Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Propylheptanol
- Analytical purity: 99.5% (GC)
- Lot/batch No.: CIW/E, Reg. No. 20 595
- Test substance No.: 94/279
- Stability under test conditions: by reanalysis after the in-life phase of the study; the stability of the test substance in the vehicle over a period of 3 hours was proven before the start of the study (as the emulsions were prepared daily and administered within this time period, the stability was guaranteed)
- Homogeneity: proven by visual inspection
- Storage condition of test material: room temperature; protected from light

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, Germany
- Age at study initiation: 42 days
- Weight at study initiation: the body weight of the males was in the range of 100 - 124 g (group mean: 110 g) and the body weight of the females was in the range of 85 - 101 g (group mean: 94 g)
- Housing: single
- Diet: ground Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by Klingentalmuehle AG, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water (from water bottles) were available ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Cremophor EL in doubly distilled water (approx. 5 mg/100 ml)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
To prepare the emulsion, Propylheptanol was weighed depending on the dose group, then the vehicle was filled up to the desired volume and subsequently mixed using an Ultra Turrax. During administration to the animals the test substance preparations were kept homogeneous with a magnetic stirrer. The test substance preparations were prepared daily.

The administration volume was 10 ml/kg body weight, based upon the latest individual body weight determination.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the beginning of the administration period the homogeneity of the test substance in the vehicle was checked at the low and high concentration; these analyses served also as concentration controls. Further concentration control analyses were carried out in samples from the mid dose drawn at the beginning of the administration period, and in samples from all doses drawn at the end of the administration period. Analytical checks of the concentrations revealed 89% - and 103% of the target concentrations. Although one value was only 89% of the target concentration (high dose at termination of the study), this was assessed as being acceptable.
Duration of treatment / exposure:
3 months
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 150, 600 mg/kg bw/day
Basis:
other: nominal
No. of animals per sex per dose:
10
Control animals:
other: yes, group #1: bi-distilled water, group #2: emulsifier (Cremophor EL in doubly distilled water (approx. 5 mg/100 ml))
Details on study design:
- Dose selection rationale:
Based upon the findings in a prenatal toxicity screening study with Propylheptanol (1580, 790 and 158 mg/kg body) the following doses were selected for the present study: 600 mg/kg body weight (as high dose with expected effects on body weight), 150 mg/kg body weight (as mid dose), 30 mg/kg body weight (as low dose)

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for morbidity or mortality twice a day from Mondays to Fridays and once a day on Saturdays, Sundays and public holidays. Additionally, comprehensive clinical examinations were carried out before treatment, less than 1 hour after application and, if the animals showed clinical signs, 3 - 4 hours after application.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was determined before the start of the administration period in order to randomize the animals. During the conduct of the study, the body weight was determined on day 0 (start of administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly over a period of 7 days
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start and towards the end of the administration period the eyes of the animals were examined for any changes using an ophthalmoscope.
- Dose groups that were examined: test groups 0, 1 and 4

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 10 animals per test group and sex
- Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets; the differential blood count was evaluated visually; clotting analyses: prothrombin time (Hepato Quick's test)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning.
- Animals fasted: No
- How many animals: 10 animals per test group and sex
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase, cyanide-insensitive Palmitoyl-CoA-oxidation, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected overnight.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (withdrawal of food and water in the metabolism cages)
- Parameters checked: volume, color, turbidity, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment

OTHER: At necropsy liver samples were collected for examinations in liver homogenate.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Means and standard deviations of each test group were calculated. As two control groups were used in this study (groups 0 and 1), comparison was performed using either group 0 as the reference control group (with which groups 1 to 4 were compared) or group 1 was taken as the reference control group (with which groups 2 to 4 were compared).
Food consumption, body weight, body weight change, food efficiency: Parametric one-way analysis using the F-test (ANOVA) (two-sided). If the resulting p-value was equal or less 0.05, a comparison of each group with the control group using the DUNNETT's test (two-sided) was performed for the hypothesis of equal means.
Clinical pathology parameters, except differential blood count: Parametric one-way analysis using the F-test (ANOVA) (two-sided). If the resulting p-value was equal or less 0.05, a comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
Urinalysis, except volume, color and turbidity: Pairwise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions.
Weight parameters: A comparison of each group with the control group using the DUNNETT's test (two-sided) was performed for the hypothesis of equal means.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY:

Dose group 4 (600 mg/kg):
- slight salivation in all males and females within one hour after administration
- urine-smeared anogenital region in 4 males and 6 females 3 - 4 hours after administration

Dose groups 2 and 3 (30 and 150 mg/kg): No treatment-related findings

No animal died during the study period.

