Registration Dossier

Administrative data

Description of key information

Repeated Dose Dermal 90d, 1 key study, NOAEL=1000 mg/kg/d for rats (equivalent or similar to OECD TG 411)
Repeated Dose Dermal 28d, 1 key study, NOAEL=500 mg/kg/d for rats (equivalent or similar to OECD TG 410)
Repeated Dose Oral 90d, read across, 1 key study NOAEL=500ppm (~34 mg/kg/day) for rats (equivalent or similar to OECD TG 408)
Repeated Dose Inhalation 90d-Testing not required based on Column 2 Annex IX.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD method 408.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic, Germantown New York
- Age at study initiation: ca. 42 days
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum



ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-72
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was incorporated into the diet at levels of 0, 500, 5000, 20000 ppm. Trial diet was prepared and analyzed for homogeneity, stability and concentration. The diet had to be stable for a minimum of three weeks. The concentration of test material in the diet had to be at or no more than 10% the level specified in the protocol.

DIET PREPARATION
- Rate of preparation of diet (frequency): one week prior to study initiation and at approximately two week intervals thereafter.
- Mixing appropriate amounts with (Type of food): Purina Certified Lab Chow
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks. Except for periods of deprivation required for hematology/serum chemistry studies and necropsy.
Frequency of treatment:
Daily for 13 weeks. Except for periods of deprivation required for hematology/serum chemistry studies and necropsy.
Remarks:
Doses / Concentrations:
500, 5000, 20000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
20/sex/dose for a total of 40 animals/dose
Control animals:
yes, plain diet
Details on study design:
N/A
Positive control:
N/A
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily



DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:


BODY WEIGHT: Yes
- Time schedule for examinations: Day after receipt and the day of their release from quarantine. Animals allocated into experimental groups were weighed immediately before the initiation of treatment and approximately weekly thereafter.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


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



OPHTHALMOSCOPIC EXAMINATION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to study initiation and during weeks 5 and 13
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals: 10/sex/dose for a total of 20 animals/dose



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to study initiation and during weeks 5 and 13
- Animals fasted: Yes
- How many animals: 10/sex/dose for a total of 20 animals/dose



URINALYSIS: Yes
- Time schedule for collection of urine: weeks 5 and 13
- Metabolism cages used for collection of urine: No
- Animals fasted: No data


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Adrenals, heart, ovaries, testes, brain, kidneys, prostate, thymus, epididymides, liver, spleen, uterus

HISTOPATHOLOGY: Yes
Adrenals, bone and marrow, brain, epididymides, esophagus, eye and optic nerve, gross lesions, heart and aorta, small and large intestine, kidneys, liver, lung, lymph nodes, mammary gland, ovaries, pancreas, peripheral nerve, pituitary, prostate and seminal vesicles, salivary gland, skeletal muscle, spleen, spinal cord, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus
Statistics:
Quantitative data were analyzed by parametric methods: analysis of variance and associated F-test followed by Tukey’s multiple comparison test, provided that there was statistical significance in ANOVA.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
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
Seven males (1 control, 6 high-dose) died or were sacrificed before the scheduled necropsy. No other clinical signs indicative of systemic toxicity were observed during the study.

HAEMATOLOGY
Statistically significant, dose-related alterations in hematology parameters reported for females included red blood cell count, hemoglobin, hematocrit, segmented neutrophils and lymphocytes. More specifically, red blood cell count, hemoglobin, and hematocrit were decreased, and segmented neutrophils were increased, at week 13 in females receiving 20,000 ppm of the test compound. Lymphocytes were reduced in 20,000 ppm females at week 1, and in females receiving 5000 and 20,000 ppm at week 13, with no significant differences observed at week 5. A linear dose-response relationship was found for all of these parameters, suggesting that the results were treatment-related. However, there were no statistically significant changes in hematology parameters among females receiving 500 ppm of the test compound, and the values in 5000 ppm females did not differ significantly from controls for any parameter other than lymphocytes. Comparison with historical reference values to further determine the significance of the results indicated that the dose-response for segmented neutrophils and lymphocytes at 20,000 ppm fell outside the normal range (10 - 90th percentile). The remaining hematology parameters were within normal ranges. No hematology effects were reported for male rats.


