1-Propanol, 2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-

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:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

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:

-

EC Number:

416-210-4

EC Name:

-

Cas Number:

128119-70-0

Molecular formula:

C14H26O2

IUPAC Name:

2-methyl-3-({1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}oxy)propan-1-ol

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- A total of 23 male and 23 female healthy CD rats of Sprague-Dawley origin (Crl:CD ® B VAF PLUS(TM)) were obtained from Charles River (UK) Ltd, Margate, Kent, England arriving on 12 January 1994.
- The rats were 28 ± 1 days old, in a weight range of 70 - 90 g on arrival. An eight-day acclimatisation period was allowed between delivery of the animals and start of treatment.
- All rats were initially caged, as far as possible, in groups of five according to sex in metal cages with wire mesh floors.
- A standard pelleted laboratory rodent diet (Special Diets Services, Rat and Mouse Maintenance Diet) and drinking water were provided ad libitum, except as noted. The batches of diet used for the study had been analysed for nutrients, possible contaminants and micro-organisms
- Animal room temperature was controlled in the range 19.0 - 22.0°C and relative humidity was controlled in the region of 37 to 57% RH. These parameters were continuously monitored using a Kent Clearspan MI05 7-day chart recorder. Air exchange was maintained at approximately 19 air changes per hour and lighting was controlled to provide 12 hours artificial light (0700 - 1900 hours) in each 24-hour period.
- The health status of all animals was monitored, by daily observation throughout the acclimatisation period, to ensure that the rats selected for final assignment to the study were satisfactory.
- Two days prior to the start of treatment each animal was weighed and forty rats were randomly allocated to four groups, each consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised. Following the commencement of treatment spare animals were removed from the study. No further investigations were performed on these animals.
- The cages (each containing five rats) constituting each group were distributed in batteries in such a manner that possible environmental influences arising from their spatial distribution was equilibrated, as far as possible, for all treatments.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- The test substance was administered by oral gavage to rats of Groups 2 to 4 inclusive, using a syringe and rubber catheter at a dose volume of 5 ml/kg .
- Control animals received corn oil by oral gavage at the same dose volume.
- Each animal received a constant dosage based on its most recently recorded bodyweight.
- Prior to dosing, the test substance formulations for Groups 2 to 4 were mixed by inversion (x20) .

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chemical stability of test substance formulations was assessed prior to the start of treatment by the Department of Analytical Chemistry at Huntingdon Research Centre.

Duration of treatment / exposure:

Twenty-eight consecutive days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

150 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:

5 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The high dosage was selected on the basis of available toxicity data and a preliminary oral toxicity investigation performed at this laboratory (HRC Schedule No . IFFI 1 47). Other levels were selected on the basis of the key dosages relative to EEC labelling requirements.

Examinations

Observations and examinations performed and frequency:

Mortality: The rat killed for humane reasons was subjected to detailed macroscopic examination in an attempt to
define the cause of death .

Clinical signs: All animals were observed four times on Day one and three times per day subsequently for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded. All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. This was to allow a post mortem examination to be undertaken during the working part of that day. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately mid-day. Any animal showing signs of severe debility or toxicosis was sacrificed for reasons of animal welfare.

Body weight: All rats were weighed prior to dosing on Day 1 (Week 0) and subsequently at weekly intervals throughout the study.

Food consumption: The quantity of food consumed in each cage was measured at weekly intervals throughout the study .

Water consumption: Daily monitoring by visual appraisal was maintained throughout the dosing period. Gravimetric measurement of water consumption was initiated during Week 3 due to a suspected treatment-related effect.

