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Description of key information

Oral repeated toxicity :  two key studies were available for evaluation of repeated exposure toxicity.
- A subchronic 90 day study was performed in rats with the analogue Pentanol (equivalent to OECD 408)- Butterworth, 1978).
Read across justification (see document attached in chapter 13): The read accross is based on the comparable pattern of the physicochemical, structural properties and toxicological profils between the two analogues (pentanol vs. heptanol). Indeed, comparative NOAELs were observed in the repeated exposure study obtained in the OECD 422 with Heptanol (at least 28 days exposure for males and females (Spézia, 2012)) showing low order of toxicity (NOAEL= 1000 mg/kg/day) as well as in the 90 day subchronic study performed with pentanol (Butterworth, 1978), see also REACH disseminated dossiers).
Based on the available data on aliphatic alcohols, it is shown that there is an inverse relationship between chain length and toxicity. The shorter chain alcohol tends to induce more pronounced effects when compared to materials with a longer chain length (i.e irritant property). We considered therefore that the 90 days study performed wth pentanol represented a worst case for assessment of heptanol repeated toxicity exposure
It was therefore considered that the use of this latter study for evaluation of the subchronic evaluation of Heptanol was justified. Indeed the same pattern of toxicity profile was obtained confirming the low order of toxicity of this aliphatic alcohols analogue category.
Furthermore a SIDS on aliphatic alcohols category (SIAM 22, 2006) support the fact that the family of aliphatic alcohols is of low order of toxicity (see justification for read across, attached document in section 13).
- An oral repeated dose toxicity study combined with the reproductive test (OECD 422) was perfomed with Heptanol in 2012 (Spézia, 2012).

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
no
Test material information:
Composition 1
Species:
rat
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: from a specified-pathogen-free breeding colony
- Weight at study initiation: 91.8 ± 2.1
- Diet (ad libitum): Spillers' Laboratory Small Animal Diet
- Water (ad libitum): tap-water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 1
- Humidity (%): 40 - 60
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
13 weeks, daily
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily during 90 days
Remarks:
Doses / Concentrations:
50, 150, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: initially, then at days 1, 2 and 6 and at intervals of not more than 1 week up to thereafter (last weighing: days 91)

FOOD CONSUMPTION CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: food and water consumptions were measured over the 24-hour period preceding the day of weighing.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after final dose, the animals were killed by exsanguination from the abdominal aorta
- Anaesthetic used for blood collection: Barbiturate
- Animals fasted: Yes, the animals were deprived of food for 24 hour before sacrifice
- Parameters examined haemoglobin content, packed cell volume and counts of erythrocytes and leucocytes. Slides were prepared from all blood samples for the counting of reticulocytes and the different types of leucocytes, but the counts were confined to the controls and the animals on the highest level of treatment except at week 2, when leucocyte counts were also made on the 150-mg/kg groups.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after final dose, the animals were killed by exsanguination from the abdominal aorta
- Animals fasted: Yes, the animals were deprived of food for 24 hour before sacrifice
- Parameters examined: at week 13, serum was analysed for urea, glucose, total protein and albumin as well as for the activities of glutamic-oxalacetic transaminase, glutamic- pyruvic transaminase and lactic dehydrogenase.

URINALYSIS: Yes
- Time schedule for collection of urine: during week 2 and 6 and from the remaining rats during week 12.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, urine was collected over a 6-hour period of water deprivation from the rats about to be killed.
- Parameters were examined: the volumes and specific gravities of these samples were measured to determine the renal concentrating ability of the rats. In addition, the samples were examined for their appearance, number of cells and content of albumin, glucose, ketones, bile salts and blood. At week 6 and 12 the concentration test was extended to include measurements of the volume and specific gravity of urine produced in a 4-hr period commencing after 16 hr without water. In addition, the renal diluting capacity was investigated by measuring the volume and specific gravity of urine produced in the first 2 hr after a water load of 25 ml/kg.

NEUROBEHAVIOURAL EXAMINATION: NoSacrifice and pathology
Sacrifice and pathology
GROSS PATHOLOGY: Yes, at autopsy all the tissues were examined for gross abnormalities and the brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum, adrenals, gonads, pituitary and thyroid were weighed.
HISTOPATHOLOGY: Yes, samples ot the above organs and of lung, lymph nodes, salivary gland, trachea, oesophagus, aortic arch, thymus, urinary bladder, colon, rectum, pancreas, uterus and skeletal muscle were preserved in 10% buffered formalin. Paraffin-wax sections of these tissues were stained with haematoxylin and eosin for microscopic examination, which was carried out on liver and kidney sections from all animals but on other types of tissue from only half of the control rats and from those given 1000 mg test substance/kg bw for 13 weeks.

