Sodium (3-hydroxy-2-pentylcyclopentyl)acetate

Endpoint summary

Description of key information

Oral: OECD 407, rat, NOAEL = 300 mg/kg bw/day, LOAEL = 1000 mg/kg bw/day, OECD 408, rat, NOEL = 300 mg/kg bw/day

Inhalation: no information available

Dermal: OECD 410, rat, NOEL = 500 mg/kg bw/day, LOAEL (local) = 1000 mg/kg bw/day or 8 mg/cm^2 (assuming 330 g bw of rat and 41.8 cm^2 body surface, 10 % exposed area)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2003-12-02 to 2004-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, according to OECD guideline 407

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Sprague-Dawley, Crl CD (SD) IGS BR

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 8 weeks old
- Weight at study initiation: 264-312 g males, 169-209 g females
- Housing: in suspended wire-mesh cages: two rats of the same sex and group
- Diet (e.g. ad libitum): A04 C pelleted maintenance diet, batch Nos. 30925, 31021 and 31118 (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum, except fasting periods
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 50±20
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% of aqueous solution

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was mixed with the required quantity of vehicle in order to achieve the concentrations of 10, 30 and 100 mg/mL and then homogenized using a magnetic stirrer. The test item dosage forms were prepared daily (stability up to 4 hours) and stored at room temperature.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day
- Lot/batch no. (if required): 101K0185

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diluted samples of dosage form were analyzed by reverse phase chromatography with MS detection (LC-MS, negative mode) after ionisation using an Electro-Spray Interface (ESI). One dilution was prepared for each sample, and one injection was performed for each final dilution. The test item peak area was determined for each sample and the corresponding concentration was calculated using the equation obtained from the calibration data. All the results are expressed as mg/mL of Mexoryl SBO.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment (if not random): the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition and allocated to groups (by sex), according to a computerized stratification procedure, so that the average body weight of each group was similar.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: once before allocation of the animals to groups, on the first day of treatment, and then once a week until the end of the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before beginning of the treatment period and at the end of the treatment period (week 4)
- Dose groups that were examined: control and high-dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 5
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined:
in peripheral blood: erythrocytes (RBC), hemoglobin (HB), mean cell volume (MCV), packed cell volume (PCV), mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), thrombocytes (PLAT), leucocytes (WBC), differential white cell count with cell morphology, neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), monocytes (M), reticulocytes (RETIC), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB).
in bone marrow: bone marow differential cell count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 5
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), inorganic phosphorus (I.PHOS), glucose (GLUC), urea (UREA), creatinine (CREAT), total bilirubin (TOT.BIL), total proteins (PROT), Albumin (ALB), albumin/globulin (A/G), total cholesterol (CHOL), triglycerides (TRIG), alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT).

URINALYSIS: Yes
- Time schedule for collection of urine: in week 5
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined:
quantitative parameters: volume, pH, specific gravity.
semi-quantitative parameters: proteins (PROT), glucose (GLUC), ketones (CETO), bilirubin (BILI), nitrites (NITR), blood (BLOOD), urobilinogen (UROB), cytology of sediment, leucocytes (WBC), erythrocytes (RBC), cylinders (CYLIN), magnesium ammonium phosphate crystals (AMM.PH), calcium phosphate crystals (CAL.PH), calcium oxalate crystals (CAL.OX.), cells (CELLS).
qualitative parameters: appearance (APP), color (COLOR)

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
HISTOPATHOLOGY: Yes (see table 1)

Statistics:

The sequence used for the statistical analyses of body weight, food consumption, hematology, blood bichemistry, urinalysis and organ weight data is presented in the Figure 1.

Clinical signs:

no effects observed

Description (incidence and severity):

except ptyalism, observed in 6/10 males and 7/10 females given 1000 mg/kg/day by gavage, during the last week of the study.

