Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 September 1992 to 05 March 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA Pesticide Assessment Guidelines, Subdivision F. Hazard Evaluation: Human and Domestic Animals 82-2 Repeated dose dermal toxicity study.
Version / remarks:
Revised Edition, November 1984
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate
EC Number:
266-358-7
EC Name:
Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate
Cas Number:
66423-13-0
Molecular formula:
C18H27ClO3
IUPAC Name:
octyl (2R)-2-(4-chloro-2-methylphenoxy)propanoate

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on species / strain selection:
The rabbit was chosen as the test species as it has been shown to be a suitable model for this type of study and is the species recommended in the test guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10 to 12 weeks old on arrival.
- Weight at study initiation: 2.0 to 2.8 kg on arrival.
- Fasting period before study: Food and water were only withdrawn overnight prior to collection of samples.
- Housing: All rabbits were caged individually in metal cages with perforated floors.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: An eight-day acclimatisation period was allowed between delivery of the animals and start of treatment. The health status of all animals was monitored, by daily observation throughout the acclimatisation period, to ensure that the rabbits selected for final assignment to the study were satisfactory.

DETAILS OF FOOD AND WATER QUALITY: The batches of diet used for the study had been analysed for nutrients, possible contaminants and microorganisms. Results of the routine physical and chemical examination of drinking water at source, as conducted usually weekly by the supplier, are made available to Huntingdon Research Centre Ltd., as quarterly summaries. There was no information available to the Study Director to indicate that any non-nutrient substance likely to influence the effect of the test material could reasonably have been expected to be present in the diet or the drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature was generally controlled in the range 15 to 21 °C, however on Day 11, minimum to maximum temperature readings of 17 to 30.5 °C were recorded as a result of animal building maintenance work.
- Humidity (%): Relative humidity was generally controlled in the region of 36 to 62 % R.H., however on Day 11, minimum to maximum humidity readings of 32 to 62 % R.H. were recorded as a result of animal building maintenance work.
- Air changes (per hr): Air exchange was maintained at approximately 19 air changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled to provide 12 hours artificial light (0700 - 1900 hours) in each 24-hour period.

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Remarks:
(the test material was moistened with distilled water to ensure good contact with the skin)
Details on exposure:
RANGE-FINDING STUDY
A group comprising one male and one female rabbit of the New Zealand White strain was treated by dermal application of the test material at a dosage of 1 000 mg/kg/day. The treatment area was a shaven region of skin measuring 12 x 8 cm (approximately 10% of the total body surface) and the test material was moistened with distilled water at a constant volume of 3 mL/rabbit/day. After dosing, the treatment site was covered by an impervious bandage for approximately six hours. Following the exposure period, the dressings were removed and the treated skin was washed with warm water (30 - 40 °C) and then gently blotted dry.

MAIN STUDY
- Two days before the start of treatment each animal was weighed and forty rabbits were randomly allocated to four groups, each consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.
- Prior to dosing on Day 1 (24 September 1992), a final health status examination revealed one of the rabbits allocated to Group 2, believed to be a female, to be in fact a very immature male. This animal was replaced before study start with an appropriate spare rabbit.
- Immediately after dosing, a male rabbit in Group 2 started to convulse violently. The animal was sacrificed immediately on humane grounds - its condition was attributed to shock brought on by the bandaging procedure (a very infrequent but recorded response in rabbits). Post mortem findings revealed congested lungs as the only notable finding. This animal was immediately replaced by an appropriate spare rabbit. Neither actions were considered to have affected the integrity of the study and the weights of replacement rabbits were similar to those of the original animals.
Following the commencement of treatment, spare animals were removed from the study. No further investigations were performed on these animals.