CLINICAL CHEMISTRY
Parameters affected at week 13 included serum urea nitrogen, uric acid, alanine aminotransferase (ALT), albumin, potassium, phosphorus, calcium, and chloride in males; and ALT, alkaline phosphatase (ALP), cholesterol, total bilirubin, total protein, albumin, globulin, sorbitol dehydrogenase (SDH), chloride, and cholinesterase in females.

A linear dose-response relationship was found with regard to ALT, uric acid, albumin, potassium, and phosphorus in males; and with regard to ALT, ALP, cholesterol, total protein, albumin, SDH, and cholinesterase in females, suggesting that these results were treatment related. However, changes observed in males at week 13 were statistically significant only at 20,000 ppm, except for a small but significant (p<0.05) decrease in chloride at 5000 and 20,000 ppm. Calcium was significantly increased in males at 500 ppm but not at higher doses; the lack of a dose-response suggests the change was not substance-related.

In females, ALT, ALP, and SDH levels were significantly increased only at 20,000 ppm, while cholesterol, protein, albumin, and cholinesterase were significantly decreased at both 5000 and 20,000 ppm. Bilirubin was decreased at 500 ppm at week 13, but was not affected at higher doses, suggesting a non-treatment related effect. A small, but statistically significant reduction in chloride levels was found at all doses at week 13, though chloride levels were unaffected at weeks 1 and 5. Chloride levels did not appear to be dose-related, and amounted to only a 2-3% reduction from controls. The A/G ratio was significantly increased at all doses at week 5, reflecting increased albumin and decreased globulin levels at this sampling interval. However, at week 13, globulin was significantly decreased only at 20,000 ppm and the A/G ratio was not affected.

When compared with historical serum reference values, the dose-response curves for male ALT, and phosphorus at 20,000 ppm, and female ALP and cholesterol at 20,000 ppm were outside normal ranges (10 - 90th percentile). The remaining serum chemistry values were within normal ranges.


ORGAN WEIGHTS
Statistically significant increases in the absolute and/or relative weight of the kidney (both sexes) and of the adrenals and liver (females) were seen at 5000ppm and higher. At 20000ppm, significant increases in absolute and/or relative weight of the liver (males) and of the spleen (females) were observed.

GROSS PATHOLOGY
The observations recorded most frequently in the animals sacrificed on schedule included enlarged lymph nodes and abnormal areas of the liver. In general, lymph node enlargement was seen in all groups with equal frequency. Abnormal areas of the liver were observed in approximately half of the females dosed at 20,000ppm. All other observations were seen sporadically and regarded as non-treatment related, spontaneous, unremarkable and/or self-explanatory.

HISTOPATHOLOGY: NON-NEOPLASTIC
At 5000ppm at higher, macrophages were observed in the lymph nodes and liver. There was no substantial difference between the lesions induced at 5000ppm and 20000ppm. The liver lesions indicated that females were more susceptible to the test material than males. The mesenteric lymph node lesions showed no sex predilection. The microscopic changes seen in the mesenteric lymph nodes and liver of rats sacrificed in extremis suggest that the onset of lesions was earlier in the mesenteric lymph nodes than in the liver. No changes were detectable at 500ppm.

Dose descriptor:
NOAEL
Effect level:
>= 500 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
<= 5 000 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
The NOAEL for rats orally exposed to the test material is 500ppm (34.6-38.4 mg/kg bw/day). Effects on organ weights were observed at the next highest dose of 5000ppm (340-380 mg/kg/day) and were limited to increases in the absolute and/or relative weight of the kidney (both sexes) and of the adrenals and liver (females). However, there was no evidence of organ dysfunction at this dose. These findings do not warrant classification of the submission substancel for target organ toxicity (repeated exposure) under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP), under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations, or under the Globally Harmonzied System of Classification and Labelling of Chemicals (GHS).
Executive summary:

A 90-day oral toxicity study of the test material was conducted in rats (20/sex/dose) at dietary concentrations of 0, 500, 5,000 and 20,000 ppm. Seven animals died or were sacrificed during the study (1 control, 6 high dose). No other clinical signs indicative

of systemic toxicity were observed during the study. Most of the clinical observations were localized effects (e.g., neck irritation and/or alopecia on neck and forepaws from rubbing or grooming, ear irritation tags, etc). Chromodacryorrhea (red tears) and reddish nasal discharge were likely the result of transitory stress from procedures such as bleeding, handling, etc.

