Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
10 Nov 1986- 9 Feb 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance propane-1,2-diyl diacetate (CAS 623-84-7). In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
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): only trade name given
- Analytical Purity: > 99.5%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Wiga, Sulzfeld, F.R.G)
- Age at study initiation: 5 weeks (at arriving)
- Weight at study initiation: 198 - 235 g (males) and 131 - 167 g (females)
- Housing: animals were individually housed in polycarbonate cages containing purified sawdust as bedding material (Woody Clean, The Broekman Institute, The Netherlands)
- Diet: RMH-8 (Hope Farms, Woerden, The Netherlands), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-19.5
- Humidity (%): 50-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
propylene glycol
Details on exposure:
At weekly intervals beginning on the day before the first application, the fur from the upper dorso-lateral area of each animal was clipped to an area of 5x7 cm and 5x5 cm for males and females, respectively, accounting for approximately 10% of the total body surface. The formulated test substance was evenly spread on porous surgical gauze (5x5 cm and 5x3.5 cm for males and females, respectively) by means of a plastic syringe. The patch of surgical gauze was fixed to aluminium foil with a thin layer of vaseline. This was similarly attached to a stroke of flexible bandage (Coban. 3M, St. Paul. USA) and was subsequently applied to the clipped area of each rat. This semi-occlusive bandage was prepared daily.

The animals were dosed daily (excluding weekends) for approximately 6 hours for four weeks, usually beginning at approximately 9.00 a.m. (light period for animals from 7.00 a.m. to 7.00 p.m.). During the 6-hour exposure period the bandage was frequently checked (approximately once per two hours). Occasionally some animals managed to remove the bandage in which case the bandage was replaced or reapplied during the exposure period. After the exposure period the bandage was removed and the remaining test substance was gently removed from the treated surface with moist tissues.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Since the test substance was miscible and stable in propylene glycol, propylene glycol (European Pharmacopoeia quality) was used as vehicle for dosing. Based on data on its stability in propylene glycol, the test substance solutions were prepared at least weekly. The test formulations contained per 100 ml vehicle 2.5, 7.5 and 25 g test substance.for the respective dose groups from low to high (i.e. 100, 300 and 1000 mg/kg body weight). Samples of the various dose concentrations were taken on day 4, 11, 18 and 25 for chemical analysis of their actual concentration. These analyses were performed on the day of sampling using a Gas Chromatographic (GC) method.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily, 6 h/day, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
The test substance was applied daily, on a 5-days-per-week basis, to the skin in graduated doses to several groups of experimental animals, one dose per group, for a period o f 28 days. During the period of application the animals were observed daily to detect signs of toxicity. Animals which died during the test were necropsied. Body weights and food consumption were measured weekly. At the end of the study blood was collected in order to perform haematology and clinical chemistry. Subsequently, all surviving animals were sacrificed and necropsied and appropriate histopathological examinations were carried out.

Examinations

Observations and examinations performed and frequency:
With the exception of days 0, 7, 21 and 28, cage-side observations were performed once daily on working days (beginning on day 1) as well as weekends until terminal sacrifice on day 28. Any deviations from normal were recorded. In particular, attention was paid to changes of skin. fur, eyes, mucous membranes, respiratory system, faeces and general appearance. On working days a mortality check was performed in the evening.

Physical examination
Once a week. i.e. on days 0 (prior to dosing), 7, 14, 21 and 28 the animals underwent a physical examination. In addition to the parameters mentioned for cage-side observations, particular attention was paid to ears, mouth, urogenital region, anus, abnormal masses, gait, general state and behaviour.

