Bicyclo[2.2.1]heptane, 2-ethyl-5-methoxy-

Endpoint summary

Description of key information

A repeated dose toxicity test was performed according to OECD TG 407. A NOAEL of 55 mg/kg bw/day (actual dose received) (male/female) was derived. At 800 mg/kg/day, several changes were noted in the parameters and tissues examined that were considered to be treatment-related. Though the effects were generally minor and the toxicological relevance remains somewhat unclear. 
 

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:

06 October 2006 and 06 February 2007

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: United States Environmental Protection Agency, Title 40 Code of Federal Regulations Part 792, Federal Register, 29 November 1983.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl,CD (SO) BR strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (U.K) Limited, Manston, Kent
- Age at study initiation: The rats were 28 ± 1 days old
- Weight at study initiation: Males weighed 125 to 141. Females weighed 110 to 125g.
- Fasting period before study: Animals were not fasted prior to study.
- Housing: The animals were housed in groups of five in cages suspended over trays lined with absorbent paper.
- Diet: A pelleted diet (Labsure LAD 1) was used (ad libitum). The diet was considered not to have any level of contaminant. Exception - food was withdrawn overnight prior to collection of blood prior to termination (Week 4) .
- Water: ad libitum mains water was supplied from polycarbonate bottles attached to the cage.
The drinking water was considered not to have any level of contaminant.
- Acclimation period: Seven days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8˚C to 21.5˚C
- Humidity (%): 54.1 to 58.2%
- Air changes (per hr): The rate of air exchange was maintained at a rate of approximately 20 air changes per hour.
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES:
From: Day1 (29 June 1988) to Day 28 (27 July 1988).

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in distilled water.

VEHICLE
- Concentration in vehicle:
3, 30 and 200 mg/ml.

- Amount of vehicle (if gavage):
5 ml/kg bodyweight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The high dosage concentration of the test substance was prepared freshly each day as a 16% w/v solution in corn oil. The intermediate (1.1% w/v) and low (0.1% w/v) dosage concentrations were similarly prepared by direct dilution of the test substance with the vehicle.

The concentration of ethyl methoxy norbornane in the final solution was quantified by gas - liquid chromatography using flame ionization detection.

The analytical procedure was validated by fortifying control vehicle ( corn oil , 1 ml ) to concentrations of 1 and 200 mq/ml ethyl methoxy norbornane, with either a solution of ethyl methoxy norbornane in carbon tetrachloride ( inclusion levels <20 mq/ml ) or with the neat standard ethyl methoxy norbornane ( inclusion levels <20 mq/ml ) , and analyzing as described in section 3 .

Procedural recoveries were determined for each inclusion level and analysed concurrently with test samples .

Determination of concentrations of ethyl methoxy norbornane in test solutions prepared for Day 1 of dosing. Representative samples ( approximately 20 ml ) of freshly prepared test solutions , received from the Department of Formulation, were thoroughly mixed by shaking and duplicate sub-samples ( 1 ml ) were analysed.

Determination of the chemical stability ethyl methoxy norbornane in corn oil solution. A freshly prepared batch of test solution, at nominal concentrations of 1 mg/ml and 200 mg/ml ethyl methoxy norbornane, was immediately sub-sampled for analysi s . The remainder of the test solution was stored at ambient temperature in the dark for 4 hours before analysis. At each occasion, duplicate sub-samples were analysed.

The mean analysed concentrations of ethyl methoxy norbornane in test solutions prepared for Day 1 of dosing and the deviation of mean results from nominal values were all within 2% of nominal concentrations .

The results of these analyses are presented in the attached analytical report.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dosage levels administered were selected on the basis of an acute oral toxicity study (HRC Report No . 88l039D/IFF 65/AC) and a seven-day preliminary oral toxicity study (HRC Schedule No. IFF/64-P) carried out at HRC .

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were performed on all animals early in each working day and again in the late afternoon to look for dead or moribund animals. At weekends a similar procedure was followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION: The quantity of food consumed in each caqe was measured at weekly intervals throughout the study.