Statistics

Student's t test and the ranking test of White

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, at autopsy all the tissues were examined for gross abnormalities and the brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum, adrenals, gonads, pituitary and thyroid were weighed.
HISTOPATHOLOGY: Yes, samples ot the above organs and of lung, lymph nodes, salivary gland, trachea, oesophagus, aortic arch, thymus, urinary bladder, colon, rectum, pancreas, uterus and skeletal muscle were preserved in 10% buffered formalin. Paraffin-wax sections of these tissues were stained with haematoxylin and eosin for microscopic examination, which was carried out on liver and kidney sections from all animals but on other types of tissue from only half of the control rats and from those given 1000 mg test substance/kg bw for 13 weeks.
Statistics:
Student's t test and the ranking test of White
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
No abnormalities in appearance or behaviour were seen during the study.

BODY WEIGHT AND WEIGHT GAIN, FOOD CONSUMPTION AND WATER CONSUMPTION
There were no significant differences between the treated and control rats in body weight or in food and water consumption (see table 1).

HAEMATOLOGY AND CLINICAL CHEMISTRY (see table 2)
Only isolated differences from the controls were seen in the results of the haematological studies. These included a lower total leucocyte count at week 2 in the male rats given 150 or 1000 mg test substance/kg bw/day and lower haemoglobin concentrations at week 13 in the male animals given 50 or 1000 mg/kg bw/day. Also there were higher percentages of reticulocytes in the male rats given 1000 mg/kg bw/day at week 2 and in the females at week 13, as well as a slightly lower percentage of lymphocytes at week 6 in the females given 1000 mg/kg bw/day. The results of the serum analyses were similar in test and control rats.

URINALYSIS
The urine was free from bile, blood, glucuse and ketones, while the concentration of albumin was similar in all groups. At week 6 there were lower cell counts in the urine of the male rats given 150 or 1000 mg test substance/kg bw/day, the differences being statistically significant. Some statistically significant differences were also apparent in the concentration tests at week 12; the specific gravity of the samples collected at 16-20 hour from females given 1000 mg/kg bw/day was higher than the control value and the volume was lower. After the same period on test, the male rats given 50 or 1000 mg/kg bw/day produced less urine in the 6-hour period without water. No differences from the controls were found in the dilution test nor at week 2 and 6 in the concentration tests.

ORGAN WEIGHTS (see table 3)
Examination of the organ weights showed some isolated differences at week 2, but none thereafter. The stomach weights in the males and females given 1000 mg n-amyl alcohol/kg bw/day were higher than those of the controls, but the difference was confined to the male rats when the values were related to body weight. Also, a higher heart weight was found in the female rats given the top level of treatment, but this was not evident when the figure was related to body weight. Relative to body weight, the spleens from the female rats dosed with 1000 mg/kg bw/day showed a low value, as did the female kidney weights, both at this and at the 150 mg/kg bw/day level.

GROSS PATHOLOGY
At autopsy, no abnormalities were seen at any dose level

HISTOPATHOLOGY: NON-NEOPLASTIC
On histological examination, protein casts and foci of calcification were found in the kidney tubules, particularly from the male animals, but the incidences were similar in the treated animals and their corresponding controls. The incidence of fatty change and inflammatory cell infiltration in the liver was again comparable in the control and treated rats. No histological changes related to the period or level of treatment were seen in any of the organs examined.





Any other information on results incl. tables
Table 1: Mean body weights and food and water consumption values of rats given daily doses of 0 - l000 mg test substance/kg bw/day for l3 weeks








CONCLUSION

Only isolated changes with no consistent pattern were found in the hematological studies, either with respect to dose-response, sex or time relationships. The reductions of hemoglobin concentration in the male rats at week 13 suggest a mild anemia, but this is not supported by other measurements. The packet cell volumes mean corpuscular hemoglobin concentrations and erythrocyte and reticulocyte counts were all within the normal limits and there were no variations in spleen weight. The low total white cell counts observed in the male animals after 2 week treatment (with 150 or 1000 mg/kg bw/day) were probably accidental findings for 2 reasons: 1) the corresponding female rats did not show a similar effect and 2) the differential white cell counts were very similar to those of the controls. The few variations observed in the reticulocyte counts were within the normal range for rats of this strain.

No specific gravity or volume measurements of the urine indicated any adverse effect on renal function in the treated groups. The two isolated reductions in relative kidney weights (after 2 weeks treatment) were neither consistent with absolute kidney weight measurements nor associated with any histopathological findings, or present after a more prolonged treatment.

The increased stomach weights found at week 2 in some rats given the top dose may be associated with a mild irritation since the test substance was administered by gastric intubation. In any case, the effect did not persist throughout the study.

The few other changes in organ weights were isolated, followed no consistent pattern and were considered to be unrelated to treatment.

The highest dose level tested can therefore be considered as the NOAEL under the conditions of the study.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Critical effects observed:
not specified
Executive summary:

CONCLUSION

Pentanol (n-Amyl alcohol) was given daily by oral intubation (7 days /week) to groups of 15 male and 15 female rats for 13 week at dose-level of 0, 50, 150 and 1000 mg/kg/day. In addition groups of five rats of each sex, with body weight dimilar to thouse of the main groups, wee given daily doses of 0, 50, 150 or 1000 mg/kg/day and were killed after 2 or 6 weeks. The n amyl alcohol was dissolved in corn oil and administered to rats at a dosage volume of 5 ml/kg. The animals were weighed initially at days 1, 2 and 6 and then at intervals of not more than 1 week up to day 91 of the study. Food and water consumptions were measure over the 24 hours perod preceding the day of weighing. At week 13 serum was analysed for urea, glucose, total protein and albumin as well as for the enzymatic activities transaminase. At autopsy, all the tissues were examined for gross abnormaties. Microscopic examination was performed on selected tissues.

