2-ethylhexyl 4-(dimethylamino)benzoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 July 1990 to 03 April 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 4-(dimethylamino)benzoate

EC Number:

244-289-3

EC Name:

2-ethylhexyl 4-(dimethylamino)benzoate

Cas Number:

21245-02-3

Molecular formula:

C17H27NO2

IUPAC Name:

2-ethylhexyl 4-(dimethylamino)benzoate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD®BR

Details on species / strain selection:

The Sprague-Dawley rat has historically been used in safety evaluation studies and is recommended by appropriate regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 42 days old
- Weight at study initiation: Males: 160.4 to 209.7 grams. Females: 138.8 to 166.9 grams.
- Housing: Singly in stainless steel hanging wire-mesh cages
- Diet: ad libitum
- Water: Tap water available ad libitum via automatic watering system
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 6 °F
- Humidity: 50 ± 20 %
- Air changes: Ten or greater air changed per hour were maintained.
- Photoperiod: A controlled diurnal cycle (12 hours of light/ 12 hours of darkness) was maintained via artificial illumination.

IN-LIFE DATES
From: 22 January 1991
To: 03 April 1991

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration is generally recognised as the primary means of developing general toxicity data.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared weekly (an additional batch was prepared on Day 4 of study, prior to the confirmation of 7-day stability) and stored at room temperature.
An appropriate quantity of test material was weighed for each dose level on an analytical balance (mg) in a weigh boat. The weighed amount was then transferred from the weigh boat into a pre-calibrated beaker. A small amount of corn oil (vehicle) was used to rinse the weigh boat and the rinse was added to the contents of the beaker. Corn oil was added to the beaker until approximately half the final volume was achieved. A stir bar was then added to the container and the contents were mixed on a magnetic stir plate for 1 to 2 minutes. The stir bar was then removed and the mixture was adjusted to the desired final volume with corn oil. The stir bar was placed back into the beaker and the mixture was subsequently stirred for an additional 2 to 3 minutes to achieve a homogeneous solution. Solutions were stirred during dosing.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no.: 9F 01:17 and 0B 28 06:05 (Supplied by Mazo-Lerch Food Distributors)
- Purity: Assumed to be 100 %

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A routine analysis was conducted on each batch of the test solution (all groups including Group 1). Room temperature stability was confirmed for 1, 2, 3, 7 and 14 days (performed concurrently with the study).
All dosing solutions, all dose levels, were within 4 % of their respective target concentrations. For the lowest and highest concentrations used, dosing solutions of the test material were found to be stable for at least 14 days when stored refrigerated.

This method describes the procedure for the determination of the test material in corn oil in the range of 10 to 700 mg/mL. The test material and corn oil solutions were dissolved in and diluted with hexane. Quantitation was achieved by gas chromatography using a flame ionisation detector (FID).

- Preparation of calibration standards (Target: 1.0 mg/mL stock standard)
Approximately 100 µL of the test material was accurately weighed into a 100 mL volumetric flask then dissolved and dilute to volume with hexane. Calibration standards were prepared at concentrations of 80.0, 100.0, 120.0 and 160.0 µg/mL.

INSTRUMENT PARAMETERS
Instrument: Hewlett-Packard 5880A GC equipped with a FID and 5880A terminal, or equivalent
Column: 30 M X 0.53 mm megabore DB-1 column
Gas Flow: 25 mL/min
Temperature: Oven: 210 °C; Injection port: 210 °C; Detector: 220 °C
Injection Volume: 2 µL
Chart Speed: 0.25 cm/min
Attenuation: 2 to 5

CALCULATIONS
Nanograms detected were determined through linear regression or taken directly from the chromatogram.
mg/mL found = ng detected x calculation factor

Calculation factor = [(A x B) / ( C x D x E)] x (1 / 1000)
where:
A = Final volume Dilution # 1 (mL)
B = Final volume Dilution # 2 (mL)
C = Aliquot taken for Dilution # 1 (mL)
D = Aliquot taken for Dilution # 2 (mL)
E = µL injected

