Registration Dossier

Toxicological information

Repeated dose toxicity: dermal

Currently viewing:

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:
27.01. 1997- 19-07.1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guidelines followed but no statement of GLP. Restriction due to the fact that the study was conducted on read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): [EC number 425-620-2, read cross material]
- Expiration Date: November 2001
- Physical state: Amber liquid
- Analytical purity: pure, no adjustments in the dose calculation were made
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
other: Crl:CD®BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston Facility, Stone Ridge, New York
- Age at study initiation: Males: Approximately 7-8 weeks, Females: Approximately 9-10 weeks
- Weight at study initiation: Males: 246.3 to 270.2 grams, Females: 197.9 to 245.9 grams
- Fasting period before study: no data
- Housing: Single housed during the test period, Caging: Suspended stainless steel and wire mesh with absorbent paper below cages
- Diet (e.g. ad libitum): PMI Certified Rodent Diet Meal 5002, ad libitum
- Water (e.g. ad libitum): Automatic Watering System, ad libitum
- Acclimation period: 13 days; animals were examined for viability at least once daily

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-22.3
- Humidity (%): 30 to 70 percent relative humidity
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light (0700 to 1900 Hours) and 12 hours dark (1900 to 0700 Hours) by automatic timer.

IN-LIFE DATES: From: January 28, 1997 To March 11, 1997

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: the dorsal surface from the shoulder region to the lumbar region
- % coverage: approximately 10% of the body surface
- Type of wrap if used: COBAN wrap
- Time intervals for shavings or clipplings: at least once weekly

REMOVAL OF TEST SUBSTANCE
- Washing : gentle wipe of the exposure site with peanut oil and a paper towel
- Time after start of exposure: approximately 6 hours after exposure

TEST MATERIAL
- Amounts applied :100mg/kg, 300mg/kg, 1000 mg/kg


USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, The animal was wrapped with COBAN to prevent ingestion of the test material.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
1000 mg/kg (satellite group)
Basis:
nominal per unit area
No. of animals per sex per dose:
Group 1 ( control group)- 0 mg/kg (sham dose )- 5 males and 5 females
Group 2- 100 mg/kg- 5 males and 5 females
Group 3- 300 mg/kg- 5 males and 5 females
Group 4- 1000 mg/kg- 5 males and 5 females
Group 5 (satellite recovery group) - 1000 mg/kg- 5 males and 5 females
Control animals:
yes, sham-exposed
Details on study design:
Dose levels were selected based on findings of previously conducted studies with this material including an acute dermal toxicity study (LD50 > 2000 mg/kg) and 28-day oral toxicity study (no signs of overt toxicity at the limit dose level of 1000 mg/kg).

All animals were examined for abnormalities prior to dose initiation and those determined to be unsuitable for inclusion on this study because of poor health, outlying body weights, or other abnormalities were excluded from selection. Study animals were selected from the suitable animals by a computer generated sorting program and their weight variation within their sex was within ±20% of the mean body weight by sex.

Fifty Crl:CD®BR rats were divided into 5 groups of 5 male and 5 female rats each. The test material was applied to the clipped, unabraded dorsal surface of the rats 7 days per week for 28 days. Group 1 served as a control group. Groups 2, 3, and 4 received 100, 300, and 1000 mg/kg of the test material, respectively. Group 5 served as a satellite recovery group. The satellite animals were treated with test material at 1000 mg/kg and then observed for 14 days after treatment for reversibility, persistence, or delayed occurrence of toxic effects. Individual doses were adjusted weekly based on the most recent body weights.

Clinical observations were performed daily throughout the study and dermal responses were evaluated at specified intervals according to the Draize method. Body weight was recorded pretest, at dose initiation (Day 0), on Days 7, 14, 21 and 27, on the day of death, and on Days 35 and 41 for the satellite animals. Food consumption was measured weekly. Hematology and serum chemistry samples were taken from all animals on Day 28 and from all surviving satellite animals on Day 42. A full macroscopic postmortem examination was performed on all animals and required organs were preserved. Selected organs were weighed at study termination. A range of tissues was examined microscopically.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations were made as to the nature, onset, severity, and duration of toxicological signs daily during the study.

DERMAL IRRITATION : Yes
- Time schedule for examinations: Dermal evaluations were performed prior to dosing on Days 0, 1, 3, 7, 10, 14, 17, 21 and 24. Dermal evaluations also were performed after sleeve removal on Day 0 and prior to blood collection on Day 28. Additional dermal evaluations were performed for the satellite animals on Days 31, 35, 38 and 42. All evaluations were performed according to the Draize method.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded prior to initiation of dosing for group allocation. Body weights also were recorded on the day of initiation of dosing (Day 0), on Days 7, 14, 21, and 27, and on the day of death. Additionally, body weights were recorded for the satellite animals on Days 35 and 41.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was measured on Days 7, 14, 21 and 27 during the test period. Food consumption also was measured for the satellite animals on Days 35 and 41. The Weeks 4 and 6 food consumption values were based on 6 days due to fasting of the animals on Days 27 and 41 for blood collection on Days 28 and 42, respectively.

