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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study report which meets basic scientific principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
Only two dose levels were used instead of the recommended three, and application was open rather than semi-occluded.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MRD-80-51
- Substance type: technical product
- Physical state: oily liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Lot/batch No.: ECA-6654
- Expiration date of the lot/batch: no data
- Stability under test conditions: evidently assessed, but no data provided
- Storage condition of test material: at room temperature

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HARE Rabbits For Research, Marland Breeding Farms, Inc., New Jersey
- Age at study initiation: no data
- Weight at study initiation: males: mean 2.1 kg, range 1.7-2.8 kg; females: mean 2.3 kg, range 1.6-2.9 kg
- Fasting period before study: no data
- Housing: Individually in elevated stainless steel cages
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-26
- Humidity (%): 43-75
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 July 1980 To: 8 September 1980

Administration / exposure

Type of coverage:
open
Vehicle:
other: Primol 185
Details on exposure:
TEST SITE
- Area of exposure: ~6.5 x 5.0 cm
- % coverage: no data
- Time intervals for shavings or clippings: clipped on Monday and Thursday of each week throughout the study

REMOVAL OF TEST SUBSTANCE
- Washing (if done): gently wiped with paper towels
- Time after start of exposure: ~6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): up to 2 ml/kg bw
- Concentration (if solution): 5 and 25% (w/v)
- Constant volume used: yes


VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount(s) applied (volume or weight with unit): 2 ml/kg bw


USE OF RESTRAINERS FOR PREVENTING INGESTION: no, but Elizabethan collar fitted
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sent to sponsor, no data provided
Duration of treatment / exposure:
27-30 days
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2 ml/kg bw of a 0, 5 or 25% solution
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
0, 70 or 350 mg/kg bw/day of MRD-80-51
Basis:
other: nominal per unit body weight; corrected for 5 applications per week
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: selected on the basis of a previous toxicity study (Dermal irritation screen in rabbits, Bio/dynamics Project No. 80-2483)
- Rationale for animal assignment (if not random): randomly distributed into control and treatment groups in an attempt to equalize mean group body weights
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once pretest, weekly durinq treatment and terminally (after fastinq).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: no data

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:
- Time schedule for examinations: no data

OPHTHALMOSCOPIC EXAMINATION: not examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: ~2 weeks prior to treatment and at study termination (on one of days 27-30 of treatment)
- Anaesthetic used for blood collection: no
- Animals fasted: no data
- How many animals: all survivors
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: ~2 weeks prior to treatment (and 1 week prior, in the case of plasma and erythrocyte cholinesterase), and at study termination (on one of days 27-30 of treatment)
- Animals fasted: no data
- How many animals: all survivors
- Parameters checked in table 1 were examined.

URINALYSIS: not examined

NEUROBEHAVIOURAL EXAMINATION: not examined

OTHER: brain cholinesterase levels were determined at study termination.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes - external surface, all orifices, cranial cavity, carcass, external and cut surface of the brain and spinal cord, thoracic, abdominal and pelvic cavities and their viscera, and cervical tissues and organs.

HISTOPATHOLOGY: Yes (see table 2) - these tissues were taken from all animals in the study and preserved and examined microscopically from all control and high-dose animals. Slides of the testes from all low-dose males were also examined microscopically (based on the findings of the high-dose evaluations). Slides of all gross lesions and tissue masses taken at necropsy were prepared and examined microscopically from all animals in the study. Slides of all tissues were examined microscopically from all animals either dying spontaneously or killed in extremis during the course of the study.
Other examinations:
none
Statistics:
A range of statistical methods were employed, including Bartlett's, ANOVA, Dunnett's, Kruskal-Wallis, Dunn's rank sum and Jonckheere's.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Two high-dose males and one low-dose male died during the course of the study, and 1 male and 1 female at the high dose were killed in a moribund condition. At the high dose 7 males and 8 females were slightly or moderately emaciated, while 4 males and 3 females at the low dose showed slight emaciation (which became moderate in 1 male and 1 female from week 3). This compared with a single control male consistently showing slight emaciation throughout the study. There was a greater incidence of lacrimation and nasal discharge at the high dose (and to a lesser extent, lacrimation at the low dose), compared with controls, as well as increased staining of the ano-genital area. The high test concentration produced thickening of the outer skin layer from week 3 or 4 of treatment in all surviving rabbits and, by week 4, exfoliation of the outer layer and roughening of the inner layer in 1 male and 8 of the 9 surviving females. No such effects were observed at the low test concentration. At both test concentrations, there were marked, dose-related increases in the incidence and severity of erythema, oedema, atonia, desquamation, fissuring, eschar formation and exfoliation.

