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Repeated dose toxicity: dermal

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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
duplication of group size (10 instead of 5 per sex and dose); satellite group: 5 per sex and dose.
Principles of method if other than guideline:
Group sizes were increased to improve the biostatistical power.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
liquid: viscous
Details on test material:
- Manufacturers identification: Novares LA 300
- Substance type: organic
- Test material is 'Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol' (OAPP), EC list number 700-960-7 (assigned manually to validated substances from inquiries by ECHA). Originally the substance phenol, methylstyrenated, CAS No. 68512-30-1, EC No. 270-966-8 was submitted for registration. Subsequent to substance validation, the identity of the substance was changed by ECHA.
- for additional information see respective study record
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares LA 300 (phenol, methylstyrenated)
- Lot/batch No.: 28166
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light

Test animals

Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF (Specified Pathogen Free) breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 205 ± 12 g (m); 163 ± 9 g (f)
- Fasting period before study: no
- Housing: all the study proceeded in the SPF (Specified Pathogen Free) animal house of CETA under conditions according to SOP No. 12. Animals were housed in SPF animal room, 2-3 rats of the same sex (during acclimatisation period) or individually (during treatment period) in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood.
- Diet: free access to food (ad libitum; complete pelleted diet for rats and mice in SPF breeding (ST 1 BERGMAN); the diet was sterilised before use.
- Composition of the diet: wheat, oats, fish meal powder, dried snail-clover, soya extracted groats, wheat sprouts, dehydrated yeast, calcium carbonate, vitamin and mineral complex
- Nutrient content of the diet: Crude protein - min. 21%, Drip - max. 14%, Fat - min. 3%, Fiber - max. 4.1%, Ash - max. 7%, Calcium - min. 1%, Phosphorus - min. 0.8%, Magnesium - min. 0.2%, Sodium - max. 0.25%.
- Water (e.g. ad libitum): Free access to drinking water; ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: Relative humidity of 30 - 70%
- Air changes (per hr): approx. 15
- Photoperiod: 12 h light, 12 h darkness

Administration / exposure

Type of coverage:
occlusive
Vehicle:
olive oil
Details on exposure:
TEST SITE
Approximately 24 hours before testing, fur was shaved from dorsal area of the trunk of the test animals.
- Area of exposure: not less than 10% of the body surface (approx. 5 - 6 cm x 8 - 9 cm)
- Type of wrap: during exposure, the test substance was held in contact with the skin with porous gauze dressing and non-irritating tape for 6 hours.
- Time intervals for shavings: approx. weekly

REMOVAL OF TEST SUBSTANCE
- The treated and control groups were treated 7 days per week at the same daytime (7.00 – 9.00 am) for the period of 28 days.
- At the end of the exposure period (6 h daily), residual test substance was removed using water.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the concentrations of emulsions at all dose levels were adjusted such as to ensure the application of 1 ml per 100 g of body weight.
- The emulsion was prepared daily just before administration.
- Concentration: 160, 400, and 1000 mg test item per 10 mL of vehicle
- Constant volume used: yes (10 mL/kg bw)

VEHICLE
- Justification for use and choice of vehicle: due to the viscosity, the test substance was applied as emulsion in olive oil.
- Amount(s) applied: 1 mL olive oil per 100 g body weight (same as treatment groups)

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days; 6 h/day
Frequency of treatment:
daily (7 d/week)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
controls
Dose / conc.:
160 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups: 10 male, 10 female
Satellite groups: 5 male, 5 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Basis for selected doses: Range finding study with dose levels of 100, 250, 500, and 1000 mg/kg/day.
The doses for the dose-range finding experiment were derived from the limit dose of 1000 mg/kg/day (according to the guideline).
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All rats were examined for vitality or mortality daily during the treatment and recovery period.
The health condition and behavior were controlled daily during the check-in, acclimatisation period, during the administration and during the recovery period in groups.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of administration, then at weekly intervals
During the first part of observation, behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behaviour.
The second part was the observation during removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanness of fur around foramina.

