2-hydroxybenzoic acid 2-butyloctyl ester

Endpoint summary

Description of key information

In a key study, the subject material was tested by oral gavage in male and female rats following OECD guideline 407 and following accepted GLP standards.

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

Study period:

19 February 1998 to 8 May 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Albino (Outbrd) VAF/Plus(TM) CD (TM) (Sprague-Dawley derived)[Cr1: CD BR]

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY 12484
- Age at study initiation: 44 days
- Weight at study initiation: male, 205.1g (mean), range 193-216g; female, 162.0g (mean), range 144-172g
- Fasting period before study: no
- Housing: doubly housed in elevated, stainless-steel, wire-mesh cages during the first week of acclimation period and individually thereafter
- Diet (ad libitum): Certified Rodent Diet No. 5002 (meal), PMI Nutrition International, St. Louis, Missouri
- Water (ad libitum): tap water, Elizabethtown Water Company, Westfield, New Jersey
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 36 to 70
- Air changes (per hr): not given
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 10 April 1998 To: 8 May 1998

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Refer to Table 1. Using a syringe, the appropriate amount of test material was placed into a calibrated beaker. Vehicle was added to reach the correct final volume. The solution was stirred with a stir bas/plate for at least 10 minutes or until thoroughly mixed. Daily aliquots were placed in labeled dosing containers and stored at room temperature. Control animals received vehicle alone. Dosing solutions were prepared weekly.

VEHICLE
- Justification for use and choice of vehicle (if other than water): test article solubility
- Concentration in vehicle: see Table 1
- Amount of vehicle (if gavage): see Table 1
- Lot/batch no.: 86H0059
- Purity: 100%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Solutions for all three dose levels were assayed during Week 1 and 3 (one sample per concentration was taken and two subsamples were analyzed).

The following is a summary of dose confirmations of Hallbrite BHB (as % nominal):
Week 1: 103.3 (Group II), 94.2 (Group III), 95.7 (Group IV)
Week 3: 106.0 (Group II), 101.4 (Group III), 91.8 (Group IV)
Mean (SD): 104.7 (1.9), 97.8 (5.1), 93.8 (2.8)

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0 (control), 15, 150 and 1000 mg/kg/day
Basis:
other: nominal by gavage

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Justification for Frequency, Duration and Route of Administration:
The oral route is one of the potential routes of accidental human exposure to this test material. The frequency, duration and route is specified in the referenced guideline.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: Animals were observed, in their cages, for mortality and signs of severe toxic or pharmacologic effects twice daily.


DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Animals were removed from their cages and examined twice pretest and once weekly during the study period. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen, and external genitalia as well as evaluations of respiration and palpation for tissue masses.

Behavioral assessments were performed in conjunction with the physical examinations and included evaluations of autonomic activity, changes in gait, posture, response to handling, stereotypic and abnormal behaviors.


BODY WEIGHT:
- Time schedule for examinations: Animals were removed from their cages and weighed twice pretest, weekly during treatment and terminally (after fasting). Terminal, fasted body weights were obtained just prior to necropsy.


FOOD CONSUMPTION:
Feed was available without restriction for 7 days/week. Animals were provided full feeders weighing 570 g (including weight of feed, jar and lid). After 6 days, feeders were reweighed and the resulting weight was subtracted from the full feeder weight to obtain the grams consumed per animal over the 6-day period. Food consumption was measured (weighed) weekly, beginning one week prior to treatment.


HAEMATOLOGY:
- Time schedule for collection of blood: Blood for hematology studies was collected into tubes containing EDTA anticoagulant. Blood for coagulation studies was collected into tubes containing sodium citrate anticoagulant. Blood was collected at study termination (Week 4, test day 29).
- Anaesthetic used for blood collection: Blood was obtained from the orbital sinus (retrobulbar venous plexus) under light carbon dioxide/oxygen anesthesia.
- Animals fasted: overnight prior to blood collection
- How many animals: 5/sex/group
- Parameters checked: (Technicon H-1 Hematology System, Bayer Corp.) hemoglobin concentration, hematocrit, erythrocyte count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin concentration, total leukocyte count, absolute lymphocyte (calculated: total WBC x %lymphocyte / 100), absolute segmented neutrophils (calculated: total WBC x %segmented neutrophils / 100); (Photo-Optical Clot Detection System, COAG-A-MATE X2, Organon Teknika Corp.) prothrombin time, activated partial thromboplastin time; erythrocyte morphology (Henry, 1991).


CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Blood for clinical chemistry studies was collected into tubes with no anticoagulant, allowed to clot, and centrifuged to obtain serum. Blood was collected at study termination (Week 4, test day 29).
- Animals fasted: overnight prior to blood collection
- How many animals: 5/sex/group
- Parameters checked: (Hitachi 717, Boehringer Mannheim Corp. Automatic Analyzer) aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, glucose, cholesterol, triglycerides, total protein, albumin, globulin, albumin/globulin ratio, total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorous, gamma-glutamyl transferase.


NEUROBEHAVIOURAL EXAMINATION:
Testing was staggered over two sessions with approximately equal numbers of animals/sex/dose group in each session.

Motor Activity:
All animals in all dose groups were measured for motor activity pretest and on Week 4 using a modified version of Schulze’s procedures (Schulze, 1990). Locomotor activity of all animals was monitored using an automated Photobeam Activity System (San Diego Instruments, Inc., San Diego, CA). Sessions were 60 minutes in length; each was divided into 12, 5-minute intervals.

Functional Observational Battery:
A functional observational battery (Moser, 1989) was performed on all animals pretest and on Week 4. The observer performed evaluations “blind”. The following evaluations were performed: home cage, handling, open field, reflex assessments, grip strength, landing foot splay, hindlimb extensor strength, air righting ability, body weight, and stereotypical behavior.

Sacrifice and pathology:

At study termination (Week 4, test day 29) all animals were sacrificed by exsanguination following carbon dioxide inhalation.

Macroscopic Examinations:
Complete macroscopic postmortem examinations were performed on all animals killed at a scheduled sacrifice interval immediately after death. Examinations included all external surfaces and orifices; the external surfaces of the brain and spinal cord; the organs of the cranial, thoracic, abdominal and pelvic cavities and neck; and the remainder of the carcass for the presence of macroscopic morphologic abnormalities. Animals were fasted prior to scheduled sacrifices.

Organ Weights:
Organs listed in Table 2 were weighed for all animals at the scheduled sacrifice interval. Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other contiguous tissues in a uniform manner. Paired organs were weighed together.

Tissues Preserved and Examined Histopathologically:
The organs listed in Table 2 were prepared and examined microscopically for all animals in the control and high-dose group. Any abnormalities noted during examinations which were seen during histopathological examinations were recorded. All tissues were preserved in 10% buffered formalin. Eyes were placed first in glutaraldehyde/paraformaldehyde initially and then retained in 10% formalin. Testes and epididymides were placed in Bouin’s solution initially and then retained in 10% formalin. Lungs and urinary bladder were infused with formalin prior to their immersion into a larger volume of the same fixative. After fixation, tissues and organs from all animals were routinely embedded in parafin, cut at a microtome setting of 4 to 7 microns, mounted on glass slides, stained with hematoxylin and eosin and examined by light microscopy. Bones were decalcified in formic acid.

Statistics:

Body weight and food consumption data were initially analyzed by Bartlett’s test and if results were significant, then transformed by Blom’s Normalized Rank Test. The data was then analyzed by a standard 2-way analysis of variance and residuals tested for normality using the Shapiro-Wilk test. Further analysis, as appropriate, included Dunnett’s test, linear regression, and reanalysis using ANOVA.

Hematology, clinical chemistry, organ weights, forelimb and hindlimb grip strength and landing foot splay were analyzed by methods for multiple group analyses. Equality of means was determined by one-way ANOVA followed by multiple comparisons, if needed. Parametric procedures were used if variances were equal; otherwise, Dunnett’s test was employed. A statistical test for trend in the dose levels was also performed. The variances of two groups were tested for equality using the F test and if equal, a standard independent two-sample t-test was used to determine equality of means.

