complex reaction mass of Chinese gum rosin post reacted with acrylic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-230-1

EC Name:

-

Cas Number:

144413-22-9

Molecular formula:

Not applicable - the substance is an UVCB

IUPAC Name:

1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,4b,5,6,10,10a-decahydrophenanthrene-1-carboxylic acid; prop-2-enoic acid

Test material form:

other: pale yellow blocks

Details on test material:

- Chemical name: Rosin acid reaction products with acrylic acid hydrogenated

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
A sufficient number of male and female Sprague-Dawley CrI :CD®BR strain rats were obtained from CharIes River (UK) limited, Margate, Kent. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for eleven days during which time their health status was assessed. A total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed 179 to 214g, and the females weighed 146 to 175g, and were five to seven weeks old.
The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food and water. A pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK) was used.
Mains water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were randomly allocated to treatment groups using random letter tables, and the group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

ENVIRONMENTAL CONDITIONS
The animals were housed in air-conditioned rooms within the Safepharm Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and printouts of hourly mean temperatures and humidities were included in the study records. The temperature and relative humidity were controlled to remain within target ranges of 21 ± 2°C and 55 ± 15% respectively. Occasional deviations from these targets were considered not to have affected the purpose or integrity of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg bw/day of Arachis oil BP. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of each test material formulation. The concentration of ARAKAWA KE-604 in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The results indicate that the prepared formulations were within ±8% of the nominal concentration.

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

FUNCTIONAL OBSERVATIONS:
- Behavioural Assessments (days 1, 8, 15, 22)
- Functional Performance Tests (Motor Activity, Forelimb/Hindlimb Grip Strength) (day 22)
- Sensory Reactivity (day 22)

BODYWEIGHT:
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION:
Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

LABORATORY INVESTIGATIONS
Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

Sacrifice and pathology:

On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone (Sagatal, 60 mg/mL; May and Baker Limited, Dagenham, Essex, UK) followed by exsanguination.
All animals were subjected to a full external and internal examination, and ar macroscopic abnormalities were recorded.
The following organs removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus.

Other examinations:

HISTOPATHOLOGY:

Adrenals*, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum)*, Brain (including cerebrum, cerebellum and pons)*, Caecum*, Colon*, Duodenum*, Epididymides*, Eyes, Gross lesions*, Heart*, Ileum*, Jejunum*, Kidneys*, Liver*, Lungs (with bronchi)*, Lymph nodes (cervical and mesenteric)*, MuseIe (skeletal), Oesophagus, Ovaries*, Pancreas, Pituitary, Prostate*, Rectum*, Salivary glands (submaxillary), Sciatic nerve*, Seminal vesicles*, Skin (hind limb), Spinal cord (cervical)*, Spleen*, Stomach*, Testes*, Thymus*, Thyroid/parathyroid*, Trachea*, Urinary bladder*, Uterus*.

All tissues were despatched to Finn International, Norfolk, UK for processing. The tissues shown with "*" from all control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 pm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 15 and 150 mg/kg bw/day dose group animals. Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE 84 pathology computerisation system for tabulation and report production.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous pairwise comparisons were conducted using Dunnetts's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" test.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.
Probability values (p) are presented as follows:
p < 0.001 ***
P < 0.01 **
p < 0.05 *
P >= 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Details see "Any other information on results"

Mortality:

mortality observed, treatment-related

Description (incidence):

Details see "Any other information on results"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Details see "Any other information on results"

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Details see "Any other information on results"

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Details see "Any other information on results"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Details see "Any other information on results"

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Details see "Any other information on results"

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Details see "Any other information on results"

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Clinically observable signs of toxicity were detected among animals of either sex treated with 1000 mg/kg bw/day, which were most evident during the second half of the treatment period and more severe in females. This deterioration in physical condition was confirmed by the detailed open-field behavioural assessments, which identified similar albeit fewer changes during weeks 2, 3 and 4 of the study. Males from this treatment group also showed a reduction in bodyweight gain during the final week of treatment.
Treatment with the test material also produced minor hepatic changes with an increase in absolute and relative liver weight detected in females treated with 1000 mg/kg bw/day at terminal kill. Histopathological examinations did not reveal any evidence of microscopic liver changes although one male showed pallor of the liver at necropsy.
Blood chemical determinations revealed an increase in plasma alkaline phosphatase (AP) concentration among males from this treatment group although given that AP is such a ubiquitous enzyme a direct association with the minor liver changes could not be confirmed.
Animals of either sex treated with 150 or 15 mg/kg bw/day showed no observable clinical signs of toxicity throughout the study.

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

MORTALITY

There were no deaths during the study.

