Bis[(3,4-epoxycyclohexyl)methyl] adipate

Endpoint summary

Description of key information

Oral: Read-across to structurally similar substance

Based on the results of this study, the NOEL for oral administration of the test material to rats for a minimum of 90 days was determined to be 5 mg/kg/day for both males and females.

Based on these results, the test material does not require classification according to Directive 67/548/EEC or Regulation 67/548/EEC.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 December 1999 to 15 November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted on read-across material

Justification for type of information:

Read across to substance with the same functional groups.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

The dose sheets for the last five animals/sex in the 500 mg/kg/day group for study days 71-77 were inadvertently misplaced. Room temperature and relative humidity were inadvertently not recorded on study day 16.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

On study day 8, the 5 mg/kg/day group males were inadvertently dosed with the 50 mg/kg/day group formulation.

Principles of method if other than guideline:

Study was completed in accordance with standard OECD test guideline procedures

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD®(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 28 days old at receipt.
- Weight at study initiation: 197 - 201g (males), 144 - 151g (females)
- Housing: Animals were housed three per cage by sex for three days on arrival. Following this, all animals were housed individually in clean, wire-mesh cages suspended above cage-board.
- Diet: ad libitum, except during the period of fasting prior to blood and urine collections
- Water: Municipal water treated by reverse osmosis ad libitum.
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.1 to 29.0 °C
- Humidity: 35.3-57 %
- Photoperiod: 12-hour light/12-hour dark photoperiod

IN-LIFE DATES: From: 10 January 2000 To:11 April 2000 for 13 week necropsy and 8 May 2000 forrecover y kill at week 17. Study completed on 4 October 2001.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Each test material formulation was prepared daily to ensure homogeneous suspension. The appropriate amount of the test material was weighed into a tared, pre-calibrated, labelled storage container. While the test material was slowly stirred with a magnetic stirrer, the vehicle was added in approximately 1 mL aliquots until the volume was brought to the appropriate calibration mark for the relevant dose group. During addition of the vehicle, the storage container was occasionally swirled manually. Once a uniform preparation was achieved, the test material formulations were not stirred further. The formulations were protected from light throughout the dosing procedures and were gently swirled prior to the sampling and dosing procedures.

The test material formulations were administered orally by gastric intubation via a 16-gauge stainless steel gavage cannula as a single daily dose for 91 or 92 consecutive days. A total dose volume of 5 mL/kg was administered to each animal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dosing formulations of the test material in corn oil were analysed to determine the test material concentration by gas chromatography with a mass selective detector. The compound response was considered to be linear from 9.47 to 114 µg/mL using a 1-µL injection. The dosing formulations (1 mg/mL and 100 mg/mL) were homogeneous with respect to the test material. The mean concentrations for all strata (top, middle and bottom) ranged from 94.4 to 110 % of the target dose concentrations. The relative standard deviations (RSDs) between strata for the low and high dosing formulations were 2.1 and 0.56 %, respectively, indicating uniform dispersal of the test material in corn oil. The low and high dose formulations were stored at room temperature for 3 days and then analysed to assess stability. The mean concentrations were 98.5 and 110 % of the Time Zero concentrations, and therefore, the formulations were considered to be stable.

Duration of treatment / exposure:

91 or 92 days.

Frequency of treatment:

Daily administrations.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

The control and 500 mg/kg/day groups each consisted of 25 males and 25 females, and the 5 and 50 mg/kg/day groups each consisted of 20 males and 20 females.

Control animals:

yes, concurrent vehicle

Details on study design:

The test material was administered once daily by oral gavage to three groups (Groups 2- 4) at dose levels of 5, 50 and 500 mg/kg/day, respectively. Doses were based on an epoxy equivalent correction factor of 92.5 %. A concurrent control group (Group 1) received the vehicle, Mazola ® corn oil, on a comparable regimen. The control and 500 mg/kg/day groups each consisted of 25 males and 25 females, and the 5 and 50 mg/kg/day groups each
consisted of 20 males and 20 females. A dose volume of 5 mL/kg was used for all groups.

Groups of 15 rats/sex/dose level were assigned to the primary necropsy, and 5 rats/sex/dose level (Groups 2 and 3) or 10 rats/sex/dose level (Groups 1 and 4) were assigned to a 28-day recovery period.