Body weights and food consumption
Individual body weights were determined immediately prior to the first dosing (day 01, weekly thereafter (days 7. 14, 21 and 27) and on day 28 prior to sacrifice (fasted body weight). Individual food consumption was recorded weekly (days 7, 14, 21 and 27) and was expressed as grams food consumed per animal and as grams food consumed per 100 grams body weight per day.
Sacrifice and pathology:
Haematology and clinical chemistry
Prior to sacrifice on day 28 the animals were anaesthetized with ether and the abdominal cavity was opened. Blood was collected via the abdominal aorta by means of an infusion set provided with a winged needle (Mediwing, Argyle, Sherwood Medical Industries. Tullamore. Ireland). For haematology approximately 1.5 ml of blood were collected directly into vials containing 0.03 ml of di- and tripotassium EDTA (30% (WIV). pH 7.4). Immediately thereafter blood samples were collected in clot tubes for clinical chemistry. The vials intended for haematology were rotated at 90 rpm until transportation to 'Bergschot Centrum voor Onderzoek (BCO)' in Breda, The Netherlands for analysis. The maximum period between blood collection and transport to BCO was 5 1/2 hours.
The following haematological parameters were measured:
- Erythrocyte count (RBC) and size distribution
- Haematocrit (PCV)
- Haemoglobin concentration (HB)
- Leucocyte count. total (WBC) and differential
- Platelet count (THRO) and size distribution
The following were determined by calculation:
- Mean cell volume (MCV)
- Mean corpuscular haemoglobin (MCH)
- Mean corpuscular haemoglobin concentration (MCHC)
- Mean cell volume/erythrocyte count (MCVIRBC)
The approximately 3 ml blood samples intended for clinical chemistry were allowed to clot during 30-60 minutes. The serum was separated by
centrifugation and transferred into another tube. Serum samples were kept on ice until analysis by BCO.
The following blood chemistry values were measured:
- Alanine aminotransferase (GPT)
- Aspartate aminotransferase (GOT)
- U-Glutamyl transpeptidase (GGT)
- Creatinine (CREA)
- Urea nitrogen (BUN)
- Glucose (GLUC)
- Bilirubin (TBILL)
- Total protein (T?P)
- Albumen (ALB)
- Calcium (Cat+)
- Sodium (Na+)
- Potassium (Kt)
- Inorganic phosphate (PI)
- Chloride (C1-1
The red blood cells of the samples were counted using a "Coulter" blood cell counter, while their size distribution was determined using the "Haemalog 6000" (Technicon). Measurement of haemoglobin concentration, counting of white blood cells and subsets thereof, i.e. lymphocytes, neutrophils, large unstained cells, monocytes, eosinophils and basophils, and the counting of thrombocytes were also performed using the Haemalog 6000. The haematocrit values were determined using a microcentrifuge. All clinical chemical parameters were measured in serum samples using a "Parallel" clinical chemistry analyzer.

Post-mortem procedures
On completion of blood collection the animals were sacrificed by exsanguination. Subsequently a full gross necropsy was performed which included examination of the external surface and openings and the cranial. thoracic and abdominal cavities with their contents. Liver, adrenals, kidneys, spleen and testes were removed and weighed. The following tissues were removed and preserved in 10% formalin: Liver, spleen, kidneys, adrenals, normal and treated skin and macroscopically abnormal tissues. Fixed organs and tissues from animals of the control and high dose group were embedded in paraffin, sectioned, stained with haematoxylin and azophloxine, and examined microscopically.
Other examinations:
No additional information available.
Statistics:
Means with standard deviation were calculated for all quantitative parameters. Statistical procedures were carried out for quantitative data suspected of changes and were based on a one-way analysis of variance supplemented with a t-test (Biodata Handling with Microcomputers, Barlow. 1983). One-way analysis of variance (F-test) was used to analyse the results for overall effects of dosage. Significant differences between control and individual dosages were also assessed by using the F-test where P<0.05 was accepted as the lowest level of significance. The total food consumption represents the group mean of the sum of the individual food consumption over the entire period (day 7. 14. 21 and 27). whereas the body weight gain was obtained by calculating the group mean of the mean individual body weight gain (obtained from a linear regression analysis for the individual body weights over the entire period, i.e. day 0, 7, 14, 21 and 27).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
no effects observed
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:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
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 specified
Details on results:
Dose range finding investigation
All animals of the 100, 300 and 1000 mg/kg dose groups survived without showing any signs of test substance related toxicity. Macroscopy at necropsy on day 9 revealed congested livers in all animals of the high dose group (1000 mg/kg). No further abnormalities or changes of liver weight or kidneys weight were noted for the dose groups included in the pilot study.

Main study
Dose selection
Based on the results obtained in the dose range finding investigation, the test substance was tested at levels of 100, 300 and 1000 mg/kg
body weight/day. The dose volume was 4 ml/kg body weight for all groups. The doses were adjusted weekly for changes in body weight.

Mortality, cage-side observations and physical examinations
No test substance related mortality occurred and no evident signs of toxicity were observed during the study. Several animals of each treatment group (including the control) revealed temporary local eschar formation on the treated skin surface. This was not considered to be test substance related, but due to shaving and the way of bandaging. Two female animals (designated A5 and 87) died during exposure on day 15 and 27, respectively. In those cases the bandage was probably fixed too tight. Incidental other findings were bloody eye encrustation (once in the high dose group) and focal erythematous spots on the treated skin site (control animal).

