Registration Dossier

Administrative data

Description of key information

Based on an OECD Guideline 407 and GLP, a NOEL (No Observed Effect Level) for the read-across substance, 4-methyl-4-phenylpentan-2-ol, of 100 mg/kg bw/day was identified. The findings at 400mg/kg/day, including short term transient neurological effects in a few animals immediately post dosing, could not be seen in a longer duration study at the same dose, and increases in relative kidney weights were not accompanied by evidence of kidney damage (biochemcial or histopathogical). That´s why these findings were considered to be incidental and a NOAEL (No Observed Adverse Effect Level) for the read-across substance, 4-methyl-4-phenylpentan-2-ol, of 400mg/kg bw/day can be identified.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-03-08 to 2004 -04-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
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
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Charles River Wiga GmBH D-97320 Sulzfeld, Germany
- Age at study initiation: 41-42 days
- Weight at study initiation: males 194.4 +/- 7.4g; females 178.2 +/- 9.9g
- Fasting period before study: none
- Housing: Makrolon Type 3 cages singly housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days for males ; 6 days for females

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 -22.0
- Humidity (%): 35-65%
- Air changes (per hr): not reported-air conditioned
- Photoperiod (hrs dark / hrs light): (12/12)

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Sixty rats (30 males and 30 females) of the Wistar Crl:WI BR strain were allocated to four groups. Groups 2 and 3 each comprised 5 males and 5 females and groups 1 and 4 each comprised 10 males and 10 females. At the end of the 28 day treatment period 5 males and 5 females of groups 1 and 4 (groups 1a and 4a) were maintained, undosed, for further 14 days.
Three groups received the original test item by oral gavage at dose levels of 100, 400 and 1000 mg/kg bw/day. A fourth group received water only, and served as control. Animals were dosed once daily, and treatment continued for 28 days.
Frequency of treatment:
Daily for 28 days
Remarks:
Doses / Concentrations:
0, 100, 400, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Control 10 females and 10 males ; low dose 5 females and 5 males ; mid dose 5 females and 5 males; high dose 10 feamles and 10 males
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): The dose levels had been selected alter a pre-experiment using doses of 1000 and 300 mg/kg bw/day in 5 male and 5 female rats each for a period of 12 days.
- Rationale for selecting satellite groups:part of OECD TG 407 protocol
- Post-exposure recovery period in satellite groups:14days
- Section schedule rationale (if not random):
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:detailed off-cage clinical examination was carried out daily before administration. A second clinical examination was carried out on-cage in a period within 2 hours after administration and a third clinical examination was carried out on-cage after 2 hours after administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule for observations : detailed off-cage clinical examination was carried out daily before administration. A second clinical examination was carried out on-cage in a period within 2 hours after administration and a third clinical examination was carried out on-cage after 2 hours after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day:
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data:

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice after overnight fasting
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- Parameters checked
Erythrocyte count (RBC),
Haemoglobin concentration (HB)
Packed cell volume (HCT)
Platelet count (PLT)
Total leucocyte count (WBC)
Leucocyte differential count
Prothrombin time (PT)
Fibrinogen concentration (FIB)
Determinations were carried out on blood collected in heparin anticoagulant

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:at sacrifice after overnight fasting
- Animals fasted: Yes
Alanine aminotransferase (ALT)
Alkaline phosphatase (AP)
Aspartate aminotransferase (AST)
Albumin (Alb)
Creatinine (Crea)
Glucose (Gluc)
Total cholesterol (Chol)
Total protein (Prot)
Urea
Potassium (Pot)
Sodium (Sod)
Determinations were carried out on serum on the day of blood sampling.
The parameters ALT, Chol and Sod were also determined in the male animals of the satellite
groups at the end of the recovery period and the parameters ALT and Chol were also
determined in the female animals of the satellite groups at the end of the recovery period

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY:
The animals were killed at the end of the treatment or recovery period, respectively, by CO2
asphyxiation. All animals were examined externally. The cranial, thoracic and abdominal cavities were opened and examined macroscopically.
Organ Weights:
The following organs of all animals were weighed after trimming off fat and other contiguous tissue:
adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spieen, testes and thymus.
From bilateral organs both were weighed and reported separately.