TEST SITE
- Area of exposure: The treatment area was a shaven region of skin measuring 12 x 8 cm on the back of each rabbit.
- % coverage: Approximating to 10 % of the total body surface.
- Type of wrap if used: The treatment site was covered with an impervious bandage consisting of gauze covered with 'Elastoplast' elastic adhesive dressing backed with impervious 'Sleek' plaster.
- Time intervals for shavings or clipplings: The hair was clipped from the dorsal region of each rabbit. Hair clipping was carried out approximately twenty-four hours prior to the first application of the test material. Hair clipping was repeated as necessary during the experimental period. The skin sites were not abraded.

REMOVAL OF TEST SUBSTANCE
- Washing: The treated skin was washed with warm water (30 - 40 °C) and gently blotted dry.
- Time after start of exposure: The test material remained on the back of each rabbit for approximately six hours each day after which time the dressings were removed.

TEST MATERIAL
- Amount(s) applied: The appropriate amount of test material (equating to the correct dosage) was weighed out daily for each animal and spread evenly over the prepared skin of the rabbits.
- Constant volume or concentration used: Yes. Each animal received a constant dosage based upon its most recently recorded bodyweight. The test material was moistened with distilled water at a constant volume/dosage (0.1, 0.3 and 3 mL/rabbit/day for Groups 2, 3 and 4 respectively) to ensure good contact with the skin.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Animals were treated for either twenty-one (males) or twenty-two (females) consecutive days. Treatment of animals not scheduled for sacrifice on Day 22 was continued up to 24 hours prior to sacrifice.
Frequency of treatment:
Animals were treated once daily.
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
Five per sex per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: A high dosage of 1 000 mg/kg/day was selected on the basis of available toxicity information, in particular, rabbit acute dermal toxicity data [LD50 (rabbit) > 4 000 mg/kg bodyweight] and the results of a preliminary study.
- Fasting period before blood sampling for clinical biochemistry: Yes, food and water were withdrawn overnight prior to collection of samples.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded. All animals were checked early in each day and again in the late afternoon to look for dead or moribund animals.

DERMAL IRRITATION: Yes
- Time schedule for examinations: Local irritation was recorded immediately prior to the first daily application of the test material and subsequently daily. Local dermal reactions (erythema and oedema) resulting from treatment were assessed on a numerical basis according to a modified Draize scoring system as follows:
Erythema and eschar formation
0 = No erythema
1 = Slight erythema
2 = Well-defined erythema
3 = Moderate erythema
4 = Severe erythema (beet redness) to slight eschar formation (injuries in depth)

Oedema
0 = No oedema
1 = Slight oedema
2 = Well-defined oedema (area well-defined by definite raising)
3 = Moderate oedema (raised approximately 1 mm)
4 = Severe oedema (raised more than 1 mm and extending beyond the area of exposure)
Dermal changes other than erythema and oedema were recorded separately.