BODY WEIGHT AND WEIGHT GAIN
At the high dose the rabbits lost weight throughout the course of the study, while at the low dose body weights were only slightly below those of the controls.

HAEMATOLOGY
After 4 weeks of treatment, mean haemoglobin, haematocrit and erythrocyte values were lower than in the controls, the reductions being statistically significant at the high dose and slight at the low dose. Various other haematological changes were not considered by the investigators to be of physiologic or toxicologic significance.

CLINICAL CHEMISTRY
Mean cholesterol values were increased and albumin levels were reduced, the effects being statistically significant at the high dose and slight at the low dose. Various other clinical chemistry changes were considered by the investigators to be within normal physiological limits and not of toxicologic significance.

ORGAN WEIGHTS
There were slight to statistically significant dose-related increases in relative kidney and adrenal weights in comparison with controls, while high-dose males showed marked reductions in the absolute weights of the testes and epididymides. Other organ weight changes were considered by the investigators to be "either reflective of body weight differences or within normal physiological limits and not of toxicologic significance".

GROSS PATHOLOGY
The testes of the high-dose males were observed to be markedly smaller than those of the control males.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination confirmed the irritating skin lesions in treated rabbits at both test concentrations. The testicular effects in the high-dose males were characterized by aspermatogenesis, diffuse tubular hypoplasia and a reduced mitotic activity in the seminiferous tubules.

OTHER FINDINGS
Terminal plasma, erythrocyte and brain cholinesterase values were slightly reduced (in some cases statistically significant) in both treatment groups, in comparison with controls.

Effect levels

Dose descriptor:
LOAEL
Effect level:
ca. 70 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; body weight; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Repeated dermal applications (uncovered) of up to 2 ml/kg bw of a solution containing 0, 5 or 25% test material, equivalent to about 0, 70 or 350 mg/kg bw/day (after adjusting for exposure on 5/7 days per week), were made to the clipped skin of rabbits (10/sex/group) for around 28 days (6 hours/day, 5 days/week). This resulted in weight loss, haematology and clinical chemistry effects, organ weight changes and local irritancy at both test doses, while the high-dose males also showed evidence of testicular toxicity (including reduced absolute testis weight and tissue changes). A LOAEL of 70mg/kg bw/day was established.
Executive summary:

Groups of 10 male and 10 female New Zealand White rabbits received repeated dermal applications of up to 2 ml/kg bw of a solution containing 0, 5 or 25% of test material in Primol 185 for around 28 days (6 hours/day, 5 days/week). These doses are equivalent to 0, 100 or 500 mg/kg bw per application or about 0, 70 or 350 mg MRD-80-51/kg bw/day after adjusting for exposure on 5/7 days per week. The application site remained uncovered, but the rabbits were fitted with an Elizabethan collar to prevent ingestion of the substance. Approximately 6 hours after application, excess test material was removed with paper towels.

Some deaths occurred at the top dose during the course of treatment, and at both doses the animals became emaciated. Other effects observed, particularly at the top dose, included weight loss (at the top dose only), lacrimation, skin irritation, nasal discharge and staining of the ano-genital area. There were reductions in mean haemoglobin, haematocrit and erythrocyte values after 4 weeks, particularly at the top dose, and changes in certain clinical chemistry findings (notably increased cholesterol levels and decreased albumin levels). Relative kidney and adrenal weights were increased at both doses, and high-dose males also showed reduced absolute testis weights. Histopathological examination confirmed the irritant effects of the test substance on the skin and its testicular toxicity.

As there were treatment-related effects at both test doses (~70 and 350 mg/kg bw/day), a NOAEL could not be established in this study, and the LOAEL can be considered to be 70 mg /kg bw/day.