HISTOPATHOLOGY: Yes
Control and high dose group only. Whole organs or samples of the tissues of all animals listed below were fixed in 10 percent formaldehyde and preserved in 4 percent formaldehyde:
Adrenals Sciatic nerve, Bone marrow (from sternum), Small and large intestine (including Peyer's patches), Brain (3 sections) Spinal cord (from second trachelo-vertebra [Axis]), Epididymides, Spleen, Heart, Stomach, Kidneys, Testes, Liver, Thymus, Lungs, Thyroid, Lymph nodes (mesenteric and submandibular), Trachea, Urinary bladder, Ovaries, Uterus, Prostate.
All the listed organs from animals of the control and high dose group which were killed at the end of the administration period were examined microscopically alter staining with Hemalum-Eosin.
Paired organs were both examined

Statistics:
Group means and standard deviations were calculated for all numerical data. Sexes were analysed separately.
The statistical significance compared to the control was proved by means of the following different statistical methods. The statistical significance is
declared at the two-sided 5% level.
• Welch t-test:
- for all data of body weights, body weight gain and food consumption
- for the comparison of only two mean values at the satellite groups for the parameters of haematology (excluding differential leucocyte count), coagulation, clinical biochemistry and absolute and relative organ weights
• Dunnett test:
- for the comparison of the mean values of the three dose groups to control for the parameters of haematology (excluding differential leucocyte
count), coagulation, clinical biochemistry and absolute and relative organ weights
• Calculation of mean value, standard deviation and range:
- for the parameters of differential leucocyte count
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
None of the animals died during the course of investigation . A partly severe impairment of the general state of well-being and behaviour observed in a few animals was only short lived.
Mortality:
no mortality observed
Description (incidence):
None of the animals died during the course of investigation . A partly severe impairment of the general state of well-being and behaviour observed in a few animals was only short lived.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Neither the body weights nor the body weight gain of the animals were clearly influenced by the administration of the test item in comparison to the water control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption of the animals of the high dose group was slightly decreased in the first week, statistically significantly in three of the four cases. However all data are in the range of the historical control data in the testing facility.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
None of the haematological parameters investigated were affected by the administration of the test item .None of the coagulation parameters investigated were affected by the administration of the test item.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative organ weights of the kidneys and the liver were statistically significantly increased in the male animals of the high dose group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormalities were found in the animals.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no histological findings observed considered to be caused by the test item administration.
Details on results:
CLINICAL SIGNS AND MORTALITY
Only once (day 3) in one male animal (No. 20; DG 3: 400 mg/kg bw/day) a clinical observation (gasping breathing) was observed during the off-cage clinical examination before administration. This single sign seems not to be caused by the test item. In the course of the second clinical examination in
the period within 2 hours after administration some clinical signs concerning gait (apathy and ataxia [gait staggering or creeping locomotion]) and posture (abdominal position, coma) were observed on some days in some animals of the DG 3: 400 mg/kg bw/day and the DG 4: 1000 mg/kg bw/day. The most severe clinical signs were observed on the first administration day or in the first week of administration. It appears that the animals became accustomed to treatment with the test item.No clinical signs were observed at the third clinical examination at 2 hours after administration. This shows that the partly severe impairment of the general state of well-being and behaviour was only short-lived. The examination of sensory response, grip strength or motor activity did not show any alterations prior to the administration of the test item or at the end of the study. None of the animals died during the course of investigation.

BODY WEIGHT AND WEIGHT GAIN
Neither the body weights nor the body weight gain of the animals were clearly influenced by the administration of the test item in comparison to the water control group. Although some statistically significant differences were calculated between the groups, all data were within the range of the historical control data of the testing facility.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food consumption of the animals of the high dose group was slightly decreased in the first week, statistically significantly in three of the four cases. However all data were within the range of the historical control data of the testing facility.