% target = (mg/mL found / target mg/mL) x 100

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

Low and mid dose: 10 per sex per dose
Control and high dose: 15 per sex per dose (5 animals per dose per sex designated as recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Rats deemed suitable for the study were selected via a computerised weight-randomisation program and assigned to groups.
- Rationale for selecting satellite groups: To evaluate the reversibility, persistence or delayed occurrence of any toxic effects after a recovery period.
- Post-exposure recovery period in satellite groups: At least 4-weeks duration.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The rats were observed twice daily for mortality and moribundity. Daily cage-side observations for overt signs of toxicity were performed at the second daily mortality check. Signs were recorded by exception as they were observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A thorough physical examination was conducted at each weighing interval.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded at randomisation, prior to the start of treatment, weekly thereafter and at termination.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Measured weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: An indirect ophthalmoscopic examination was performed on each animal prior to treatment and during week four using 1 % Mydriacyl as the mydriatic agent.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At sacrifice by puncture of the orbital venous sinus. Anticoagulant was sodium citrate for APTT and PT. the anticoagulant for all other parameters was EDTA.
- Anaesthetic used for blood collection: Yes; CO2/ O2
- Animals fasted: Yes- food fasted overnight with water available
- How many animals: All that reached terminal and recovery sacrifice
- The following parameters were examined: leukocyte count (WBC), corrected leukocyte count (COR WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), platelet count (PLATELET), leukocyte differential, cell morphology, mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), reticulocyte count (RETIC), absolute reticulocyte count (A RETIC), prothrombin time (PT) and activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At sacrifice by puncture of the orbital venous sinus
- Animals fasted: Yes- food fasted overnight with water available
- How many animals: All that reached terminal and recovery sacrifice
- The following parameters were examined: sodium (SODIUM), potassium (POTAS), aspartate aminotransferase (AST), alanine aminotransferase (ALT), chloride (CHLORIDE), total protein (T PROT), albumin (ALBUMIN), calcium (CALCIUM), inorganic phosphorus (IN PHOS), total bilirubin (T BILI), blood urea nitrogen (BUN), creatinine (CREAT), glucose (GLUCOSE), gamma glutamyltransferase (GGT), globulin (GLOBULIN), alkaline phosphatase (ALK P), triglycerides (TRIGLY), albumin/globulin ratio (A/G) and total cholesterol (T CHOL).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At each scheduled sacrifice, all animals were weighed (animals were food- fasted overnight prior to necropsy), anaesthetised with sodium pentobarbital, exsanguinated, and necropsied.
The necropsy included examination of the following: external surface of the body, external surface of the brain, nasal cavity/paranasal sinuses, all orifices, cranial cavity, carcass, thoracic, abdominal, and pelvic cavities and their viscera and cervical tissues and organs.

- Organ Weights
The following selected organs from each sacrificed animal were weighed (when present) after careful dissection and trimming to remove fat and other contiguous tissue in a uniform manner: Brain (including brainstem), liver, kidneys, heart, ovaries, pituitary (post fixation), testes with epididymides, spleen and adrenals.
Organ-to-terminal body weight and organ-to-brain weight ratios were calculated.

HISTOPATHOLOGY: Yes
The following tissues (when present) were taken from each animal and preserved in 10 % neutral-buffered formalin: Sternum with bone marrow, adrenals, aorta (thoracic), brain with brainstem (medulla/pons, cerebellar cortex and cerebral cortex), colon, cecum, rectum, duodenum, ileum, jejunum, oesophagus, heart, ovaries, spleen, thymus, pancreas, kidneys, all gross lesions, liver, thyroid (parathyroids), urinary bladder, testes with epididymides, stomach, pituitary, mesenteric lymph node, sciatic nerve, uterus, lungs and trachea.
The following tissues were preserved for possible future examination if indicated by signs of toxicity or target organ involvement: Salivary glands (mandibular), mammary gland (females only), eyes, femur including articular surface, cervical spinal cord, mid-thoracic spinal cord, lumbar spinal cord, extraorbital lacrimal glands, skin, seminal vesicles, prostate, vagina and cervix and skeletal muscle.
All preserved tissues from the first list were embedded in paraffin, sectioned, stained with haematoxylin and eosin and examined microscopically from all animals in the control and high-dose groups. Gross lesions were examined microscopically from all animals. Target organs noted at the high dose were examined microscopically from all animals. Lungs, liver and kidneys were examined microscopically from all low- and mid-dose animals. For satellite animals, histopathology was performed on tissues identified as target organs in the treated groups (testes, epididymides, spleen, liver and kidney).