Only isolated changes with no consistent pattern were found in the hematological studies, either with respect to dose-response, sex or time relationships. The reductions of hemoglobin concentration in the male rats at week 13 suggest a mild anemia, but this is not supported by other measurements. The packet cell volumes mean corpuscular hemoglobin concentrations and erythrocyte and reticulocyte counts were all within the normal limits and there were no variations in spleen weight. The low total white cell counts observed in the male animals after 2 week treatment (with 150 or 1000 mg/kg bw/day) were probably accidental findings for 2 reasons: 1) the corresponding female rats did not show a similar effect and 2) the differential white cell counts were very similar to those of the controls. The few variations observed in the reticulocyte counts were within the normal range for rats of this strain.

No specific gravity or volume measurements of the urine indicated any adverse effect on renal function in the treated groups. The two isolated reductions in relative kidney weights (after 2 weeks treatment) were neither consistent with absolute kidney weight measurements nor associated with any histopathological findings, or present after a more prolonged treatment.

The increased stomach weights found at week 2 in some rats given the top dose may be associated with a mild irritation since the test substance was administered by gastric intubation. In any case, the effect did not persist throughout the study.

The few other changes in organ weights were isolated, followed no consistent pattern and were considered to be unrelated to treatment.

The highest dose level tested can therefore be considered as the NOAEL under the conditions of the study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1

Mode of Action Analysis / Human Relevance Framework

Additional information

Two key studies were available for evaluation of repeated exposure toxicity:

1/A GLP study perfomed according to OECD 422 with Heptanol (F. Spézia, 2012): The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until day 5 post‑partum (p.p.)inclusive.

This study provides information on the possible health hazards (including neurological and immunological effects) likely to arise from repeated exposure over a limited period of time. It can also indicate effects on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Three groups of ten male and ten female Sprague-Dawley rats received the test item, daily, by oral administration (gavage), before mating, during mating and, for the males, until sacrifice, for the females, throughout gestation until day 5p.p., at dose-levels of 100, 300 or 1000 mg/kg/day.

An additional group of ten males and ten females received the vehicle control, corn oil, under the same experimental conditions. The dosing volume was 5 mL/kg/day.

 

There were no unscheduled deaths in control, 100 and 300 mg/kg/day groups. At 1000 mg/kg/day, one male was sacrificed moribund (hypoactivity, loud/abdominal breathing, and ptyalism) on study day 35 most likely due to reflux at dosing.

Hypoactivity, loud/abdominal breathing and dyspnea were observed at 1000 mg/kg/day and in males only (except hypoactivity which was also noted in one female treated at 1000 mg/kg/day, during the lactation period). These clinical signs were considered to be related to the treatment with the test item. Ptyalism was considered to be related to the test item but of minor toxicological importance.

There were no biologically significant effects on mean body weight or mean body weight changes or food consuption during the premating, mating, gestation or lactation periods.

No changes were observed on either motor acitvity of the animals or hematology and blood chemistry parameters.

 The test item administration at 100, 300 or 1000 mg/kg/day did not induce any organ weight, macroscopic or microscopic changes.

Conclusion

Based on the experimental conditions of this study:

- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day,

2/A subchronic toxicity study equivalent to OECD 408 with Pentanol (Butterworth, 1978):

The read across justification document is attached in section 13.

Pentanol (n-Amyl alcohol) was given daily by oral intubation (7 days /week) to groups of 15 male and 15 female rats for 13 week at dose-level of 0, 50, 150 and 1000 mg/kg/day. In addition groups of five rats of each sex, with body weight dimilar to thouse of the main groups, wee given daily doses of 0, 50, 150 or 1000 mg/kg/day and were killed after 2 or 6 weeks. The n amyl alcohol was dissolved in corn oil and administered to rats at a dosage volume of 5 ml/kg. The animals were weighed initially at days 1, 2 and 6 and then at intervals of not more than 1 week up to day 91 of the study. Food and water consumptions were measure over the 24 hours perod preceding the day of weighing. At week 13 serum was analysed for urea, glucose, total protein and albumin as well as for the enzymatic activities transaminase. At autopsy, all the tissues were examined for gross abnormaties. Microscopic examination was performed on selected tissue.

The treatment had not demonstrable effect on body weight gain, food and water consumption, hematological values, serum and urine analyses, renal function, organ weights or histopathology. The No Observed Effect Level was 1000 mg/kg/day.

Justification for classification or non-classification

There are conclusive but not sufficient data for the classification of heptanol with regard to repeated toxicity obtained with heptanol taking into account the NOAEL of 1000 mg/kg/day.

No classification for repeated toxicity has to be applied for heptanol according to EU Directive 67/584/EEC and EU regulation (EC) No 1272/2008 (CLP).