 

Additional parameters evaluated included physical examinations, hematology, serum chemistry, ophthalmology, body weight, gross pathology, histopathology, and urinalysis. No statistically significant differences between dose groups were observed for body weights, food consumption, ophthalmology, or urinalysis.

 

Statistically significant, dose-related alterations in hematology parameters reported for females included red blood cell count, hemoglobin, hematocrit, segmented neutrophils and lymphocytes. More specifically, red blood cell count, hemoglobin, and hematocrit were decreased, and segmented neutrophils were increased, at week 13 in females receiving 20,000 ppm of the test compound. Lymphocytes were reduced in 20,000 ppm females at week 1, and in females receiving 5000 and 20,000 ppm at week 13, with no significant differences observed at week 5. A linear dose-response relationship was found for all of these parameters, suggesting that the results were treatment-related. However, there were no statistically significant changes in hematology parameters among females receiving 500 ppm of the test compound, and the values in 5000 ppm females did not differ significantly from controls for any parameter other than lymphocytes. Comparison with historical reference values to further determine the significance of the results indicated that the dose-response for segmented neutrophils and lymphocytes at 20,000 ppm fell outside the normal range (10 - 90th percentile). The remaining hematology parameters were within normal ranges. No hematology effects were reported for male rats.

 

Serum chemistry for males and females showed some statistically significant differences between untreated and treated animals. Parameters affected at week 13 included serum urea nitrogen, uric acid, alanine aminotransferase (ALT), albumin, potassium, phosphorus, calcium, and chloride in males; and ALT, alkaline phosphatase (ALP), cholesterol, total bilirubin, total protein, albumin, globulin, sorbitol dehydrogenase (SDH), chloride, and cholinesterase in females.

 

A linear dose-response relationship was found with regard to ALT, uric acid, albumin, potassium, and phosphorus in males; and with regard to ALT, ALP, cholesterol, total protein, albumin, SDH, and cholinesterase in females, suggesting that these results were treatment related. However, changes observed in males at week 13 were statistically significant only at 20,000 ppm, except for a small but significant (p<0.05) decrease in chloride at 5000 and 20,000 ppm. Calcium was significantly increased in males at 500 ppm but not at higher doses; the lack of a dose-response suggests the change was not substance-related.

 

In females, ALT, ALP, and SDH levels were significantly increased only at 20,000 ppm, while cholesterol, protein, albumin, and cholinesterase were significantly decreased at both 5000 and 20,000 ppm. Bilirubin was decreased at 500 ppm at week 13, but was not affected at higher doses, suggesting a non-treatment related effect. A small, but statistically significant reduction in chloride levels was found at all doses at week 13, though chloride levels were unaffected at weeks 1 and 5. Chloride levels did not appear to be dose-related, and amounted to only a 2-3% reduction from controls. The A/G ratio was significantly increased at all doses at week 5, reflecting increased albumin and decreased globulin levels at this sampling interval. However, at week 13, globulin was significantly decreased only at 20,000 ppm and the A/G ratio was not affected.

 

When compared with historical serum reference values, the dose-response curves for male ALT, and phosphorus at 20,000 ppm, and female ALP and cholesterol at 20,000 ppm were outside normal ranges (10 – 90th percentile). The remaining serum chemistry values were within normal ranges.

 

Statistically significant increases in the absolute and/or relative weight of the kidney (both sexes) and of the adrenals and liver (females) were observed at ≥ 5,000 ppm. At 20,000 ppm, significant increases in the absolute and/or relative weight of the liver (males) and spleen (females) were observed.

 

During necropsy of the animals sacrificed on schedule, the most frequent observation was enlarged lymph nodes and “abnormal areas” of the liver. In general, lymph node enlargement was seen in all groups with equal frequency. “Abnormal areas” of the liver were observed in approximately half of the females dosed at 20,000 ppm. Other gross observations were sporadic and non-treatment related.

 

No statistically significant changes were detectable in any of the endpoints examined at the 500ppm dose level for either sex. Based on these data, the NOAEL for this study is considered to be 500 ppm. 