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: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: samples were taken from all animals on Day 28 and from all satellite animals on Day 42
- Anaesthetic used for blood collection: Yes - methoxyflurane
- Animals fasted: Yes, overnight fast
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:samples were taken from all animals on Day 28 and from all satellite animals on Day 42
- Animals fasted: Yes, overnight fast
- How many animals: all animals

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
All animals were sacrificed via exsanguination following methoxyflurane anesthesia and subjected to a gross necropsy.

The following organs were weighed prior to placement in fixative from all animals in Groups 1, 2, 3, and 4 on Day 28 and all animals from Group 5 on Day 42 : liver, testes, brain, kidneys and adrenals.

The following tissues were removed from all animals and preserved in 10% neutral buffered formalin: adrenals, aorta, brain (3 levels), esophagus,
epididymides, exorbital lacrimal gland, eyes, femoris muscle with sciatic nerve, femur with articular surfaces, Harderian gland, heart, kidneys, large intestine (sections from colon and cecum), liver, lung (with mainstem bronchi), mammary gland (female), mesenteric lymph node, ovaries and oviducts, pancreas, pituitary, prostate, rectum, salivary glands/mandibular lymph node, seminal vesicles, skin (treated and untreated), small intestine (sections from duodenum, jejunum, and ileum), spinal cord at 3 levels (cervical, mid-thoracic, and lumbar), spleen, sternum with marrow, stomach, testes, thymus, thyroid (parathyroids), trachea, urinary bladder, uterus (corpus, cervix), all gross lesions.

The following tissues from animals in Groups 1, 4, and 5 were processed, sectioned, stained (hematoxylin and eosin) and examined microscopically: liver, kidneys, testes, adrenals, skin (treated and untreated) gross lesions. The liver, kidneys, skin (treated and untreated), and gross lesions from the low and mid-dose group animals also were processed and examined microscopically.






Other examinations:
No data
Statistics:
Statistical evaluation of equality of means was done by an appropriate one way analysis of variance and a test for ordered response in the dose groups. First, Bartlett's Test was performed to determine if the dose groups had equal variance. If the variances were equal, the testing was done using parametric methods, otherwise nonparametric techniques were used.

For the parametric procedures, a standard one way ANOVA using the F distribution to assess significance was used. If significant differences among the means were indicated, Dunnett's Test was used to determine which treatment groups differed significantly from control. In addition to the ANOVA, a standard regression analysis for linear response in the dose groups was performed. The regression also tested for linear lack of fit in the model.

For the nonparametric procedures the test of equality of means was performed using the Kruskal-Wallis Test. If significant differences among the means were indicated, Dunn's Summed Rank Test was used to determine which treatment groups differed significantly from the control. In addition to the Kruskal-Wallis Test, Jonckheere's Test for monotonic trend in the dose response was performed.

Bartlett's Test for equal variance was conducted at the 1% level of significance. All other tests were conducted at the 5% and 1% level of significance.

The statistical t-test was used to compare the satellite group's main study termination and recovery termination hematology and clinical chemistry values. The t-test was used to compare the satellite group's Day 42 relative organ weight with the control group's Day 28 relative organ weight and also to compare the satellite group's Day 42 relative organ weight with the high dose group's Day 28 relative organ weight.

The statistical t-test also was used to compare the high dose and satellite animals to substantiate their equivalence in order to accurately evaluate the recovery effect.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
incidental findings of occasional sores or scabs among males and one mid dose female with an umbilical hernia but these signs were concerned as a no treatment- related. Most animals survived to terminal sacrifice on Day 28 or 42.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
transient dermal irritation observed in several treated females
Mortality:
no mortality observed
Description (incidence):
incidental findings of occasional sores or scabs among males and one mid dose female with an umbilical hernia but these signs were concerned as a no treatment- related. Most animals survived to terminal sacrifice on Day 28 or 42.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
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:
CLINICAL SIGNS AND MORTALITY
Most animals survived to terminal sacrifice on Day 28 or 42 (satellite) and there were no treatment-related clinical signs of toxicity in the animals at any dose level. Incidental findings were limited to occasional sores or scabs among males, and one mid dose female with an umbilical hernia.
One control group female was found dead on Day 27. She was not observed with any abnormalities during the study.