HAEMATOLOGY
None of the haematological parameters investigated were affected by the administration of the test item.
Statistically significant increases in platelet count were recorded for females treated with 400 or 1000 mg/kg/day. This seems to be incidental, caused by an incidental low control value, because the values of the treated animals are within the range of the historical control data of the testing facility (655 - 1255 - 103/L). The values of the leucocyte differential count were also all within the normal range for the strain of rat used. None of the coagulation parameters investigated were affected by the administration of the test item. Statistically significant increases in fibrinogen were also recorded for females treated with 400 mg/kg/day. This change also seems to be incidental, caused by an incidental low control value, because the values of the treated group are within the range of the historical control data of the testing facility (2.06 - 3.55 g/L).

CLINICAL CHEMISTRY

None of the ,clinical-biochernical parameters investigated was affected by the administration of the test item.
The alanine aminotransferase activity (ALT) in the female animals of the high dose group was statistically significantly increased when compared with the controls. Consequently this parameter was also examined in the satellite groups. On the latter occasion the activity of this enzyme was indicated as statistically significantly increased in the male animals. Both effects were very small and seem to be incidental, because the values were within the range of the historical control data of the testing facility (males: 50.2 - 82.5 U/1; females: 38.4 - 75.3 U/1).
The cholesterol level in the male animals of all dose groups was also statistically significantly decreased in comparison with the controls and consequently was also examined in the satellite group animals. On the latter occasion a less significant difference was observed. It is concluded that the statistically significantly decreased values are incidental, because the values were within the range of the historical control data of the testing facility (43.8 - 87.0 mg/dl).
The sodium level in the male animals of the middle and high dose groups was reported as statistically significantly increased and, consequently, was also determined for the satellite group animals. On the latter occasion a less significant effect was observed. The increase in this parameter was considered incidental, caused by an incidental low control value, because the values were in the range of the historical control data of the testing facility (114 - 155 mmo1/1).


ORGAN WEIGHTS
The absolute and relative organ weights of the kidneys and the liver were statistically significantly increased in the male animals of the high dose group. The values exceed also the confidence range of the historical control data of the testing facility (males, absolute organ weights, kidney: 1035 -1768 mg, liver: 8596 - 16770 mg; relative organ weights, kidney: 0.33 - 0.44 %, liver: 2.9 - 4.4 %). The relative organ weights of the right kidney were also statistically significantly increased in the male animals of the middle dose group. In the female animals a statistically significant increase of organ weights could only be observed in the relative organ weights of the liver in the animals of the high dose group. This statistically significant increase of the relative organ weights of liver could also be observed in the female animals of the satellite group. Two further statistically significant differences were indicated (absolute organ weights in the female animals, heart and left ovary, low dose group). The values are in the borderline range or exceed the confidence range of the historical control data in the testing facility (heart: 801 - 1087; ovary: 54 - 81 mg). Nevertheless this seems to be incidental, because there is no dose dependence and the statistical significance was not confirmed by the relative organ weights and/or by the right organ in the case of the ovary. None of the other absolute or relative organ weights investigated were affected by the administration of the test item.


GROSS PATHOLOGY

All organs and body cavities of all animals were examined with respect to the location, colour, shape and size under predominant consideration of adrenals, bone marrow (from sternum), brain, epididymides, heart, kidneys, liver, lungs, lymph nodes (mesenteric and submandibular), ovaries, prostate, sciatic nerve, small and large intestine (including Peyer' s patches), spinal cord (from second trachelo-vertebra [Axis]), spieen, stomach, testes, thymus, thyroid, trachea, urinary bladder and uterus. Not any abnormality was found in the animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no histological findings observed caused by the test item administration. The few histological findings observed are considered incidental and they are in the physiological range of the animals used.


