Benzoic acid, 4-[(1-oxodecyl)oxy]-

Endpoint summary

Description of key information

Oral administration to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, for 28 days resulted in no deaths, no clinical symptoms after daily and weekly (weeks 1, 2 and 3) observations, no clinical symptoms during the functional observational battery, no effects on food consumption or body weight development, no effects upon hematology, clinical biochemistry or urinalysis parameters, no changes in mean absolute or relative organ weights, no effects upon sperm motility, morphology and count, and no macroscopical or microscopical findings of toxicological relevance. 
Based on the results of this study, 1000 mg/kg body weight/day was established as the no-observed-effect-level (NOEL) and as the no-observed-adverse-effect-level (NOAEL) for DOBA. 

Key value for chemical safety assessment

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP and OECD guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 60 males and 60 females. Groups 1 and 4: 10 males and 10 females each: Groups 2 and 3: 5 males and 5 females each
- Age (at Delivery): Ca. 7 weeks
- Body Weight Range (at Acclimatization): 212 to 239 g (mean 221 g) in males and 130 to 158 g (mean 147 g) in females
- Identification (during Acclimatization): Cage card and tail mark (later ear tattoo)
- Identification (during Treatment): Cage card and individual ear tattoo
- Randomization: Randomly allocated to groups by body weight.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, and are considered not to have any influence on the study. Therefore, these data are not reported but are retained at Harlan Laboratories Ltd. The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch nos. 73/11 and 69/11) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batches were analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of respective samples were performed.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

DOSE FORMULATIONS

The test item was used as supplied by the Sponsor. The purity was not corrected.

The dose formulations were prepared weekly. Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study, the stability of the test item formulations was considered to be sufficient to justify weekly preparation.
DOBA was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures were stirred using a magnetic stirrer and used at room temperature (20 ± 5 °C).

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS

The dose formulations were stored in glass beakers at room temperature (20 ± 5 °C).

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
- Frequency of Administration: Daily.
- Dose Levels:0 mg/kg/day (Group 1, vehicle control), 50 mg/kg/day (Group 2), 200 mg/kg/day (Group 3) and 1000 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Wistar rats
- Dose Volume: 5 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (Group 1, vehicle control), 10 mg/mL/day (Group 2), 40 mg/mL/day (Group 3) and 200 mg/mL/day (Group 4)
- Duration of Pre-Randomization Phase: 1 day
- Duration of Acclimatization Phase: 6 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD

The analysis was performed by Harlan Laboratories Ltd. using a HPLC method provided by the Sponsor.

After experimental start and during week 3, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 hour and 8 days).

The samples were delivered to the analytical department at ambient temperature (Harlan Laboratories Ltd., Analytics, Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The test item was used as analytical standard.

Dose formulation samples (primary samples or duplicates) were discarded upon written confirmation by the study director after acceptance of the draft report.

RESULTS

The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by linear regression analysis. The regression coefficients (R2) calculated were found to be better than 0.99

The DOBA peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of DOBA and, therefore, the absence of the test item in the vehicle control samples (corn oil) was confirmed.

The contents of DOBA ranged from 97.7% to 105.5% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of DOBA in the preparations was approved because single results found did not deviate more than 2.9% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item DOBA and corn oil as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
200 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Group 1 (0 mg/kg bw/day): 10 males and 10 females
Group 2 (50 mg/kg bw/day): 5 males and 5 females
Group 3 (200 mg/kg bw/day): 5 males and 5 females
Group 4 (1000 mg/kg bw/day): 10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this oral toxicity study was to assess the cumulative toxicity of DOBA when administered daily to rats by gavage for a period of 28 days. The reversibility of treatment-related changes was assessed after a treatment-free 14-day recovery period. This study should provide a rational basis for toxicological risk assessment in man.

In this subacute toxicity study, DOBA was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, corn oil, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment (Allocation A animals). Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed (Allocation B animals).

Positive control:

Not required

Observations and examinations performed and frequency:

VIABILITY/MORTALITY

Observations for viability / mortality were recorded twice daily.

DAILY OBSERVATIONS

The animals were observed for clinical signs once before commencement of administration as well as daily on days 1 - 28 (twice daily during days 1 - 3) during the treatment period and once daily during days 1 to 14 of the recovery period.

WEEKLY BEHAVIORAL OBSERVATIONS

The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter. More details on the investigations can be found in the section “Any other information on materials and methods incl. tables” below.

FUNCTIONAL OBSERVATIONAL BATTERY

During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.

- Grip Strength: Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

- Locomotor Activity: Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.

FOOD CONSUMPTION

The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

BODY WEIGHTS

Body weights were recorded once during pre-randomization, weekly during acclimatization, treatment and recovery periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.