Mortality:

no mortality observed

Description (incidence):

except ptyalism, observed in 6/10 males and 7/10 females given 1000 mg/kg/day by gavage, during the last week of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Slightly higher body weight gain was recorded in males treated by oral route, without dose-relationship.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Slightly lower erythrocyte count, hemoglobin and mean cell hemoglobin concentrations as well as higher mean cell volume were noted at the end of the treatment period in males given 1000 mg/kg/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The only treatment-related difference consisted of slightly higher triglyceride level in animals given 1000 mg/kg/day.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher liver weights were noted in animals given 1000 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Liver enlargement was noted in 2/10 males and 1/10 females given 1000 mg/kg/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver changes consisted of hepatocellular hypertrophy among animals given 1000 mg/kg/day.

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
No unscheduled death occured during the study. No relevant clinical signs were noted, except ptyalism, observed in 6/10 males and 7/10 females
given 1000 mg/kg/day by gavage, during the last week of the study. This sign, commonly noted when a test item is administered by gavage, was not considered as an adverse effect.

BODY WEIGHT AND WEIGHT GAIN
Slightly higher body weight gain was recorded in males treated by oral route, without dose-relationship. This slight effect was not considered as an adverse effect.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ophthalmological findings.

HAEMATOLOGY
Slightly lower erythrocyte count, hemoglobin and mean cell hemoglobin concentrations as well as higher mean cell volume were noted at the end of the treatment period in males given 1000 mg/kg/day.

CLINICAL CHEMISTRY
The only treatment-related difference consisted of slightly higher triglyceride level in animals given 1000 mg/kg/day.

URINALYSIS
Lower urinary pH value was noted in animals given 300 and 1000 mg/kg/day. Since there were no other associated findings this effect was not considered as toxicologically significant.

ORGAN WEIGHTS
Higher liver weights were noted in animals given 1000 mg/kg/day.

GROSS PATHOLOGY
Liver enlargement was noted in 2/10 males and 1/10 females given 1000 mg/kg/day by oral route.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver changes consisted of hepatocellular hypertrophy among animals given 1000 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Conclusions:

No adverse effects were observed at 100 and 300 mg/kg/day. At 1000 mg/kg/day, ptyalism was observed in both sexes and some hematological (lower erythrocyte count, hemoglobin and mean cell hemoglobin concentrations and higher mean cell volume) and blood biochemical (higher triglyceride level) changes were noted. Microscopic examination showed hepatocellular hypertrophy in males and females, correlating with higher liver weights and liver enlargement among these animals.
Consequently, under the experimental conditions of the study, taking into account the slight change in the hemoglobin concentration in females given 300 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) by oral route is considered as 300 mg/kg/day.

Executive summary:

The objective of this GLP and OECD guideline 407 conform study was to evaluate the potential toxicity of the test item Mexoryl SBO, following daily oral (gavage) administration to rats for 4 weeks. The oral route was selected since it is presumed to ensure an absorption at least equal to that expected from percutaneous absorption of the test item in man. Three treated groups of 10 male and 10 female Sprague-Dawley rats received the test item daily, by gavage at the dose-level of 100, 300 or 1000 mg/kg/day. One additional group of 10 males and 10 females received the vehicle alone (0.5 % aqueous carboxymethylcellulose) under the same experimental conditions (i.e. by gavage) and acted as oral control group. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded once a week. Ophthalmological examinations were carried out before and at the end of the treatment period. Hematological and blood biochemical investigations and urinalysis were performed at the end of the treatment period. 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 all the control and high-dose groups (groups 1 and 4) and on all macroscopic lesions (all groups). No unscheduled death occurred during the study. No relevant clinical signs were noted. Slightly higher body weight gain was recorded in males treated by oral route, without dose-relationship. This slight effect was not considered as an adverse effect. The food consumption was considered to be unaffected by treatment with the test item. There were no treatment-related ophthalmological findings. Slightly lower erythrocyte count, hemoglobin and mean cell hemoglobin concentrations as well as higher mean cell volume were noted at the end of the treatment period in males given 1000 mg/kg/day. The only treatment-related difference consisted of slightly higher triglyceride level in animals given 1000 mg/kg/day. Lower urinary pH value was noted in animals given 300 and 1000 mg/kg/day. Since there were no other associated findings this effect was not considered as toxicologically significant. Higher liver weights were noted in animals given 1000 mg/kg/day. Liver enlargement was noted in 2/10 males and 1/10 females given 1000 mg/kg/day. Liver changes consisted of hepatocellular hypertrophy among animals given 1000 mg/kg/day. Under the experimental conditions of the study, taking into account the slight change in the hemoglobin concentration in females given 300 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) by oral route is considered as 300 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant and guideline conform subacute and subchronic repeated dose toxicity study