Parameters evaluated included clinical observations, body weights, food consumption, clinical pathology (haematology, serum chemistry and urinalysis), ophthalmology, vaginal cytology and spermatogenic endpoints. Complete necropsies were performed on all animals and selected organs were weighed. Selected tissues were examined microscopically at the primary and recovery necropsies.

Positive control:

10 animals/sex not assigned to the study from the same shipment of animals (with body weights closest to the group mean for animals placed on the study) were maintained in the animal room throughout the study. These animals were used as positive controls.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Clinical observations were performed on all animals just prior to dosing and approximately one to two hours following dosing. Each animal received a detailed physical examination weekly, beginning 10 days prior to the test article administration and just prior to the scheduled necropsy. When appropriate, explicitly defined scoring systems were used. Signs noted included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture and response to handling, as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards) were also recorded. Signs such as skin lesions and hair loss were also recorded at this time. The absence or presence of findings was recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded approximately weekly, beginning 10 days prior to test material administration. Mean body weights were calculated for each study week and mean body weight changes were calculated for the corresponding intervals. A final body weight (fasted) was recorded for each animal on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Individual food consumption was measured approximately weekly, beginning 10 days prior to test material administration (study week -2). Food consumption was not measured from the day of randomisation to study day 0. Food intake was calculated as g/animal/day for the corresponding body weight intervals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ocular examinations were conducted on all animals prior to the initiation of dosing and during study week 12. All ocular examinations were conducted using an indirect ophthalmoscope, preceded by mydriasis.
- Dose groups that were examined: All dose groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: All time points. During Weeks 5, 13 and 17
- Anaesthetic used for blood collection: Not required - blood obtained from tail vein
- Animals fasted: Yes - overnight
- How many animals: 10 animals/sex/group assigned to the primary necropsy during study week 5 and on all animals assigned to the scheduled necropsies (study weeks 13 and 17).
- Parameters checked: Total Leukocyte Count, Erythrocyte Count, Haemoglobin, Haematocrit, Mean Corpuscular Volume, Mean Corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Platelet count, Prothrombin time, Activate Partial Thromboplastin Time, Differential Leukocyte Count Percent and Absolute, Platelet estimate and Red Blood Cell Morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: All time points. During Weeks 5, 13 and 17
- Animals fasted: Yes - overnight
- How many animals: 10 animals/sex/group assigned to the primary necropsy during study week 5 and on all animals assigned to the scheduled necropsies (study weeks 13 and 17)
- Parameters checked: Albumin, Total Protein, Globulin, Albumin/Globulin Ratio, Total Bilirubin, Urea Nitrogen, Creatinine, Alkaline Phosphatase, Alanine Aminotransferase, Aspartate Aminotransferase, Gamma Glutamyltransferase, Glucose, Total Cholesterol, Calcium, Chloride, Phosphorus, Potassium, Sodium, Creatine Kinase, Lactate Dehydrogenase, Direct Bilirubin, Indirect Bilirubin, Sorbitol Dehydrogenase

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to the day blood samples were collected during Weeks 5, 13 and 17.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: Specific Gravity, PH, Urobilinogen, Total Volume, Urine Creatinine, Urine N-acetyl-ß-glucosamidase, Osmolality, Colour, Appearance, Protein, Glucose, Ketones, Bilirubin, Occult Blood, Leukocytes, Nitrites, Microscopy of Sediment.

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes-A complete necropsy was conducted on all animals. The necropsy included, but was not limited to, examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY: Yes
- A full standard guideline tissue list was preserved at necropsy for histopathological examination. All tissues were examined for the control and high dose group killed at week 13. Microscopic examination was also conducted on the kidneys, liver, lungs, nasal tissues and gross lesions in the 5 and
50 mg/kg/day groups at the primary necropsy.

For animals in the recovery groups, microscopic examination was conducted on the liver in the control, 50 and 500 mg/kg/day groups and nasal tissues in the control, 5, 50 and 500 mg/kg/day groups.

