Cyclohexane-1,2-dicarboxylic anhydride

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:

From February 17,2010 to July 6,2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexane-1,2-dicarboxylic anhydride

EC Number:

201-604-9

EC Name:

Cyclohexane-1,2-dicarboxylic anhydride

Cas Number:

85-42-7

Molecular formula:

C8H10O3

IUPAC Name:

octahydro-2-benzofuran-1,3-dione

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polynt Lot No. T208210033
- Manufacturing date: 11 Feb 2010
- Expiration date of the lot/batch: 11 Feb 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep container tightly closed. Keep in a dry, cool and well ventilated place.
- Storage at testing facility 5 years

Test animals

Species:

rat

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River (UK) Ltd.
- Age at study initiation:47 to 54 days of age
- Weight at study initiation:245 to 308 g for males and 162 to 207 g for females.
- Housing: barriered rodent facility.The cages were made of a polycarbonate body with a stainless steel mesh lid. Wood based material was used as bedding and was sterilised by autoclaving and changed at appropriate intervals each week. Cages, food hoppers and water bottles were changed at appropriate intervals.The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the t ransference of such agents between rooms. Before the study commenced, the room was cleaned and disinfected. Each animal room was kept at pos itive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Diet : ad libitum. free access to a standard rodent diet (Rat and Mouse No. l Maintenance Diet), except when urine was being collected and ovemight before routine blood sampling. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: ad libitum. Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes, except when ur ine was being collected.
- Acclimation period:12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19 to 23°C
- Humidity (%):40 to 70%
- Photoperiod 12/24 cycle dark/light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance, HHPA, was prepared for administration as a series of graded concentrations in the vehicle. For each concentration the required amount of test material was weighed out and melted in an oven at 60°C. Approximately 50% of the required amount of vehicle was added to the test material and mixed using a spatula (the formulation was warmed to 37°C to aid dissolution). The formulation was made up to the required volume with the remaining vehicle and warmed again to 37°C. Following magnetic stirring for a minimum of five minutes, the formulation was divided into aliquots using a syringe. All containers were purged with Nitrogen. Concentration were made in ascending order. The test substance was used as supplied. All formulations were prepared freshly each week and stored refrigerated (approximately 4°C)

VEHICLE
- Justification for use and choice of vehicle : Test substance is unstable in aqueous media.
- Concentration in vehicle: volume-dose of 5 mL/kg bodyweight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method involved dilution using acetone followed by gas chromatographic analysis with flame ionisation detection (FID). Sample concentrations were determined with reference to an external standard prepared at a concentration of 60 μg/mL.
At specified intervals during treatment (first week and last week), freshly prepared test formulations were sampled (4 × 1 mL, accurately weighed). Duplicate samples were analysed. The concentrations of hexahydrophthalic anhydride (HHPA) in the test formulations analysed during the study and the deviation of mean results from nominal values were within applied acceptance limits of +10%/-15% of nominal concentration, confirming accurate formulation. Individual results were within 2% of the mean value, confirming the precision of analysis.

Duration of treatment / exposure:

28 days (4 weeks) + 14 days (2 weeks) recovery

Frequency of treatment:

All animals were dosed in sequence of cage-number within each group, once each day at approximately the same time each day, seven days per weekfor 28 days.

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
300 mg/kg bw
Basis:
actual ingested

Doses / concentrations 4

Remarks:

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

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Control and high dose
- Post-exposure recovery period in satellite groups: 2 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
SENSORY ACTIVITY AND GRIP STRENGHT: Yes
MOTOR ACTIVITY: Yes
MORTALITY: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION: Yes
HAEMATOLOGY: Yes
BLOOD CHEMISTRY: Yes
URINALYSIS: Yes
NECROPSY AND HISTOPHATOLOGY: Yes
ORGAN WEIGHTS: Yes
HISTOLOGY: Yes
LIGHT MICROSCOPY: Yes

Sacrifice and pathology:

Animals killed during the study and those surviving until the end of the scheduled treatment or recovery period were killed by carbon dioxide asphyxiation and subsequent exsanguination. The sequence in which the animals were killed after completion of treatment or recovery was selected to allow satisfactory inter-group comparison.

Statistics:

The following data types were analysed at each timepoint separately: Grip strength and motor activity, Bodyweight, using gains over appropriate study periods, Blood chemistry, haematology and urinalysis, Organ weights.
The following sequence of statistical tests was used for grip strength, motor activity, bodyweight, organ weight and clinical pathology data: A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. For comparisons involving two groups only, t-tests were used. For all other comparisons the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For comparisons involving two groups only, Wilcoxon’s rank sum tests were used. For all other comparisons the H1 approximate test, the nonparametric equivalent of the F1 test was applied.
For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate. The treatment comparisons were made on adjusted group means in order to allow for differences in bodyweight which might influence the organ weights.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See comments below

Mortality:

mortality observed, treatment-related

Description (incidence):

See comments below

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

Description (incidence and severity):