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Quality of whole database:

No information is available.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Quality of whole database:

No information is available.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2008-10-21 to 2009-10-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to GLP guideline 410

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

Application period amended

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation: 384 - 444 g males, 231 -261 g females
- Fasting period before study: no
- Housing: in individual suspended wire-mesh cages
- Diet (e.g. ad libitum): All animals had free access to SsniffR/M-H pelleted maintenance diet, batch Nos. 1860828 and 7691203 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly. During periods of fasting food was removed but the animals were not deprived of water.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

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

Type of coverage:

semiocclusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: from 45 to 50 cm^2 in males and from 30 to 35 cm^2 in females
- % coverage: 10
- Type of wrap if used: a film
- Time intervals for clipplings: at least 4 hours before dosing and at least once a week

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with tap water and dried with cotton wool
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.236, 0.472 and 0.943 mL/kg/day
- Concentration (if solution): 0, 250, 500 and 1000 mg/kg/day
- Constant volume or concentration used: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 250, 500, 1000 mg/kg/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were specified by the Sponsor based on the results of a previous study (CIT/Study No. 26883 TSR) in which the test item, was administered daily by the oral route (gavage) at dose-levels of 100, 300 or 1000 mg/kg/day, or by the cutaneous route at 100 mg/kg/day for 4 weeks. In this previous study, the No Observed Adverse Effect Level (NOAEL) for the oral route was considered to be 300 mg/kg/day, and the No Observed Effect Level (NOEL) for the cutaneous route was considered to be higher than 100 mg/kg/day. When the test item was applied by the cutaneous route at 100 mg/kg/day, no overt toxic effects were observed. Scabs, associated with very slight or well-defined erythema, were transiently observed in some animals. Since these signs were observed transiently and with a similar incidence in control and treated animals, they were considered to be related to treatment with the vehicle rather than treatment with the test item.

- Rationale for animal assignment (if not random): during the acclimation periods, the required number of animals (20 males and 20 females for phase I and 10 males and 10 females for phase II) were selected according to body weight and clinical condition. They were then allocated to groups (by sex) according to a
computerized stratification procedure, so that the average body weight of each group was similar.

- Section schedule rationale (if not random): no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: before each daily treatment (including before dosing on day 1)

BODY WEIGHT: Yes
- Time schedule for examinations: once before group allocation, on the fist day of treatmentand on days 4, 8, 11 and 14.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 15
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: Erythrocytes (RBC), Hemoglobin (HB), Mean cell volume (MCV), Packed cell volume (PCV), Mean cell hemoglobin concentration (MCHC), Men cell hemoglobin (MCH), Thrombocytes (PLT), Leucocytes (WBC), Differential white cell count with cell morphology: neutrophils (N), eosinophils (E), basophils (B), lymphocytes and large unstained cells (L+LUC), monocytes (M); Reticulocytes (RTC), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Fibrinogen (FIB).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 15
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (I. PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL.), Total proteins (PROT), Albumin (ALB),Albumin/globulin ratio (A/G), Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT).