Statistics:

Standard one-way analysis of variance (ANOVA) was used to analyse: Absolute body weight (0, 1, 2, 3, 4, 5, 6, 7, 8), body weight change (Weeks 0 to 4 and 5 to 8), absolute food consumption (1, 2, 3, 4, 5, 6, 7, 8), total food consumption (Weeks 1 to 4), clinical pathology parameters (except cell morphology) and organ weight data of the control groups in a statistical comparison to the data for the same sex of the treated groups.
If variances of the data were heterogeneous, a series of transformations was performed on the data to achieve homogeneity prior to statistical analyses. When the series of transformations was not successful in achieving variance homogeneity, analyses were performed on rank-transformed data. The criterion for significance of group comparisons was at the 5 % two-tailed probability level.
The term "significant" is used to indicate a statistically significant difference between the control group and the specified treated group of the same sex.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was noted in the Group 4 males and females at the time of cage-side observations. This sign was occasionally noted also prior to and/or during dosing in some Group 4 animals.
Urine stains, alopecia and sores were the only clinical signs noted at the weekly physical examinations during the dosing phase. Alopecia and sores were the only clinical signs noted during the recovery period.
Only urine stains in the Group 4 females during the dosing period may have been treatment related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female from Group 1 died after the Week 4 blood collection; since death followed blood collection it was considered to be accidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistical evaluations showed a significant decrease in the Group 4 male mean body weight (weeks 3 to 8) and body weight change (weeks 0 to 4).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase was noted for the Group 4 female mean food consumption at Week 3 and a decrease at Week 7. However no biologically significant effect on food consumption was noted in any of the treatment groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Findings noted at Week 4 were anterior synechia and retinal linear atrophy in one Group 2 male. At the termination of this study, there did not appear to be any ophthalmoscopic abnormalities related to the compound or dose level. The ophthalmoscopic observations noted are considered to be incidental findings.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The changes were suggestive of a mild hepatic involvement that was reversible in all cases by week 9. Evaluation of the haematology data at Week 5 revealed minimal, but significant decreases in red cell mass (erythrocyte count, haemoglobin and haematocrit) in the Group 4 animals, except for the erythrocyte count value in the males. APTT was significantly decreased in Group 4 males. Significant increases in percent and absolute reticulocyte counts were noted in the Group 4 females, indicative of an appropriate regenerative response.
Correlative evidence of an erythrocytic regenerative response was noted in the erythrocyte morphology data, wherein there was a tendency for an increase in severity grade for polychromasia in the Group 4 females. By the Week 9 recovery interval, the aforementioned hematologic changes had completely resolved. The remaining statistically significant differences in the haematology data were felt to be incidental to the administration of the test material, based on the low magnitude of the change and/or the lack of a dose response.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The changes were suggestive of a mild hepatic involvement that was reversible in all cases by week 9. Statistical evaluation of the clinical chemistry data at Week 5 revealed minimal but significant decreases in globulin and increases in albumin to globulin ratio in the Group 4 males and females. Triglyceride and gamma glutamyltransferase in the Group 4 females and total bilirubin in the Group 4 males were significantly increased. Statistically significant differences noted for other parameters were thought to be incidental to test material administration based on the low magnitude of the change, the occurrence only at the recovery interval and/or the lack of a dose response.
At Week 9, a statistically significant increase was noted for alanine aminotransferase only. No statistical differences were noted at Week 9 for any other clinical chemistry parameters.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Evaluation of the terminal sacrifice absolute organ weight data exhibited a significant increase in liver weights for the Group three females and Group 4 males and females. Significantly decreased mean absolute testis weight (with epididymides) and pituitary weight was noted in the Group 4 males. The mean absolute spleen weight was significantly increased in the Group 4 females. In the recovery animals significant decreases were noted only in the Group 4 males for absolute mean terminal body, kidney and testis (with epididymides) weights.
Evaluation of the organ-to-terminal body weight data from the terminal sacrifice showed significant increases in the spleen (Group 4 males and females), kidney (Group 4 males) and liver (Group 4 males and Group 3 and 4 females). The testis (with epididymides) weight was significantly decreased in the Group 4 males. A significant increase was noted in the Group 4 female pituitary weight at the recovery sacrifice.
Evaluation of the organ-to-brain weight data at terminal sacrifice revealed significant increases in the spleen (Group 4 female), kidney (Group 4 female) and liver (Group 2 and 4 males and Group 3 and 4 females). Significant decreases were noted in the testis (with epididymides) and pituitary of the Group 4 males at the terminal sacrifice. At the recovery sacrifice a significant decrease was noted in the kidney weight for the Group 4 males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