CLINICAL OBSERVATIONS

Animals of either sex treated with 1000 mg/kg bw/day developed clinical signs from Day 3 onwards including hunched posture, noisy respiration, increased salivation around the time of dosing and up to one hour after dosing and red/brown staining and wetting of the external body surface; isolated incidents of pilo-erection, gasping respiration and vocalisation were also apparent in two females from this treatment group. Observations were mostly confined to the second half of the treatment period and were slightly more evident among females.

An isolated incident of increased salivation approximately two minutes after dosing was detected in one male treated with 150 mg/kg bw/day on Day 4 of the study. lncreased salivation around the time of dosing is often reported following the oral administration of a test material formulation and is usually attributed to an unpalatable or locally irritant test material rather than an indication of test material toxicity.

There were no clinically observable signs detected among females treated with 150 mg/kg bw/day or animals of either sex from the remaining treatment group.

BEHAVIOURAL ASSESSMENTS

Detailed open-field observations did not detect any behavioural changes that could be convincingly attributed to test material neurotoxicity. However, behavioural assessments confirmed the signs detected clinically among 1000 mg/kg bw/day animals of either sex with observations of hunched posture, laboured and noisy respiration and increased salivation noted during Weeks 2, 3 and 4 of the study.

All remaining intergroup differences in behavioural scores were considered to be the result of normal variation among rats of the strain and age employed in the study and were considered to be of no toxicological significance.

FUNCTIONAL PERFORMANCE TESTS

There were no treatment-related changes in functional performance parameters measured that could convincingly be attributed to test material toxicity. Females treated with 1000 mg/kg bw/day showed a slight but statistically significant increase in grip strength; however, increased grip strength is unlikely to indicate a toxicologically important change.

SENSORY REACTIVITY ASSESSMENTS

Sensory reactivity assessment did not reveal any treatment-related changes. All inter and intra group differences in sensory scores were considered to be normal variation for rats of the species and strain used and, therefore, was considered to be of no toxicological significance. Statistical analysis of startle reflex data did not reveal any significant intergroup differences.

BODYWEIGHT

Males treated with 1000 mg/kg bw/day showed a statisticatly significant reduction in bodyweight gain during the final week of treatment compared with controls. Females treated with 1000 mg/kg bw/day and animals of either sex from the remaining treatment groups showed a similar bodyweight gain to controls throughout the study.

FOOD CONSUMPTION

There was no adverse effect on dietary intake during the study. Treated animals showed a similar dietary intake to controls throughout the study. Food efficiency (the ratio of bodyweight gain to dietary intake) was slightly reduced for males treated with 1000 mg/kg bw/day during the final week of treatment.

WATER CONSUMPTION

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

HAEMATOLOGY

There were no treatment-related changes detected in the haematological parameters measured.

Females treated with 1000 mg/kg bw/day showed slight but statistically significant reductions in haemoglobin and haematocrit levels when compared with controls. Examination of the individual data revealed unusually low individual values in one 1000 mg/kg bw/day female but in the absence of any histopathological correlates or effects on other haematology parameters, these minimal intergroup differences (p<0.05) were considered to be of no toxicological significance.

BLOOD CHEMISTRY

Males treated with 1000 mg/kg bw/day showed statistically significant increases in plasma total protein and alkaline phosphatase concentration when compared with controls. Several of the individual values for either parameter were outside the normally expected respective ranges for rats of the strain and age used. Females treated with 1000 mg/kg bw/day and animals of either sex from the remaining treatment groups showed no treatment related changes in the blood chemical parameters measured.

Males treated with 15 mg/kg bw/day showed a statistically significant reduction in plasma total protein concentration when compared with controls. In the absence, however, of a similar change at the 1000 and 150 mg/kg bw/day dose levels, a dose relationship could not be established and accordingly this intergroup difference was disregarded.

Males treated with 1000 mg/kg bw/day showed a statistically significant increase in plasma creatinine concentration but in the absence of a concomitant change in plasma urea concentration or any supporting histopathological evidence of renal dysfunction, this was considered to be of no toxicological importance.

Males from this treatment group also showed a slight but statistically significant reduction in plasma chloride concentration when compared with controls. The level of significance achieved was minimal (p<0.05) and in the absence of any imbalances in other plasma electrolytes, this isolated intergroup difference was considered to be of no toxicological significance.

Females treated with 1000 mg/kg bw/day showed slight but statistically significant increases in plasma phosphorus and bilirubin concentration when compared with controls. All of the values were with in the normal expected respective ranges for rats of the strain and age used and in the absence of any toxicological correlates, these minimal intergroup differences (p<0.05) were considered to be of no toxicological importance.

