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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 1996
Deviations:
yes
Remarks:
behaviour was not examined
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
no determination of T4/TSH levels, F1 offspring and females killed after day 4 of lactation, no sperm analysis
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the closed bottle at room temperature
- Stability under test conditions: verified at the beginning and at the end of the test
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crj: CD (SD) IGS
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Tsukuba Breeding Center of Charles River Laboratories Japan, Inc.
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 297.7 – 427.7 g after acclimatization and 403.0 – 356.5 g after grouping; Females: 205.5 – 297.8 g after acclimatization and 254.9 – 218.6 g after grouping for females
- Fasting period before study: no
- Housing: animals were single-housed in metal wire-mesh flooring cages (220 x 270 x 190 mm)
From day 14 post coitum females were housed in a plastic reproduction cage for rats (350 x 400 x 180 mm), to which paper pulp chips (ALPHA-dri manufactured by Kasho Company Limited) were supplied appropriately as bedding. Moreover, the temperature and humidity of the breeding room was set at 23.5-25.5 °C and 45-70 %, respectively during the breeding period.
- Diet: pellets (CE-2 manufactured by Clea Japan, Inc.), ad libitum
- Water: tap water (Hadano City Waterworks Bureau), ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 50 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test article was weighed, dissolved in corn oil and administered within 8 days after the preparation.

VEHICLE
- Concentration in vehicle: 0.4, 2 and 10%(w/v), respectively in the low, mid and high dose groups
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: V8P7069
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
It was confirmed in a previous acute oral study that the prepared test article formulation (0.40, 2.00, 10.0 % (w/v)) contained the test article at the predetermined concentration, respectively. Each 2 mL of the prepared samples were volume-adjusted with n-propanol and measured by high performance liquid chromatography (HPLC; analytical column: Inertsil ODS-2 [4.6 mm i.d. x 150 mm, particle size 5 μm]; mobile phase: water/acetonitrile [60 : 40]). Their concentrations were obtained by using a calibration curve constructed from the measured values (MBA: 10-50 μg/mL).
Duration of treatment / exposure:
- Males: at least 42 days (2 weeks premating, 2 weeks during mating, 2 weeks after the completion of mating period)
- Females: at least 40 days (2 weeks premating, throughout mating periods up to 2 weeks, then throughout pregnancy up to day 4 of lactation after parturition; females copulated without parturition were dosed until the day corresponding to day 24 of gestation).
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a 14-day repeated-dose toxicity study, 5 animals per sex and per dose group were orally dosed at 0, 250, 500 and 1000 mg/kg bw/day. Males and females showed salivation after administration from 500 mg/kg bw/day. High dose males and females exhibited significantly depressed food consumption (there was also a tendency toward retarded body weight gain in males). Hematological examination revealed a significant decrease in platelet count for high dose females. Blood biochemical examination showed significant increases in alkaline phosphatase activity for males and GPT activity for males and females as well as a tendency toward an increase in the GOT activities for males and females, in the high dose group.
At autopsy, thickening, edema, and reddish or dark spots of the surface of gastric mucosa were found in high dose males and females, in mid dose females and in low dose males. Additionally, an increase in the absolute liver weight was seen for males and females from 500 mg/kg bw/day onwards, as well as in relative liver weights in males from 500 mg/kg bw/day onwards and females from the 250 mg/kg bw/day onwards.

The high dose for the present study was therefore determined to be 500 mg/kg bw/day, to induced apparent toxicity but no mortality; the medium dose was set at 100 mg/kg bw/day and the low dose was 20 mg/kg bw/day (obtained by dividing the medium dose by a common ratio of 5).
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: More than once a day before and after administration during the dosing period.

BODY WEIGHT: Yes
- Time schedule for examinations: on days 1 (starting day of administration), 7, 14, 21, 28, 35 and 42 of administration as well as on day of autopsy for all males, and on days 1, 7 and 14 of administration for all females. In addition, females that required some time to mate were weighed also on days 21 and 28 of administration. Body weights were also determined on days 0, 7, 14 and 20 of gestation for females copulated, and on days 0 and 4 of lactation as well as on day of autopsy for females with parturition. Females without parturition, that were confirmed to have copulated, were weighed on the day corresponding to day 25 of gestation (day of autopsy).