The slides for nasal turbinate sections were peer-reviewed for determination of the nature and significance of the epithelial degeneration (and regeneration following the recovery phase)

Other examinations:

Determination of Oestrous Cycle: Vaginal smears for determination of the stage of oestrus were obtained from all females once daily beginning 22 or23 days prior to the primary necropsy. The average cycle length was calculated for complete oestrous cycles (i.e., the total number of returns to metoestrus [M] or dioestrus [D] from oestrus [E] or prooestrus [P]) beginning 22 or 23 days prior to the primary necropsy. The final vaginal smear for each female was collected on the day of necropsy.

Spermatogenic Analysis - Motility/Viability Assessment, Morphology Assessment and Enumeration of Epididymal and Testicular Sperm numbers and Sperm Production Rate - investigations completed immediately following euthanasia. A differential count of 200 spermatozoa/rat was used to determine morphological differences. The left testis and epididymis from each male at the primary necropsy evaluated for sperm numbers and sperm production rate using the method described by Blazak et al.

Organ Weights
The following organs were weighed from all animals at the scheduled
necropsies:
Adrenals
Brain
Epididymides (total and cauda)
Heart
Kidneys
Liver
Ovaries (with oviducts)
Prostate
Spleen
Testes
Thymus
Thyroid glands with parathyroids
Uterus (with cervix).
Paired organs were weighed together. Organ to final body weight and organ to brain weight ratios were calculated.

Statistics:

All analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5% comparing the control group to the treatment groups (by sex). Body weight, body weight change, food consumption, clinical data, oestrous cycle data, organ weight data, epididymal and testicular sperm numbers and sperm production rates were subjected to a one-way analysis of variance (ANOVA), followed by Dunnett's test if the ANOVA revealed statistical significance. The percentage of motile spermatozoa and the percentage of sperm with normal morphology were analysed by the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences, followed by the Mann-Whitney U-Test comparing the control and test article-treated groups if the ANOVA revealed statistical significance (p<0.05).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related clinical observations consisted of evidence of increased salivation (clear material around the mouth and on the forelimbs and ventral neck) and yellow material on various body surfaces (urogenital area, anogenital area, hindlimbs and ventral neck/trunk) in the 500 mg/kg/day group males and females. These findings were primarily noted at the 1-hour post-dosing observation, and less frequently noted prior to dosing and during detailed physical examination. Evidence of increased salivation and yellow material on various body surfaces were first noted on study days 8 and 9, respectively, and, once noted, were observed sporadically throughout the remainder of the dosing period.
- No test material-related clinical observations were noted during the recovery period.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsies (study weeks 13 and 17).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights were lower in the 500 mg/kg/day group males throughout the study when compared to the control group. The lower mean body weights were considered to be related to test material treatment. At the end of the dosing period (study week 13), mean body weight in the 500 mg/kg/day group males was approximately 7 % lower than the control group. For males, weight gain was significantly reduced in the 500 mg/kg/day group during study week 5 to 6 and study week 11 to 12. There were no clear treatment-related differences in mean absolute body weight for the females in any group.
Some statistically significant differences were noted when body weight gains for the control and test material-treated groups were compared; however, these differences were not considered test material-related. Weight gain for males in the 50 and 500 mg/kg/day groups was lower than the control group for the week preceding the initiation of dosing. However, as these differences did not result in significantly lower mean body weights compared to the controls at the time of the first dose, they were considered to be random biological variability.
For females, a decrease in mean weight gain (approximately 19 % less than the control group) was noted for the high dose group during the first week of dosing. This difference from the control group was considered to be statistically significant and resulted in a reduction in mean cumulative weight gain (statistically significant at study weeks 1 and 3) which remained throughout the dosing period.
Overall weight gain during the recovery period was similar across doses for males and females and there was no clear evidence of the high dose group males returning to control mean level.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related changes were noted in food consumption. However, some statistically significant differences were noted when the control and treated groups were compared. A slight decrease in food consumption was observed in the 500 mg/kg/day group females during study week 0 to 1 when compared to the control group. In addition, higher food consumption was observed in the 500 mg/kg/day group males during study weeks 3 to 4 and 5 to 6 and in the 50 mg/kg/day group males during study week 12 to 13. These differences from the control group were slight and no trends were apparent. Therefore, these changes were not attributed to test material administration. There were no other remarkable changes in food consumption during the dosing or recovery.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No oculopathic changes indicative of a test material-related effect were observed at any dose level.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test material-related changes in haematology parameters in any treatment group. However, several statistically significant differences were observed when the control and test material-treated groups were compared. At the study week 5 evaluation, mean white blood cell count was increased in the 500 mg/kg/day group males, mean red blood cell count was increased in the 50 mg/kg/day group males and mean absolute lymphocyte counts were increased in the 5 mg/kg/day group males.
- At the study week 13 evaluation, increased mean white blood cell count was observed in the 5 mg/kg/day group males, decreased mean MCHC was noted in the 500 mg/kg/day group males and mean prothrombin times were increased in the 50 and 500 mg/kg/day group males and the 50 mg/kg/day group females. Mean absolute lymphocyte count was higher in the 5 mg/kg/day group males and mean percent and absolute neutrophil counts were lower and mean percent lymphocyte counts were higher in the 50 and 500 mg/kg/day group females.
- All of the changes observed occurred sporadically, were without apparent dose-related response, were of minor magnitude and were inconsistent between the sexes. Consequently, the haematological differences observed were considered to be of normal biological variation and were not test material related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in serum chemistry parameters considered to be related to the toxicity of the test material consisted of elevated urea nitrogen and phosphorus levels and reduced cholesterol levels (statistically significant in the 500 mg/kg/day males only) in the 50 and 500 mg/kg/day groups and elevated direct bilirubin and sorbitol dehydrogenase levels in the 500 mg/kg/day group.
Mean urea nitrogen (BUN) levels were increased in the 50 and 500 mg/kg/day group males and females at the study week 5 and 13 evaluations when compared to the control group.
Mean phosphorus levels were increased in the 500 mg/kg/day group males and females at the study week 5 evaluation (statistically significant for males only) and in the 50 and 500 mg/kg/day group males and females at the study week 13 evaluation when compared to the control group (statistically significant).
Mean creatine kinase levels were decreased in the 500mg/kg/day group in males at the study week 5 evaluation and in a dose related manner at the study week 13 evaluation when compared to the control group. These decreases were determined to be statistically significant.
Mean serum cholesterol concentrations were decreased in the 50 and 500 mg/kg/day males at the study week 5 and 13 evaluations when compared to the control group, however, difference was only statistically significant in the 500mg/kg/day group only. In females, mean serum cholesterol concentrations were decreased in the 50 mg/kg/day group at the 13 week evaluation.
Mean direct bilirubin and sorbitol dehydrogenase levels in the 500 mg/kg/day group males and females were increased (usually statistically significant) at the study week 5 and 13 evaluations when compared to the control group.
In addition to the above observations, several statistically significant differences were observed when the test material-treated groups were compared to the control group. In males, mean calcium, sodium and lactate dehydrogenase levels differed from the control group values at different study intervals in various groups. Mean creatine kinase levels in the 5 and 50 mg/kg/day group males were decreased at the study week 13 evaluation when compared to the control group.
In females, mean total protein, globulin, bilirubin (indirect and total), glucose, calcium and potassium levels differed from the control group values at different study intervals in various groups. However, these changes observed in females occurred sporadically, did not occur in a dose related manner, were of minor magnitude, did not display consistency between sexes and were biologically irrelevant. As no macroscopic or microscopic changes correlated with these serum chemistry alterations, the serum chemistry differences were considered to be spurious, the result of biological variation and not test material-related.
There were no treatment-related effects on serum chemistry parameters following the recovery period. The only statistically significant differences from the control group at study evaluation week 17 (recovery period) consisted of increased mean potassium and indirect bilirubin levels for the 5mg/kg/day group females and an increased mean lactate dehydrogenase value for the 500mg/kg/day males. These changes however, did not occur in a dose-related manner, were inconsistent between sexes, were biologically irrelevant and/or did not have any corresponding macroscopic or microscopic findings.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in urinalysis parameters considered to be related to test material treatment consisted of decreased mean urine pH in the 500 mg/kg/day group males and females and decreased mean urine creatinine levels in the 500 mg/kg/day group males. Urine pH was decreased in the 500 mg/kg/day group males and females at the study week 5 and 13 evaluations (usually statistically significant). Urine creatinine levels for males in the 500 mg/kg/day group were decreased at the study week 5 and 13 evaluations (statistically significant). However, when adjusted for time and body weight, the effect was not apparent.
When the control and test material treated groups were compared, some statistically significant differences were observed. A lower mean creatinine was observed in the 5mg/kg/day group males at study week 5 evaluation, higher mean urobilinogen was noted in the 500 mg/kg/day group males at the study week 5 evaluation and higher mean osmolality was noted in the 500 mg/kg/day group females at the study week 13 evaluation. However, these changes were not considered to be test material related as they were only noted at a single evaluation, were not consistent between the sexes and there were no correlating microscopic findings.
During the recovery period, all urinalysis values for all dose groups of both sexes were similar to the control group values.