DERMAL IRRITATION (if dermal study): yes, examined

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: yes
Once every week, the remainder of pellets was weighed in each cage. Average values were calculated for each week of the study.
The food consumption for each animal/day was calculated from the average values of each group.

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: yes
Weight gains were computed as arithmetic mean per group and day.

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice per week

OPHTHALMOSCOPIC EXAMINATION: not examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of test period (day 29 of study of main groups; day 42 of study of satellite groups)
- Anaesthetic used for blood collection: yes (light ether narcosis)
- Animals fasted: yes (18 h before blood sampling)
- How many animals: no data
- Parameters checked in table No. 2 were examined

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of test period
- Animals fasted: yes (18 h before blood sampling)
- How many animals: no data
- Parameters checked in table No. 3 were examined

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of test period
- Metabolism cages used for collection of urine: yes
- Animals fasted: no data
- Parameters checked in table No. 4 were examined

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: saily at the end of the exposure period (main groups) or at the end of the recovery period (satellite groups)
- Dose groups that were examined: all groups
- Battery of functions tested: The sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated, and motor activity assessment was conducted. Moreover, the individual observations of grip strength were performed using dynamometer. Measurements were made on: 1) forelegs, 2) hindlegs, 3) all four legs.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy of the cranial, thoracic and abdominal cavities; organs were collected for weighing and further histological examination.
(absolute and relative weights of lungs, liver, kidneys, adrenals, testes, epididymides, brain and pituitary gland)

HISTOPATHOLOGY: Yes; organs of control animals and animals treated with the highest dose level (1000 mg/kg/day) were examined:
Normal and treated skin, lung, liver, kidneys, spleen, brain, pituitary gland, testes, epididymides (both fixed in Davidson solution), as well as ovaries
and accessory genital organs, adrenal glands, heart and target organs (organs showing gross lesions or changes in size).
Statistics:
ANOVA test - Analysis of Variance (QC.Expert 2.5) - at the significance level of 0.05 was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. The respective control groups with vehicle were compared with the three treated groups, and the respective satellite controls with vehicle were compared with the treated satellite groups.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
single incidental effects without any dose-response-relationship, not toxicologically relevant
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
see details below (clinical signs); local effects, not toxicologically relevant
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
see details below: variations in parameters are minor and sporadic, unrelated to dose.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
see below for details
Urinalysis findings:
no effects observed
Description (incidence and severity):
see below for details
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
see below details
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below for details: minor, not toxicologically relevant
Gross pathological findings:
no effects observed
Description (incidence and severity):
see below for details: sporadic changes noted, none in recovery groups
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
see below for details: only in females changes in kidney and urethra, not considered treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY:
Males: One animal at the dose level of 400 mg/kg/day showed red secretion around the nose or eyes.
In one animal at the dose level of 160 mg/kg bw/day diarrhoea was observed.
Slight skin erythemas of the application area were recorded at the dose level of 400 mg/kg bw/day (one male in the 3rd week), and marked erythemas at the dose level of 1000 mg/kg bw/day (1 male in the 1st week, 4 males and 2 satellite males in the 3rd week and 2 males and 1 satellite male in the 4th week).
Females: At the dose level 400 mg/kg bw/day, slight skin erythemas of the application area were recorded only in one animal in the 3rd week.
At the dose level of 1000 mg/kg bw/day, this change of application area was observed in a few females during the whole application:
The following number of affected females during the test period showed dermal irritation: 6 females and 1 satellite female in the 1st week, 1 female in the 2nd week, 5 females and 3 satellite females in the 3rd week, and 3 females and 2 satellite females in the last week of application.
No animal died during the test period.

BODY WEIGHT AND WEIGHT GAIN:
In both sexes no significant differences between the treatment groups and the control groups were observed during the whole test period.