See comments below

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Mortality, clinical signs
There were 5 treatment-related mortalities during the course of the study, predominantly during the first two weeks of treatment. For three females and one male at 1000 mg/kg/day, and one male at 300 mg/kg/day common findings of impaired breathing or macroscopic changes in the stomach were observed. Microscopic examination commonly revealed stomach lesions including epithelial hyperplasia and/or degeneration of the foveolar region and hyperkeratosis, epithelial hyperplasia, submucosal inflammation and/or erosion in the non glandular region. In addition, inflammatory exudate in the airways and degeneration/necrosis/erosion of the epithelium were evident in the nasal turbinates. These deaths were considered most likely attributable to aspiration of formulation droplets during the extubation of the dosing catheter causing local irritation to the airways, rather than evidence of systemic toxicity. One further mortality, at 100 mg/kg/day, was considered not to be related to treatment.
Treatment-related signs observed at routine physical examination were restricted to noisy respiration (rales) in 2 males and 2 females at 1000 mg/kg/day, and those in relation to dose administration were restricted to a low incidence of noisy respiration (rales) after dosing among females receiving 1000 mg/kg/day. Signs of chin rubbing and associated salivation observed after dosing at 1000 mg/kg/day were considered a non-specific response to the formulation.
The bodyweight gain, food consumption, sensory reactivity and motor activity of males and females was considered unaffected by treatment at all dose levels investigated.

Haematology, clinical chemistry and urinalysis
Haematological assessments after 4 weeks of treatment revealed high neutrophil, eosinophil, monocyte and large unstained cell concentrations, and as a result high total white blood cell concentrations, among males receiving 1000 mg/kg/day. Males at 1000 mg/kg/day showed high alkaline phosphatase, plasma urea, creatinine and cholesterol concentrations and plasma gamma-glutamyl transpeptidase concentration was low. Plasma phosphorous concentrations were slightly low in all groups of treated females, although
no dose response was evident. At 1000 mg/kg/day, slightly high plasma sodium was apparent among males and females, chloride levels were high among females, and calcium concentrations were high and potassium levels low among males.
Males and females at 1000 mg/kg/day showed high mean urine volumes after 4 weeks of treatment.

Macroscopic pathology
Macroscopic changes after 4 weeks of treatment among males and females receiving 1000 mg/kg/day included a high incidence of stomach changes. At 300 mg/kg/day, 3 males and 2 females showed similar findings in the stomach. Microscopic examination revealed epithelial hyperplasia of the foveolar region, submucosal inflammation of the glandular region, hyperkeratosis, epithelial hyperplasia, epithelial vacuolation, mucosal and submucosal inflammation and erosion of the non glandular region in the majority of animals receiving 1000 mg/kg/day, and at a slightly lower incidence/severity in the 300 mg/kg/day group. These changes in the stomach were considered to be indicative of irritation by the test material and not systemic toxicity.

Microscopic pathology
Microscopic findings in the kidneys, cortical tubular basophilia, often accompanied by cortical tubular dilatation, was observed in most study groups, including Control males, with a higher incidence and severity in animals given 1000 mg/kg/day. This finding was most prominent in female decedents. Unilateral papillary inflammation/necrosis of the kidney, associated with papillary epithelial hyperplasia, was also seen in 1 male after 4 weeks of treatment at 1000 mg/kg/day.

Recovery groups
After 2 weeks recovery there was no indication of a persistent or latent effect of treatment upon haematological or urinalysis parameters, organ weights, nor were there any macroscopic abnormalities considered to reflect previous treatment, however, slightly high potassium and slightly low calcium concentrations were observed among males that had previously received 1000 mg/kg/day. A lower incidence and severity of most of the microscopic stomach changes was seen in males and females in the 1000 mg/kg/day group, although several of the findings, such as mucosal and submucosal inflammation and erosion of the non glandular region, were not observed in these animals, indicating full recovery after two weeks off-dose. Slight unilateral tubular basophilia in the kidney was recorded in 1 male previously given 1000 mg/kg/day.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic effects

2

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Death/humane kill following respiratory impairment associated with inflammatory cells in the respiratory epithelium of the nasal turbinates

3

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: microscopic stomach changes

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it was concluded that a dose level of 300 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for systemic toxicity. The No Observed Effect Level (NOEL) for death/humane kill following respiratory impairment associated with inflammatory cells in the respiratory epithelium of the nasal turbinates was 100 mg/kg/day. The dose of 100 mg/kg/day was also the NOEL for microscopic stomach changes considered to be indicative of irritation by the test material.

Executive summary:

The oral toxicity of hexahydrophthalic anhydride (HHPA) over an exposure period of 4 consecutive weeks and recovery from any potential treatment-related effects over a period of 2 consecutive weeks has been investigated. Methods were in accordance with OECD/EU test guidelines. It was concluded that a dose level of 300 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for systemic toxicity. The No Observed Effect Level (NOEL) for death/humane kill following respiratory impairment associated with inflammatory cells in the respiratory epithelium of the nasal turbinates was 100 mg/kg/day. The dose of 100 mg/kg/day was also the NOEL for microscopic stomach changes considered to be indicative of irritation by the test material