Clinical pathology: Food was withdrawn overnight prior to collection of samples. Blood was withdrawn under light ether anaesthesia from the orbital sinus of all rats prior to termination (Week 4).
- Haematology: The following parameters were analysed with an Ortho ELT - 1 500 analyser, using standard Ortho methodology: Packed cell volume, Haemoglobin, Red blood cell count, Absolute indices: Mean corpuscular haemoglobin concentration (Calculated: Hb (g/dl) x 100 / PCV (%)), Mean corpuscular volume: (Calculated : PCV (%) x 10 / RBC (1E6/mm3)), Platelet count, Total white blood cell count. The following estimations were measured using the appropriate methodology: Thrombotest (Method of Owren, P . A . (Lancet, 1959 , ii, 754)), Differential white blood cell count - namely: Neutrophils, Lymphocytes, Eosinophils, Basophils, and Monocytes. The percentage distribution of each cell type was determined by standard microscopy of a blood smear stained with modified Wright's stain counting 100 cells . Percentage values were then converted to absolute values by computer, inevitably involving a "rounding off" in a proportion of the results . Hence, the measured total WBC may differ slightly from the calculated total for the differential count. Additionally, blood film slides were examined for morphological abnormalities. If abnormal cells were observed when examining the stained slides, their presence were recorded and included in the haematology appendix. No abnormalities were seen that were considered to be treatment-related.
-Biochemistry: The following parameters were analysed with a Hitachi 737 Clinical Chemistry analyser: Glucose, Total protein, Albumin, Globulin, Albumin/Globulin ratio, Urea nitrogen, Creatinine, Alkaline phosphatase (AP), Glutamic-pyruvic transaminase (Alanine aminotransferase), Glutamic-oxaloacetic transaminase (aspartate aminotransferase), Total bilirubin, Sodium, Potassium, Calcium, Chloride, Inorganic phosphorus, Cholesterol

Sacrifice and pathology:

Termination: After 28 days of treatment (Day 29 ) all animals surviving treatment were killed by carbon dioxide asphyxiation and a complete autopsy undertaken. The order of sacrifice was determined using pre-set cage sequence. Specified organs were weighed and relevant tissue samples were fixed for microscopic examination.

Organ weight: The following organs from each animal were dissected free of fat and weighed: adrenals, brain, kidneys, liver, ovaries, spleen, and testes (with epididymides)

Macroscopic pathology: The macroscopic appearance of the tissues of all rats was recorded and samples of the following tissues were preserved in 10 % buffered formalin: adrenals*, aorta, brain (medullary, cerebellar and cortical sections), caecum, colon, duodenum, eyes (Davidson's fluid as fixative), femur (with joint), head, heart*, ileum, jejunum, kidneys*, larynx, liver*, lungs, lymph nodes (cervical and mesenteric sections), mammary glands, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spleen*, sternum (for bone and marrow sections), stomach, testes including epididymis * (Bouin's as fixative), thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus (with cervix), vagina, any macroscopically abnormal tissue*
* Tissues required for histopathological examination for rats from Groups 1 and 4

Microscopic pathology: Fixed tissue samples required for microscopic examination w ere prepared by embedding in paraffin wax (mp 56°C); sections were cut at 4 µm (with the exception of testes which were cut at 2 µm) and stained with haematoxylin and eosin. Microscopic examination of prepared slides (from tissues indicated under Macroscopic pathology) was carried out for all animals that died during the study, in an attempt to ascertain the cause of death and all rats of Group 1 (Control group ) and Group 4 (High dosage group) killed on Day 29. Examinations were extended, following documented approval from the Sponsor, to include the liver (males and females) and kidneys (males only) for rats of the intermediate and low dosage groups.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following sequence of statistical tests was used for bodyweight gains, organ weight and clinical pathology data and selected tissues examined microscopically:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75 %) , the proportion of values different from the mode was analysed by Fisher's exact
test followed by Mantel 's test for a trend in proportions. Otherwise:
- Bartlett's test was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1 % level , a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
- If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out followed by Williams' test for a dose-related response.
- If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wall is analysis of ranks was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test).
Covariate analysis of organ weight data (with final body weight as covariate) was also performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10% level of significance.
Significant differences between control animals and those treated with the test substance were expressed at the 5 % (* P < 0.05) or 1 % (** P < 0.01 ) level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