FOOD CONSUMPTION:
In both sexes no significant differences between the treatment groups and the control groups were observed during the whole test period.

FOOD EFFICIENCY:
Males: During the 1st week of application, a slightly decreased food efficiency was recorded at the lowest dose level. From the 2nd week to the 4th week of application, the food efficiency revealed no significant differences at all dose levels compared with the control group.
Females: In the 1st week of application, an increased food efficiency was recorded at all dose levels. Relatively well-balanced conversion was recorded from the 2nd and 3rd week of application. In the last week of application, a decrease in food efficiency was recorded at all dose levels.
Satellite groups: In the 1st week of the test period, the food efficiency was increased in the treated group of both sexes. From the 2nd week to the end of the application period and during the recovery period no sicnificant differences were observed.

WATER CONSUMPTION:
The average water consumption of the treated groups of both sexes revealed no significant differences in comparison with the control groups during the whole application period except in the female group of the lowest dose level, it was lower than in the control group during the whole study.
Satellite males: The water consumption of both groups was not signifcant different during the whole application and recovery period.
A slight decrease was recorded only in the 3rd week of application.
Satellite females: In the treated group, the water consumption was higher than in the control group during the whole application period and the two weeks of recovery (the increase especially during treatment can be explained by the higher mean body weight of the treated group).

HAEMATOLOGY: see Report, Table 14 and 15
Males: Changes in the haemocoagulation system were recorded. A slightly shortened prothrombin time (PT) was measured at the dose levels of 400 and 1000 mg/kg bw/day with statistical significance only at the middle dose.
The concentration of fibrinogen was statistically significantly increased at the dose level of 1000 mg/kg bw/day.
Total leucocyte count was slightly decreased (without statistical significance) at all dose levels.
Other measured parameters were similar to the control group.
Females: A shortened prothrombin time (PT) was measured at all dose levels, but with statistical significance only at the dose level of 400 mg/kg bw/day.
Total leucocytes count was decreased with statistical significance at the dose level of 160 mg/kg bw/day.
A slightly decreased mean corpuscular volume was recorded at all treated groups but without statistical significance.
Other measured parameters were similar to the control group.
Satellite groups: A statistically significant increase in the value of haematocrit was recorded in the treated group in both sexes.
Other parameters of red blood cell - total erythrocyte count and MCV - were slightly increased without statistical significance.
A statistically significant decrease in the platelet count was recorded in the treated females.
Total leucocyte count of treated males was insignificantly decreased.
Fibrinogen concentration of the treated males was statistically significantly increased.
A shortened PT was recorded without statistical significance. All other measured parameters were similar to the control group.

CLINICAL CHEMISTRY: see Report, Table 16 and 17
In both sexes an increased activity of ALP was recorded at the dose levels of 400 (males) and 1000 mg/kg bw/day (males and females), but with statistical significance only at the high dose level.
All treated groups showed an increased value of bilirubin without statistical significance and without evidence of a dose-response relationship (in females only at the dose level of 1000 mg/kg bw/day).
In males at the dose level of 160 mg/kg bw/day, a significantly decreased concentration of total protein together with an insignificantly decreased content of albumine was measured. In females the total protein content was significantly decreased at the dose levels of 160 and 400 mg/kg bw/day.
Only in male animals a significantly increased concentration of glucose was observed.
Only in females a decrease of ALT activity (significant at the dose levels of 160 and 1000 mg/kg bw/day), a decrease of anorganic phosphorus (signifcant at the dose level of 1000 mg/kg bw/day), and a slight increase of the value of urea at the dose levels of 400 and 1000 mg/kg bw/day (without significance) were determined.
All other measured parameters were similar to those of the control groups.