URINALYSIS: Yes
- Time schedule for collection of urine: week 3 (end of the treatment period)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined: volume (Volume), pH (pH), Specific gravity (SP.GRAV), Proteins (PROT), Glucose (GLUC), Ketones (CETO), Bilirubin (BILI), Nitrites (NITR), Blood (haemoglobin) (BLOOD), Urobilinogen (UROB), Cytology of sediment: leucocytes (WBC), erythrocytes (RBC), cylinders (CYLIN), magnesium ammonium phosphate crystals (AMM.PH), calcium phosphate crystals (CAL.PH), calcium oxalate crystals (CAL.OX.), epitelial cells (CELLS); Appearance (APP), Color (COLOR).

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 2)
HISTOPATHOLOGY: Yes (see Table 2)

Statistics:

please see Table 1

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

females

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 specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

higher urea and creatinine levels in females were compared with literature data and considered to be within the normal fluctuation range.

Urinalysis findings:

no effects observed

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:

not specified

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

A mammary carcinoma was recorded in a female treated at 1000 mg/kg/day. The process leading to tumor demands several weeks or months. A relationship to treatment given for 2 weeks was therefore excluded.

Details on results:

CLINICAL SIGNS AND MORTALITY
No effects

BODY WEIGHT AND WEIGHT GAIN
No effects

FOOD CONSUMPTION
No effects

FOOD EFFICIENCY
No data

WATER CONSUMPTION
No data

OPHTHALMOSCOPIC EXAMINATION
No data

HAEMATOLOGY
No effects

CLINICAL CHEMISTRY
Higher urea and creatinine levels in females were compared with literature data and considered to be within the normal fluctuation range.

URINALYSIS
No effects

NEUROBEHAVIOUR
Not examinated

ORGAN WEIGHTS
No effects

GROSS PATHOLOGY
No effects

HISTOPATHOLOGY: NON-NEOPLASTIC
No data

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
A mammary carcinoma was recorded in a female treated at 1000 mg/kg/day. The process leading to tumor demands several weeks or months. A relationship to treatment given for 2 weeks was therefore excluded.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: very slight to well-defined erythema, were observed in 3/5 females, and scab formations at the application site were observed in one female

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: very slight to well-defined erythema in 3 out of 5 females and scab formations at the application site in one female

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

8 mg/cm² per day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: assuming 330 g bw of rat and 41.8 cm^2 body surface, 10 % exposed area

Critical effects observed:

not specified

Conclusions:

Under the experimental conditions of this study, the NOEL was considered to be 1000 mg of Mexoryl SBO/kg bw/day in males and 500 mg of Mexoryl SBO/kg bw/day in females. The local LOAEL was considered to be 1000 mg of Mexoryl SBO/kg bw/day in females.

Executive summary:

The GLP compliant study was based on the OECD TG 410, however, the potential toxicity of the test item following daily cutaneous application to rats was conducted during 2 weeks and not during 21/28 days as it is recomended in the guideline.

During phase I, three treated groups (groups 2 to 4) of five male and five female Sprague-Dawley rats received the test item by daily cutaneous application at dose-levels of 250, 500 or 1000 mg/kg/day for 2 weeks. The test item was supplied "ready-to-use" and administered under dosage-volumes of 0.236, 0.472 and 0.943 mL/kg/day, respectively. A group of five males and five females received the control item (purified water) under the same experimental conditions and acted as a control group (group 1). Due to a technical issue with the material used for the administration to group 3 males and to group 4 males and females, and based on the absence of toxic effects noted in the males at 1000 mg/kg/day during phase I, an additional control group (group 5) and an additional high-dose group (group 6) were treated under the same experimental conditions as groups 1 and 4 (phase II). The results of group 4 animals are presented for information, but the group 6 results are considered to be the representative results of daily administration at 1000 mg/kg/day. The dosage form was applied to the dorsum, over an area of approximately 10% of the total body surface. The animals wore a protective collar over a period of at least 6 hours after each application in order to avoid ingestion of the test item. After the exposure period, the application site was cleaned using tap water and dried with cotton wool. The animals were checked twice daily for mortality, and clinical signs were observed once a day. The body weight of each animal was recorded once before the beginning of the study, then on the first day of treatment and on days 4, 8, 11 and 14. Food consumption was recorded once a week during the study. On completion of the treatment periods, hematology, blood biochemistry and urinalysis investigations were performed before sacrifice and the animals were examined macroscopically. Designated organs were weighed and selected tissues were preserved. A microscopic examination of designated organs was performed.