CLINICAL LABORATORY INVESTIGATIONS

After 4 Weeks: 22-Mar-2012 (allocation A and B)
After 6 Weeks: 05-Apr-2012 (allocation B)

Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.

Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage.

The following hematology parameters were determined:

- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Reticulocyte count
- Reticulocyte maturity index (low, medium, high fluorescence)
- Leukocyte count, total
- Differential leukocyte count:
- Neutrophils
- Eosinophils
- Basophils
- Lymphocytes
- Monocytes
- Large unstained cells
- Platelet count
- Methemoglobin
- Heinz bodies
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Phospholipids
- Aspartate aminotransferase
- Alanine aminotransferase
- Lactate dehydrogenase
- Alkaline phosphatase
- Bile acids
- Gamma-glutamyl-transferase
- Creatine kinase
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

The following urine parameters were determined:

- Urine volume (18 hour)
- Specific gravity (relative density)
- Color
- Appearance
- pH value
- Nitrite
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Erythrocytes
- Leukocytes

Sacrifice and pathology:

TERMINATION AND NECROPSY
Sacrifice:

After 4 Weeks of Treatment: 22-Mar-2012 (allocation A)
After 6 Weeks of Recovery: 05-Apr-2012 (allocation B)

All allocation A and B animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

A blood sample was taken from each animal by heart puncture (ca. 2 mL) into appropriate tubes (Vacutainer glass tubes containing SST-Gel and Clot Activator or the equivalent) for serum preparation and cooled. Following centrifugation, the serum samples were divided in two aliquots and transferred to plastic (polypropylene) tubes. These samples were stored at approximately -80 °C and protected from light. In the absence of findings that would be indicative of thyroid changes, analysis of the samples was considered unnecessary.

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution unless indicated otherwise.

- Adrenal glands
- Aorta
- Bone (sternum, femur including joint)
- Bone marrow (femur)
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in Bouin's solution)
- Esophagus
- Eyes w/optic nerve (fixed in Davidson's solution)
- Harderian gland (fixed in Davidson's solution)
- Heart including auricles
- Ileum, with Peyer's patches
- Jejunum with Peyer's patches
- Kidneys
- Larynx
- Lacrimal gland, exorbital
- Liver
- Lungs, filled w/formalin at necropsy
- Lymph nodes – mesenteric and mandibular
- Mammary gland area
- Nasal cavity
- Ovaries
- Pancreas
- Pharynx
- Pituitary gland
- Seminal vesicles/Prostate gland incl. coagulating glands
- Rectum
- Salivary glands - mandibular, sublingual
- Sciatic nerve
- Skeletal muscle
- Skin
- Spinal cord - cervical, midthoracic, lumbar
- Spleen
- Stomach
- Testes (fixed in Bouin's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Tongue
- Trachea
- Urinary bladder, filled w/formalin at necropsy
- Uterus, with oviducts, cervix and vagina
- All gross lesions

ORGAN WEIGHTS

The following organs from allocation A and B animals were weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.

- Adrenal glands
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Epididymides (fixed in Bouin's solution)
- Heart including auricles
- Kidneys
- Liver
- Ovaries
- Pituitary gland
- Seminal vesicles/Prostate gland incl. coagulating glands
- Spleen
- Testes (fixed in Bouin's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Uterus, with oviducts, cervix and vagina

The terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios as well as organ to brain weight ratios were determined.

SPERM ANALYSIS

- Motility: At necropsy of males after 4 weeks, a sperm sample from the left caudal epididymis was obtained from control and treated males. The sample was diluted with a pre-warmed (about 37-40 °C) physiological medium, and shortly after being obtained, one hundred sperm was counted in duplicates (two sperm samples) microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.

- Morphology: In males of groups 1 and 4 after 4 weeks, the sperm in the original physiological medium for motility determination was used for morphological assessment after Eosin staining and fixation. The slides were stored at room temperature until analysis. The remaining fixation was stored at 4°C for maximal 2 weeks for possible re-evaluation. 500 sperms per sample were evaluated microscopically and classified into the following categories:
A: Normal, complete sperm
B: Normal head, abnormal tail
C: Normal head only, tail detached
D: Abnormal head only, tail detached
E: Abnormal head, normal tail

The percentages of categories A to E were determined.

- Sperm Count: In males of groups 1 and 4 after 4 weeks, the left testis and the left cauda epididymis were taken for determination of homogenization-resistant sperm (HRS) in testes and epididymis. The testes and cauda epididymis were frozen at -20 ± 5 °C pending evaluation. For evaluation the testes and cauda epididymis were thawed and processed separately. Testes and cauda epididymis were weighed and placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm heads were counted microscopically using a modified Neubauer chamber. Dilution factors for each sample were documented in the raw data.

HISTOTECHNIQUE

All organs and tissues samples as listed above were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.