At 1000 mg/kg/day, paddling of the forepaws was seen in one male and one female rat on a few occasions during the study. Lethargy was seen in all male rats at this dosage on Day 10 accompanied by cage grid biting in two rats. These signs were transient and, at the incidence and severity seen in this study, were considered to be a result of discomfort following the dosing procedure.
One female rat receiving 1000 mg/kg/day was of thin appearance from Day 22 to termination. All the female rats receiving 1000 mg/kg/day were noted to be of thin appearance on Day 27 (and also 28 and 29. The thin appearance of these rats at this time was probably attributable to the overnight starvation prior to the bleed on Day 27.
Red/brown staining around the urogenital region was recorded on Day 21 for two female rats and on Days 23 and 24 for receiving 1000 mg/kg/day. At the low incidence seen in this study this sign was considered to be incidental and not of toxicological importance.
Fur loss was noted during the study for most rats receiving 1000 mg/kg/day, however, in the absence of any histopathological findings this was considered to be caused by the rats fighting and not an effect of treatment.
Increased salivation after dosing and associated wet fur was seen on the majority of occasions throughout the study in rats dosed at 1000 mg/kg/day. This sign was occasionally accompanied by red/brown perioral staining. An increase in salivation following dosing is commonly observed in orally dosed rat studies and is considered to be a result of the unpalatability of the test substance and is, therefore, not of toxicological importance. The greasy fur seen from Day 6 to termination for rats dosed at 1000 mg/kg/day was linked to administration of the vehicle, corn oil. In rats receiving 150 mg/kg/day, greasy fur was seen from Day 23 to termination with occasional incidences of increased salivation in a few rats. At 15 mg/kg/day and in the control group, greasy fur was seen from Day 23 to termination.

Additional information on clinical signs can be found in Appendix 5 of the attached study report. (Page 141 to 189)

Mortality:

no mortality observed

Description (incidence):

One rat receiving 150 mg/kg/day, was sacrificed on humane grounds on Day 2. Clinical signs prior to sacrifice, were shallow and noisy respiration, lethargy and cold to the touch. A post mortem revealed a perforated oesophagus and thoracic cavity that contained coagulum and ingesta. Death was considered to be a result of an intubation error. All other findings for this animal were considered to be incidental. There were no further mortalities during the study.

Additional information on mortality can be found in Appendix 5 of the attached study report. (Page 141 to 189)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no differences from control in bodyweights or bodyweight gains that were considered to be related to treatment.
- In males, body weight was 12, 15 and 1% higher at 15, 150, and 1000 mg/kg bw, respectively.
- In females, body weight was 6, 6, and 12% lower at 15, 150, and 1000 mg/kg bw, respectively.

Additional information on body weights can be found in Table 1 (Page 122) of the attached study report.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences from control in food consumption that were considered to be treatment-related. Higher than control food consumption was noted in male rats receiving 150 mg/kg/day. This is reflected in the higher than control bodyweights for these animals.

Additional information on food consumprion can be found in Table 2 (Page 123) of the attached study report.

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption (as measured during Week 3) was markedly higher tin male and female rats
- In males, water consumption was 4, 17 and 31% higher at 15, 150, and 1000 mg/kg bw, respectively.
- In females, water consumption was 7, 31, and 221% higher at 15, 150, and 1000 mg/kg bw, respectively.

Additional information on water consumption can be found in Table 3 (Page 124) of the attached study report.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no differences from control that were considered to be related to treatment. The lymphocyte count (and hence total white blood cell count) was statistically significantly lower than control for male rats treated at 1000 mg/kg/day.
- Total white blood count was 13 and 30% lower at 150, and 1000 mg/kg bw, respectively.
- Lymphocyte count was 16 and 30% lower at 150, and 1000 mg/kg bw, respectively.
There was considerable individual variation and overlap of values between the groups and this difference from control was considered to have arisen by chance.