No treatment-related findings were noted at 250 or 500 mg/kg/day. During phase II, the following findings at 1000 mg/kg/day were considered to be test item-related: local reactions, including very slight to well-defined erythema, were observed in 3/5 females, and scab formations at the application site were observed in one female. At this dose-level, increased urea (+17%) and creatinine (+10%) levels were recorded in the females. However applicant consider these urea and creatinine data as not treatment-related, since literature source (Handbook of toxicology) show the fluctuation of these parameters within a normal permissible range. Microscopic findings consisted of acanthosis, observed in most of the control and treated animals; it was most probably secondary to clipping and thus of no toxicological importance.

No treatment-related findings were recorded at 250 or 500 mg/kg/day. At 1000 mg/kg/day test item-related findings of minimal to slight intensity at the application site were recorded in females only. Consequently, under the experimental conditions of this study, the NOEL (No Observed Effect Level) was considered to be 1000 mg of Mexoryl SBO/kg/day in males and 500 mg of are not part of the disclosed dossier information by ECHA/kg/day in females. The local LOAEL was considered to be 1000 mg of Mexoryl SBO /kg bw/day in females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

two reliable studies are available. In the supporting study the dosage range is too low.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2008-10-21 to 2009-10-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to GLP guideline 410

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

Application period amended

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation: 384 - 444 g males, 231 -261 g females
- Fasting period before study: no
- Housing: in individual suspended wire-mesh cages
- Diet (e.g. ad libitum): All animals had free access to SsniffR/M-H pelleted maintenance diet, batch Nos. 1860828 and 7691203 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly. During periods of fasting food was removed but the animals were not deprived of water.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

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

Type of coverage:

semiocclusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: from 45 to 50 cm^2 in males and from 30 to 35 cm^2 in females
- % coverage: 10
- Type of wrap if used: a film
- Time intervals for clipplings: at least 4 hours before dosing and at least once a week

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with tap water and dried with cotton wool
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.236, 0.472 and 0.943 mL/kg/day
- Concentration (if solution): 0, 250, 500 and 1000 mg/kg/day
- Constant volume or concentration used: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 250, 500, 1000 mg/kg/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were specified by the Sponsor based on the results of a previous study (CIT/Study No. 26883 TSR) in which the test item, was administered daily by the oral route (gavage) at dose-levels of 100, 300 or 1000 mg/kg/day, or by the cutaneous route at 100 mg/kg/day for 4 weeks. In this previous study, the No Observed Adverse Effect Level (NOAEL) for the oral route was considered to be 300 mg/kg/day, and the No Observed Effect Level (NOEL) for the cutaneous route was considered to be higher than 100 mg/kg/day. When the test item was applied by the cutaneous route at 100 mg/kg/day, no overt toxic effects were observed. Scabs, associated with very slight or well-defined erythema, were transiently observed in some animals. Since these signs were observed transiently and with a similar incidence in control and treated animals, they were considered to be related to treatment with the vehicle rather than treatment with the test item.

- Rationale for animal assignment (if not random): during the acclimation periods, the required number of animals (20 males and 20 females for phase I and 10 males and 10 females for phase II) were selected according to body weight and clinical condition. They were then allocated to groups (by sex) according to a
computerized stratification procedure, so that the average body weight of each group was similar.