Cato Research, Durham, North Carolina, performed analysis of motor activity counts. Sets of counts over 12 measured intervals gave a profile of each animal and a multivariate model was used to determine if profiles for different groups had the same shape (the parallel hypothesis). Based on these comparisons, the Wilks’ Lambda test, two-way ANOVA or Dunnett’s t-test were employed.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

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:

CLINICAL SIGNS AND MORTALITY
Excessive salivation was observed in one female rat treated with 1000 mg/kg/day of Hallbrite BHB during the second week of the study. During Week 3, excessive salivation was observed in two males and two females treated wiht 1000 mg/kg/day. One of these females also exhibited slight red stains on the snout during Week 3. A third female exhibited lacrimation during Week 3. There were no treatment-related findings during Week 4.

No mortality was observed during the study. All animals survived to their scheduled necropsy.

BODY WEIGHT AND WEIGHT GAIN
There were no effects on body weight values or body weight gains related to the administration of Hallbrite BHB. Body weight gains among the males treated with 150 or 1000 mg/kg/day were smaller than controls during Week 1 and/or Week 2, but these differences did not persist into Weeks 3 and 4.

Mean body weight change (g) for Weeks 1, 2, 3 and 4:
Males:
Group I (control): 60.2, 107.0, 154.2, and 194.0
Group II: 54.2, 93.4, 137.8, 173.2
Group III: 47.8 (pGroup IV: 47.2 (p
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The mean food consumption values of the rats treated with Hallbrite BHB were generally comparable to control group values. The few values which were statistically significantly different from the control values were not considered to be related to the administration of Hallbrite BHB.

HAEMATOLOGY
The mean protrombin and activated partial protromboplastin times were increased among the animals treated with 1000 mg/kg/day Hallbrite BHB. This effect was more pronounced in males than in females. Other hematological values for treated animals were considered comparable to those of the control animals and within the range for normal variability.

Mean protrombin (PT) and activated partial protromboplastin times (APTT) (sec) were as follows for the various dose groups:
Males, PT: 11.4 (control), 11.7, 11.7, 19.0 (pMales, APTT: 23.9 (control), 25.9, 26.9, 35.4 (pFemales, PT: 9.9 (control), 9.6, 9.7, 11.2 (not sig)
Females, APTT: 19.7 (control), 19.3, 20.0, 24.6 (p
CLINICAL CHEMISTRY
The mean globulin (GLOB) values of male animals treated with 1000 mg/kg/day of Hallbrite BHB were decreased in comparison to those of the controls. As the mean albumin (ALB) values were comparable among the groups, the albumin/globulin (A/G) ratios for the animals treated at 1000 mg/kg/day were also increased relative to control rats. Alanine aminotransfersase (ALT) was increased and blood urea nitrogen (BUN) was decreased for male rats versus control values. In addition, the mean triglyceride (TRI) value for female rats treated with 1000 mg/kg/day was increased in comparison to the control value. As these changes in clinical chemistry values were not accompanied by microscopic changes in any of the organs/tissues examined, they were not considered to be biologically significant. Other clinical chemistry values for treated rats were considered comparable to those of the control animals.

GLOB (g/dL), males only: 1.7, 1.7, 1.7, 1.2 (pA/G ratio, males only: 2.8, 2.8, 2.8, 4.2 (pALT (IU/L), males only: 35, 36, 35, 45 (pBUN (mg/dL), males only: 12.7, 10.2, 11.3, 9.9 (pTRI (mg/dL), females only: 33, 34, 44, 67 (p

NEUROBEHAVIOUR
Motor Activity:
There was no indication of a neurotoxic effect of Hallbrite BHB based on motor activity evaluations.

Functional Observational Battery:
There was no evidence of a neurotoxic effect of Hallbrite BHB based on a functional observational battery of tests. No abnormalities in neuromuscular or sensorimotor activity, or autonomic or central nervous system function were detected after 4 weeks of treatment with 15, 150, and 1000 mg/kg/day of Hallbrite BHB.

ORGAN WEIGHTS
The mean organ weight values of the rats treated with Hallbrite BHB were comparable to those of the control animals.