 

The conversion of NOAEL from ppm in the diet to mg/kg bw/day was calculated using the concentration of the test substance in the diet, the food consumption factor and the body weight factor. On this basis, the NOAEL for the study is calculated to be 34.6 to 38.4 mg/kg/day.

Classification for target organ toxicity (repeated exposure) under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP), under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations, or under the Globally Harmonzied System of Classification and Labelling of Chemicals (GHS).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
34.6 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: modern GLP study with no significant protocol deviations
Qualifier:
according to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Crl:CD (SD) rats (a total of 45 male and 45 female) were received from Charles River (UK)
Ltd. The rats were ordered at 29 to 35 days of age and within a weight range of 118 to 145 g
for males and 108 to 135 g for females. On receipt all animals appeared healthy and a sample
of 15 males and 15 females were weighed. All animals were found to be within the
acceptable weight range for this study.
On arrival, the animals were removed from the transit boxes and allocated to study cages.
Using the sequence of cages in the battery, one animal was placed in each cage. Each sex
was allocated separately.
Each animal was assigned a number and identified uniquely within the study by a tail tattoo.
Each cage label was colour-coded according to group and was numbered uniquely with cage
and study number, as well as the identity of the occupant.
Before the start of treatment, one male with non-resolving ophthalmic lesions and one female
with a bodyweight at the extreme of the weight range were replaced with spare animals of
suitable weight from the same batch.
The animals were allowed to acclimatise to the conditions described below for 12 days before
treatment commenced. For those animals selected for this study, their age at the start of
treatment was 41 to 47 days and their bodyweights were in the range of 212 to 261 g for
males and 164 to 213 g for females.
The spare animals were removed from the study room after treatment commenced.
2.3.2 Animal housing, diet and water supply
Animals were housed inside a barriered rodent facility (Building 8, Room 0816). The
facility was designed and operated to minimise the entry of external biological and chemical
agents and to minimise the transference of such agents between rooms. Before the study the
room was cleaned and disinfected.
Each animal room was kept at positive pressure with respect to the outside by its own supply
of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature
and relative humidity controls were maintained within the range of 19 to 230C and 40 to 70%
respectively and monitored continuously. There were no deviations from these ranges.
Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours
continuous dark per 24 hours.
Alarms were activated if there was any failure of the ventilation system, or temperature limits
were exceeded. A stand-by electricity supply was available to be automatically brought into
operation should the public supply fail.
The animals were housed individually. The cages were made of a polycarbonate body with a
stainless steel mesh lid. Softwood bark-free fibre was used as bedding provided at a depth of
2 cm (estimated by eye) and was sterilised by autoclaving and changed at appropriate
intervals each week. Cages, food hoppers and water bottles were changed at appropriate
intervals.
The cages constituting each group were blocked together by sex and the groups were
dispersed in batteries so that possible environmental influences arising from their spatial
distribution were equilibrated, as far as was practicable. Additionally, batteries of cages were
rotated around the room at weekly intervals to further minimise possible spatial variations.
The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1
Maintenance Diet) except overnight before routine blood sampling. This diet contained no
added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles
fitted with sipper tubes.
Each animal was provided with an Aspen chew block for environmental enrichment. The
soft white untreated wood blocks were provided throughout the study and were replaced
when necessary. A plastic shelter (tunnel) was also supplied for environmental enrichment;
and removed from the cage during periods of occlusion after administration and when an
Elizabethan collar was fitted, during the first five weeks of treatment. From the sixth week of
treatment, in an attempt to reduce the animal tampering with the semi-occlusive dressing,
nestlets (bedding material) were supplied to each cage, and were replaced when necessary,
and plastic shelters (tunnels) were provided at all times, with the exception of the 30 minute
period when an Elizabethan collar was worn. Following administration, two pellets of diet
were placed in the animal cage as forage food.
Each batch of diet was analysed routinely by the supplier for various nutritional components
and chemical and microbiological contaminants. Supplier's analytical certificates were
scrutinised and approved before any batch of diet was released for use. The quality of the
water supply is governed by regulations published by the Department for Environment, Food
and Rural Affairs. Certificates of analysis were received routinely from the water supplier
and the suppliers of the bedding, nestlets and Aspen chew blocks. Since the results of these
