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Repeated dose toxicity: dermal

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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 24 to December 20, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD Guideline 410; Study under EPA GLP Regulation 40 CFR &92;

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
14 days exposure period, followed by 14 days recovery period; no separate limit test performed;
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: colorless liquid
Details on test material:
See information in the field "Confidential details on test material"

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)IGS BR
Sex:
male/female

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test articles were applied seven days per week for two weeks to the shaved intact dorsal skin of each rat. The application sites were covered with gauze, occluded with plastic wrap and secured with non-irritating tape for a period of six hours per exposure. Following the exposure period, the application sites were gently washed with a solution of non-
irritating soap and disposable paper towels to remove residual test article. Each test article group consisted of 10 males and 10 females. Dosage levels were 100, 300 and 1000 mg/kg/day. The test article was administered undiluted based on specific gravity at varying dosage volumes. A concurrent control group received deionized water on a comparable regimen. The animals were examined twice daily for mortality and moribundity.

Clinical and dermal observations were performed daily prior to and following exposure and once each day of the recovery period. Detailed physical examinations were performed weekly. Ophthalmological examinations were performed prior to dosing and during study weeks 1 and 3. Clinical pathology evaluations (hematology, serum chemistry and urinalysis) were performed prior to the scheduled primary necropsy. Complete necropsies were performed for all animals and selected organs weighed from animals at the primary and recovery necropsies. Selected tissues were examined microscopically.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Six (6) hours per day, seven (7) days per week for two (2) weeks;
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw;
Basis:
nominal per unit area
No. of animals per sex per dose:
10 male animals per dose;
10 female animals per dose;
excluding satelitte animals;
Control animals:
yes
Details on study design:
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
no data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, before and after exposure, once daily in recovery period;
DERMAL IRRITATION (if dermal study): Yes, mild;
- Time schedule for examinations: daily, and at necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, selected organs;
Other examinations:

Body weights

Individual body weights were recorded twice weekly, beginning
approximately 10 days prior to test article administration. Mean body weight
changes were calculated for each corresponding interval.

A final (fasted) body weight was collected for each animal on the day of
scheduled necropsy.

Food consumption

Individual food consumption was measured weekly, beginning
approximately 10 days prior to test article administration. Food intake was
calculated as g/animal/day for the corresponding body weight intervals.


Clinical pathology

Blood and urine samples for clinical pathology evaluation were collected
from all surviving animals at the time of the primary necropsy (study week 2). Blood was collected from a lateral tail vein or the retro-orbital sinus. Urine was collected overnight using metabolism cages. The animals were fasted overnight prior to collection of blood samples. Clinical pathology methods, procedures and references are presented in Appendix F of the attached complete study report, as is the list of the clinical parameters in hematology, serum chemistry, and urinanalysis, examined in this study (Study report DBE WIL 385001, (2000), pages 28 & 29).

Ophthalmological examinations

Ocular examinations were conducted on all animals prior to initiation of test
article administration (study week -1) and during study week 1. Even though no ocular changes attributed to test article administration were noted at study week 2, a recovery examination was conducted (study week 3). All ocular examinations were conducted using an indirect ophthalmoscope (or other equivalent suitable equipment), preceded by pupillary dilation with an appropriate mydriatic agent.
Statistics:
All analyses were conducted using two-tailed tests for significance levels of
5% and 1 % comparing the vehicle control and test article-treated groups to the control group by sex. All means are presented with standard deviations and the number of sampling units (N) used to calculate the means. Body weight, body weight change, food consumption and efficiency,
clinical laboratory and absolute and relative organ weight data were subjected to a one-way analysis of variance (ANOVA), followed by Dunnett's test if the ANOVA revealed statistical significance (p<0.05).

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:
no effects observed
Clinical biochemistry findings:
no effects observed
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Clinical observations and survival

Except for the deaths two animals (one male, one female, thought to be not treatment related) there were no test article-related deaths. There were no test article-related clinical observations. Clinical findings observed in the test article-treated groups were noted similarly in the control group, were not present in a dose-related manner, were noted at a low incidence, typically in single animals and/or were those types of clinical signs commonly observed in rats of this strain and age.

Dermal observations

Low incidences of test article-related erythema and/or edema, generally graded as very slight, were observed for DMA, DMS, DMG and DBE treatment groups in at least one sex. Erythema was observed for one to four males in each of the 300 and 1000 mg/kg/day DMA and DBE groups, the 1000 mg/kg/day DMS group and all three DMG groups. For females, erythema was noted for one to five animals in all DMA and DMG groups and for the 100 and 1000 mg/kg/day DBE groups. Erythema was not observed for females treated with DMS at any dose level. Edema was limited to single males in the 1000 mg/kg/day DMA, DMS and DMG groups and one female in the 1000 mg/kg/day DMG group. Erythema and edema were not observed until the second week of dosing for any dose group and were not observed in the control group. The most prevalent sign of dermal irritation observed during the dosing period was eschar (scab) formation, mostly focal. Focal eschar formation (scabbing) was typically first observed near the end of the first week or beginning of the second week of dosing. This finding was observed at higher incidences than in the control group for all groups treated with DMA, DMG and DBE and the 300 and 1000 mg/kg/day DMS male groups. The number of animals with focal eschar/eschar was greater in the DMG and DBE groups than in the DMA and DMS groups. However, the most severe scores recorded were for male no. 41569 (1000 mg/kg/day DMS group), which was observed with eschar for eight consecutive days beginning on study day 6, reduced to focal eschar on the day of primary necropsy. In addition, fissuring was noted for one 1000 mg/kg/day DMA group female on the final day of the dosing period. The only other dermal finding was desquamation, generally first observed during the second week of dosing. Desquamation was noted for all groups, but with the highest incidence in the test article-treated groups. This increased incidence was considered to be possibly test article-related for most dosage levels in both sexes for DMA, DMG and DBE. The following table summarizes the dermal findings during the dosing period:

Males

Control

DMA

DMS

DMG

DBE

Dermal Finding

1

2

3

4

5

6

7

8

9

10

11

12

13

Erythema

0

0

1

2

0

0

2

1

4

4

0

1

2

Edema

0

0

0

1

0

0

1

0

0

1

0

0

0

Focal EscharlEschar

1

2

7

8

1

4

4

9

9

9

7

7

6

Desquamation

4

5

9

9

6

6

5

8

9

8

9

10

9

Females

Cntrol

DMA

DMS

DMG

DBE

Dermal Finding

1

2

3

4

5

6

7

8

9

10

11

12

13

Erythema

0

1

2

2

0

0

0

1

3

5

1

0

3

Edema

0

0

0

0

0

0

0

0

0

1

0

0

0

Focal Eschar/Eschar

2

4

4

4

0

2

0

6

6

7

5

2

10

Fissuring

0

0

0

1

0

0

0

0

0

0

0

0

0

Desquamation

4

6

7

6

1

3

4

8

9

8

6

7

9

DMA, dimethyl adipate; DMS, dimethyl succinate; DMG, dimethyl glutarate; DBE, dibasic ester.

Body weights

 

Mean body weights were unaffected by dermal exposure to DBE. Occasional statistically significant variations from the control group in mean body weight gain and/or mean cumulative gain were noted in the test article-treated groups. However, no consistent effects were observed and mean body weights were comparable to the control throughout the study.

 

Food consumption

 

Food consumption was unaffected by exposure to any of the test articles. Statistically significant changes in food efficiency were only observed during the recovery period and were considered random occurrences.

Clinical pathology

No test article-related effects on hematologic parameters were observed. A few statistically significant (p 0.05) differences from the control group were noted; however, the changes were not observed in a dose-related manner or were not of sufficient magnitude to be toxicologically relevant.

Serum chemistry

 

There were no test article-related changes in serum chemistry parameters. Slight increases (p 0.05) in mean sorbitol dehydrogenase levels were noted in the 300 mg/kg/day DMG and 1000 mg/kg/day DBE group females when compared to the control group. However, the changes were slight, present in only one sex, changes in other liver enzymes measured (i.e., ALP, ALT, AST or GGT) were not observed and there were no corresponding macroscopic or microscopic findings in the liver. Therefore, these changes were mostlikely random occurrences unrelated to test article treatment. Other statistically significant (p 0.05) changes from the control group were not observed in a dose-related manner and/or were not observed with

sufficient magnitude to be toxicologically relevant.

Urinalysis

No test article-related effects on urinalysis parameters were noted. Specific gravity was decreased (p 0.05) 100 mg/kg/day DBE group females. These changes were slight and not observed in a dose-related manner. Therefore, a relationship to test article exposure was not apparent.

Ophthalmalogical examinations

No oculopathic lesions indicative of a toxic effect were observed. All findings were typical for this species and strain.

Anatomic pathology

A pale liver was noted for the 300 mg/kg/day DBE group male that was found dead on study day 10. There were no internal macroscopic findings for the 100 mg/kg/day DBE group female that was found dead. At the study week 2 primary necropsy, scabbing of the treated skin was observed for one 300 mg/kg/day DMA group male, one 1000 mg/kg/day DMS group male, two 1000 mg/kg/day DMG group males and one male in each of the DBE groups. Findings for the treated skin of females consisted of scabbing for one 1000 mg/kg/day DMA group female and dark red  areas for one 1000 mg/kg/day DBE group female. No microscopic findings correlated with the dark red areas noted for the 1000 mg/kg/day DBE group female. Scabbing of the treated skin was not observed for any animals at the study week 4 recovery necropsy.  No other test article-related macroscopic findings were noted at the scheduled necropsies.

Organ weight

No changes attributed to test article exposure were observed in organ weights. Mean and relative (to final body weight and brain weight) lung weights were increased (p 0.05) in the 100 mg/kg/day DBE group females.

Microscopic Examination

The cause of death for the 300 mg/kg/day DBE group male and 100 mg/kg/day DBE group female could not be determined microscopically.  It was considered not treatment related.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the no-observed effect level (NOEL) for systemic toxicity of DBE when administered dermally to male and female rats for 14 consecutive days was 1000 mg/kg/day.
Executive summary:

A 14-day dermal toxicity study was performed in the rat in accordance with the OECD Guideline 410 and under GLP Regulations. There were no test article or treatment related deaths or clinical observations following administration of DBE for 14 consecutive days. Minimal to mild dermal irritation was observed for all treatment levels near the end of the first week or during the second week of dosing. Scabbing of the treated skin was observed at necropsy for one male in each of the DBE-treatment groups and dark red areas on the treated skin was observed for one female in the 1000 mg/kg/day DBE group; however,  microscopic findings did not indicate a generalized reaction of the treated skin to DBE. There were no other test article-related microscopic findings. Body weights and food consumption were unaffected by DBE treatment. There were no test article-related changes in hematology, serum chemistry, urinalysis, ophthalmoscopy, neurobehaviour or organ weight parameters. Based on these study results, the no-observed-effect level (NOEL) for systemic toxicity for dermal application of DBE for 14 consecutive days was considered to be 1000 mg/kg/day.