BODY WEIGHT: Yes
- Time schedule for examinations: All rabbits were weighed prior to dosing on Day 1 (Week 0) and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION:
- Food consumption: The quantity of food consumed by each rabbit was measured at weekly intervals throughout the study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was withdrawn from the median artery of the ear of all rabbits prior to termination. Male and female samples were collected on consecutive days (Days 20 and 21 respectively).
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, food and water were withdrawn overnight prior to collection of samples.
- How many animals: All rabbits
- Parameters checked: The following parameters were analysed with an Ortho ELT-1500 Analyser, using standard Ortho methodology: Packed cell volume (PCV), Haemoglobin (Hb), Red blood cell count (RBC).
Absolute indices:
Mean corpuscular haemoglobin concentration (MCHC), Calculated: Hb (g/dL) x 100 / PCV (%)
Mean corpuscular volume (MCV), Calculated: PCV (%) x 10 / RBC (x10^6/mm³)
Mean corpuscular haemoglobin (MCH), Calculated: Hb (g/dL x 10 / RBC (x10^6/mm³)
Platelet count (Plts) and Total white blood cell count (WBC)
The following estimations were measured using the appropriate methodology:
Thrombotest (1T) - Method of Owren, P.A. (Lancet, 1959, ii, 754), Differential white blood cell count (Diff) - namely: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M).
The percentage distribution of each cell type was determined by standard microscopy of a blood smear stained with modified Wright's stain counting 100 cells. Percentage values were then converted to absolute values by computer inevitably involving a "rounding off'' in a proportion of the results. Hence, the measured total WBC may differ slightly from the calculated total for the differential count.
Additional blood film slides were prepared and examined for morphological abnormalities: Polychromasia, Hypochromasia, Anisocytosis, Rouleaux formation, Separate film report (generated for additional abnormalities).
The collected blood samples were divided into tubes containing EDTA anticoagulant for haematological investigations and citrate anticoagulant for coagulation tests.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was withdrawn from the median artery of the ear of all rabbits prior to termination. Male and female samples were collected on consecutive days (Days 20 and 21 respectively).
- Animals fasted: Yes, food and water were withdrawn overnight prior to collection of samples.
- How many animals: All rabbits
- Parameters checked: The following parameters were analysed with an Hitachi 737 Clinical Chemistry Analyser:
Glucose - using BCL Test Kit (hexokinase mediated), Total protein, Albumin (Alb), Globulin (Glob), Calculated: Total protein (g/dl) minus Alb (g/dl), Albumin/Globulin ratio (A/G), Calculated from Total protein and Albumin concentrations, Urea nitrogen (Urea Nitr), Creatinine, Alkaline phosphatase (AP) - reaction temperature 30 °C, Glutamic-pyruvic transaminase (GPT), also known as 'alanine aminotransferase (ALT)' - using BCL Test Kit, reaction temperature 30 °C, Glutamic-oxaloacetic transaminase (GOT), also known as 'aspartate aminotransferase (AST)' - using BCL Test Kit, reaction temperature 30 °C, Total bilirubin (Bilirubin), Sodium (Na), Potassium (K), Calcium (Ca), Chloride (Cl), Inorganic phosphorus (P), Cholesterol (Chol).
The collected blood samples were divided into tubes containing heparin anticoagulant for biochemical tests.

URINALYSIS: Yes.
- Time schedule for collection of urine: Overnight urine samples were collected for all rabbits prior to termination. As no overnight urine samples were obtained for various rabbits from all four groups, resampling occurred on the night prior to sacrifice.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, food and water were withdrawn overnight prior to collection of samples.
- Parameters checked: The following estimations were measured using the appropriate methodology:
Volume (Vol), pH - using pH meter, Specific Gravity (SG) - using Atago UR-1 Refractometer, sample compared with water (nominal value of 1 000)
Protein - using Roche Cobas Centrifugal Analyser, utilising modified method of Macart, M. and Gerbaut, L. (Clin. Chim. Acta, 1984, 141, 77)
The following tests were also performed using qualitative indicators of analyte concentration:
Total reducing substances (TRS), Glucose (Gluc), Ketones (Ket), Bile pigments (Bile Pigs), Urobilinogen (Uro-bi), Haem pigments (Haem Pigs - positive or negative finding only).
Microscopic examination of urine samples was not performed as the turbidity of rabbit urine collected overnight prevents accurate assessment of cellular deposits.
The colour and appearance of samples was not recorded in the raw data. This deviation from protocol was not considered to affect the validity of the results as any abnormalities would have been noted.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
All animals were randomly killed on Day 22 (males) or Day 23 (females) by means of an intravenous overdose of pentobarbitone sodium and a complete gross necropsy undertaken. This necropsy included observations of all external surfaces, orifices and cranial cavity, the external and cut surfaces of brain, all viscera and glands, and the carcass. Specified organs were weighed and relevant tissue samples were fixed for microscopic examination.

GROSS PATHOLOGY: Yes.
The following organs from each animal were weighed as soon after dissection as possible to avoid drying: Adrenals, brain, heart, kidneys, liver, ovaries, spleen, testes (with epididymides), thyroid (with parathyroid).

