Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The publication lists no information on guideline/s and GLP but the report contains sufficient data for interpretation of study results.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2003

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The 13-week subchronic toxicity study was performed with groups of 12 Sprague-Dawley rats/sex/dose. Rats received semi-occluded daily dermal applications of DPO for 6 h/day. All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the diethyl phthalate (DEP) vehicle at concentrations to administer 0, 100, 300 or 1000 mg DPO/kg body weight/day.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
DPO used in this study was an equal blend from three sources (Dow Chemical U.S.A., Midland, MI, USA; Haarmann & Reimer, GmbH, Holzminden, Germany; Haarmann & Relmer, GmbH also supplied a sample from INOWTAL), each had a purity of > 99%.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Sprague-Dawley CD rats were obtained from Charles River (UK) Ltd (Margate, Kent, UK).

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
other: diethyl phthalate
Details on exposure:
Four groups of 12 male and 12 female Sprague-Dawley rats were used. The hair on their backs was clipped before dosing and then twice weekly. Three groups were dosed daily with DPO via the dermal route of application using a semiocclusive dressing for a period of 6 h/day for 13 weeks. Doses of 2 ml/kg were applied to gauze squares ( ~ 4 cm x 4 cm) with an aluminium foil back. These patches were placed on the shaved skin on the animals' backs and held in place by a semi-occlusive dressing (50 mm Micropore, 3M) and tape (50 mm Blenderm, 3M). The gauze squares and dressing were removed after 6 h, and the dosed area was washed with DEP to remove the non-absorbed DPO. The rats were dosed at a constant volume of 2 ml/kg body weight and concentrations in DEP were calculated to achieve dose levels of 100, 300 or 1000 mg/kg body weight/day.
One control group (12 males/12 females) received DEP at a dose volume of 2 ml/kg.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day
Doses / concentrations
Remarks:
Doses / Concentrations:
Solvent control, 100, 300 or 1000 mg/kg body weight/day
Basis:
other: nominal
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The 13-week subchronic toxicity study was performed with groups of 12 Sprague-Dawley rats/sex/dose. Rats received semi-occluded daily dermal applications of DPO for 6 h/day. All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the diethyl phthalate (DEP) vehicle at concentrations to administer 0, 100, 300 or 1000 mg/kg bw/day.

During the study, the animals were assessed for general clinical signs, skin irritation (erythema, eschar, edema and thickening), body weight, food consumption and water consumption. During week 13, samples from 10 males and 10 females from all groups were taken for urinalysis, hematology and clinical blood chemistry parameters.

After 13 weeks of dosing, animals were killed by CO2 narcosis, and necropsied. Selected organs were examined and weighed. Histopathologic
examinations were performed on major tissues from all animals in the control and high-dose groups; kidneys from all dose groups were examined histopathologically.

Hematology, clinical chemistry, organ weight and body weight data were statistically analyzed for homogeneity of variance by using the 'F-max' test. If the group variances appeared homogeneous a parametric ANOVA was used, and pair wise conparisons were made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, a non-parametric test such as Kruskal Wallis ANOVA was used. Organ weights were also analyzed conditional on body weight (i.e. covariance and relative analyses). Histology data were analyzed using Fisher's Exact Probability test.
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
During the study, the animals were assessed for general clinical signs, skin irritation (erythema, eschar, edema and thickening), body weight, food consumption and water consumption.
Sacrifice and pathology:
During week 13, samples from 10 males and 10 females from all groups were taken for urinalysis, hematology and clinical blood chemistry parameters. After 13 weeks of dosing, animals were killed by CO2 narcosis, and necropsied. Selected organs were examined and weighed. Histopathologic
examinations were performed on major tissues from all animals in the control and high-dose groups; kidneys from all dose groups were examined histopathologically.
Other examinations:
No data
Statistics:
Hematology, clinical chemistry, organ weight and body weight data were statistically analyzed for homogeneity of variance by using the 'F-max' test. If the group variances appeared homogeneous a parametric ANOVA was used, and pair wise conparisons were made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, a non-parametric test such as Kruskal Wallis ANOVA was used. Organ weights were also analyzed conditional on body weight (i.e. covariance and relative analyses). Histology data were analyzed using Fisher's Exact Probability test.