various analyses did not provide evidence of contamination that might have prejudiced the
study, they are not presented.
No other specific contaminants that were likely to have been present in the bedding, nestlets,
chew blocks, diet or water were analysed, as none that may have interfered with or prejudiced
the outcome of the study was known.
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
Treated animals received the test substance as a dermal administration followed by six hours
semi-occlusion of the dermal test site. Administration was performed on five days of each
week during Weeks 1 to 12 and daily thereafter, until the day of dispatch to necropsy in
Week 14. Individual dose volumes were measured using a micropipette (Days 1-3 only) or a
1 mL syringe (graduated to 0.01 mL). The test substance was initially applied to the middle
of the application site (6x6 cm; approximately 10% of the body surface area) to ensure the same area of skin was treated, and distributed evenly, as far as possible across the site, using
the tip of the micropipette (Days 1-3 only) or a metal spatula.
The control (sham-dosed) group was similarly treated, with the absence of the test substance.
All animals were dosed in sequence of animal/cage-number within each group. The volume
administered to each animal was calculated from the most recently recorded scheduled
bodyweight.
A record of the weight of each formulation dispensed and the amount remaining after dosing
was made. The balance of these two weights was compared with the predicted usage as a
check that the doses had been administered correctly. It is considered that any discrepancy in
the weight of material used was not significant.
Immediately after administration, unmedicated gauze dressing was held in position around
the trunk with a cotton wool pad, an open woven bandage and a tubigrip bandage. Sufficient
tension was used to ensure that the dose remained in contact with the skin. The dressing was
used to maximise the potential for absorption of the test material across the dermal barrier
and minimise the risk of oral ingestion.
After no less than six hours, the semi-occlusive dressing was carefully removed. To reduce
the risk of oral ingestion and to prevent residual test material being moved to the skin
adjacent, during grooming, the test site was cleansed with copious quantities of warm tap
water (approximately 30-35oC) and a mild, dilute soap (Simple™), using cotton wool. The
test site and surrounding hair were dabbed-dry with disposable paper towels. Fresh tubigrip
was used for each administration.
After the application site had been washed and dried an Elizabethan collar was fitted to each
animal for no less than 30 minutes. This procedure ensured that the application site was
completely dry before possible grooming could occur. The test site then remained
non-occluded until the next administration.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Substance applied neat. No need for analytical validation.
Duration of treatment / exposure:
6 hours / day, 5 days/week for 13 weeks
Frequency of treatment:
6 hours / day, 5 days/week for 13 weeks
Remarks:
Doses / Concentrations:
125 mg/kg bw/day
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal per unit area
No. of animals per sex per dose:
15 animals per sex per dose.
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
Serial observations
Dated and signed records of all activities relating to the day by day running and maintenance
of the study within the animal unit as well as to the group observations and examinations
outlined in this experimental procedure were recorded in the Study Day Book. In addition,
observations relating to individual animals made throughout the day were recorded.
All observations described below were performed in animal/cage number sequence except
where otherwise indicated.
2.4.1 Clinical observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment. Any deviation from normal was recorded at the time in respect of nature and
severity, date and time of onset, duration and progress of the observed condition, as
appropriate.
Daily (on the five days the animals were treated/sham dosed) during the first week of
treatment and twice weekly (during the five day/week period when the animals were
treated/sham dosed) from Week 2 of treatment until termination, detailed observations were
recorded at the following times in relation to dose administration:
Immediately before dosing X
Immediately after dosing on return of the animal to its cage
On completion of dosing of each group
Between one and two hours after completion of dosing (bandage application)
As late as possible in the working day (following removal of all Elizabethan
collars) X
In addition, a more detailed weekly physical examination was performed on each animal to
monitor general health.
On each day of treatment all animals were assessed visually at approximately 3 to 4 hours
after dose administration in order to ensure that the dressings were still in place (see
Deviations from protocol).
X The dermal application site of each animal was examined for signs of irritancy before each
administration (see Deviations from protocol) and at removal of the semi-occlusive dressing,
according to the Draize, J.H. (1965) scoring system, as follows:
Erythema and eschar formation:
No erythema 0
Slight erythema 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4
Oedema formation
No oedema 0
Slight oedema 1
Well-defined oedema (area well-defined by definite raising) 2
Moderate oedema (edges raised approximately 1 mm) 3
Severe oedema (raised more than 1 mm and extending beyond the area of exposure) 4