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):

Test material-related organ weight changes consisted of higher mean liver weights (usually statistically significant) in the 50 and 500 mg/kg/day group males and females and higher mean kidney weights in the 500 mg/kg/day group males and females at the study week 13 necropsy.
In the 50 and 500mg/kg/day males, and females, mean absolute and relative liver weights were increased at necropsy in week 13. The differences from the control group were statistically significant with the exception of absolute liver weight and liver weight relative to brain weight.
In males and females in the 500mg/kg/day dose group, mean absolute and relative kidney weights were increased at study week 13 necropsy in comparison to the control group. These differences observed were considered to be statistically significant compared to the control group.
When the control and test-material treated groups were compared, several statistically significant differences were observed. Mean absolute prostate weight was decreased in the 5mg/kg/day group males and mean thryoid weight relative to final body weight was increased in the 500mg/kg/day group males at the study week 13 necropsy. As no dose-related trends were apparent and there were no correlating macroscopic or microscopic changes, these organ weight changes were considered to be a result of normal biological variation and unrelated to test material administration.
No test material-related differences in organ weights were observed for any dose group of both sexes following the recovery period. Thymus weights were significantly increased in the 50mg/kg/day males, however, this increase was not considered to be treatment related as a similar effect was not evaluated at any previous time-point evaluation or at any higher concentrations.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test material-related macroscopic findings were noted in the liver of 3/15 males in the 500 mg/kg/day group at the study week 13 necropsy.
Pale livers were observed in 3/15 males in the 500 mg/kg/day group. Pale livers were not noted in any other group of males, including the control group, and were not noted in any female group. No other test material-related gross findings were observed at either necropsy. All other macroscopic changes were considered to be spontaneous and/or incidental in nature and unrelated to test material administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test material-related microscopic changes were observed in the liver of the 50 and 500 mg/kg/day group males and females at the primary necropsy and in the nasal tissues of 50 and 500 mg/kg/day males and females at the study week 13 (primary) and 17 (recovery) necropsies.
At the study week 13 necropsy, in both males and females in the 50 and 500mg/kg/day dose groups, fine hepatocellular vacuolation, in a periportal distribution, was observed with increased incidence.
The primary lesion observed in the nasal tissues in the 50 and 500 mg/kg/day group males and females were degeneration of olfactory epithelium. Some of these olfactory epithelial degenerative changes persisted at the recovery evaluation in males and females at the aforementioned dose groups, however, some regeneration of the olfactory tissue was also observed.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

OTHER FINDINGS
Oestrous Cycle Data: No test material-related changes in oestrous cycle were observed.
Spermatogenic Evaluations: No test material-related effects on spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate and sperm motility and morphology) were observed at any dose level.

Key result

Dose descriptor:

NOEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

The NOAEL of 5 mg/kg bw/day was determined based on systemic effects observed in the high dose group – increased liver and kidney weights, reduced bodyweight gains and lower food consumption, which were consistent with similar findings observed at the same dose level in the pre-natal developmental toxicity study. The liver effects are considered to be adaptive, indicating a response to a mild toxic insult; the changes were reversible within the recovery phase and showed no dose response relationship. The renal weight change was not associated with any clinical pathology or microscopic changes that could clarify the cause of this effect. Kidney weight changes were also restricted to the high dose group.