HISTOPATHOLOGY

Slides of all organs and tissues listed below collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist.

- Adrenal glands
- Bone marrow (femur)
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in Bouin's solution)
- Eyes w/optic nerve
- Heart including auricles
- Ileum, with Peyer's patches
- Jejunum with Peyer's patches
- Kidneys
- Liver
- Lungs, filled w/formalin at necropsy
- Lymph nodes – mesenteric and mandibular
- Ovaries
- Pituitary gland
- Seminal vesicles/Prostate gland incl. coagulating glands
- Rectum
- Sciatic nerve
- Skeletal muscle
- Spinal cord - cervical, midthoracic, lumbar
- Spleen
- Stomach
- Testes (fixed in Bouin's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Trachea
- Urinary bladder, filled w/formalin at necropsy
- Uterus, with oviducts, cervix and vagina
- All gross lesions

Attempts were made to correlate gross observations with microscopic findings.

A peer-review of the histopathology phase report was performed. The findings of the study pathologist and the peer-reviewing pathologist compared favorably.

Statistics:

The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, sperm analysis, organ weights and ratios as well as macroscopic findings:

- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test.

Clinical signs:

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:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

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:

not examined

Details on results:

1. IN-LIFE DATA

VIABILITY/MORTALITY

All animals survived the scheduled treatment or recovery periods.

DAILY OBSERVATIONS

There were no clinical symptoms observed during the daily observations in the males during the treatment or recovery periods.

In females treated with 1000 mg/kg/day, breathing noises were noted in one female on days 6-10 of the treatment period. In view of the transient nature of this finding, it was considered to be unrelated to the treatment with the test item.

WEEKLY BEHAVIORAL OBSERVATIONS

There were no clinical symptoms noted during the weekly behavioral observations performed during weeks 1, 2 and 3.

FUNCTIONAL OBSERVATIONAL BATTERY

There were no clinical symptoms noted during the functional observational battery performed during week 4.

- Grip Strenght: The mean fore- and hind limb grip strength of the test item-treated animals compared favorably with those of the controls.

- Locomotor Activity: The mean locomotor activity of the males treated with 1000 mg/kg/day was significantly increased during the first measurement interval (0-10 minutes), but compared favorably with the control males during all subsequent measurement intervals. This finding was considered to be unrelated to the treatment, as a similar difference was not seen in the females at this dose level.

FOOD CONSUMPTION

There were no effects upon the absolute or relative mean daily food consumption during the treatment and recovery periods.

BODY WEIGHTS

There were no effects upon mean body weights or mean body weight gain during the treatment and recovery periods.


2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

None of the statistically significant differences noted in the hematology parameters of the males and females after the treatment period were considered to be of toxicological relevance. All values were either unrelated to dose or within the ranges of the historical control values.

After the recovery period, the hematology parameters of all males and all females compared favorably.

CLINICAL BIOCHEMISTRY

None of the statistically significant differences noted in the clinical biochemistry parameters of the males and females after the treatment period were considered to be of toxicological relevance. All values were either unrelated to dose or within the ranges of the historical control values.

After the recovery period, the clinical biochemistry parameters of all females compared favorably. The mean alanine aminotransferase activity was significantly reduced in males previously treated with 1000 mg/kg/day, and exceeded the lower range of the historical control data. However, reductions in these parameters are not associated with toxicological changes and were therefore considered to be incidental.

URINALYSIS

There were no test item-related findings at any dose level. In males treated with 1000 mg/kg/day, the pH of the urine was significantly lower than that of the control males at the end of the treatment period, but remained within the range of the historical control data and considered to be incidental.

The urinalysis parameters of the test item-treated females compared favorably with those of the controls during the treatment and recovery periods.


3. PATHOLOGY

ORGAN WEIGHTS

The mean absolute and relative organ weights of the test item-treated males compared favorably with those of the control males after four weeks’ treatment. The mean absolute organ weights and organ-to-brain weight ratios of the test item-treated females were unaffected by treatment after four weeks.

The mean brain-to-body weight ratios of the females treated with 50 mg/kg/day and 1000 mg/kg/day were significantly elevated (p<0.05) when compared with the control females. However, these differences were considered to be related to the lower mean terminal body weights at 50 mg/kg/day (p<0.05) and 1000 mg/kg/day rather than any organ-specific effect.

After the recovery period, the mean absolute pituitary weights were significantly increased (p<0.01) in males when compared with controls. In the absence of any morphological changes, this finding was considered to be incidental.

MACROSCOPICAL FINDINGS

There were no gross lesions that could be attributed to treatment with the test item. All gross lesions recorded were considered to be within the range of normal background alterations.

SPERM ANALYSIS

Sperm cells evaluated for motility (allocated to classifications progression, stationary and not motile) did not indicated differences between control males and test item-treated males. At 200 mg/kg/day, the number of progressive and non-motile sperm differed significantly from the control males. In the absence of a dose-response relationship, these findings were considered to be incidental.