The macroscopic appearance of the tissues of all rabbits was recorded and samples of the following tissues were preserved in 10% buffered formalin: Adrenals, aorta, brain (medullary, cerebellar and cortical sections), caecum, colon, duodenum, eyes (Davidson's fluid as fixative), gall bladder, heart, ileum, jejunum, kidneys*, larynx, liver*, lungs*, lymph nodes (cervical and mesenteric), mammary gland, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, salivary gland, sciatic nerve, skeletal muscle, skin (treated and untreated)*, spleen, sternum (for bone and marrow section), stomach, testes (including epididymides), thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus, vagina, any macroscopically abnormal tissues*.
* Tissues required for histopathological examination for rabbits from Groups 1 and 4.

HISTOPATHOLOGY: Yes
Fixed tissue samples required for microscopic examination were prepared by embedding in paraffin wax (m.p. 56 °C); sections were cut at 4 μm and stained with haematoxylin and eosin.
Microscopic examination of prepared slides (from tissues indicated under Macroscopic pathology) was carried out for all rabbits of Group 1 (control group) and Group 4 (high dosage group) killed on Days 22 (males) or 23 (females).
Examinations were extended to include the spleens from all female rabbits following an apparent decrease in the weight of this organ for females from the intermediate and high dosage groups.
Slides of treated skin from all rabbits of Group 2 and 3 (low and intermediate dosage groups respectively) were also prepared as a contingency to cover possible changes at the high dosage.
Statistics:
All statistical analyses were carried out separately for males and females.
The following sequence of statistical tests was used for bodyweight gains, weekly food consumption, organ weight and clinical pathology data:
(i) If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75 %), the proportion of values different from the mode was analysed by Fisher's exact test followed by Mantel's test for a trend in proportions. Otherwise:
(ii) Bartlett's test was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1 % level, a logarithmic transformation was tried to see if a more stable variance structure could have been obtained.
(iii) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a oneway analysis of variance was carried out followed by Williams' test for a dose-related response.
(iv) If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test).
Organ weight analysis was initially carried out using analysis of variance as outlined above on absolute organ weights. Covariate analysis of organ weight data (with final bodyweight as covariate) was also performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10 % level of significance.
Significant differences between control animals and those treated with the test material have been expressed at the 5 % (* P < 0.05) or 1 % (** P < 0.01) level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related clinical signs.
Notable clinical findings were limited to transient cases of changes in faeces production (loose faeces, soft faeces and few faeces) and a thin-looking appearance in rabbits from all four groups. These changes are commonly seen in laboratory rabbits and at the frequency observed were not considered to be important.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Slight erythema was observed as early as Day 2 among rabbits treated at 1 000 mg/kg/day with reactions progressing to well-defined erythema with slight or well-defined oedema in eight animals by Day 8; slight erythema only was recorded in the remaining two males. Thereafter, these responses were generally maintained to the end of the study although well-defined erythema with moderate oedema was recorded for one male between Days 10 and 14 and for one female between Days 10 and 16. Additional reactions comprised cases of beige staining of the treatment site (not enough to prevent assessment of erythema) from as early as Day 3 and lasting to 19 days plus treatment site dryness and desquamation (sloughing) of the stratum corneum generally recorded during all three weeks and lasting up to 18 days in the majority of rabbits. Less common cases of cracking and/or hardening of the treatment site, lasting up to 8 days during the middle part of the study, were recorded for two males and up to two females; one further male also showed spots on the treatment site on Days 17 and 18.
Although slightly less marked than above, irritation reactions for rabbits treated at 100 mg/kg/day ranged from slight erythema with or without slight oedema from as early as Day 2 or 3 to well-defined erythema with slight or well-defined oedema in seven animals by Day 8; slight erythema only was recorded in the remaining three females. Thereafter, these reactions were maintained, generally to the end of the study, although a slight degree of amelioration was recorded during Week 3 for three males. Additional reactions comprised beige staining of the treatment site during Weeks 2 and 3 and lasting up to 11 days for three males and three females plus sloughing from as early as Day 6 and lasting up to 15 days for all rabbits.