Satellite groups:
Males: A significantly decreased value of urea was recorded in the treated group. The apparent effect is not treatment-related but due to
the inexplicably high mean urea level observed in the urine of the concurrent control group.
Furthermore, a significantly increased activity of AST, a decreased activity of ALT an increased concentration of bilirubin, an increased concentration of chloride and a decreased concentration of potassium with statistical significance was recorded in the treated group.
Females: A statistically significant decrease in the concentration of calcium and potassium were recorded in the treated group.
The value of total protein in the treated group was slightly decreased (without statistical significance).
All other measured parameters were similar to the control group. All parameters were within the historical ranges of average control values.

URINALYSIS: see Report, Table 18 and 19
No statistically significant changes in urine were recorded at any dose level, except a statistically significant decrease in urine pH of females at the dose level of 160 mg/kg/ bwday.
Presence of protein, blood and leucocytes was recorded in the urine of one male at the dose level of 400 mg/kg bw/day.
Presence of protein in the urine of two males and leucocytes in the urine of one male were detected at the dose level of 1000 mg/kg bw/day.
The urine of three males at the highest dose level showed white clouding.
Satellite groups: No significant intergroup differences were observed. The urine of one treated male showed blood.
Presence of leucocytes was recorded in the urine of three control and two treated males.

NEUROBEHAVIOUR: see Report, Table 12 and 13 (Functional observation)
The reaction to approach, touch, the reaction to noise, and the reaction to pain and light (pupillary reflex) were the same in all treated groups of males and not different from the control groups. The values of grip strength of forelegs and hind legs of all groups showed no significant differences.
All inter- and intra-group differences in scores were considered to be a result of normal variation for the rats of the used strain and of age, and were of no toxicological importance.

ABSOLUTE ORGAN WEIGHTS:
In male animals at the dose levels of 400 and 1000 mg/kg bw/day, the absolute weight of the pituitary gland was significantly increased. Male animals at these dose levels also showed a slightly increased weight of liver (not significant).
Female animals showed a slightly increased weight of liver at the dose level of 400 mg/kg bw/day.
Further significant intergroup differences were not observed.
Satellite groups: The treated males showed a decreased body weight at the end of the recovery period. Hence, the absolute weight of all organs was slightly lower than in the control males, but only the absolute weight of epididymides in treated males was significantly decreased. In females no significant differences were observed.

RELATIVE ORGAN WEIGHTS:
In male animals a statistically significant dose-dependant increase in the relative weight of liver was recorded at the dose levels of 400 and
1000 mg/kg bw/day.
Also the relative weight of the pituitary gland was significantly increased at the dose levels of 400 and 1000 mg/kg bw/day.
Female animals at the dose levels of 400 and 1000 mg/kg bw/day showed as well a slightly increased relative weight of liver (not significant).
Further significant intergroup differences were not observed.
In the satellite groups no significant deviations were found.

GROSS PATHOLOGY: see Report, Table No. 24 and 25 for details
The macroscopic examination revealed few pathologic changes in both sexes in all groups.
In the satellite groups no macroscopic recognisable pathologic changes were found.

HISTOPATHOLOGY: NON-NEOPLASTIC: see Report, Table 26 and 27 for details
At the application area no significant changes were found in all examined groups and both sexes.
Only in female animals, the most frequent changes were corticomedullary mineralisation of the kidneys and hydrometra of the uterus, without evidence of an association to treatment, since there was a remarkable background of these effects, while reduced or absent in the recovery groups (see also Attached Document, Table 26 and 27).

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall systemic effects
Remarks on result:
other: highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The dermal administration of the test substance Novares LA 300 (Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol) to rats for a period of 28 consecutive days did not cause unacceptable pathological changes at the site of administration. No significant changes, which indicate organ dysfunction, were observed in clinical biochemistry, haematology, and urinalysis parameters, and in histopathological examination at any dose level.
Based on these results, 1000 mg/kg bw/day, the highest dose tested, can be considered as a dermal NOAEL(28d, systemic) exhibiting no toxicologically significant effects in male and female rats.