2,2'-[6-(4-methoxyphenyl)-1,3,5-triazine-2,4-diyl]bis{5-[(2-ethylhexyl)oxy]phenol}

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-May-2003 to 14-Aug-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): FAT 70’884/C
- Other names (as cited in study report): FAT 70’884/B, FAT 70’884, TINOSORB® S, CGF-C-1607
- Physical state: yellowish powder
- Analytical purity: >=97.0%
- Purity test date: 9 April 2003
- Lot/batch No.: 02464CL3
- Expiration date of the lot/batch: 26 February 2008

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France (l’Arbresle, France)
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 221 g (range: 201 to 240 g) for the males and 166 g (range: 145 to 178 g) for the females
- Fasting period before study: None reported
- Housing: individually in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm); autoclaved sawdust
- Diet (e.g. ad libitum): A04 C pelleted maintenance diet [SAFE, Villemoisson, Epinay-sur-Orge, France], ad libitum
- Water (e.g. ad libitum): tap (filtered with a 0.22 µm filter), ad libitum- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

IN-LIFE DATES: From: 6 May 2003; To: 14 Aug 2003

Administration / exposure

Type of coverage:

open

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on exposure:

TEST SITE
- Area of exposure: dorsum
- % coverage: 10
- Type of wrap if used: none
- Time intervals for shavings or clipplings: at least 4 hours before dosing, and once per week during treatment.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): cotton swab moistened with water
- Time after start of exposure: before each dosing

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.5 mL/kg-day
- Concentration (if solution): 100, 200, and 400 mg/mL
- Constant volume or concentration used: yes
- For solids, paste formed: yes (cream-like consistency)

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility / stability

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, in a satellite group only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of samples taken from each dosage form (including the control) prepared for use in weeks 1, 2, 6 and 13 was determined by HPLC.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

with protective collar

No. of animals per sex per dose:

10 (principal), plus 3 satellite animals per sex per dose group.

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose-levels were selected on the basis of the results of a preliminary 2-week toxicity study performed in the same species (CIT/Study No. 25455 TSR) at the dose-level of 1000 mg/kg/day, in which no overt signs of toxicity or cutaneous reactions were noted. Therefore, according to international guidelines, the dose-level of 1000 mg/kg/day was chosen as the high dose-level.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Before each application up to week 4, then once weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: once each week

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, once each week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all animals before the first treatment, then the vehicle only (group 2) and high dose without collar (group 5) groups at the end of the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at end of study
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: yes
- How many animals: all principal animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at end of study
- Animals fasted: yes
- How many animals: all principal animals


URINALYSIS: Yes
- Time schedule for collection of urine: at end of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: yes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at end of study
- Dose groups that were examined: vehicle only (group 2) and high dose without collar (group 5)
- Battery of functions tested: Functional observation battery (FOB), including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature.

MONITORING OF ESTROUS CYCLE: Yes
- Time schedule for examinations and animals: 14 consecutive days during weeks 11 and 12 for each female

TOXICOKINETICS: Yes
- Time schedule for examinations: each satellite animal on day 8 and on one occasion in weeks 6 and 13, just before the dosing.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

ORGAN WEIGHTS: Yes
The body weight of all animals killed at the end of the treatment period was recorded before sacrifice and the organs were weighed wet as soon as possible after dissection.
Adrenals
Brain (including medulla/pons cerebellar and cerebral cortex)
Epididymides
Heart
Kidneys
Liver
Ovaries (with oviducts)
Spleen
Testes
Thymus
Thyroids with parathyroids

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- tissues for animals of the vehicle control and high-dose I and II groups killed at the end of the treatment period,
- all macroscopic lesions of all the animals of the low- and mid-dose groups killed on completion of the treatment period.
Macroscopic lesions
Adrenals
Aorta
Brain (including medulla/pons cerebellar and cerebral cortex)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes with Harderian glands
Femoral bone with articulation
Heart
Ileum (with Peyers patches)
Jejunum
Kidneys
Liver
Lungs with bronchi
Lymph nodes (mandibular and mesenteric)
Mammary gland area
Optic nerves
Ovaries (with oviducts)
Pancreas
Pituitary gland
Prostate
Rectum
Salivary glands (sublingual and submandibular)
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin (1 level on treated area and 1 level on non treated area)
Spinal cord (cervical, thoracic and lumbar)
Spleen
Sternum with bone marrow
Stomach with forestomach
Testes
Thymus
Thyroids with parathyroids
Tongue
Trachea
Ureters
Urinary bladder
Uterus (horns and cervix)
Vagina

Statistics:

The statistical analyses of body weight, food consumption, hematology, blood biochemistry, urinalysis and organ weight data was performed by comparison of treated groups (groups 3 to 6) and vehicle control group (group 2) and by comparison of untreated group (group 1) and group vehicle control group (group 2).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, non-treatment-related