Additional information on haematology can be found in Table 4 (Page 125) of the attached study report.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Lower glucose levels (49%) and higher albumin (7%) and globulin (15%) and hence total protein (13%) levels achieved statistical significance in comparison with controls for male rats receiving 1000 mg/kg/day.
The remaining statistically significant differences from control for rats receiving 1000 mg/kg/day: 1.4% higher sodium (males), 5.5 and 1.9% higher calcium (males and females, respectively), 2.0 and 3% lower chlorine (males and females, respectively), 16% lower glutamic oxaloacetic transaminase (females) and 100% higher bilirubin levels (females), were all within the expected ranges for rats of this age and strain and not considered to be related to treatment.
There were no statistically significant differences from control in rats dosed at 150 or 15 mg/kg/day.

Additional information on biochemistry can be found in Table 5 (Page 126) of the attached study report.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly higher than control liver weights (bodyweight adjusted) were recorded for male (33%) and female (66%) rats treated at 1000 mg/kg/day. There were no other differences from control in the organ weights measured.

Additional information on organ weight can be found in Table 6 (Page 127 and 128) of the attached study report.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed at termination revealed the following changes:
Skin: Fur loss in 3/5 male rats and 4/5 female rats treated at 1000 mg/kg/day Bornafix, compared to none in the control group and, in the absence of any histopathological change, was considered to be caused by fighting rather than an effect of treatment.
Liver: Enlargement in 3/5 male rats and 5/5 female rats treated at 1000 mg/kg/day, compared to none in the control group.
There were no other findings of note.

Additional information on gross pathology can be found in Table 7 (Page 129 and 130) of the attached study report.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings:
- Liver - Centrilobular and midzonal hepatocyte enlargement in all rats receiving 1000 mg/kg/day
- Kidneys - A dose-related incidence and degree of eosinophilic inclusions in proximal tubular epithelium in male rats from all dose levels.

Additional information on microscopic pathology can be found in Table 9 (Page 131 and 133) of the attached study report.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

The analytical results confirm that the doses were accurately formulated for Day 1 of the toxicity study. The results also confirm that specimen formulations were stable for a period representing the time from preparation to completion of dosing

Treatment-related findings:

	Control		15 mg/kg/day		150 mg/kg/day		1000 mg/kg/day
	Male	Female	Male	Female	Male	Female	Male	Female
Liver
Centrilobular and midzonal hepatocyte enlargement
Total	0	0	0	0	0	0	5**	5**
Minimal	0	0	0	0	0	0	5**	3
Moderate	0	0	0	0	0	0	0	2
Kidneys
Eosinophilic inclusions in proximal tubular epithelium
Total	0	0	2	0	5**	0	5**	0
Trace	0	0	2	0	1	0	0	0
Minimal	0	0	0	0	4*	0	0	0
Moderate	0	0	0	0		0	5**	0
Number of animals	5	5	5	5	5	5+	5	5

* P<0 . 05

** P<0 . 01

+ Includes one intercurrent death

 

DISCUSSION

There was a treatment-related effect in the liver of male and female rats from the high dosage group. Centrilobular or midzonal hepatocyte enlargement, with associated enlarged liver and increased liver weight was recorded. Associated minor biochemical changes (lower glucose, higher protein) were also recorded. This liver finding is and probably related to metabolism of the test substance. 

The microscopic finding in the kidneys of all treated groups of male rats, a dosage-related incidence of eosinophilic inclusions in proximal tubular epithelium, is characteristic of light  hydrocarbon nephropathy syndrome. This syndrome is specific to male rats and is a common finding with compounds of this nature. This finding, whilst treatment-related, is not considered predictive for a similar effect in man. 

Additional treatment-related findings for high dosage group rats were occasional signs indicative of discomfort following dosing and an increase in water consumption during Week 3. The higher water consumption, also seen for male and female rats treated at 150 mg/kg/day was thought to be related to the unpalatability of the test substance. Other evidence of unpalatability was seen (e.g. increased salivation following dosing), therefore the increased water consumption was not considered to be  toxicologically important.

For rats of the intermediate dosage group, 150 mg/kg/day, the only treatment-related findings were an increase in water consumption, which was not considered toxicologically important, and the light hydrocarbon nephropathy syndrome for male rats which is not considered to be relevant to man. In the low dosage groups, 15 mg/kg/day, the light hydrocarbon nephropathy syndrome in males was the only treatment-related finding.