Reactions were limited for rabbits treated at 10 mg/kg/day to cases of slight erythema as early as Day 3 in seven animals and lasting up to 20 days along with slight oedema in one male for 8 days. The remaining three rabbits showed no response throughout the study. Additional reactions comprised sloughing for one male and one female during the middle part of the study.
No dermal reactions were recorded for control rabbits.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Intergroup variation in actual bodyweights and bodyweight gains revealed no disturbances that were considered to be related to treatment with the test material. Analysis of individual values shows that mean differences from controls can be attributed to natural variation in these parameters.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Intergroup variation in food consumption revealed no disturbances that were considered to be related to treatment with the test material. Analysis of individual values shows that mean differences from controls can be attributed to natural variation in these parameters.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increases in monocyte counts for males treated at 1 000 mg/kg/day and in basophil values for females receiving 100 and 1 000 mg/kg/day in comparison with controls (P < 0.01 and P < 0.05 respectively) were small in nature and can be attributed to chance.
Intergroup variation in the remaining parameters measured revealed no disturbances that were considered to be related to treatment with the test material.
Analysis of blood slides revealed a low occurrence of slight polychromasia and/or slight anisocytosis (9/40 incidences) among study animals. This finding is not uncommon among young laboratory rabbits and was not considered to be important.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant (P < 0.05) decrease in urea nitrogen levels was recorded for all female rabbits treated with the test material in comparison with the controls. Cholesterol values were also statistically (P < 0.05) lower for female rabbits treated at 100 or 1 000 mg/kg/day. These differences in urea nitrogen and cholesterol were not dosage-related and, as wide variation is commonly seen in laboratory rabbits, statistical significance was considered to have arisen by chance.
There were no other statistically significant differences from the controls for remaining biochemical parameters measured.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Results revealed a statistically significant increase in protein levels for males treated at 1 000 mg/kg/day in comparison with controls (P < 0.05). Reduced urine volume (P < 0.05 or P < 0.01) was also recorded for all females treated with the test material. Both these changes can be attributed to chance with the latter finding arising as a result of a large urine output from two control females.
Intergroup variation in the remaining parameters measured revealed no disturbances that were considered to be related to treatment with the test material.
No overnight samples were obtained for 4/40 rabbits. Some success in obtaining a specimen (2/4 rabbits) occurred following repeat overnight sampling for these animals on the night prior to sacrifice. Results were essentially similar to those recorded at first analysis.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significantly (P < 0.05) lower than control absolute spleen weights were recorded for female rabbits treated at 100 or 1 000 mg/kg/day. This finding was not dosage-related and following histopathological examination was considered to be of no toxicological importance.
There were no other significant differences in organ weights between control and treated groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed at termination revealed no changes that were attributable to treatment with the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes were found in the treated skin samples. These were as follows:
Minimal diffuse acanthosis was found in the treated skin samples of three males and three females treated at 1 000 mg/kg/day and also in two females treated at 10 mg/kg/day. This change was not present in rabbits treated at 10 mg/kg/day.
Spleens from all rabbits showed minimal or moderate congestion with the majority of control animals showing a moderate degree. These findings were considered to be of no toxicological importance.
All other findings were considered to be incidental in origin and of no toxicological significance.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
RANGE-FINDING STUDY
Clinical signs
Soft faeces were recorded for the male rabbit on Day 1 and for the female on Days 1, 2 and 7 to termination; the latter animal also had diarrhoea on Days 5 and 6. No other clinical findings were seen throughout the study.

