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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 2003 to 20 Sep 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
Jul 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The Japanese Ministry of Health and Welfare (MHW) Guidelines 1986 for a 28-day repeat dose oral toxicity study as required by the Japanese Chemical Substances Control Law 1973 of the Ministry of International Trade and Industry (METI)
Version / remarks:
1986
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000
Version / remarks:
Jul 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
449-360-4
EC Name:
-
Cas Number:
647828-16-8
Molecular formula:
C18H32O
IUPAC Name:
decahydro-2,2,6,6,7,8,8-heptamethyl-2H-Indeno[4,5-b] furan
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD) IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: 5-6 weeks
- Weight at study initiation: Male: 131 - 184 g and female: 127 - 156 g
- Housing: 5 animals of same sex/cage in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet: Certified pelleted diet. Ad libitum
- Water: Mains drinking water from polycarbonate bottles. Ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 May 2003 To: 20 Sep 2003

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test substance was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test substance formulations were determined. Results showed the formulations to be stable for at least 14 days.

DIET PREPARATION:
- Rate of preparation of diet: weekly
- Storage temperature of food: 4 °C in the dark

VEHICLE:
- Concentration in vehicle: 3.75, 37.5 and 250 mg/mL
- Amount of vehicle: 4 mL/kg. The volume of test and control material administered to each animal was based on the most recent body weight 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 substance formulation and were analysed for concentration of test substance. The concentration of the test substance in the formulations was determined by gas chromatography (GC) using an external standard technique. The results indicate that the prepared formulations were within ± 10 % of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
Intermediate dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose group and Recovery high dose group
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of the 14-day range finding study.
- Rationale for animal assignment: Animals were randomly allocated to treatment groups using random letter tables. Group mean body weights were determined to ensure similarity between the treatment groups.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
Not included.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: Immediately before dosing and 5 hours (on working days) or 1 hour (in weekends) after dosing during the working week. Animals were observed immediately before dosing and 1-hour after dosing at weekends. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends).
- Cage side observations checked: Overt signs of toxicity, ill-health or behavioural change.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Prior to start of treatment and on days 7, 13, 21 and 26, and also during week 4. Observations were carried out approximately 2 hours after dosing on each occasion.
- Signs of functional/behavioural toxicity were assessed through the following parameters: Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

BODY WEIGHT:
- Time schedule for examinations: Day 0 (before start of treatment) and on days 7, 14, 21 and 28 and, in the case of recovery group animals, on days 35 and 42. Weight was also determined after termination.

FOOD CONSUMPTION:
- At weekly intervals per cage group.

FOOD EFFICIENCY:
- Food efficiency was determined.

WATER CONSUMPTION:
- Time schedule for examinations: Daily per cage group.

HAEMATOLOGY:
- Time schedule for collection of blood: Day 28 for normal treatment and control groups, day 42 for recovery groups.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All anmials
- Parameters checked: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), Total leucocyte count (WBC), neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Platelet count (PLT), Reticulocyte count (Retic). Cresyl blue stained slides were prepared but reticulocytes were not assessed. Prothrombin time (CT) was assessed by ‘Hepato Quick’ and Activated partial thromboplastin time (APTT) was assessed by ‘Preci Clot’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Day 28 for normal treatment and control groups, day 42 for recovery groups.
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Urea, Glucose, Total protein (Tot. Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (P), Gamma glutamyltranspeptidase (yGT), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Triglycerides (Tri), Total cholesterol (Chol), Total bilirubin (Bili).

URINALYSIS:
- Time schedule for collection of urine: During week 4 for normal treatment and control groups, during week 6 for recovery groups.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Volume, Specific Gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Reducing Substances, Blood.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Week 4
- Dose groups that were examined: All animals
- Battery of functions tested: Sensory activity (to auditory, visual and proprioceptive stimuli, parameters measured: grasp response, touch escape, vocalisation, pupil reflex, toe pinch, blink reflex, tail pinch, startle reflex, finger approach) / grip strength / motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY:
On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all surviving animals were killed by intravenous over-dose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY:
The following tissues were preserved: 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*, Oesophagus, Ovaries*, Pancreas, Pituitary, Prostate*, Rectum*, Salivary glands (submaxillary), Sciatic nerve*, Seminal vesicles*, Skin (hind limb), Spinal cord (cervical)*, Spleen*, Ileum*, Jejunum*, Kidneys*, Liver*, Lungs (with bronchi)*, Lymph nodes (cervical and mesenteric)*, Muscle (skeletal), Stomach*, Testes*, Thymus*, Thyroid/parathyroid*, Trachea*, Urinary bladder*, Uterus*. The tissues marked with * were stained (HE) and examined microscopically.