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to autopsy on the day after day 42 of administration for all males, on the day after day 4 of lactation for females with parturition, and on the day corresponding to day 25 of gestation for females copulated without parturition
- Anaesthetic used for blood collection: Yes (pentobarbital sodium)
- Animals fasted: Yes; 18 - 24 hours
- How many animals: all
- Parameters examined: reticulocyte percentage (control and high dose), red blood cell count (RBC), white blood cell count (WBC), platelet count, hemoglobin content (Hb), mean corpuscular volume (MCV), hematocrit (Ht), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), differential white blood count, prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: subsequent to the blood sampling for the purpose of hematological examination; blood samples were collected again from the abdominal caudal vena cava by using heparin as an anticoagulant
- Animals fasted: Yes
- How many animals: all
- Parameters examined: total protein, albumin, total cholesterol, glucose, blood urea nitrogen (BUN), creatinine, alkaline phosphatase activity (ALP), GOT activity, GPT activity, γ-GTP activity, triglyceride, inorganic phosphorus (Inorg. phos.), total bilirubin, calcium, A/G ratio, sodium, potassium, chlorine.

URINALYSIS: Yes
- Time schedule for collection of urine: week 6 of administration
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: by collecting approximately 4-hour urines or freshly voided urines; pH, occult blood, protein, sugar, urobilinogen, ketone bodies, and bilirubin were examined through the test paper method, as well as color tone and turbidity by visual inspection, respectively.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- Mating: Copulation and time to copulation was checked based on the presence or absence of vaginal plug and sperms in vaginal smear every morning.

- Observation of parturition and lactation state:
For animals, of which the parturition state could be monitored, their conditions were observed.
As to animals of which the parturition state could not be directly observed, the presence or absence of parturition problems, such as parturition difficulties, delays etc., was assessed and recorded based on clinical signs after parturition.
Parturition was checked between 9 a.m. and 11 a.m. For animals which were confirmed to have completed parturition, that day was defined as parturition day. For animals which completed parturition after 11 a.m., the following day was regarded as parturition day.
For all cases that were confirmed to have completed parturition, the gestation length (number of days from day 0 of gestation to parturition day) was calculated, and also the birth rate was obtained. After parturition, the lactation state was observed daily.
Oestrous cyclicity (parental animals):
For all females the estrous cycles were observed (baseline and during prepairing) based on the vaginal smears, in order to obtain the average number of estrous days and also estrous types.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups (stillborn and liveborn), postnatal mortality, presence of gross anomalies/external malformations, pup weights (on days 0 and 4 of lactation)
all pups were subjected to autopsy on day 4 of lactation

GROSS EXAMINATION OF DEAD PUPS:
yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on the day after day 42
- Maternal animals: All surviving animals on the day after day 4 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- The number of corpora lutea of pregnancy (no data derived from non-pregnant animals) and number of implantation sites were counted, respectively in ovaries and uterus under a stereoscopic microscope.

ORGAN WEIGHTS
- Brain, heart, thymus, liver, kidneys, spleen and adrenal glands (in addition, testes and epididymes for males) were weighed, and the respective organ weights to body weight (relative weights) were obtained. In females, ovaries and uterus were weighed.