Dermal response
Irritation reactions progressed from slight erythema for both rabbits with slight oedema in the male on Day 3 to well-defined erythema with slight or well-defined oedema by the end of the study. These reactions were accompanied to termination by dryness and desquamation (sloughing) of the stratum corneum from Day 5 or 6 and beige staining on the dose site from Day 7; the latter observation did not prevent assessment of dermal scoring.

Bodyweight and food consumption
Satisfactory bodyweight gains and food consumption were recorded for both rabbits throughout the study.

Terminal procedures
Post mortem revealed no left kidney and an apparently enlarged gall bladder for the male as the only macroscopic abnormalities. Liver and spleen weights for the latter animal appeared slightly low while the right kidney weight appeared slightly raised; liver, spleen and kidney weights for the female were all unremarkable.

The results of this investigation with the test material indicated that 1 000 mg/kg/day was a suitable high dosage choice for the twenty-one day study in the rabbit. The proposed dosages for the latter study were 0, 10, 100 and 1 000 mg/kg/day.

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 systemic effects observed at the highest dose tested.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study it was considered that 1 000 mg/kg/day represents a NOAEL in the rabbit in terms of systemic toxicity while a no effect level for dermal irritation was not established.
Executive summary:

The repeated dose toxicity of the test material via the dermal route was assessed according to OECD Test Guideline 410 and in compliance with GLP.

This study was performed to assess the cutaneous and systemic toxicity of the test material to the rabbit. The test material was administered by dermal application once daily, to groups of five male and five female rabbits for twenty-one (males) or twenty-two (females) consecutive days, at fixed dosages of 10, 100 and 1 000 mg/kg/day. The test material was moistened sufficiently with distilled water to ensure good contact with the skin. A further group of rabbits (five males and five females) was held as a concurrent control receiving distilled water alone. Bodyweights, food consumption and clinical observations were recorded during the study. Blood samples for clinical investigations were taken on Day 20 (males) or Day 21 (females) and animals were killed and examined macroscopically on Day 22 (males) or Day 23 (females). Histological examination of specified tissues was then initiated.

The following comments in relation to real or possible treatment-related findings are made in summary:

Among rabbits treated at 1 000 mg/kg/day, slight erythema was observed as early as Day 2 with reactions progressing to well-defined erythema with slight or well-defined oedema in eight animals by Day 8; slight erythema only was recorded in the remaining two males. Thereafter, these responses were generally maintained to the end of the study although well-defined erythema with moderate oedema was recorded for one male and one female between Days 10 and 16. Additional reactions comprised cases of beige staining of the treatment site (not enough to prevent assessment of erythema) from as early as Day 3 and lasting to 19 days plus treatment site dryness and desquamation (sloughing) of the stratum corneum generally recorded during all three weeks and lasting up to 18 days in the majority of rabbits. Less common cases of cracking and/or hardening of the treatment site, lasting up to 8 days during the middle part of the study, were recorded for two males and up to two females; one further male also showed spots on the treatment site on Days 17 and 18.

Among rabbits treated at 100 mg/kg/day, irritation reactions ranged from slight erythema with or without slight oedema from as early as Day 2 or 3 to well-defined erythema with slight or well-defined oedema in seven animals by Day 8; slight erythema only (developing to well-defined erythema) was recorded in the remaining three females. Thereafter, these reactions were maintained, generally to the end of the study, although a slight degree of amelioration was recorded during Week 3 for three males. Additional reactions comprised beige staining of the treatment site during Weeks 2 and 3 and lasting up to 11 days for three males and three females plus sloughing from as early as Day 6 and lasting up to 15 days for all rabbits.

Among rabbits treated at 10 mg/kg/day, reactions comprised slight erythema as early as Day 3 in seven animals and lasting up to 20 days along with slight oedema in one male for 8 days. The remaining three rabbits showed no response throughout the study. Additional reactions comprised sloughing for one male and one female during the middle part of the study.

Microscopic pathology: Minimal diffuse acanthosis was recorded in the treated skin samples of 3/5 males and 3/5 females at 1 000 mg/kg/day and 2/5 females at 100 mg/kg/day.

Remaining parameters, namely clinical signs, bodyweight, food consumption, biochemistry, haematology, urinalysis, organ weights and macroscopic pathology, were not affected by the dermal application of the test material at 10, 100 or 1 000 mg/kg/day.

Under the conditions of the study it was considered that 1 000 mg/kg/day represents a NOAEL in the rabbit in terms of systemic toxicity while a no effect level for dermal irritation was not established.