ORGAN WEIGHTS:
Weight was measured for the following tissues: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus.
Statistics:
- Group mean values and standard deviations were calculated where appropriate.
- Haematological, blood chemical, organ weight (absolute and relative to terminal body weight), weekly body weight gain and quantitative urinalytical 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 Dunnett's test.
- Recovery group data was analysed in a two-tailed t-test incorporating Levene's test for homogeneity of variance. Where Levene's test showed unequal variances among either non-recovery or recovery group data, the affected parameters were analysed using non-parametric methods: Kruskal-Wallis ANOVA and mann-Whitney 'U' test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Increased salivation in the high dose group (male/female). Episodes of increased salivation around the time of dosing was detected in either sex treated with 1000 mg/kg bw/day from day 14 onwards, regressing in recovery 1000 mg/kg bw/day animals following cessation of treatment. One female of the intermediate dose group showed increased salivation on day 23. This effect is regarded as a local effect due to e.g. local irritation or the taste of the substance and therefore not of toxicological relevance.
Incidental findings were confined to red/brown staining of the fur around the eyes observed during the final week of dosing in one 1000 mg/kg bw/day male. No other treatment-related effects were noted.

For more details see Table 1 (Page 52 – 57) and Table 2 (Page 58 – 63) of the attached study report.
Mortality:
no mortality observed
Description (incidence):
There were no deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased body weight gain in the high dose group (female). Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain in comparison with controls during week 2 and week 4 of the study. Females treated with 15 or 150 mg/kg bw/day also showed similar significant reductions during week 4. The changes in week 4 could be related to the decrease in food consumption in the same week. No other treatment-related effects were noted.

For more details see Table 9 (Page 80), Table 10 (Page 81), Table 11 (Page 82) and Table 12 (Page 83) of the attached study report.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant treatment-related effects were noted. A slight decrease in food consumption was noted in week 4 for all treated females. This could be caused by the taste of the test substance.

For more details see Table 13 (Page 84) and, Table 14 (Page 85) of the attached study report.
Food efficiency:
no effects observed
Description (incidence and severity):
No significant treatment-related effects were noted.

For more details see Table 15 (Page 86) and Table 16 (Page 87) of the attached study report.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No significant treatment-related effects were noted.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increased (1.7 fold) prothrombin time in the high dose group (male) was observed. At the mid- and high dose no increase was observed. Aetiology of this isolated finding is uncertain, but an effect on liver integrity cannot be discounted as prolonged clotting times may indicate an inhibitory effect on the synthesis of clotting factors of which the liver is the major site.

No other significant treatment-related effects were noted.

For more details see Table 17 (Page 88) and Table 18 (Page 89) of the attached study report.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cholesterol in males is 10, 24, and 31% increased at 15, 150 and 1000 mg/kg bw, respectively, reaching statisical significance at the mid high dose.

Cholesterol in females is 7, 40, and 60% increased at 15, 150 and 1000 mg/kg bw, respectively, reaching statisical significance at the mid and high dose. The statistically significant increase in plasma cholesterol levels in females treated with 1000 mg/kg bw/day is accompanied with a statistically significant 2.8-fold increase in gamma glutamyl transpeptidase. The toxicological significance of the increase at 150 mg/kg bw is dubious as histopathological evidence of obstructive liver damage is absent.

The remaining statistically significant intergroup differences detected were considered to be of no toxicological importance.

For more details see Table 19 (Page 90) and Table 20 (Page 91) of the attached study report.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant treatment-related effects were noted.

For more details see Table 21 (Page 92) and Table 22 (Page 93) of the attached study report.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No significant treatment-related effects were noted.

For more details see Table 3 (Page 64 – 68), Table 4 (Page 69 – 73), Table 5 (Page 74), Table 6 (Page 75), Table 7 (Page 76 - 77) and Table 8 (Page 78 – 79) of the attached study report.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver effects:
- Absolute males: Non-statistically significant 13, 23, and 35% increase at 15, 150 and 1000 mg/kg bw. In the recovery group, an increase of 13% was observed.
- Absolute females: -2, 22, and 40% increase at 15, 150 and 1000 mg/kg bw. Statistically significance was attained at the high dose. In the recovery group, a statistically significant increase of 12% was observed.
- Relative males: 1, 12, and 31% increase at 15, 150 and 1000 mg/kg bw. Statistically significance was attained at the high dose. In the recovery group, a statistically significant decrease of 12% was observed.
- Relative females: 4, 18, and 41% increase at 15, 150 and 1000 mg/kg bw. Statistically significance was attained at the mid and high dose. In the recovery group, a statistically significant increase of 16% was observed.
These liver effect are more often seen following treatment with xenobiotics and therefore, considered adaptive and of no toxicological significance.