2.4.2 Mortality
Debilitated animals were observed carefully and killed for reasons of animal welfare where
necessary. A complete necropsy was performed in all cases as described below.
2.4.3 Bodyweight
The weight of each rat was recorded one week before treatment commenced (Week -1), on
the day that treatment commenced (Week 0), weekly throughout the treatment period and
before necropsy. More frequent weighings were instituted, when appropriate, for animals
displaying ill-health, so that the progress of the observed condition could be monitored.
These data are not reported here.
2.4.4 Food consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded for the week before treatment started (Week -1), and each week throughout the
treatment period until Week 13 (forage food (two pellets offered at dose administration) was
included in the calculations. Animals were fed ad-libitum during Week 14 until termination.
From these records the mean weekly consumption per animal (g/rat/week) was calculated for
each cage.
2.4.5 Ophthalmic examination
Before treatment commenced, the eyes of all animals allocated to the study (including spare
animals) were examined by means of a binocular indirect ophthalmoscope. One rejected
male was replaced with a male that had no adverse ocular abnormality, selected from the
spare animals for the study. During Week 13 of treatment the eyes of all animals of Groups 1
(Sham control) and 4 (1000 mg/kg/day) were similarly examined.
Prior to each examination, the pupils of each animal were dilated using tropicamide
ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber,
iris (pupil dilated), lens, vitreous and fundus were examined.
As no treatment-related changes were observed, the examination was not extended to animals
of Groups 2 or 3 (125 or 500 mg/kg/day).
Sacrifice and pathology:
Macroscopic pathology
All animals were subject to a detailed necropsy.
After a review of the history of each animal, a full macroscopic examination of the tissues
was performed. All external features and orifices were examined visually. The cranial roof
was removed to allow observation of the brain, pituitary gland and cranial nerves. After
ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and
pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position,
morphology or interaction was recorded.
The requisite organs were weighed and external and cut surfaces of the organs and tissues
were examined as appropriate. Any abnormality in the appearance or size of any organ and
tissue was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.
2.5.3 Organ weights
The following organs, taken from each animal killed after 13 weeks of treatment, were
dissected free of adjacent fat and other contiguous tissue and the weights recorded:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
* Weighed after partial fixation
Spleen
Testes
Thymus
Thyroid with parathyroids^
Uterus with cervix
Bilateral organs were weighed together. Organ weights were also adjusted for terminal
bodyweight, using the weight recorded before necropsy.

Histology
For those animals specified in the Pathology section, the relevant tissues were subject to
histological processing.
Tissue samples were dehydrated, embedded in paraffin wax, sectioned at approximately four
to five micron thickness and stained with haematoxylin and eosin.
Those tissues subject to histological processing included the following regions:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Femur with joint - longitudinal section including articular surface, epiphysial
plate and bone marrow
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Liver - section from all main lobes
Lungs - section from two major lobes, to include bronchi
Spinal cord - transverse and longitudinal section at the cervical, lumbar and
thoracic levels
Sternum - included bone marrow
Stomach - included keratinised, glandular and antrum in sections
Thyroid - included parathyroids in section where possible
Uterus - uterine body with cervix section
For bilateral organs, sections of both organs were prepared. A single section was prepared
from each of the remaining tissues required for microscopic pathology.