Based on the effects observed in the liver, the NOAEL was determined to be 150 mg/kg/day.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD 407, GLP), the NOAEL was determined to be 150 mg/kg bw/day, based on severe increased liver weight.

Executive summary:

This study was performed to assess the systemic toxicity of Bornafix to the rat according to OECD TG 407 and following GLP. The substance was administered by oral gavage, once daily, to groups of five male and five female rats for 28 consecutive days at 15, 150 and 1000 mg/kg/day. The test material was prepared as solutions in corn oil at concentrations of 0.3, 3.0 and 20% w/v. A further group of rats (five males and five females ) was held as a concurrent control receiving the vehicle alone. Bodyweights, food consumption and clinical observations were recorded during the study. Blood samples were taken on Day 27 and all animals were killed and examined macroscopically on Day 29. Histopathological examination of the tissues was then initiated.

Results: Clinical signs: There were no treatment-related mortalities. Signs indicative of discomfort following dosing (occasional incidences of paddling of the forepaws and isolated occurrences of lethargy and cage grid biting) were noted among rats treated at 1000 mg/kg/day. Water consumption: Markedly higher than control for female rats treated at 1000 mg/kg/day. Water consumption for males at 1000 mg/kg/day and males and females treated at 150 mg/kg/day was also higher than for controls. Body weight: No toxicological relevant effects on body weight, body weight gain or food consumption were noted during dosing of the substance. Haematology: No toxicological relevant haematology effects were seen in males and females except a slight decrease in total white blood cells in the high dose males (30%) because of lower leucocytes. Biochemistry: Lower glucose levels and higher albumin and globulin (and hence total protein) levels achieved statistical significance in comparison with controls for male rats receiving 1000 mg/kg/day. Other statistically significant differences from control were all within the expected ranges for rats of this age and strain and not considered to be related to treatment. Organ weights: Statistically significantly higher than control relative liver weights were recorded for male (33%) and female (66%) rats treated at 1000 mg/kg/day. Macroscopic pathology: Liver enlargement was seen in most rats receiving 1000 mg/kg/day. Microscopic pathology: Centrilobular or midzonal hepatocyte enlargement in the liver of male and female rats receiving 1000 mg/kg/day. A dose-related degree and incidence of eosinophilic inclusions in proximal tubular epithelium in the kidneys of male rats from all dose groups, which were considered to related alpha-hydrocarbon nephropathy.

Discussion: There was a treatment-related effect in the liver of male and female rats from the high dosage group. Centrilobular or midzonal hepatocyte enlargement, with associated enlarged liver and increased liver weight was recorded. Associated minor biochemical changes (lower glucose, higher protein) were also recorded. This liver finding is and probably related to metabolism of the test substance. The microscopic finding in the kidneys of all treated groups of male rats, a dosage-related incidence of eosinophilic inclusions in proximal tubular epithelium, is characteristic of light  hydrocarbon nephropathy syndrome. This syndrome is specific to male rats and is a common finding with compounds of this nature. This finding, whilst treatment-related, is not considered predictive for a similar effect in man. Additional treatment-related findings for high dosage group rats were occasional signs indicative of discomfort following dosing and an increase in water consumption during Week 3. The higher water consumption, also seen for male and female rats treated at 150 mg/kg/day was thought to be related to the unpalatability of the test substance. Other evidence of unpalatability was seen (e.g. increased salivation following dosing), therefore the increased water consumption was not considered to be  toxicologically important. For rats of the intermediate dosage group, 150 mg/kg/day, the only treatment-related findings were an increase in water consumption, which was not considered toxicologically important, and the light hydrocarbon nephropathy syndrome for male rats which is not considered to be relevant to man. In the low dosage groups, 15 mg/kg/day, the light hydrocarbon nephropathy syndrome in males was the only treatment-related finding.

Conclusion: Based on the effects observed in the liver, the NOAEL was determined to be 150 mg/kg/day.