- Section schedule rationale (if not random): no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: before each daily treatment (including before dosing on day 1)

BODY WEIGHT: Yes
- Time schedule for examinations: once before group allocation, on the fist day of treatmentand on days 4, 8, 11 and 14.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 15
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: Erythrocytes (RBC), Hemoglobin (HB), Mean cell volume (MCV), Packed cell volume (PCV), Mean cell hemoglobin concentration (MCHC), Men cell hemoglobin (MCH), Thrombocytes (PLT), Leucocytes (WBC), Differential white cell count with cell morphology: neutrophils (N), eosinophils (E), basophils (B), lymphocytes and large unstained cells (L+LUC), monocytes (M); Reticulocytes (RTC), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Fibrinogen (FIB).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 15
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (I. PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL.), Total proteins (PROT), Albumin (ALB),Albumin/globulin ratio (A/G), Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT).

URINALYSIS: Yes
- Time schedule for collection of urine: week 3 (end of the treatment period)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined: volume (Volume), pH (pH), Specific gravity (SP.GRAV), Proteins (PROT), Glucose (GLUC), Ketones (CETO), Bilirubin (BILI), Nitrites (NITR), Blood (haemoglobin) (BLOOD), Urobilinogen (UROB), Cytology of sediment: leucocytes (WBC), erythrocytes (RBC), cylinders (CYLIN), magnesium ammonium phosphate crystals (AMM.PH), calcium phosphate crystals (CAL.PH), calcium oxalate crystals (CAL.OX.), epitelial cells (CELLS); Appearance (APP), Color (COLOR).

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 2)
HISTOPATHOLOGY: Yes (see Table 2)

Statistics:

please see Table 1

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

females

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 specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

higher urea and creatinine levels in females were compared with literature data and considered to be within the normal fluctuation range.

Urinalysis findings:

no effects observed

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:

not specified

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

A mammary carcinoma was recorded in a female treated at 1000 mg/kg/day. The process leading to tumor demands several weeks or months. A relationship to treatment given for 2 weeks was therefore excluded.

Details on results:

CLINICAL SIGNS AND MORTALITY
No effects

BODY WEIGHT AND WEIGHT GAIN
No effects

FOOD CONSUMPTION
No effects

FOOD EFFICIENCY
No data

WATER CONSUMPTION
No data

OPHTHALMOSCOPIC EXAMINATION
No data

HAEMATOLOGY
No effects

CLINICAL CHEMISTRY
Higher urea and creatinine levels in females were compared with literature data and considered to be within the normal fluctuation range.

URINALYSIS
No effects

NEUROBEHAVIOUR
Not examinated

ORGAN WEIGHTS
No effects

GROSS PATHOLOGY
No effects

HISTOPATHOLOGY: NON-NEOPLASTIC
No data

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
A mammary carcinoma was recorded in a female treated at 1000 mg/kg/day. The process leading to tumor demands several weeks or months. A relationship to treatment given for 2 weeks was therefore excluded.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: very slight to well-defined erythema, were observed in 3/5 females, and scab formations at the application site were observed in one female

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: very slight to well-defined erythema in 3 out of 5 females and scab formations at the application site in one female

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

8 mg/cm² per day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: assuming 330 g bw of rat and 41.8 cm^2 body surface, 10 % exposed area

Critical effects observed:

not specified

Conclusions:

Under the experimental conditions of this study, the NOEL was considered to be 1000 mg of Mexoryl SBO/kg bw/day in males and 500 mg of Mexoryl SBO/kg bw/day in females. The local LOAEL was considered to be 1000 mg of Mexoryl SBO/kg bw/day in females.

Executive summary:

The GLP compliant study was based on the OECD TG 410, however, the potential toxicity of the test item following daily cutaneous application to rats was conducted during 2 weeks and not during 21/28 days as it is recomended in the guideline.