Pathology
2.6.1 Light microscopy
Microscopic examination was performed as follows:
All tissues preserved for examination (as specified above) were examined for all
animals of Groups 1 (Sham control) and 4 (1000 mg/kg/day) sacrificed on
completion of the scheduled treatment period and for all animals killed during the
study. Additionally, the lungs were examined for all animals of Groups 2
(125 mg/kg/day) and 3 (500 mg/kg/day) sacrificed on completion of the scheduled
treatment period.
Tissues reported at macroscopic examination as being grossly abnormal were
examined in line with current practice.
Findings were either reported as "present" or assigned a severity grade. In the latter case one
of the following five grades was used - minimal, slight, moderate, marked or severe. A
reviewing pathologist undertook a peer review of the microscopic findings.
Other examinations:
2.4.6 Haematology, peripheral blood
During Week 13 of treatment, blood samples were obtained from all animals after overnight
withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane
and blood samples were withdrawn from the sublingual vein.
Blood samples (nominally 0.5 mL) were collected into tubes containing EDTA as
anticoagulant and examined for the following characteristics using a Bayer Advia 120
haematology analyser:
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Reticulocyte count (Retic)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Total white cell count (WBC)
Differential WBC count
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Pit)
Morphology flags were generated by the Advia 120 analyser. The most common
morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were
categorised as follows:
= no abnormalities detected
+ = slight
++ = moderate
+++ = marked
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by
light microscopy, in the case of flags from the Advia 120 analyser. Confirmation or a written
description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate
anticoagulant and examined in respect of:
Prothrombin time (PTP) - using an ACL 3000 Plus analyser and IL PT-Fibrinogen
reagent
Activated partial thromboplastin time (APTT) - using an ACL 3000 Plus Analyser
and IL APTT reagent

2.4.7 Blood chemistry
At the same time and using the same animals as for peripheral haematology, lurther blood
samples (nominally 0.7 mL) were collected into tubes containing lithium heparin as
anticoagulant. All tubes were mechanically agitated for at least two minutes and the sample
subsequently centrifuged at 2000 g for 10 minutes in order to separate the plasma. After
separation, the plasma was examined in respect of:
Using a Roche P Modular Analyser:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Urea
Creatinine (Great)
Glucose (Glue)
Total cholesterol (Choi)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and
analysed albumin concentration.
Statistics:
See below
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
dermal irritation
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Critical effects observed:
not specified
Conclusions:
No adverse effect level is the maximum dose tested, 1,000 mg/kg bw/day
Executive summary:

The systemic toxic potential of MCP2484 (a base oil), to Crl:CD(SD) (Sprague-Dawley) rats

by dermal (semi-occluded) administration, on five days of each week, to Sprague-Dawley

rats for 13 weeks was assessed at doses of 125, 500 or 1000 mg/kg/day.

One animal was killed for welfare reasons following six weeks of treatment as, following

attempts to remove the semi-occlusive bandage, it had caused physical trauma to its paws and

head, which was considered to be an exaggerated individual response to the restrictive

semi-occlusive dressing and not a result of treatment with MCP2484.

There were no clinical signs or signs related to administration and there were no dermal

reactions attributable to the test substance. Some animals in the Sham Control or treated

groups showed evidence of trauma, limited to the edges of the test site, including cuts,

encrustations, abrasions or red staining to feet (forelimbs and hindlimbs/feet) and a few had

scratches on the test site, which were considered attributable to attempts by the animal to

remove the bandage. In cases where the semi-occlusive bandage was removed, it was reapplied.

Data shows that there was a higher incidence of bandage removal in animals treated

with MCP2484 than in the Sham Control group; however, as there was no relationship with

dose and no evidence of a dermal response caused by the test substance, it was considered

that the physical nature of the test substance aided attempts to remove the bandage.

Bodyweight gain was considered to be unaffected by treatment and there was no effect of

treatment on food consumption and no ophthalmic change. It was considered that there was

no toxicologically important effect of treatment on haematology or chemical constituents of

the blood in Week 13, or organ weights following 13 weeks of treatment.

There were no histopathological findings related to treatment and assessment of the lungs of

all animals revealed no evidence of infection.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

One key study for repeated dermal exposure for the submission substance was identified. Read-across studies for the structural analogue, reaction products of 1 -hexadecene and naphthalene, were also identified and used to assess repeated dose toxicity through oral exposure. There were no studies identified for repeated dose toxicity for inhalation exposure.