BODY WEIGHT AND WEIGHT GAIN:

Dose group 4 (600 mg/kg):
- impairment of body weight in males, resulting in reduced values of 13 % (compared to control group 0) or 14% (compared to control group 1) on day 91
- impairment of body weight change in males, resulting in reduced values of 22% (compared to control group 0) or 25% (compared to control group
1) on day 91
- decreased mean terminal body weight in male rats (- 16.7% or - 14.1% when compared with control groups 0 or 1) and in female rats (- 8.2% when compared with control group 1)
These findings were assessed as being substance-related.

In high dose females, statistically significantly decreased values of body weight were obtained on days 42, 56, and 70 when compared to test group 1, only. This was related to the fact, that the body weight of the vehicle control was slightly higher than the body weight of the water control. The statistically significant deviations observed in females were therefore assessed as being incidental and not substance-related. Also, the single statistically significant difference in high dose females in the body weight change on day 42 (compared to test group 1) was assessed as being incidental.

Dose groups 2 and 3 (30 and 150 mg/kg): No treatment-related findings

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):

Dose group 4 (600 mg/kg):
- impairment of food consumption in males (- 18%; compared to both control group 0 and control group 1) and females (- 10%; compared to both control group 0 and control group 1); this was assessed as being substance-related;

Dose groups 2 and 3 (30 and 150 mg/kg): No treatment-related findings

FOOD EFFICIENCY:

Food efficiency was statistically significantly impaired in high dose males on days 63 (compared to test group 0), 91 (compared to test group 1), in mid dose males on day 77 (compared to test group 0), in high and mid dose females on day 35 (compared to test group 1) and in high dose females on day 42 (compared to test group 1). Due to the lack of a dose-response relationship and the only isolated significances, this was assessed as being incidental.

OPHTHALMOSCOPIC EXAMINATION: No substance-related effects were observed.

HAEMATOLOGY / CLINICAL CHEMISTRY:

Dose group 4 (600 mg/kg):
- decrease in platelets, globulins and cholesterol in both sexes and in triglycerides in males, only
- increase in albumin, cyanide-insensitive palmitoyl-CoA-oxidation in the serum of both sexes

Dose groups 2 and 3 (30 and 150 mg/kg): No treatment-related findings

URINALYSIS

Dose group 4 (600 mg/kg):
- decrease in urinary specific gravity in the females
- increase in urinary volume and transitional epithelial cells in the females, only, and increase in squamous epithelial cells in the urine of both sexes

Dose groups 2 and 3 (30 and 150 mg/kg): No treatment-related findings

ORGAN WEIGHTS

Dose group 4 (600 mg/kg):
- increased mean absolute and relative liver weights in male (absolute weight: + 26.2% / + 28.1%, relative weight: + 47.6% / + 53.8%) and female rats (absolute weight: + 31.2% / 24.6%; relative weight: + 37.6% / 35.7%; when compared with either control group); indicative for peroxisomal proliferation

Comparison of dose group 4 with control group 0:
Due to the significantly decreased mean terminal body weight of the males of dose group 4, the mean absolute brain weight was also significantly (p Due to the significantly decreased terminal body weight in male rats of dose group 4, significantly increased (p < 0 .01) mean relative weights of the kidneys (+ 10.4%), testes (+ 21.7%), epididymides (+ 14.8%), and brain (+ 10.3%) were recorded. In females of group 4, the mean relative weight of the adrenal glands was significantly (p
Comparison of dose group 4 with control group 1:
Due to the significantly decreased mean terminal body weight in group 4, the mean absolute brain weight was also significantly (p Due to the significantly decreased terminal body weight in male rats of dose group 4, significantly increased (p
Dose group 3 (150 mg/kg):
- increased mean relative liver weight in female rats (+ 7.5% / + 6.1%; when compared with either control group), revealing dose response relationship, but indicative for peroxisomal proliferation
Comparison of dose group 3 with control group 0:
In dose group 3 of the female rats, the mean relative kidney weight was slightly but significantly (p The other mean relative weight parameters did not show significant differences when compared with control group 0.

Comparison of dose group 3 with control group 1:
The mean weights of the kidneys and the epididymides were significantly increased (p
Dose group 2 (30 mg/kg):
No significant differences when compared with control group 0.

Comparison of dose group 2 with control group 1:
In female rats of dose group 2 (30 mg/kg), only, the mean weights of kidneys (p
GROSS PATHOLOGY: no treatment-related findings

HISTOPATHOLOGY:
Treatment-related microscopic findings were noted in the liver (male and female rats), thyroid glands (male rats, only), and pituitary gland (male rats, only).