DERMAL OBSERVATIONS
All males were free of erythema and/or edema throughout the study with the exception of one satellite male observed with very slight erythema on Day 28. Additionally, desquamation was observed in one 300 mg/kg male on Day 7 and in one satellite animal on Day 28. [See attached Table 2 ( males)].
Very slight to slight edema was noted in several treated females on Day 3 and very slight edema was noted in one satellite female on Day 21 [See attached Table 2 (females- edema)]. Very slight to well defined erythema was noted in several animals on Day 3 and/or Day 7, and in one satellite animal on Days 10, 17, 21, and 24 [ See attached Table 2 (females- erythema)].

BODY WEIGHT AND WEIGHT GAIN
All animals displayed increases in body weight over their initial values. There were no statistically significant differences in mean body weight or mean body weight change between treated and control animals of either sex. Mean body weight of the satellite animals (not included in statistical analysis) appeared comparable to controls and other treated groups.

FOOD CONSUMPTION
There were no statistically significant differences in mean food consumption between treated and control animals of either sex. Mean food consumption of the satellite animals (not included in statistical analysis) appeared comparable to controls and other treated groups.

HAEMATOLOGY
There were no statistically significant differences in quantitative or semi-quantitative hematological parameters between treated and control animals of either sex at main study termination (Day 28). Mean hematological values of the satellite animals (not included in statistical analysis) also appeared comparable to controls and/or to other treated groups on Day 28.

Following the recovery period, there were several statistically significant differences in semi-quantitative hematological parameters in satellite group animals There was a statistically significant decrease in mean corpuscular hemoglobin concentration in the male satellite from Day 28 to Day 42. This small difference (<4%) was not considered clinically significant.

SERUM CHEMISTRY
There were no statistically significant differences in serum chemistry parameters between treated and control animals of either sex at main study termination, with the exception of a small increase (p¿0.05) in mean potassium of the high dose males compared with controls. This minor difference (<11%) were not considered clinically significant. Mean serum chemistry values of the satellite animals (not included in statistical analysis) appeared comparable to controls and/or other treated groups on Day 28.

Following the recovery period, there were several statistically significant differences in serum chemistry parameters of the satellite group animals from Day 28 to Day 42. These included statistically significant increases in mean glucose and calcium in the males; mean total protein and albumin in the females; and a decrease in aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase in males; and mean sodium in the females. These differences were not considered clinically significant since they were limited to the recovery phase, and they occurred in the absence of other corroborating clinical or histopathological effects. The absence of significant findings at main study termination further supports the conclusion that these findings were spurious and unrelated to the test material.

ORGAN WEIGHTS
There were no statistically significant differences in mean absolute organ weight, organ to body weight ratio, or organ to brain weight ratio between the treated and control males at main study termination.

There were statistically significant decreases in mean absolute liver weights for the low, mid, and high dose females when compared with the controls. However, there were no statistically significant differences in mean organ to body weight ratio or organ to brain weight ratio between the treated and control females at main study termination. Thus, the decreased absolute liver weights were attributed to differences in terminal body weight rather than an adverse effect. Additionally, following recovery, the female satellite animals did not show any statistically significant differences in liver weight when compared with the controls or the high dose group.

Following recovery, there were several small, but statistically significant differences in relative organ weights. These included a statistically significant decrease in mean testes to body weight and brain to body weight ratios of the satellites on Day 42 compared with controls on Day 28 or the high dose group on Day 28. Additionally, there was a statistically significant decrease in mean brain to body weight ratio of the satellite females compared to the high dose. These differences were not considered biologically significant since they were limited to the recovery phase, and they occurred in the absence of other corroborating clinical or histopathological effects. The absence of significant findings at main study termination further supports the conclusion that these findings were spurious and unrelated to the test material.

GROSS PATHOLOGY
There were no gross postmortem observations which were judged to be related to the test material, and the majority of animals were free of observable abnormalities at termination. Postmortem findings were limited to single or low incidences of sores/scabs; folder, adhered liver; discolored liver, thymus, or mandibular lymph nodes; distended uterus; dilated renal pelvis; and/or a mass in the liver.

HISTOPATHOLOGY: NON-NEOPLASTIC

There were no microscopic changes observed in any of the organs or tissues examined to indicate any systemic toxicity or cutaneous irritation from the dermal application of the test material.