FOOD EFFICIENCY: Weekly food efficiency (bodyweight gain/food intake) was not calculated.

WATER CONSUMPTION
- Time schedule for examinations: Water intake was visually observed daily.

OPHTHALMOSCOPIC EXAMINATION: Not applicable

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period.
- Anaesthetic used for blood collection: No data:
- Animals fasted: Yes
- How many animals: Five males and five females
Routine haematological parameters were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period.
- Anaesthetic used for blood collection: No data:
- Animals fasted: Yes
- How many animals: Five males and five females
Routine biochemical parameters were examined.

URINALYSIS:
Not applicable

NEUROBEHAVIOURAL EXAMINATION: No
Not applicable

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see tables)
All animals were subjected to a full external and internal macroscopic examination and any abnormalities were recorded.

HISTOPATHOLOGY: Yes (see tables)
All control and high dose animals were subjected to a full histological examination and low and intermediate group animals were routinely subjected to examination of liver and spleen.

Other examinations:

MORTALITY DATA
ORGAN WEIGHTS
MICROSCOPIC EXAMINATION

Statistics:

Statistical analyses

All statistical analyses were carried out separately for males and females.

Bodyweight data were analysed using weiqht gains .

The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data:

(i) If the data consisted predominantly o f one particular value (relative frequency of the mode exceeded 75%), the proportion of values different from the mode was analysed by appropriate methods. Otherwise:

(ii ) Bartlett's test (1) was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.

(ill) I f no significant heterogeneity was detected a one-way analysis of variance was carried out . If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks (2 ) was used.

(iv ) Analyses of variance were followed by Student's It' test and Williams' test (4 ) for a dose-rel ated response, although only the one thought more appropriate for the response pattern observed was reported . The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the It' test and Williamsl test (Shirley's test (3)).

For organ weight data, where appropriate, analysis of covariance was used in place of analys is of variance in the above sequence. The final bodyweight was used as covariate in an attempt to allow for differences in bodyweight which may have influenced the organ weights.

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):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

RESULTS

CLINICAL FINDINGS (see appended Appendix 6)

There were no mortalities.

Increased salivation following dosing and hunched posture were observed during the study period for all rats receiving ethyl methoxy norbornane, 800 mg/kg/day . These clinical findings, which persisted for one to several days , were not observed for control rats or for rats receiving ethyl methoxy norbornane, 5 or 55 mg/kg/day.

Following dosing on Day 1, greasy fur was recorded for all rats including those in the control group. In all instances, greasy fur persisted until termination.

BODYWEIGHT CHANGES (Figure 2, Table 1, Appendix 1)

Bodyweight gains of rats receiving ethyl methoxy norbornane were similar to those of control animals during the four-week treatment period.

FOOD CONSUMPTION (Table 2, Appendix 2)

Slightly higher food consumption was recorded during Week 1 to 4 for male and female rats receiving ethyl methoxy norbornane, 800 mg/kg/day in comparison with controls.

Food consumption for rats receiving ethyl methoxy norbornane, 5 or 55 mg/kg/day was considered similar to that of controls.

HAEMATOLOGY (Table 3, Appendix 3)

Haematological investigations were carried out during Week 4 for all rats.

In comparison with control animals higher lymphocyte counts, resulting in higher total white blood cell (WBC) counts, were recorded for male and female rats receiving ethyl methoxy norbornane, 800 mg/kg/day. Statistical significance (p<0.05 or p<0.01) was achieved in most instances. Lymphocyte and total WBC counts for rats receiving ethyl methoxy norbornane, 5 or 55 mg!kg/day were similar to those of the controls.

Thrombotest times for female rats receiving ethyl methoxy norbornane were significantly lower (p<0.05 or p<0.01) than those of the controls. However. The apparent trend to lower female thrombotest times was very low in magnitude, was not observed for male rats and was not, therefore, considered to be of toxicological importance.

In all other instances, haematological parameters for rats receiving ethyl methoxy norbornane were similar to those receiving corn oil.