Dermal: One 28 -day study and one 90 -day study were used to evaluate the potential hazard following repeated dermal exposure to the submission substance. Based on these data, the submission substance is expected to have a low order of repeated dose toxicity by the dermal route. Testing in both studies was performed in a manner similar or equivalent to currently established OECD guidelines. Following 28 days of dermal exposure to the submission substance, the NOAEL for rats was determined to be 500 mg/kg/day.  Statistically significant differences were found between the serum chemistry data from the control animals and the highest dose group for inorganic phosphorous and uric acid in males and for glucose and aspartate aminotransferase (AST) in females. When the historical serum reference values were taken into consideration however, only the dose-response curve for female AST at 2000 mg/kg/day fell outside the normal range. A minor adrenal vacuolation was also observed in 3/10 males treated at the highest dose.

Following 90 days of dermal exposure to the registered substance, the NOAEL for rats was determined to be 1000 mg/kg/day. There were no toxicologically relevant effects at any dose level or any sex in this study.

 

Oral: The submission substance is expected to have a low order of repeated dose toxicity by the oral route of exposure based on read-across data. Testing was performed in a manner similar or equivalent to currently established OECD guidelines. Following 90 days of oral exposure to a structural analogue (reaction products of 1 -hexadecene and naphthalene) the NOAEL for rats was determined to be 500ppm (34.6-38.4 mg/kg bw/day). Effects on organ weights were observed at the next highest dose of 5000ppm (340-380 mg/kg/day) and were limited to increases in the absolute and/or relative weight of the kidney (both sexes) and of the adrenals and liver (females). However, there was no evidence of organ dysfunction (including no abnormal histopathology) at any dose. Overall, the limited effects observed were not indicative of significant toxicity and occurred at doses above the guidance values for classification.

 

Inhalation: In accordance with Column 2 of REACH Annex IX, repeated dose toxicity testing via the inhalation route is not necessary for the submission substance as this exposure route is not the most appropriate route of administration, having regard to the likely route of human exposure. 

Read Across Justification

Category or groups of chemicals that have similar structural features generally are expected to have similar properties as well as similar toxicological characteristics that are predictable across the group in a homologous or structurally analogous series.  Therefore, the use of surrogates having close structural similarities and the same chemical group functionality as in the case of the alkylated naphthalenes would be appropriate for read-across for“a mixture of isomers of: mono-(2-tetradecyl)naphthalenes; di-(2-tetradecyl)naphthalenes; tri-(2-tetradecyl)naphthalenes”.  

“A mixture of isomers of: mono-(2-tetradecyl)naphthalenes; di-(2-tetradecyl)naphthalenes; tri-(2-tetradecyl)naphthalenes”is a C14-alkylated naphthalene and is also described as MCP 2484, and MCP 968 in this document.  

“Reaction products of 1-hexadecene and naphthalene” is a C16-alkylated naphthalene and is also described as MCP 917. “Reaction products of 1-hexadecene and naphthalene” is already registered under REACH.

Achemical category groupis a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity (i.e., structural analogues). Under REACH, Annex XI allows for the chemical safety assessment of chemicals by the use of this chemical category group or analog approach; see Chapter R.6.2. of the ECHA guidance document (ECHA 2008) for discussion. Based on the close chemical similarity between“a mixture of isomers of: mono-(2-tetradecyl)naphthalenes; di-(2-tetradecyl)naphthalenes; tri-(2-tetradecyl)naphthalenes”and its alkylated naphthalene analogues, we believe that the read-across data for“reaction products of 1-hexadecene and naphthalene” are adequate to support hazard characterization ofthe submission substance.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
At least two studies are available to assess the effects of the registered substance upon repeated oral exposure. One read across 90d oral gavage study, and a two-generation reproductive toxicity study by the dietary route.
In the read across study, increases in the absolute and relative kidney and adrenal weights were observed at and above the 5000ppm dose (approx. 34.6mg/kg/d).
In the two-generation reproductive toxicity study, there were no changes in absolute or relative kidney or adrenal weights for the F0 (parental) generation males or females, which had been dosed for 10 weeks prior to commencement of pairing, with males treated for approximately 19 weeks and females at least 17 weeks (assuming copulatory plug was observed on day 1 of mating). However, the two-generation study did not assess hematology, which was affected by treatment in the read across study.

Justification for classification or non-classification

The limited effects observed following oral and dermal administration of either the submission substance or a structural analogue were not indicative of significant toxicity and occurred at doses above the guidance values for classification. Therefore, these findings do not warrant classification of the test material for target organ toxicity (repeated exposure) under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP), under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations, or under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).