During phase I, three treated groups (groups 2 to 4) of five male and five female Sprague-Dawley rats received the test item by daily cutaneous application at dose-levels of 250, 500 or 1000 mg/kg/day for 2 weeks. The test item was supplied "ready-to-use" and administered under dosage-volumes of 0.236, 0.472 and 0.943 mL/kg/day, respectively. A group of five males and five females received the control item (purified water) under the same experimental conditions and acted as a control group (group 1). Due to a technical issue with the material used for the administration to group 3 males and to group 4 males and females, and based on the absence of toxic effects noted in the males at 1000 mg/kg/day during phase I, an additional control group (group 5) and an additional high-dose group (group 6) were treated under the same experimental conditions as groups 1 and 4 (phase II). The results of group 4 animals are presented for information, but the group 6 results are considered to be the representative results of daily administration at 1000 mg/kg/day. The dosage form was applied to the dorsum, over an area of approximately 10% of the total body surface. The animals wore a protective collar over a period of at least 6 hours after each application in order to avoid ingestion of the test item. After the exposure period, the application site was cleaned using tap water and dried with cotton wool. The animals were checked twice daily for mortality, and clinical signs were observed once a day. The body weight of each animal was recorded once before the beginning of the study, then on the first day of treatment and on days 4, 8, 11 and 14. Food consumption was recorded once a week during the study. On completion of the treatment periods, hematology, blood biochemistry and urinalysis investigations were performed before sacrifice and the animals were examined macroscopically. Designated organs were weighed and selected tissues were preserved. A microscopic examination of designated organs was performed.

No treatment-related findings were noted at 250 or 500 mg/kg/day. During phase II, the following findings at 1000 mg/kg/day were considered to be test item-related: local reactions, including very slight to well-defined erythema, were observed in 3/5 females, and scab formations at the application site were observed in one female. At this dose-level, increased urea (+17%) and creatinine (+10%) levels were recorded in the females. However applicant consider these urea and creatinine data as not treatment-related, since literature source (Handbook of toxicology) show the fluctuation of these parameters within a normal permissible range. Microscopic findings consisted of acanthosis, observed in most of the control and treated animals; it was most probably secondary to clipping and thus of no toxicological importance.

No treatment-related findings were recorded at 250 or 500 mg/kg/day. At 1000 mg/kg/day test item-related findings of minimal to slight intensity at the application site were recorded in females only. Consequently, under the experimental conditions of this study, the NOEL (No Observed Effect Level) was considered to be 1000 mg of Mexoryl SBO/kg/day in males and 500 mg of are not part of the disclosed dossier information by ECHA/kg/day in females. The local LOAEL was considered to be 1000 mg of Mexoryl SBO /kg bw/day in females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

8 mg/cm²

Study duration:

subacute

Species:

rat

Quality of whole database:

two reliable studies are available. In the supporting study the dosage range is too low.

Additional information

Oral

The repeated oral toxicity of the test item, Mexoryl SBO, was evaluated in rats according to OECD TG 407 (Haag 2004). Three treated groups of 10 male and 10 female Sprague-Dawley rats received the test item daily, by gavage at the dose levels of 100, 300 or 1000 mg/kg bw/day. The control group of 10 males and 10 females received the vehicle alone (0.5 % aqueous carboxymethylcellulose). No unscheduled death occurred during the study. No relevant clinical signs were noted. Slightly higher body weight gain was recorded in males treated by oral route, without dose-relationship. This slight effect was not considered as an adverse effect. The food consumption was considered to be unaffected by treatment with the test item. There were no treatment-related ophthalmological findings. Slightly lower erythrocyte count, hemoglobin and mean cell hemoglobin concentrations as well as higher mean cell volume were noted at the end of the treatment period in males given 1000 mg/kg/day. The only treatment-related difference consisted of slightly higher triglyceride level in animals given 1000 mg/kg/day. Higher liver weights were noted in animals given 1000 mg/kg/day. Liver enlargement was noted in 2/10 males and 1/10 females given 1000 mg/kg/day. Liver changes consisted of hepatocellular hypertrophy among animals given 1000 mg/kg/day. Under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) by oral route is considered as 300 mg/kg/day.