BIOCHEMISTRY (Table 4, Appendix 4 )

Biochemical investigations were also carried out for all rats during Week 4.

In comparison with control animals, significantly higher (p<0.01) urea nitrogen levels were recorded for male rats receiving ethyl methoxy norbornane, 800 mg/kg/day. Cholesterol levels for female rats in this high dosage group were similarly significantly higher (p<0.01) than those of the controls.

Several changes in electrolyte levels were also recorded amongst rats receiving ethyl methoxy norbornane. Calcium levels for both male and female rats receiving ethyl methoxy norbornane, 800 mg/kg/day and for female rats receiving ethyl methoxy norbornane, 55 mg/kg/day were significantly higher (p<0.05 or p<0.01) than those of the controls.

Similar shifts to significantly higher (p<0.05) potassium (male rats) and inorganic phosphorus (female rats) levels were recorded for animals in the high dosage group in comparison with controls.

Chloride levels for male rats receiving ethyl methoxy norbornane, 800 mg/kg/day were significantly lower (P
No other chanqes in biochemical parameters were noted that were considered to be related to treatment with ethyl methoxy norbornane.

ORGAN WEIGHT ANALYSIS (Table 5, Appendix 5)

In comparison with animals receiving corn oil, significantly higher adjusted liver weights (p<0.01) were recorded for both male and female rats receiving ethyl methoxy norbornane, 800 mg/kg/day.

Adrenal weights for both male and female rats in this high dosage group were similarly higher than those of the controls with statistical significance being achieved (p<0.05) for male rats.

Kidney, testis and ovary weights for treated rats were similar to those of the control s .

MACROSCOPIC PATHOLOGY (Table 6, Appendix 6)

Macroscopic pathology revealed enlargement of the liver in two male and two female rats receiving ethyl methoxy norbornane, 800 mg/kg/day. Pallor of the kidneys was also noted for two male rats in this high dosage group.

Unilateral/bilateral hydronephrosis of the kidneys and distension of the ureters were recorded amongst female rats receiving ethyl methoxy norbornane, 800 mg/kg/day.

The incidence of all other findings listed in Table 6 was considered to fall within the background range of macroscopic changes.

MICROSCOPIC PATHOLOGY (Table 7, Appendix 6)

Histopathological findings for individual animals are described, in detail, in Appendix 6 and all lesions are presented in Table 7. The following comments were made in summary:

Treatment-related changes were found in the kidneys and liver .

These are described below:

Kidneys

Eosinophilic inclusions were seen in the cortical tubular epithelium in all male rats receiving ethyl methoxy norbornane 800 or 55 mg/kg/day and in 4 of the 5 male rats receivinq ethyl methoxy norbornane, 5 mg/kg/day. The deqree of this change appeared dosage-related.

Cytoplasmic rarefaction of periportal hepatocytes was found in 4 male and 2 female rats receivinq ethyl methoxy norbornane, 800 mg/kg/day.

This change was not seen in rats receiving 55 or 5 mg/kg/day nor in rats from the control group.

In addition to the above treatment-related changes, renal pelvic dilatation was found in 3 female rats receiving ethyl methoxy norbornane, 800 mg/kg/day. This change was also found in one female rat from each of the groups receiving 55 or 5 mg/kg/day, one male receiving ethyl methoxy norbornane, 55 mg/kg/day and one female from the control group. Two of the 3 female rats with renal pelvic dilatation from the group receiving ethyl methoxy norbornane, 800 mg/kg/day also showed an associated dilatation of the ureters.

Renal pelvic dilatation is not uncommon amongst rats of this strain but the significance of the increase in incidence and degree of this finding in female rats receiving ethyl methoxy norbornane, 800 mg/kg/day, when compared with rats from the control group is not clear.

All other histopathological changes were considered incidental in origin and of no toxicological significance.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

A repeated dose toxicity test was performed according to OECD TG 407. A NOAEL of 55 mg/kg bw/day (actual dose received) (male/female) was derived. At 800 mg/kg/day, several changes were noted in the parameters and tissues examined that were considered to be treatment-related. Though the effects were generally minor and the toxicological relevance remains somewhat unclear.
 