UNSCHEDULED DEATHS
No gross lesions were observed in the Group 1 female that was the only unexpected death.

TERMINAL SACRIFICE
Gross lesions were as follows:
-Lung: Dark area (one Group 2 female);
-Spleen: Raised area (one Group 4 female) and granular/pitted/rough (two Group 2 females);
-Liver: enlarged (one Group 4 male), mass (two Group 2 males), mottled (one Group 3 female), irregularly shaped (one Group 2 male) and pale area (one group 1 male and 1 Group 2 male);
-Kidney: Pale (one Group 3 male) and dilated pelvis (one Group 1 male and two Group 4 males);
-Glandular stomach: Dark area (three Group 4 males, two Group 3 females and one Group 4 female);
-Testis: Soft (one Group 2 male and four Group 4 males), small (eight Group 4 males) and unequally sized (one Group 2 male);
-Epididymis: Small (one Group 4 male), pale area (two Group 3 males) and unequally sized (one Group 2 male)
-Uterus: Distended (one Group 3 female) and fluid filled lumen (one Group 3 female);
-Vagina: With mass (one Group 2 female);
- Abdominal adhesion: one Group 2 male; and
-Sore on foot/paw: one Group 1 male.

RECOVERY ANIMALS
-Liver: Dark area (one Group 1 male);
-Kidney: Dilated pelvis (one Group 1 male) and fluid in pelvis (one Group 1 male);
-Testis: Small (one group 1 male and three Group 4 males)
-Thymus: Firm area (one Group 4 male);
-Skin alopecia: one Group 4 female; and
-A Sore: one Group 4 male.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

SUMMARY
Microscopic evaluation revealed changes in the testis (1000 mg/kg/day), epididymis (300 and 1000 mg/kg/day), spleen (1000 mg/kg/day males; 300 and 1000 mg/kg/day females) and liver (1000 mg/kg/day) of rats sacrificed after at least 4 weeks of test material administration. Changes were observed in the epididymis only of the Group 4 (1000 mg/kg/day) that were held for an additional 4 week recovery period without test material administration before sacrifice.
Testicular changes in animals at the terminal sacrifice were increased in incidence and severity in the Group 4 males. However the incidence and severity was reduced at the recovery sacrifice. Most epididymal changes noted at the terminal sacrifice were also noted after the recovery phase in the Group 4 males. Splenic changes noted at the terminal sacrifice were substantially reduced or not observed after the recovery period. Centrolobular hypertrophy was observed in the liver of the Group 4 animals at the terminal sacrifice.