Examination of sperm cell morphology showed a slightly higher normal sperm count in males treated with 1000 mg/kg/day, but a significantly reduced number of normal sperm cells with heads only. This finding is not associated with a test item-related morphological finding and was considered to be of no toxicological relevance.

MICROSCOPIC FINDINGS

All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

- Vaginal Estrus Stages : No specific direction of estrus cycle was observed in animals treated with 1000 mg/kg/day, and therefore it was judged that there were no treatment-related effects on the estrus cycles.

- Other Findings: The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related effects were observed in either males or females up to and including 1000 mg/kg bw/day, the highest dose level used.

Critical effects observed:

not specified

Conclusions:

Oral administration of DOBA to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, for 28 days resulted in no deaths, no clinical symptoms after daily and weekly (weeks 1, 2 and 3) observations, no clinical symptoms during the functional observational battery (including mean grip strength and mean locomotor activity), no effects on food consumption or body weight development, no effects upon hematology, clinical biochemistry or urinalysis parameters, no changes in mean absolute or relative organ weights, no effects upon sperm motility, morphology and count, and no macroscopical or microscopical findings of toxicological relevance.

Based on the results of this study, 1000 mg/kg body weight/day of DOBA was established as the no-observed-effect-level (NOEL) and as the no-observed-adverse-effect-level (NOAEL) for DOBA.

Executive summary:

GENERAL 

In this subacute toxicity study, DOBA was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, corn oil, only.

 

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

 

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during acclimatization, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem.

 

MORTALITY / VIABILITY

 

All animals survived the scheduled treatment or recovery periods.

 

CLINICAL SIGNS (DAILY AND WEEKLY)

 

There were no test item-related clinical symptoms observed during the daily observations in the males or females during the treatment or recovery periods.

 

There were no clinical symptoms noted during the weekly behavioral observations performed during weeks 1, 2 and 3.

 

FUNCTIONAL OBSERVATIONAL BATTERY

 

There were no clinical symptoms noted during the functional observational battery performed during week 4.

 

GRIP STRENGTH

 

The mean fore- and hind limb grip strength of the test item-treated animals compared favorably with those of the controls.

 

LOCOMOTOR ACTIVITY

 

There were no test item-related differences in the mean locomotor activity of treated rats and control rats.

 

The mean locomotor activity of the males treated with 1000 mg/kg/day was significantly lower during the first measurement interval (0-10 minutes), but compared favorably with the control males during all subsequent measurement intervals. This finding was considered to be unrelated to the treatment, as a similar difference was not seen in the females at this dose level.

 

FOOD CONSUMPTION

 

There were no effects upon the absolute or relative mean daily food consumption during the treatment and recovery periods.

 

BODY WEIGHTS

 

There were no effects upon mean body weights or mean body weight gain during the treatment and recovery periods.

 

CLINICAL LABORATORY INVESTIGATIONS

 

- Hematology: none of the statistically significant differences noted in the hematology parameters of the males and females after the treatment period were considered to be of toxicological relevance. After the recovery period, the hematology parameters of all males and all females compared favorably.

 

- Clinical Biochemistry: none of the statistically significant differences noted in the clinical biochemistry parameters of the males and females after the treatment period were considered to be of toxicological relevance. All values were either unrelated to dose or within the ranges of the historical control values. After the recovery period, the clinical biochemistry parameters did not show any late effects of the test item treatment.

 

- Urinalysis: there were no test item-related findings at any dose level.

 

ORGAN WEIGHTS

 

The mean absolute and relative organ weights of the test item-treated males compared favorably with those of the control males after four weeks’ treatment. The mean absolute organ weights and organ-to-brain weight ratios of the test item-treated females were unaffected by treatment after four weeks’ treatment and two weeks’ recovery.

 

MACROSCOPIC / MICROSCOPIC FINDINGS

 

There were no gross lesions that could be attributed to treatment with the test item. All gross lesions recorded were considered to be within the range of normal background alterations. All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

 

SPERM ANALYSIS

 

There were no test item-related changes in the sperm morphology, motility or count.

 

CONCLUSION

 

Based on the results of this study, 1000 mg/kg body weight/day of DOBA was established as the no-observed-effect-level (NOEL) and as the no-observed-adverse-effect-level (NOAEL).

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:

reliable without restrictions - well performed GLP and OECD guideline study

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
only one study available

Justification for classification or non-classification

Oral administration at doses up to 1000 mg/kg/day, for 28 days resulted in no substance related deaths or symptoms of toxicity leading to a NOAEL of 1000 mg/kg b.w.. Since these findings do not meet the criteria for classification according to the rules laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008, classification is not warrantable.