The subchronic study was conducted to determine the toxicity profile of Mexoryl SBO in Wistar rats, when administered through oral gavage for a period of 90 consecutive days (Gohel 2013). The methods followed were as per OECD testing guideline No 408 (September 21, 1998). 40 male and 40 female Wistar rats were randomly divided into four groups of 10 male and 10 female rats. Animals were treated with Mexoryl SBO at the dose levels of 100, 300 and 1000 mg/kg bw/day for a period of 90 consecutive days through oral gavage. Concurrent control group animals were dosed with the vehicle (0.5% CMC) alone. The administration of the test item Mexoryl SBO to male and female Wistar rats produced no mortality. There were no toxicologically significant effects related to treatment with the test item on body weight, body wight gain, food consumption or ophthalmoscopic examination, neurobehavioural observations and functional observational battery testing. No treatment-related clinical signs were observed in low and mid-dose groups animals throughout the study period. Animals treated at 1000 mg/kg bw/day showed signs of mild to moderate salivation intermittently from week 9 till end of the treatment. The salivation was considered related to treatment with the test item but not supported with any other findings or observations, hence was considered as non adverse. No treatment related changes were observed in hematology and urinalysis parameters of Mexoryl SBO-treated groups when compared with the control group. Changes were observed in blood clinical parameters (i.e. Alkaline Phosphatase, total bilirubin and triglyceride levels) of Mexoryl SBO treated males and females given 1000 mg/kg bw/day. They were associated with higher mean liver weights (both absolute and relative) and microscopic lesions in liver consisting of varying degree of hepatocellular vacuolation (both microvsicular and macrovesicular, mostly periportal). Therefore, these liver changes were considered of toxicological significance at 1000 mg/kg bw/day. On the basis of the findings obtained in the present study, and specifically the liver changes observed at 1000 mg/kg bw/day, the No Observed Effect Level (NOEL) of this 90-day oral toxicity study on Mexoryl SBO was established at 300 mg/kg bw/day in male and females Wistar rats.

Inhalation 

Inhalation is not considered to be a likely route of exposure and therefore, it is not deemed necessary to conduct a repeated dose inhalation test.

Dermal

The repeated dermal toxicity of the test item Mexoryl SBO was evaluated in rats according to OECD TG 410 (Rousseau 2009). Instead of 21/28 study duration, as recommended in the test guideline, the study was conducted during 2 weeks. Three treated groups (groups 2 to 4) of five male and five female Sprague-Dawley rats received the test item by daily cutaneous application at dose-levels of 250, 500 or 1000 mg/kg/day for 2 weeks. A group of five males and five females received the control item (purified water) under the same experimental conditions and acted as a control group (group 1). The dosage form was applied to the dorsum, over an area of approximately 10% of the total body surface. No treatment-related findings were noted at 250 or 500 mg/kg/day.

The following findings at 1000 mg/kg/day were considered to be test item-related: local reactions, including very slight to well-defined erythema, were observed in 3/5 females, and scab formations at the application site were observed in one female.

Consequently, under the experimental conditions of this study, the NOEL was considered to be 1000 mg/kg bw/day in males and 500 mg/kg bw/day in females. The local LOAEL was considered to be 1000 mg/kg bw/day or 8 mg/cm^2 in females. In the reliable supporting study (Haag 2004), the NOEL was determined above 100 mg/kg bw/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

only one GLP compliant and guideline conform subacute repeated dose toxicity study via oral route. A suporting subchronic study confirms the results

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

GLP compliant and guideline conform (exept study duration) study

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

GLP compliant and guideline conform (except study duration) study

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Oral

Based on the above stated assessment of the specific target organ toxicity potential after repeated oral exposure of Mexoryl SBO, the compound does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) or according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.

Dermal

Based on the above stated assessment of the specific target organ toxicity potential after repeated dermal exposure of Mexoryl SBO, the compound does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) or according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.