Dose group 4 (600 mg/kg):
- diffuse hypertrophy (grade 2) of the liver cells in male and female rats (indicative for peroxisomal proliferation)
- loss of fatty infiltration of the liver cells in male rats (In females, there was also no indication of fat storage, however, this finding was anyhow a very rare event even in control females of dose group 0 (two females were affected with minimal (= grade 1) fatty infiltration, only))
- diffuse hypertrophy (grade 2) of the follicular cells of the thyroid glands in seven male rats
- vacuolation of basophilic (thyrotropic) cells (grade 1, grade 2 or grade 3) in the glandular part of the pituitary gland of three male rats (that had slight diffuse hypertrophy of the follicular cells of the thyroid glands)

Dose group 3 (150 mg/kg):
- diffuse hypertrophy (grade 1) of the liver cells in one female rat (indicative for peroxisome proliferation)

Dose group 2 (30 mg/kg): no treatment-related findings

In the testes of treated and control males, focal tubular atrophy was recorded in every animal. The severity of this lesion was graded minimal (grade 1) in the majority of cases. In two males of the control group, slight (grade 2) tubular atrophy was recorded. When excluding the cases with minimal and slight tubular atrophy, the incidence in groups 0 to 4 is 1/2/0/0/0. As judged from light microscopy, no alteration of spermatogenesis was noted. Higher grades of tubular atrophy affecting spermatogenesis were only noted in control males of group 0. The etiology of this lesion is not known, the spontaneous nature, however, is undoubted.

OTHER FINDINGS:
There are some additional statistically significant intergroup differences in the results of clinical pathology testing. These deviations are marginal, incidental or inconsistent, when compared with the other sex, or lack dose-response relationship. Accordingly, these findings are considered to be of no toxicological significance.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no effects observed
Dose descriptor:
LOAEL
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: peroxisomal proliferation
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: effects due to peroxisomal proliferation: increased mean relative liver weight, diffuse hypertrophy of the liver cells at 150 mg/kg bw

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Body weight (g)
mg/kg 0# 0## 30 15 600
Males
Day 0 109.8 111.0 109.7 110.2 109.7
Day 28 206.1 215.8 215.7 205.2 197.4
Day 56 253.9 265.6 271.2 257.0 232.0
Day 91 284.6 293.7 302.1 285.3 246.6**

Females
Day 0 92.8 94.2 94.0 93.6 93.1
Day 28 142.3 145.0 142.5 142.2 142.3
Day 56 164.7 171.0 164.8 163.4 160.2
Day 91 174.5 180.5 175.0 175.5 171.2

* p<0.05, ** p <0.01 Dunnett's test + Anova
# = control; ## = vehicle control

Food consumption (g)
Mg/kg 0# 0## 30 15 600
Males
Day 7 15.3 15.6 15.6 15.6 15.3
Day 28 17.5 18.3 18.1 17.1 16.0*
Day 56 17.4 18.0 18.3 17.3 16.5
Day 91 15.8 15.7 15.8 15.1 12.9**

Females
Day 7 12.7 13.2 13.0 12.7 12.5
Day 28 13.7 13.7 13.3 13.8 12.4**
Day 56 13.8 14.0 13.4 13.5 12.9*
Day 91 10.7 11.3 1.1 11.6 10.4

* p<0.05, ** p <0.01 Dunnett's test + Anova
# = control; ## = vehicle control

Clinical pathology (including hematology, urinalysis)
Males day 87
mg/kg                     0#       0##       30       150       600
PLT (Giga/l)             641       617       614      611       540**
Pal C0A (Mu/mg P)       4.50       5.21     5.49     6.64      43.27**
Alb (G/L)              36.11       35.31    35.80   38.03     41.86**
Glob (G/L)     33.97  33.38 34.07 33.52 28.24**
Trig (G/L)      2.60   2.34     2.21 2.24 1.76**
Chol (mM/L)      2.31   2.22     2.22     2.03* 1.38**
Urine ml     4.3  3.9     3.6      2.7 5.0
Females day 88
mg/kg 0# 0## 30 15 600
PLT (Giga/l) 608 605 598 614 428**
Pal C0A (Mu/mg P) 3.27 4.07 4.63 3.93 22.63**
Alb (G/L) 35.18 35.52 35.27 35.83 38.19**
Glob (G/L) 31.55 31.74 31.38 31.11 28.51
Trig (G/L) 1.09 1.06 0.97 0.97 1.47*
Chol (mM/L) 3.02 3.03 2.96 2.73 2.24**
Urine ml 1.7 1.5 2.4 2.1 5.2
* p<0.05, ** p <0.01 Dunnett's test + Anova
# = control; ## = vehicle control

Organ weights
Males
mg/kg 0# 0## 30 15 600
Liver weight
Absolute (g) 7.87 7.75 7.99 7.83 9.93**
Relative (%) 3.00 2.88 2.86 2.99 4.4**
Females
Liver weight
Absolute (g) 4.23 4.53 4.32 4.61 5.64**
Relative (%) 2.67 2.70 2.68 2.87* 3.67**
* p<0.05, ** p <0.01 Dunnett's test + Anova
# = control; ## = vehicle control


Applicant's summary and conclusion