RESULTS
Capsular inflammation in the spleen (300 and 1000 mg/kg/day males; 100, 300 and 1000 mg/kg/day females) was observed in low incidence and with low severity in animals sacrificed immediately following cessation of compound administration. The biological significance of this change is uncertain.
Testicular changes in animals sacrificed immediately following cessation of compound administration consisted of atrophy of the germinal epithelium, hypospermia, presence of syncytial cells and lumenal cellular debris (Table 1). Both the incidence and severity (based on number of animals examined) were increased in 1000 mg/kg/day animals. These changes can be observed with low incidence in normal male rats; however, their presentation and the presentation of related changes in the epididymis of 1000 mg/kg/day rats allowed separation of spontaneous occurrences from test-material-related occurrences. Following a 4-week recovery period, the incidence and severity of these changes were reduced, indicating that the changes are reversible at this point (Table 2).
Changes observed in the testis of one 1000 mg/kg/day male were considered due to incomplete recovery rather than an ongoing test-material effect. Epididymal changes in animals sacrificed immediately following cessation of test material administration consisted of hypospermia with lumenal cellular debris (1000 mg/kg/day) and oedema with inflammatory changes (300 mg/kg/day) (Table 3). Most of these changes were also observed in the 1000 mg/kg/day animals following the 4-week recovery period (Table 4).
The inflammatory changes in recovery animals were similar to those observed in concurrent controls. The hypospermia, lumenal cellular debris, and single incidence of oedema were considered static rather than continuing effects, and because the testis does recover, these effects should dissipate with a longer recovery phase.
At necropsy, soft, small, and/or unequally sized testes and epididymides were frequently observed. These macroscopic changes were considered a manifestation of the microscopic changes previously described. The pale areas described in the epididymides of 2 animals were considered manifestations of the granulomatous inflammation observed microscopically. The statistically significant lower average testis with epididymis weight observed in 1000 mg/kg/day animals sacrificed immediately following cessation of compound administration was considered primarily due to the testicular changes in this group.
Test material-related increases in the incidence and the severity of pigment accumulation were observed in the spleen of 1000 mg/kg/day males and of 300 and 1000 mg/kg/day females sacrificed immediately following cessation of test-compound administration (Table 5). These differences were substantially reduced after the 4-week recovery period and, at that point, could not be definitively ascribed to test material administration.
Test material-related increases in the incidence and the severity of extramedullary haematopoiesis were observed in the spleen of 1000 mg/kg/day females sacrificed immediately following cessation of test material administration (Table 6). Less remarkable increases were observed in similarly treated males. Differences in splenic extramedullary haematopoiesis between 0 and 1000 mg/kg/day rats of either sex were not observed after the recovery period.
Statistically significant increased average splenic weights in 1000 mg/kg/day animals were considered related to the pigment accumulation and the anaemia-related haematological response described above.
Centrolobular hypertrophy was observed in the liver of 1000 mg/kg/day terminal sacrifice animals (Table 7). Both the incidence and the severity were low. Similar hypertrophy was observed in three 100 mg/kg/day males; however, there was concurrent localised granulomatous inflammation with mineralisation in two of these animals that may account for the hypertrophy.
In spite of the low incidence and severity of this change, higher averages noted for the absolute liver weight, relative to body weight ratio and relative to brain weight ratio in the 1000 mg/kg/day groups (all of which were statistically significant) support classifying this hypertrophy as a test material-related change.
Capsular inflammation was observed in the spleen of animals sacrificed immediately following cessation of test material administration (Table 8). Both the incidence and severity were low; however, this is not a common observation, and the absence of its occurrence in concurrent control animals leaves the biological significance of this inflammation uncertain. Capsular inflammation was

DISCUSSION
The testicular changes in Group 4 males sacrificed immediately following cessation of compound administration consisted of atrophy of the germinal epithelium, hypospermia, presence of syncytial cells and lumenal cellular debris. Epididymal changes in these animals consisted of hypospermia with lumenal cellular debris. These histopathological observations are consistent with the gross pathological observations of soft and small testis; and the significantly decreased absolute testis/epididymis weight, testis/epididymis- to-terminal body weight and testis/epididymis to- brain weight ratios observed at necropsy. Following the 4 week recovery, the incidence and severity of the testis and epididymal changes were reduced, indicating that the changes were reversible.
Increases in the incidence and the severity of pigment accumulation were observed in the spleen of Group 4 males and Group 3 and 4 females sacrificed after 4 weeks of treatment with the test material. These differences were substantially reduced after the recovery period. Increases in the incidence and the severity of extramedullary haematopoiesis were also observed in the spleen of the Group 4 animals. The extramedullary haematopoiesis was not observed after the recovery period. These histopathological changes correlated well with the increased average splenic weights in the Group 4 animals sacrificed at Week 4 and the anaemia-related haematological response previously described. The biological significance of the common observation of capsular inflammation observed in animals sacrificed at Week 4 is uncertain.
Centrolobular hypertrophy was observed in the liver of Group 4 animals sacrificed at Week 4. Both the incidence and the severity were low. Increases in the absolute liver weight, liver-to-terminal body weight and liver-to-brain weight ratios at sacrifice support classifying this hypertrophy as treatment related. Neither the centrolobular hypertrophy nor the increased liver weights were observed after the recovery period.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No neoplastic changes were observed.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: on testes and epididymis