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:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Desquamation noted in males given 500 and 1000 mg/kg/day was no longer observed after 10 days of dosing and was not observed in females. This finding was explained by the fact that residues of the test item on the application site sometimes resembled desquamation. Scabs were most frequently noted in treated males. Although, this finding did not appear in controls, no dose-relationship could be observed. For half of the concerned animals, the presence of this lesion did not exceed one week and was observed during 2 to 10 weeks for the other half. This finding was considered not related to the test item because the scabs were limited in frequency, did not occur in both high-dose groups, and readily resolved during the dosing period for most of the animals. Very slight erythema was noted in treated animals with a very low incidence and during a very short period. Thus, this finding was not considered to be related to treatment with test item. Areas of hair loss were observed in males and females of all groups with a low incidence. Since this hair loss was located outside the treated area (left and right forelimbs, abdomen, thorax or neck), it was not considered to be due to the treatment with the test item but considered to be of spontaneous occurrence. No signs of systemic toxicity were noted.
A slightly lower mean body weight gain was noted in males given 1000 mg/kg/day with a protective collar. Since, this was not observed in males given 1000 mg/kg/day without protective collar and not observed in females, a relationship to treatment with the test item is not indicated.
The estrous cycle stage was not affected at any dose-level since normal cyclicity was observed in all females.
Compared to vehicle control group, a slightly higher mean cell volume was noted in males with collar given 1000 mg/kg/day (52.4 fL vs. 49.9 fL) and in females of both groups given 1000 mg/kg/day (55.3 and 54.8 fL vs. 53.5 fL, respectively). A slightly lower mean cell hemoglobin concentration was observed in males given 250 mg/kg/day (35.6 g/dL vs. 36.4 g/dL) and in animals given 500 and 1000 mg/kg/day (both groups) (males: 35.4; 35.4 and 35.4 g/dL vs. 36.4 g/dL; females: 35.9, 35.8 and 35.9 g/dL vs. 36.5 g/dL). Since these differences were slight, with individual values in the physiological range and no histopathological findings were noted, they were considered to be of no toxicological significance.
The plasma levels of the test item measured in animals given 1000 mg/kg/day with or without collar were very low and indicated that the test item was not bioavailable by cutaneaous route. Because of the very low levels in the high-dose groups, the samples for lower dose groups were not analyzed. On the untreated and treated areas of skin, the main microscopic changes were minimal to slight hyperkeratosis sometimes with parakeratosis, acanthosis, spongiosis and empty hair follicles. As the incidence and severity of these findings were not dose-related, often lower in the animals of the high-dose group wearing a protective plastic collar than in the animals of the high dose group without protective collar, and without prominent differences between the untreated and treated areas, these skin microscopic changes were considered to be unrelated to any irritant potency of the test item and most likely due to mechanical injuries incurred during dose-site preparation (clipping, cleaning etc...).

Effect levels

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 effects

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Mortality

No deaths occurred during the study.

Clinical signs

No cutaneous or systemic effects related to treatment with the test item were noted.

Detailed clinical examination and functional observation battery

There were no treatment-related changes in autonomic, physiological or neurotoxicological parameters. Motor activity was not affected by treatment with the test item.

Body weight and food consumption

No relevant differences were observed in the body weight gain between control and treated animals. The food consumption was considered to be unaffected by treatment with the test item.

Ophthalmology

No treatment-related ophthalmological findings were observed at the end of the treatment period.

Estrous cycle

The estrous cycle stage was not affected at any dose-level.

Hematology and blood biochemistry

There were no differences of toxicological significance between control and treated animals in any hematological or blood biochemical parameter.

Urinalysis

There were no relevant differences between control and treated animals for qualitative and quantitative parameters.

Plasma levels of the test item

The plasma levels of the test item measured in animals given 1000 mg/kg/day with or without collar were very low and indicated that the test item was not bioavailable by cutaneous route.

Organ weight

There were no treatment-related differences in organ weight between control and treated animals.

Macroscopic post-mortem examination

No treatment-related necropsy findings were noted.

Microscopic examination

There were no treatment-related microscopic findings.

Applicant's summary and conclusion

Conclusions:

The test item, FAT 70’884/C (batch No. 02464CL3), was administered dermally to Wistar Han rats at the dose-levels of 250, 500 and 1000 mg/kg/day for 13 weeks. No clinical signs, hematological, blood biochemical, urinary or histopathological findings were observed at any dose-level. Plasma levels indicated that the test item was not bioavailable. Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) of the test item is >= 1000 mg/kg/day.

Executive summary:

Four treated groups of 13 male and 13 female Wistar Han rats (including three satellite males and three satellite females) received the test item, FAT 70’884/C (batch No. 02464CL3), daily by cutaneous application at the dose-levels of 250, 500, 1000 (without protective collar) or 1000 mg/kg/day (with protective collar) for 13 weeks. An additional group of 10 males and 10 females received the vehicle alone (polyethylene glycol 400) under the same experimental conditions and acted as a control group. Animals of the second high-dose group (given 1000 mg/kg/day) wore a protective plastic collar over at least a 6-hour period after each application. After the exposure period, the application site of these animals was cleaned with tap water and dried with absorbent paper.

The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed before the beginning of the treatment period and then once a week. Body weight and food consumption were recorded once a week during the study. Neurotoxic evaluation using functional observation battery (FOB) was carried out at the end of the treatment period on animals of the vehicle control and high-dose groups (without collar). Ophthalmological examinations were performed on animals of the vehicle control and high-dose groups (without collar). The estrous cycle stage was determined for 14 consecutive days during weeks 11 and 12 for each female. Hematology, blood biochemical investigations and urinalysis were performed at the end of the treatment period. Satellite animals were allocated for the determination of the test item plasma levels and were sampled prior to dosing, on day 8 and weeks 6 and 13. On completion of the treatment period, the animals were killed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from animals of the vehicle control and the two high-dose groups and on all macroscopic lesions in the low- and mid-dose groups.

No clinical signs, hematological, blood biochemical, urinary or histopathological findings were observed at any dose-level. Plasma levels indicated that the test item was not bioavailable. Consequently, under the experimental conditions of the study, the No Observed Effect Level (NOEL) of the test item is 1000 mg/kg/day or above.