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:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the DEP vehicle at concentrations to administer 0, 100, 300 or 1000 mg/kg bw/day for 13-weeks. In male rats exposed to the high dose, there was a slight reduction in body weight, and a statistically significant increase in liver, brain and kidney weight relative to body weight. Absolute and relative liver weight was increased at 300 mg/kg body weight/day. In female rats, relative liver weight was increased at both 300 and 1000 mg/kg body weight/day and absolute liver weight was increased at 1000 mg/kg body weight/day. No gross or histopathological abnormalities were identified in liver, kidney or other organs.

At all dose levels, slight skin reactions were observed at a greater incidence in treated than the control animals and showed a dose response. Desquamation was present in the control, low-, mid- and high-dose groups at incidences of 33, 54, 83 and 96%, respectively. Erythema was not seen in control animals, but was present in treated rats in incidence of 38, 42 and 67%; 100, 300, 1000 mg/kg body weight/day, respectively. In the high dose rats, one had skin thickening and two had edema.

Effect levels

open allclose all
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: At 300 mg/kg/d, increases in absolute and relative liver weight were observed in both sexes, but without histopathological findings reported for the liver (or other organs).
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOEL
Remarks:
local
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Increased rate of erythema was observed at all dose levels, but erythema Draize scores were not provided. Also, no histopathological changes in the skin were noted. Desquamation noted in all study animals, including controls.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The 13-week study showed limited effects on body or organ weights or both in both sexes exposed to 300 or 1000 mg/kg body weight/day by dermal exposure. Histopathological findings were not present in any of the organs examined.

These results are consistent with the lack of toxicity observed in an oral toxicity study that was performed by Johnson et al. (1992). They administered DPO in the feed at dietary concentrations of 200, 1000 and 5000 ppm for 13 weeks. The top dose was equal to approximately 500-600 mg/kg body weight/day. The study by Johnson et al. (1992) found no compound-related effects on body weight, food consumption, hematology, serum chemistry, urinalysis, necropsy finding or histopathological examination of tissues and organs. There were some changes in body weight and associated changes in organ weights at the high dose level. These effects, however, were discounted because they were caused by a decrease in food consumption resulting from the decreased palatability of the diet at the 5000 ppm level.

In this study, male rats exposed to 1000 mg/kg body weight/day exhibited a statistically significant increase in relative brain weight, but absolute brain weight was

unaffected. A relative but not absolute brain weight is a result of body weight reduction and lacks biological significance (Feron et al., 1973; Oishi et al., 1979).

The increases in liver weights at the two higher doses in both sexes was not accompanied by any evidence of gross or histopathological findings of toxic effects, such as lesions or serum enzyme changes. Such increases in liver weight are commonly the result of microsomal enzyme induction and represent a physiological adaptation rather than an adverse effect (Glaister, 1986).

Increased relative kidney weight was observed only in the high-dose males, but not in any of the female groups. In addition, the increased relative kidney weight did not demonstrate a dose-related increase, and was not associated with evidence of renal dysfunction or histopathological lesions. Taking all these finding together, the kidney weight changes appear to be questionable and lack biological significance. There were adverse effects on the skin at all dose levels. Subchronic exposure produced limited effects.

Applicant's summary and conclusion

Conclusions:
The systemic no-observed-efect level (NOEL) in this study is 100 mg/kg/day. Adaptive changes were observed at 1000 mg/kg/day. Organ weight changes were judged to lack biological significance and the no-observed adverse-effect level (NOAEL) was determined to be 1000 mg/kg/day by the authors.
Executive summary:

Diphenyl ether (DPE) was investigated in a dermal subchronic toxicity. The 13-week study was performed with groups of 12 Sprague-Dawley rats/sex/dose that received semi-occluded daily dermal applications of DPO for 6 h/day. All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the DEP vehicle at concentrations to administer 0, 100, 300 or 1000 mg/kg/day. At the high dose level, there was a slight reduction in body weight gain in males (13%) that was not reported to be statistically significant, increase in albumin (5-6%) and phosphate (10-15%) levels in both sexes, a reduction of cholesterol in females (14%), an increase in kidney (17%) and brain (8%) weights in males, and an increase in liver weight (18-19%) in both sexes. No histopathological lesions were seen in any organ examined, including skin. At 300 mg/kg body weight/day, the only notable findings were an increase in liver weight (10%) in both sexes and a slight increase in albumin (5%) in females. In addition, increased rate of skin irritation reactions at the site of application was observed in all DPO dose groups (Draize scores not given). The systemic no-observed-effect level (NOEL) in this study is 100 mg/kg/day. The systemic findings were judged to lack biological significance and the no-observed-adverse-effect level (NOAEL) was determined to be 1000 mg/kg/day.