Executive summary:

A repeated dose toxicity test was performed according to OECD TG 407.

The test material was administered by gavage to three groups, each of five male and five female Crl,CD (SD) BR strain, for twenty-eight consecutive days, at dose levels of 5, 55 and 800 mg/kg/day. For rats receiving doses of Neoproxen at 800 mg/kg/day, several changes were noted in the parameters and tissues examined that were considered to be treatment-related. The WBC counts were increased 30 and 60% for males and females, respectively at 800 mg/kg bw. Some kidney related indicators such as higher urea nitrogen (ca 20% males only) and increase in potassium levels (< 10% males only). An increase in cholesterol (20-30% in females only) was seen. Other biochemical changes that were seen are increases in phosphate (ca 10% in females only) and Ca levels (<5%).

The kidney showed some eosinophilic inclusions in the corticular tubular epithelium of males only with increased dosage, which is a common finding in the male rat and related to alpha hydrocarbon nephropathy.

At 800 mg/kg bw macroscopically unilateral/bilateral hydronephrosis of the kidneys and distension of the ureters in females was seen. Microscopically some renal dilatation in the females is seen 3 vs 1/5), which are not uncommon in this strain of rats. The toxicological relevance of these macro –and microscopic findings remain unclear. and have therefore been taken into account in deriving the NOAEL.

In addition, the liver was affected at showing increase in weight (20-30% both males and females) and it was enlarged. Cytoplasmic rarefaction of periportal hepatocytes were seen in 4 males and 2 females at this dose of 800 mg/kg bw. Though these liver changes may be due to adaptation these effects may be considered in deriving the NOAEL in a 28-day test because the toxicological relevance may not be fully clear at this time of exposure. A NOAEL of 55 mg/kg bw/day (actual dose received) (male/female) was derived.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

55 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1. Test has been carried out according to OECD TG 407 and according to GLP.

Additional information

A repeated dose toxicity test was performed according to OECD TG 407.

The test material was administered by gavage to three groups, each of five male and five female rats Crl,CD (SD) BR strain, for twenty-eight consecutive days, at dose levels of 5, 55 and 800 mg/kg/day. For rats receiving doses of Neoproxen at 800 mg/kg/day, several changes were noted in the parameters and tissues examined that were considered to be treatment-related. The WBC counts were increased 30 and 60% for males and females, respectively at 800 mg/kg bw. Some changes in kidney related indicators such as higher urea nitrogen (ca 20% males only) and increase in potassium levels (< 10% males only) were noted. An increase in cholesterol (20-30% in females only) was seen. Other biochemical changes that were seen are increases in phosphate (ca 10% in females only) and Ca levels (<5%).

The kidney showed some eosinophilic inclusions in the corticular tubular epithelium of males only with increased dosage, which is a common finding in the male rat and related to alpha hydrocarbon nephropathy.

At 800 mg/kg bw macroscopically unilateral/bilateral hydronephrosis of the kidneys and distension of the ureters in females was seen. Microscopically some renal dilatation in the females is seen (3 vs 1/5), which are not uncommon in this strain of rats. The toxicological relevance of these macro –and microscopic findings remain unclear. and have therefore been taken into account in deriving the NOAEL.

In addition, the liver was affected at showing increase in weight (20-30% both males and females) and it was enlarged. Cytoplasmic rarefaction of periportal hepatocytes were seen in 4 males and 2 females at this dose of 800 mg/kg bw. Though these liver changes may be due to adaptation these effects may be considered in deriving the NOAEL in a 28-day test because the toxicological relevance may not be fully clear at this time of exposure. A NOAEL of 55 mg/kg bw/day (actual dose received) (male/female) was derived.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only this test is available.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Based upon the results obtained in the OECD 407 study Neoproxen does not need to be classified for repeated dose toxicity under DPD 67/548/EC and the CLP Regulation (EC) No 1272/2008.