
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: haematology
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: clinical chemistry
Dose descriptor:
NOEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: organ weights
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: gross pathology
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Repeated dermal application of DETDA at 1.0 mg/kg resulted in very slight irritation of no biologic significance. Application of 10 and 100 mg/kg resulted in mild to moderate local irritation and open wounds were observed in skin of some rabbits treated at 100 mg/kg. No other significant overt toxicity was observed in treated rabbits.

Decreases in serum calcium and sodium concentrations were observed in some treated groups of either sex; however, the electrolyte concentrations were not reduced sufficiently to interfere with normal physiologic processes.

No abnormalities that could be related to treatment were observed at gross necropsy. Histopathologic examination of fixed tissues revealed dermatitis in treated skin; no other lesions that could be related to treatment werc observed.

Repeated application of DETDA at 1.0, 10 or 100 mg/kg resulted in no significant systemic effects. Application of 10 and 100 mg/kg resulted in significant local effects on treated skin; these effects were more pronounced at the highest dosage. No significant local effects were observed after application of DETDA at 1.0 mg/kg.