For more details see Table 23 (Page 94), Table 24 (Page 95), Table 25 (Page 96) and Table 26 (Page 97) of the attached study report.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant treatment-related effects were noted.

For more details see Table 27 (Page 98) and Table 28 (Page 99) of the attached study report.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver: Centrilobular hepatocyte enlargement was observed in both sexes of the 1000 and 150 mg/kg bw/day treatment groups. Two male rats receiving 15 mg/kg bw/day of the test substance were also affected. Though this effect is commonly observed when animals are exposed to xenobiotics and possibly overloading the metabolic pathway this has been taken into account for deriving the NOAEL. This effects is recovering after 14 days without treatment.
Centrilobular hepatocyte enlargement
- Control: 0/5 in males and 0/5 in females
- Low: Minimal in 2/5 males and in 0/5 females
- Mid: Minimal in 5/5 males and in 3/5 females
- High: Slight in 5/5 males and Minimal in 5/5 females

Thyroid glands: Follicular cell hypertrophy was more prevalent and observed with higher grades of severity (slight to moderate) for rats of both sexes dosed at 1000 mg/kg bw/day. A similarly higher prevalence of the condition was observed for male rats dosed at 150 mg/kg bw/day but was not explicitly seen at the low dose level. There were no differences in incidence or grades of severity for follicular cell hypertrophy between recovery control and recovery 1000 mg/kg bw/day animals indicating complete regression of the condition at the end of the recovery period. The thyroid effects are considered to be due to increase of T4 metabolism (due to increase of liver metabolic activity) and therefore decrase of T4 in the systemic regulation which then causes a trigger for the thyroid to increase its activity to produce more T4.

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.

For more details see Table 29 (Page 100 – 101) and Table 30 (Page 102 - 104) of the attached study report.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight; haematology; clinical chemistry, liver pathology

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
presumably yes

Applicant's summary and conclusion

Conclusions:
The no observed adverse effect level (NOAEL) of the test substance was considered to be 15 mg/kg bw/day based on a sub-acute repeated dose oral toxicity study that was performed in accordance with OECD TG 407
Executive summary:

The key study was a 28-day repeated dose toxicity study according to OECD TG 407 and under GLP conditions that was performed to determine the effect of Amber Xtreme on rats. Doses of 1000, 150 and 15 mg/kg bw/day were administered by oral gavage. A satellite high dose group was allowed to recover for 14 days after treatment. The following observations were made during the treatment and recovery period: clinical signs, body weight, food and water consumption. Upon termination several haematology, clinical chemistry and urine parameters were checked for all animals. At necropsy, organs were weighed and prepared for histopathology.


No mortality was observed throughout the study. Episodes of increased salivation around the time of dosing were detected primarily in the high dose group, as well as some incidental findings like red/brown staining of the fur around the eyes. A statistically significant reduction in bodyweight gain was observed in the high dose group for females. In absence of effects on body weight this was not considered adverse. Furthermore, a statistically significant increase in prothrombin time was detected in males treated with 1000 mg/kg/day (+68%). A statistically significant increase in plasma cholesterol levels was observed in males and females treated with 1000 mg/kg/day: -31% and +60%, respectively. In females a 2.8x increase in gamma glutamyl transpeptidase was seen in the high dose. (Absolute and) relative liver weight increase statistically significant were observed for male and female animals of the high dose group (+31 and +41%, respectively) and in females of the mid-dose group (+18.5%). Slight centrilobular hepatocytes hypertrophy was seen in males at 1000 mg/kg bw (5/5) and in females in the mid and high dose group (3/5 and 5/5, respectively. Recovery was apparent after 14 days. Slight follicular cell hypertrophy was observed in the thyroid glands of both sexes of the high dose group 4/5 males and 5/5 females. No other treatment-related effects were noted.


The results of this study show that 28-day gavage treatment of rats with 1000 mg/kg bw/day Amber Xtreme showed increased prothrombine time in males at the high dose. There were alteration of clinical chemistry for cholesterol in both sexes and gamma GT in females. These effects were associated with increased relative liver weights accompanied with centrilobular liver hypertrophy at 1000 mg/kg bw. At the mid dose level of 150 mg/kg bw/day similar effects were noted but to a lesser extent. The No Observed Adverse Effect Level (NOAEL) was set at 15 mg/kg bw/day based on dose related finding in the liver resulting in increased liver weights starting in the mid dose with accompanying increased liver parameters and centrilobular hypertrophy.