The observation of nasal epithelial degeneration extended to the intermediate dose group (2/15 males and 3/15 females affected by minimal degeneration). The effects were more pronounced and in greater incidence in the high dose group. The unusual nature of the response – nasal damage caused after oral cannula administration – appears to have been raised by the study director and the nasal tissue slides were specifically subjected to peer review by a second pathologist. While the pathologists agreed a form of wording to describe the pathology, it is clear from the pathology statement that identification of the cause or the toxicological significance of this nasal finding was not agreed by the pathologists. However, no additional investigations have been disclosed to provide any new information that could address the mode of activity/cause of induction of nasal change. 

It has been postulated that refluxing of the oral dose could affect the nasal turbinates but there is no corroborating evidence in the report to suggest any difficulties during the dose administration or recording of any clinical signs post-dosing that could suggest the animals were discomforted or were involuntarily removing the dose depot by reflux action. Nasal epithelial degeneration is normally associated with inhalation of toxic fumes. The vapour pressure of the test material is 2 x 10E-3Pa, and the administered dose was formulated in corn oil. It is considered highly improbable that the high dose animals were consistently exposed to toxic/caustic vapours from this oral preparation (the acute inhalation study showed no indication of nasal damage, nasal irritation or production of nasal exudates when animals were exposed to a 5.5 mg/L atmosphere for 4 hours nose-only exposure). Since it is not possible from the available study data to determine a mode of action or aetiology for the nasal change, conclusions regarding the systemic or local nature of the effect cannot be drawn.

The nasal epithelial changes were effectively the critical data on which the NOAEL derivation was based. The cause was not established, the persistence and limited recovery can be gauged by the incidence of degenerative and regenerative change present at termination following the recovery phase. It has been assumed that the nasal change is a systemic effect and extended in a dose related manner to affect the high dose group moderately and the intermediate group minimally and was not present in any of the low dose group animals. 

Assuming the systemic derivation of the change at 50 and 500 mg/kg bw/day means the oral NOAEL of 5 mg/kg bw/day is the point of departure for derivation of systemic DNEL values. In view of the likelihood of the nasal findings being idiopathic or localised in derivation, use of the low dose NOAEL is highly conservative and ensures any tier 1 risk assessments will err on the precautionary side.

Conclusions:

Based on the results of this study, the no-observed-effect level (NOEL) for oral administration of the test material to rats for a minimum of 90 days was 5 mg/kg/day for both males and females. Based on these results, the test material does not require classification according to Directive 67/548/EEC or Regulation 67/548/EEC.

Executive summary:

The repeated dose toxicity of the test material was investigated in accordance with the standardised guideline OECD 408, under GLP conditions.

In the study conducted by Padgett (2001), the toxicity potential of the test material, was evaluated following 90 day oral (gavage) administration to Crl:CD (SD)IGS BR rats. The test material was administered once daily by oral gavage to three groups of rats at dose levels of 5, 50 and 500mg/kg/day. A concurrent control group received the vehicle, corn oil, on a comparable regimen. The control and 500 mg/kg/day groups each consisted of 25 males and 25 females, and the 5 and 50 mg/kg/day groups each consisted of 20 males and 20 females. A dose volume of 5mL/kg was used for all groups.

Observations included clinical observations, body weights, food consumption, clinical pathology (haematology, serum chemistry and urinalysis), ophthalmology, vaginal cytology and spermatogenic endpoints. Complete necropsies were performed on all animals and selected organs were weighed. Selected tissues were examined microscopically at the primary and recovery necropsies.

There were no mortalities reported. There were no test material-related changes in food consumption or haematology parameters, oculopathic changes or in the oestrous cycle or spermatogenic endpoints observed. Increased salivation and yellow material on the fur were observed primarily after dosing in the 500 mg/kg/day group males and females. Body weights of the males from the 500 mg/kg/day group were decreased, although not statistically significant, throughout the study. Following the 13 week dosing period, effects were noted in the kidney, liver and olfactory epithelium of the nasal tissues. Slight effects in absolute and relative kidney weights in the 500 mg/kg/day group and correlating serum chemistry and urinalysis changes in the 50 and 500 mg/kg/day groups were observed. There were no histopathologic changes noted in the kidneys. In the 50 and 500mg/kg/day dose groups, slight effects were observed in absolute and relative liver weights as well as liver function, accompanied by minimal to mild histopathological changes. Degeneration of the olfactory epithelium in the nasal tissues was seen in the 50 and 500 mg/kg/day group males and females but not in any of the control group animals.