ORGAN WEIGHTS

Females treated with 1000 mg/kg bw/day showed a statistically significant increase in liver weight, both absolute and relative to terminal bodyweight, when compared with controls.

Males treated with 1000 mg/kg bw/day and animals of either sex from the remaining treatment groups showed no treatment-related changes in the organ weights measured.

Females treated with 1000 mg/kg bw/day also showed a statistically significant reduction in absolute brain weight whilst males at this dose level showed reductions in absolute epididymide and spleen weight when compared with controls. There were, however, no concomitant reductions in the weights of these organs when expressed relative to terminal bodyweight and accordingly these intergroup differences were considered to be of no toxicological significance.

Females treated with 1000 mg/kg bw/day also showed a reduction in relative thymus weight when compared with controls but in the absence of

any histopathological correlates, this minimal intergroup difference (p<0.05) was considered to be of no toxicological significance.

NECROPSY

One male treated with 1000 mg/kg bw/day showed pallor of the liver at terminal kill.

There were no treatment-related macroscopic findings detected at necropsy among females treated with 1000 mg/kg bw/day or animals of either sex at the remaining dose levels.

The sole remaining finding was identified as dark areas on the lungs of one female treated with 15 mg/kg bw/day but since this finding showed no

dose-relationship it was considered to be of no toxicological significance.

HISTOPATHOLOGY

No treatment-related changes were observed.

- Spleen: A comparison of severity grades for extramedullary haemopoiesis between control and treated male rats would tend to suggest an effect of the test material by way of a reduced severity of the condition in relation to treatment. However, the severity of the condition amongst controI male rats was generally greater than might normally be expected for untreated rats maintained at this facility for the experimental period. Any indications of a group response to treatment must, therefore, be regarded as spurious.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Applicant's summary and conclusion

Conclusions:

Repeated oral administration of the test material, Arakawa KE-604, to rats by gavage for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day resulted in treatment-related changes at a dose level of 1000 mg/kg/day. There were no treatment-related changes detected among animals of either sex treated with 150 or 15 mg/kg/day. The "No Observed Effect Level" (NOEL) was therefore considered to be 150 mg/kg/day.

Executive summary:

The study was designed to investigate the systemic toxicity of the test material. It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (Adopted 27 July 1995).

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD®BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from 1000 mg/kg/day and control animals was performed.

Mortality

There were no deaths detected during the study.

Clinical observations

Clinical signs developed among animals of either sex treated with 1000 mg/kg bw/day from Day 3 onwards and included hunched posture, noisy respiration, increased salivation around the time of dosing and up to one hour after dosing and red/brown staining and wetting of the external body surface; isolated incidents of pilo-erection, gasping respiration and vocalisation were also apparent in two females fom this treatment group. There were no clinically observable signs of toxicity detected among animals of either sex treated with 150 or 15 mg/kg bw/day.

Behavioural Assessment

Detailed behavioural assessment confirmed the signs detected clinically among 1000 mg/kg bw/day animals of either sex with observations of hunched posture, laboured and noisy respiration and increased salivation noted during Weeks 2, 3 and 4 of the study.

Open-field observation did not reveal any treatment-related behavioural changes among animals of either sex treated with 150 or 15 mg/kg bw/day.

Functional Performance Tests

There were no treatment-related changes in functional performance that could convincingly be attributed to test material toxicity.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity detected during the study.

Bodyweight

Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in bodyweight gain during the final week of treatment when compared with controls.

Females treated with 1000 mg/kg bw/day and animals of either sex from the remaining treatment groups showed a similar bodyweight development to controls troughout the study.

Food consumption

There was no adverse effect on food consumption detected during the study.

Water consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differencees.

Haematology

There were no treatment-related changes detected in the haematological parameters measured.

Blood Chemistry

Males treated with 1000 mg/kg bw/day showed a statistically significant increase in plasma total protein and alkaline phosphatase concentration when compared with controls.

Females treated with 1000 mg/kg bw/day and animals of either sex at the remaining dose levels showed no treatment-related changes in the blood chemical parameters measured.

Organ Weights

Females treated with 1000 mg/kg bw/day showed a statistically significant increase in liver weight, both absolute and relative to terminal bodyweight, when compared with controls.

Males treated with 1000 mg/kg bw/day and animals of either sex at the remaining dose levels showed no treatment-related changes in the organ weights measured.

Necropsy

One male treated with 1000 mg/kg bw/day showed pallor of the liver at terminal kill.

There were no other toxicologically significant macrosopic findings detected at necropsy.

Histopathology

There were no treatment-related microscopic changes detected.