Dose descriptor:
NOEL
Effect level:
ca. 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: In the animals of the low dose group no effects were observed. Therefore a NOEL of 100 mg/kg bw/day can be determined.
Dose descriptor:
NOAEL
Effect level:
ca. 400 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:
The daily oral administration of the read-across substance, 4-methyl-4-phenylpentan-2-ol, at doses of 100, 400 or 1000 mg/kg bw/day to rats for a period of 28 days caused, particularly in the animals of the high dose group, a severe although short impairment of general well-being and behaviour. The slightly decreased food consumption in the first week could be connected with this short impairment of general well-being and behaviour. The aformentioned results show that the administration of the test item caused individually distinct effects on the central nervous system without further consequences as demonstrated by the physical condition of the animals, particularly those in the high dose group. A slight effect was also observed in the animals of the middle dose group. It would also appear that the female animals are more sensitive than the male animals. In the animals of the low dose group these effects were not observed. Therefore a NOEL (No Observed Effect Level) of 100 mg/kg bw/day can be identified. The increase of liver and kidney weights is considered to be the result of a hyperplasia caused by an increased metabolic activity for the metabolism and/or elimination of the test item. Cytotoxicity can be excluded because an increase of systemic enzyme activities was not observed and there were no macroscopic pathological or histological findings in these two organs.
Executive summary:

Sixty rats (30 males and 30 females) of the Wistar Crl:WI BR strain were allocated to four groups. Groups 2 and 3 each comprised 5 males and 5 females and groups 1 and 4 each comprised 10 males and 10 females. At the end of the 28 day treatment period 5 male and 5 female of groups 1 and 4 (groups 1 a and 4a) were maintained, undosed, for further 14 days. Three groups received the read-across substance, 4-methyl-4-phenylpentan-2-ol, by oral gavage at dose levels of 100, 400 and 1000 mg/kg bw/day. A fourth group received water only, and served as control. Animals were dosed once daily, and treatment continued for 28 days.

The animals were observed daily for mortality or visible clinical signs of reaction to treatment. Assessments of sensory reactivity (auditory, visual and proprioceptive stimuli), grip strength and motor activity were carried out prior to administration, in the last week of dosing and in the last week of the treatment-free period. Body weight and food consumption were recorded weekly. Haematological parameters (erythrocyte count, haemoglobin concentration, packed cell volume, platelet count, leucocyte count and differential leucocyte count), coagulation parameters (prothrombin time and fibrinogen concentration) and clinical biochemistry parameters of the serum (alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, protein, albumin, glucose, cholesterol, urea, creatinine, sodium and potassium) were determined after an ovemight fasting prior to killing at the termination of the study. At termination all animals were subjected to a macroscopic pathological assessment, the organ weights of the adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spieen, testes and thymus were recorded and these and a range of other organs and tissues were preserved for further histopathological examinations. The histopathological examination was performed on tissues from animals of the control group and the high dose group.

None of the animals died during the course of investigation. In a period within 2 hours after administration some clinical signs related to gait (apathy and ataxia [gait staggering or creeping locomotion]) and posture (abdominal position, coma) were observed on some days in some animals of the DG 3: 400 mg/kg bw/day and the DG 4: 1000 mg/kg bw/day, especially in the first week of administration. The general well-being and the behaviour of the animals were not influenced by administration of the test item in the time outside of the period of 2 hours after administration.

The absolute and relative organ weights of the kidneys and the liver were statistically significantly increased in the male animals of the high dose group. The relative organ weights of the right kidney were also statistically significantly increased in the male animals of the middle dose group. In the female animals a statistically significant increase of organ weights could only be observed in the relative organ weights of the liver in the animals of the high dose group. This statistically significant increase of the relative organ weights of liver could also be observed in the female animals of the satellite group. No pathological macroscopic findings were observed. No pathological histological findings caused by the test item administration were observed. There were no other treatment related finding in any of the other observations made during the study.