Body weights and food consumption
For group mean body weight no statistically significant test substance related effect was observed. For group mean food consumption and group mean food consumption/100 g body weight/day also no test substance related differences were observed.

Organ weights
There were no statistically significant test substance related effects with respect to group mean organ weights and group mean organ/body weights.

Haematology and clinical chemistry
With respect to the group mean haematology data the MCV and MCV/RBC showed a tendency to increase in male animals of all dose groups which reached statistical significance for the high dose animals only (P<0.05). No other evident test substance related effect was observed. The measured male and female values were comparable for the control group and the treatment groups. In addition, the obtained values are in general agreement with those reported in the literature (Charles River Laboratories, Technical Bulletin, 1984).

With respect to the group mean clinical chemistry data no statistically significant test substance related effect was observed in male or female animals, except for blood urea nitrogen that was decreased in male animals of the high dose group (P<0.05). The obtained values are in general agreement with those reported in the literature. Serum g-glutamyl transpeptidase was slightly increased (5 IU/L) in animal B8 and total bilirubin was increased (30 µmol/L) in animal B2.

Pathology report
Macroscopic examination of all animals at necropsy revealed no test substance related gross abnormalities. Incidentally, among animals of all treatment groups, hydronephrosis was found which is generally considered as a congenital abnormality in this rat strain. Other gross pathology findings were petechiae of the stomach (observed once in the control group). Microscopic examination of adrenals, kidneys, liver, spleen and normal and treated skin from animals of the control and high dose group revealed no evident test substance related abnormalities.

Effect levels

Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects noted in the high dose group following dermal application for 28 days

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1 Mean Hematology Data for Male Rats following Repeated Dermal Dosing in a 28-Day Study with PGDA

   Mean + Standard Deviation            
 Dosage (mg/kg)  RBC (/PL)  HB (MMOL/L)  PCV (L/L)  MCV (FL)  MCV/RBC (10E-3/E2)
 8.09 + 0.44  9.6 +0.4  0.44 + 0.02  54.2 + 2.0  6.7 + 0.6
 100  8.07 + 0.26  9.8 + 0.4  0.45 + 0.02  55.3 + 1.5  6.9 + 0.3
300  7.87 + 0.36   9.7 + 0.2  0.44 + 0.01  55.8 + 1.5  7.1 + 0.5
 1000  7.83 + 0.21  9.7 + 0.4  0.44 + 0.01  56.8 + 1.0  7.3 + 0.3

N = 6 for each value

Table 2 Summary of Hematology Historical Control Data from Male Rats at Charles River

         8 -12 Weeks of Age 13 -22 Weeks of Age       
 Test  N  Mean  Range  N  Mean  Range
 RBC, 106/ul  549  7.96  7.77 - 8.19  464  8.34  7.89 - 8.90
 Hematocrit, %  549  43.93  41.20 - 47.30  464  43.25  28.30 - 49.20
 Hemoglobin, g/dl  549  15.07  14.40 - 16.00  464  15.35  14.70 - 16.60
 MCV, fl  544  55.17  53.00 - 59.50  464  54.01  51.70 - 58.40

Report titled "Charles River-Clinical laboratory Parameters for the CD Rat (March 2006)" found at http://www.criver.com/en-us/prodserv/bytype/resmodover/baselinecolony/pages/baselinedatabydate.aspx

Applicant's summary and conclusion

Conclusions:
Repeated dermal dosing of the test material for 28 days up to a level of 1000 mg/kg body weight caused no adverse health probLems. The no observed adverse effect level is 1000 mg/kg/day.
Executive summary:

Four groups of Sprague-Dawley rats, each comprising 6 males and 6 females, received daily (5 days/week) dermal doses of the test material for 28 days at levels of 0 (control), 100, 300, and 1000 mg/kg body weight, respectively. OECD test guideline 410 was followed, applying good laboratory practice.

No test substance related mortality occurred moreover no evident signs of systemic toxicity were observed in any of the treatment groups. Temporary local eschar formation on the treated skin side of several animals among all groups was induced ,by shaving in combination with the way of bandaging and not by the test substance. No test substance related effects on body weight gain and food consumption were found. Organ weights, organ/body weight ratios, haematology and clinical chemistry data, and gross and microscopic pathology revealed no test substance related changes.

Based on these findings it was concluded that repeated dermal dosing of the test substance for 28 days up to a level of 1000 mg/kg body weight caused no adverse health problems. The no observed adverse effect level is 1000 mg/kg/day.