Microscopic examination of the treated area of the skin revealed acanthosis and hyperkeratosis of the epidermis in rats of all groups, including controls. Other accompanying changes were variable amounts of hyperplasia of sebaceous glands and focal dermal inflammation. There was essentially no difference in incidence or severity of these changes in the skin of the control rats or 100, 300, or 1000 mg/kg of the test material. As the changes occurred at similar incidences and severity in both control and treated animals, these changes were considered secondary to the repeated shaving of the skin, and/or wrapping and taping of the treatment site. The untreated skin also displayed microscopic changes but this was considered to be due to migration of the test material from the treated skin and/or the repeated shaving and wrapping of this area of the skin. The test material did not increase the severity of any changes and thus, was not considered to have caused any cutaneous irritating effects. Following recovery, the treated skin showed similar types of changes, but the severity and/or incidence was decreased indicating reversibility.

All other microscopic changes observed were considered to have occurred spontaneously and to have been incidental and unrelated to treatment.

The most common incidental finding was a minimal amount of multifocal mononuclear inflammatory cell infiltrations in the liver. The incidence and severity of this common incidental finding was not affected by the test material. Additionally, focal subcapsular necrosis was observed in the liver of a few rats of the various groups, including two control rats. This type of necrosis is consistent with that which has been associated with wrapping of the abdomen for dermal studies.

Incidental findings in the kidneys included single or low incidences per affected group of focal cortical tubular degeneration, pelvic dilation, increased cytoplasmic eosinophilia in cortical tubules, pyelonephritis, multifocal mineralization, focal chronic nephritis, a medullary cyst, and focal mononuclear-cell infiltrations.


Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse systemic findings were recorded up to the limit dose of 1000 mg/kg/day

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, topical application of the test material under conditions of this study elicited no signs of systemic toxicity. The only significant finding was transient dermal irritation observed in several treated females. There were no adverse effects with respect to clinical signs, body weights, body weight changes, food consumption, clinical parameters, organ weights, postmortem observations, or microscopic changes. Accordingly, a No Observable Adverse Effect Level (NOAEL) was established at 1000 mg/kg of the test material in both male and female rats.
Executive summary:

The comparison of overall physicochemical and toxicity profiles for tested and analogue materials indicates it is appropriate to apply read-across data from the structural surrogate when considering the repeated dose toxicity data.

This study was conducted to evaluate the potential toxicity of the test material following repeated dermal application to fifty Crl:CD®BR rats for a minimum of 28 days. Animals were divided into 5 groups: Group 1 -control group, Groups 2, 3, and 4 received 100, 300, and 1000 mg/kg of the test material, respectively, Group 5- satellite recovery group that was treated with test material at 1000 mg/kg and then observed for 14 days after treatment for reversibility, persistence, or delayed occurrence of toxic effects. Clinical observations were performed daily throughout the study and dermal responses were evaluated at specified intervals according to the Draize method. Body weight was recorded pre-test, at dose initiation (Day 0), on Days 7, 14, 21 and 27, on the day of death, and on Days 35 and 41 for the satellite animals. Food consumption was measured weekly. Haematology and serum chemistry samples were taken from all animals on Day 28 and from all surviving satellite animals on Day 42. A full macroscopic post-mortem examination was performed on all animals and required organs were preserved. Selected organs were weighed at study termination. A range of tissues was examined microscopically.

Most animals survived to terminal sacrifice on Day 28 or 42 (satellite) and were free of treatment-related clinical signs of toxicity. Slight transient dermal irritation was the only significant finding observed, and was limited to very slight to well-defined erythema, very slight to slight oedema, and/or desquamation in several treated group females primarily during Week 1. There were no biologically meaningful differences among the groups with respect to mean body weights, body weight gain, food consumption, haematological parameters, serum chemistry parameters, absolute organ weights, organ-to-body weight ratios, or organ-to-brain weight ratios. Similarly, there were no meaningful changes in these parameters following the recovery period. 

There were no gross post-mortem observations or microscopic changes which were judged to be related to the test material. There were no microscopic changes considered to be the result of any irritating effect or systemic toxicity of the topically applied test material. The most notable microscopic changes consisted of acanthosis (thickening) and hyperkeratosis of the epidermis, sebaceous gland hyperplasia and focal dermal inflammation in the treated areas of skin of male and female animals from all groups, including the control. As the changes occurred at similar incidences and severity in both control and treated animals, these changes were considered secondary to the repeated shaving of the skin, and/or wrapping and taping of the treatment site and not test material-related. All other microscopic changes were considered typical to those that occur spontaneously in young laboratory rats of this age and strain and not treatment-related.

 

In conclusion, topical application of the test material under conditions of this study elicited no signs of systemic toxicity. The only significant finding was transient dermal irritation observed in several treated females. There were no adverse effects with respect to clinical signs, body weights, body weight changes, food consumption, clinical pathology parameters, organ weights, post-mortem observations, or microscopic changes. Accordingly, a No Observable Adverse Effect Level (NOAEL) was established at 1000 mg/kg of the test material in both male and female rats.