A NOEL (No Observed Effect Level) for the read-across substance, 4-methyl-4-phenylpentan-2-ol, of 100 mg/kg bw/day was identified. The findings at 400mg/kg/day, including short term transient neurological effects in a few animals immediately post dosing which were not seen in a longer duration study, and increases in relative kidney weights not accompanied by evidence of kidney damage (biochemcial or histopathogical), were not considered to to be significant and a NOAEL (No Observed Adverse Effect Level) for the read-across substance, 4-methyl-4-phenylpentan-2-ol, of 400mg/kg bw/day can be identified.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
494.32 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One study with the read-across substance conducted according to OECD test guideline 407 and allocated a Klimisch score of 1. Taking into account the molecular weight correction between source and target substance (178.27 vs. 220.31), the NOAEL for 1,3-Dimethyl-3-phenylbutyl acetate is 494.32 mg/kg.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The daily oral administration of the read-across substance, 4-methyl-4-phenylpentan-2-ol, at doses of 100, 400 and 1000 mg/kg bw/day to rats for a period of 28 days caused, in particular in the animals of the high dose group, moderately severe, albeit short, impairment of the general state of well-being and behaviour. The following effects can be deemed as caused by the test item administration:

- In the period within 2 hours after administration some clinical signs relating to gait (apathy and ataxia [gait staggering or creeping locomotion]) and posture (abdominal position, coma) were observed on some days in some animals of the mid dose group (400 mg/kg bw/day) and the high dose group (1000 mg/kg bw/day), especially during the first week of administration.

- The food consumption of the animals of the high dose group was slightly decreased in the first week of administration.

- The absolute and relative organ weights of the kidneys and the liver were statistically significantly increased in the male animals of the high dose group. The relative organ weights of the right kidney were also statistically significantly increased in the male animals of the middle dose group. In the female animals a statistically significant increase of organ weights could only be observed in the relative organ weights of the liver in the animals of the high dose group. This statistically significant increase of the relative organ weights of the liver could also be observed in the female animals of the satellite group.

The general well-being and the behaviour outside of the period of 2 hours after administration were not influenced by administration of the test item. The body weight gain and the haematological, coagulation and clinical-biochemical parameters were not influenced by administration of the test item. No substance-dependent macroscopic pathological and histological findings were observed. The slightly decreased food consumption in the first week could be connected with the short impairment of the general state of well-being and behaviour. The above mentioned results show that the test item administration caused a short-term neurobehavioural effect to some individuals of the high dose group and also the middle dose group without any further pathological consequences. It appears that the female animals are more sensitive than the male animals. In the animals of the low dose group these effects were not observed.

Therefore a NOEL (No Observed Effect Level) for the read-across substance, 4-methyl-4-phenylpentan-2-ol, of 100 mg/kg bw/day can be determined.

The increase of kidney weights at 400 mg/kg/day is considered to be the result of an increased metabolic activity for the metabolism and/or elimination of the test item. Cytotoxicity can be excluded since an increase of systemic enzyme activities was not observed and there were no macroscopic pathological or histological findings in the tissue.

The findings at 400 mg/kg/day, including short term transient neurological effects in a few animals immediately post dosing which were not seen in a longer duration study and an increase in relative kidney weights not accompanied by evidence of kidney damage (biochemical or histopathogical), were not considered to be significant and a NOAEL (No Observed Adverse Effect Level) for the read-across substance, 4-methyl-4-phenylpentan-2-ol, of 400 mg/kg/day can be identified.

Taking into account the molecular weight correction between source and target substance (178.27 vs. 220.31), the NOAEL for 1,3-Dimethyl-3-phenylbutyl acetate is 494.32 mg/kg.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A GLP study conducted with the read-across substance in accordance with OECD TG407 guidelines

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
A suitable repeat-dose study, although not via the predominant route of exposure, is already available

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
A suitable repeat-dose study, although not via the predominant route of exposure, is already available

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
A suitable repeat-dose study, although not via the predominant route of exposure, is already available

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
A suitable repeat-dose study, although not via the predominant route of exposure, is already available

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

Justification for classification or non-classification

The NOAEL of 400 mg/kg bw/day for the read-cross substance, 4 -methyl-4 -phenylpentan-2 -ol, is outside of the classification criteria for Specific Target Organ Toxicity - Repeat Exposure (STOT-RE) of 300 mg/kg/day for a 28-day study and so 1,3 -dimethyl-3 -phenylbutyl acetate is also not classified under CLP for this endpoint.