Following a 28 day recovery period, the mean body weight of males in the 500mg/kg/day dose group was lower than the controls although weight gain during the same period was similar. Olfactory epithelial degeneration was observed in both males and females from the 50 and 500 mg/kg/day groups, although at a lower incidence. In addition, regenerative changes were also evident in the olfactory epithelium.

Based on the results of this study, the NOEL for oral administration of the test material to rats for a minimum of 90 days was determined to be 5 mg/kg/day for both males and females.

Based on these results, the test material does not require classification according to Directive 67/548/EEC or Regulation 67/548/EEC.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read across to substance with the same functional groups.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See 'remark'

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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 repeated dose toxicity of the test material was investigated in accordance with the standardised guideline OECD 408, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

In the study conducted by Padgett (2001), the toxicity potential of the test material, was evaluated following 90 day oral (gavage) administration to Crl:CD (SD)IGS BR rats. The test material was administered once daily by oral gavage to three groups of rats at dose levels of 5, 50 and 500 mg/kg/day. A concurrent control group received the vehicle, corn oil, on a comparable regimen. The control and 500 mg/kg/day groups each consisted of 25 males and 25 females, and the 5 and 50 mg/kg/day groups each consisted of 20 males and 20 females. A dose volume of 5mL/kg was used for all groups.

Observations included clinical observations, body weights, food consumption, clinical pathology (haematology, serum chemistry and urinalysis), ophthalmology, vaginal cytology and spermatogenic endpoints. Complete necropsies were performed on all animals and selected organs were weighed. Selected tissues were examined microscopically at the primary and recovery necropsies.

There were no mortalities reported. There were no test material-related changes in food consumption or haematology parameters, oculopathic changes or in the oestrous cycle or spermatogenic endpoints observed. Increased salivation and yellow material on the fur were observed primarily after dosing in the 500 mg/kg/day group males and females. Body weights of the males from the 500 mg/kg/day group were decreased, although not statistically significant, throughout the study. Following the 13 week dosing period, effects were noted in the kidney, liver and olfactory epithelium of the nasal tissues. Slight effects in absolute and relative kidney weights in the 500 mg/kg/day group and correlating serum chemistry and urinalysis changes in the 50 and 500 mg/kg/day groups were observed. There were no histopathologic changes noted in the kidneys. In the 50 and 500 mg/kg/day dose groups, slight effects were observed in absolute and relative liver weights as well as liver function, accompanied by minimal to mild histopathological changes. Degeneration of the olfactory epithelium in the nasal tissues was seen in the 50 and 500 mg/kg/day group males and females but not in any of the control group animals.

Following a 28 day recovery period, the mean body weight of males in the 500 mg/kg/day dose group was lower than the controls although weight gain during the same period was similar. Olfactory epithelial degeneration was observed in both males and females from the 50 and 500 mg/kg/day groups, although at a lower incidence. In addition, regenerative changes were also evident in the olfactory epithelium.

Based on the results of this study, the NOEL for oral administration of the test material to rats for a minimum of 90 days was determined to be 5 mg/kg/day for both males and females.

Based on these results, the test material does not require classification according to Directive 67/548/EEC or Regulation 67/548/EEC.

Justification for classification or non-classification

Based on the results of the read-across study, (Padgett, 2001), the test material, was determined to have an NOEL of 5 mg/kg/day for both males and females. Based on these results, the test material does not require classification according to Directive 67/548/EEC or Regulation 67/548/EEC - even at the next higher dose level of 50 mg/kg bw/day the effects observed in liver and kidneys were transient and reversible and did not indicate serious morphological disturbance or irreversible target organ toxicity. While dose-related effects on nasal epithelium were identified, the mode of toxic action or causation of nasal changes following oral administration via cannula were not established but the low vapour pressure for the active substance suggests the inhalation route was not the primary exposure route for eliciting the epithelial change.