GROSS PATHOLOGY
There were no treatment related gross macroscopic abnormalities reported. Those incidental findings reported are consistent with the incidence of normal background variations in this strain of rat.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment related microscopic findings. Those observed were comparable in incidence and severity in rats from treatment and control groups or the occurrence was sporadic. Such incidental findings have been seen in rats of this age used in other studies.

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

Critical effects observed:

no

Conclusions:

The oral administration of Hallbrite BHB to rats at a dose of 1000 mg/kg/day for 28 days resulted in occasional increases in salivation and increases in prothrombin and activated partial thromboplastin times. Other clinical chemistry changes in males included decreased globulin and blood urea nitrogen and increased alanine aminotransferase at a dose level of 1000 mg/kg/day and in females increased triglycerides at the 1000 mg/kg/day dose level. The clinical chemistry changes were not accompanied by any observed microscopic changes in any organs/tissues examined and were thus considered not to be biologically significant. There were no changes in body weights, food consumption, motor activity levels, functional observational batteries, organ weights or macroscopic and microscopic pathology evaluations related to the administration of Hallbrite BHB. Based on these results, the No-Observable-Effect-Level (NOEL) was considered to be 150 mg/kg/day.

Executive summary:

Hallbrite BHB was administered orally, via gastric intubation, to 30 Sprague-Dawley CD rats (5/sex/group) at dose levels of 15, 150 or 1000 mg/kg/day for a period of 28 days. Control animals received the vehicle (corn oil) at the same dose volume as administered to treated animals. Physical observations, body weights and food consumption measurements were performed on all animals and at selected intervals during the treatment period. Neurobehavioral studies were performed on all animals pretest and at study termination. Hematology and clinical chemistry evaluations were performed on all animals at study termination. Excessive salivation was observed in some animals up to and including Week 3 but these effects were absent by Week 4. Of significance, mean prothrombin and activated partial thromboplastin times were increased among the animals treated with 1000 mg/kg/day. There were no changes in body weights, food consumption, motor activity levels, functional observational batteries, organ weights or macroscopic and microscopic pathology evaluations related to the administration of Hallbrite BHB. Based on these results, the NOEL was considered to be 150 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The quality of the key study for repeated dose oral toxicity in rats was sufficient to fulfill the requirements for this endpoint.

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

The subject material was administered orally, via gastric intubation, to Sprague-Dawley CD rats (5/sex/group) at dose levels of 15, 150 or 1000 mg/kg bw/day for 28 days. Control animals received the vehicle (corn oil) at the same dose volume as administered to treated animals. Physical observations, body weights and food consumption measurements were performed on all animals and at selected intervals during the treatment period. Neurobehavioral studies were performed on all animals at pretest and at study termination. Excessive salivation was observed in some animals up to and including week 3 but these effects resolved by week 4. Clinical chemistry changes noted in male rats at the highest dose included decreased globulin and blood urea nitrogen and increased alanine aminotransferase. Increased triglycerides were reported in female rats at the highest dose level. The clinical chemistry changes noted were not accompanied by any microscopic changes in any organs/tissues and were thus not considered to be biologically relevant. Of significance, mean prothrombin and activated partial thromboplastin times were increased among the animals treated at the highest dose level of 1000 mg/kg bw/day. There were no significant changes in body weights, food consumption, motor activity levels, functional observational batteries, organ weights. Both macroscopic and microscopic pathological evaluations revealed no significant effects of test material administration. The NOEL level in the study was considered to be 150 mg/kg bw/day.

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

The subject material was tested by oral gavage in male and female rats following OECD guideline 407 and following accepted GLP standards.

Justification for classification or non-classification

Mean prothrombin and activated partial thromboplastin times were increased among male and female rats treated at the highest dose level of 1000 mg/kg bw/day in a 28 -day oral gavage study. These were the only biologically relevant effects noted in this study and these were completely absent at the next lower dose level of 150 mg/kg bw/day. These hematological effects did not result in mortality or pathological changes.

For the CLP Regulation the test material would not be rated as R48 (Danger of serious damage to health by prolonged exposure). Similarly, the test material would not be rated for “Specific Target Organ Toxicity – Repeated Exposure” according to EU CLP (Regulation (EC) No. 1272/2008).