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

testes

other: epididymis

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL under the conditions in this study was 300 mg/kg/day based on histopathological observations in the testis/epididymis at 1000 mg/kg/day. The effets on spleen and liver were considered to be not adverse.

Executive summary:

A study was carried out to evaluate the short term repeated dose toxicity of the test material using methodology equivalent to the standardised guideline OECD 407 under GLP conditions.

Groups of 10 male and ten female Sprague-Dawley rats were exposed to the test material in corn oil at dose levels of 0, 100, 300 and 1000 mg/kg/day for 28 days. The test material was administered daily via gavage. A further group of 5 animals per sex were dosed with the control and the high dose and served as recovery animals.

After 4 weeks of dose administration, 10 rats of each sex from each group were sacrificed and necropsied. To evaluate the reversibility, persistence, or delayed occurrence of any toxic effects, 5 rats of each sex in Groups 1 and 4 were sacrificed and necropsied after a recovery period of at least 4-weeks duration. Criteria evaluated for potential toxicity included mortality, clinical observations, body weight, food consumption, clinical pathology, organ weight data, as well as ophthalmoscopic, gross pathology and histopathology examinations.

There was no treatment-related mortality during the study. Treatment-related clinical observations included salivation in Group 4 males and females and urine stains in Group 4 females. The test material treatment significantly depressed mean body weights in Group 4 males during the 3rd and 4th weeks of treatment. This body weight depression continued throughout the recovery phase of the study. Mean body weight change was also significantly reduced for Weeks 0 to 4 in this group. No biologically significant effect on food consumption was noted in any of the treatment groups.

Administration of the test material resulted in minimally toxic effects on certain haematologic and serum chemistry parameters in only the Group 4 animals. These changes were suggestive of a mild hepatic involvement that was reversible in all cases by Week 9. The changes included decreases in red cell mass (erythrocyte count, haemoglobin and haematocrit) in Group 4 males and females and significantly depressed APTT in Group 4 males. Significant increases in percent and absolute reticulocyte counts were noted in Group 4 females, indicative of a regenerative response. Clinical chemistry data revealed minimal, but significant, decreases in globulin and increases in albumin to globulin ratio in the Group 4 males and females. Triglyceride and gamma glutamyltransferase in Group 4 females and total bilirubin in Group 4 males were also increased.

Rats receiving the test material for 4 weeks and then sacrificed exhibited treatment-related changes in the testis (Group 4 males), epididymis (Group 3 and 4 males), spleen (Group 3 females and Group 4 males and females) and liver (Group 4 males and females). Treatment related changes were observed only in the epididymis (Group 4 males) of rats receiving the test material for 4 weeks and then allowed to recover for 4weeks.

In conclusion, oral administration of the test material in rats at doses of 0, 100, 300 and 1000 mg/kg/day resulted in no mortality. Mild to moderate reversible toxicity was demonstrated as evidenced by effects on body weight, clinical signs, clinical and gross pathology, organ weights and histopathology. Target organs appeared to include testis/epididymis, spleen and the liver. The no-observable-effect-level (NOEL) under the conditions in this study was 100 mg/kg/day for both the males and females based on histopathological observations in the testis/epididymis and/or spleen at 300 mg/kg/day. The NOAEL under the conditions in this study was 300 mg/kg/day based on histopathological observations in the testis/epididymis at 1000 mg/kg/day. The effets on spleen and liver were considered to be not adverse.