HISTOPATHOLOGY
- Brain, pituitary gland, spinal cord, digestive tract, liver, kidneys, adrenal glands, spleen, pancreas, heart, thymus, thyroid gland, trachea, lungs, urinary bladder, mesenteric lymph nodes, mandibular lymph nodes, sciatic nerve and femur marrow, in addition, seminal vesicle and prostate ventral lobe, as well as vagina, ovaries and uterus were fixed in 10 % phosphate-buffered formalin. Lungs were fixed by infusion. Testes and epididymes were fixed and preserved in Bouin's fluid, and then moved into 10 % phosphate-buffered formalin for long-term storage. The fixed organs were paraffin sectioned in the usual manner and stained with hematoxylin and eosin, in order to conduct histopathological examination.
Uterus and vagina for 8 females having failed to conceive or having total embryonic resorption in the high-dose group and seminal vesicle and prostate for male mating partners of such females were additionally paraffin-sectioned in the usual manner and stained with hematoxylin and eosin, in order to conduct histopathological examination.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
Histopathological graded data and the sum of positive grades were subjected to significance test respectively by using Mann-Whitney U test (level of significance: 5 %) and Fisher’s one-sided exact test (level of significance: 5 %), in comparison with the control group.
Other data, i. e. body weights, food consumptions, hematological and blood biochemical values in the cases subjected to regular autopsy, and organ weights underwent first a test for uniformity of variance (level of significance: 5 %) through Bartlett’s method, by defining the values obtained from individual rat or the average values per litter as one sample. If the variance was uniform, one-way analysis of variance (level of significance: 5 %) was conducted. And if any significance was found between the groups, multiple comparisons were performed through Dunnett’s method (level of significance: 5 %). In contrast, in the cases where the variance was 0 in any group and where the variance was not uniform, Kruskal-Wallis rank test (level of significance: 5 %) was carried out. If any significance was seen between the groups, multiple comparisons were performed through Dunnett’s test (level of significance: 5 %).
As to the copulation and conception rates, Fisher’s exact test was conducted (level of significance: 5 %).
Reproductive indices:
Copulation index (no. animals copulated/no. animals mated x 100)
Fertility index (no. animals pregnant /no. animals copulated x 100)
Implantation index (no. implantation sites/ no. corpora lutea x 100)
Gestation index (no. females with live pups/no. pregnant females x 100)
Offspring viability indices:
Delivery index (no. pups born / no. implantation sites x 100)
Birth index (no. live pups d0 / no. implantation sites x 100)
Live birth index (no. live pups / no. pups born x 100)
Viability index (no. live pups on day 4 / no. live pups on day 0 x 100)
Sex ratio on day 0 (no. male live pups on day 0 / no. live pups on day 0 x 100)
Sex ratio on day 4 (no. male live pups on day 4 / no. live pups on day 4 x 100)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
12/13 males and 11/13 females of the high dose group showed transient salivation.
Mortality:
no mortality observed
Description (incidence):
No dead or moribund animals were seen in any sex and treatment group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Males: slightly increased (104%) at both 100, 500 mg/kg bw/day compared to the vehicle control values (not statistically significant) at the end of the study;
- Females:
(pre-)mating: slightly increased body weights (107% at day 14) at 500 mg/kg bw/day compared to the vehicle control values (not statistically significant);
gestation: significantly increased body weights on days 0 and 7 of gestation (113% and 108% of control respectively) at 500 mg/kg bw/day; marked individual differences not reaching statisitcal significance after day 14 of gestation in this dose group;
lactation: significantly increased body weights on day 0 of lactation at 100 and 500 mg/kg bw/day (108% and 115% of control respectively), slight increase on day 4 of lactation (104% of control) at 500 mg/kg bw/day (not statistically significant).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Males: significantly increased only on day 35 at 100 mg/kg bw/day; significantly increased from day 7 onward at 500 mg/kg bw/day
- Females: significantly increased during the premating period (days 7 and 13) at 500 mg/kg bw/day
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
platelet count: significant decrease in males at 500 mg/kg bw/day and in females at 100 and 500 mg/kg bw/day (no effects were noted on prothrombin time or activated partial thromboplastin time)
red blood cell count: significant decrease in males at 100 and 500 mg/kg bw/day
mean corpuscular volume: significant increases in females at 100 mg/kg bw/day
mean corpuscular hemoglobin: significant increases in females at 100 mg/kg bw/day

No significant changes in hematocrit value and hemoglobin content (males/females), reticulocyte count and the histopathological examination of bone marrow; spleen did not show any changes related to hematological changes. Therefore, all the changes in the erythrocyte were considered by the authors to occur accidentally.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A/G ratio: significant increases in males at 500 mg/kg bw/day
A/G ratio: significant decreases in females at 100 mg/kg bw/day
GOT activity: significant increases in males at 500 mg/kg bw/day
Inorganic phosphorus: significant increases in males at 500 mg/kg bw/day
Albumin: significant decreases in females at 100 mg/kg bw/day
Chloride: significant increases in females at 100 mg/kg bw/day
Glucose: significant increase in females at 500 mg/kg bw/day
Calcium: significant decrease in females at 500 mg/kg bw/day

Females changes (A/G ratio, albumin, chloride) were not dose-dependent and therefore considered not to be test substance related. Male changes (A/G ratio, inorganic phosphorus) were - although statistically significant - considered to be of no toxicological relevance because their were only marginal in magnitude, not observed in females or changes noted in females were reciprocal in nature.
See RDT entry (in 7.5.1) for selected clinical biochemistry parameters.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related findings were noted at 500 and 100 mg/kg bw/day (dose-dependent incidence):
- Stomach: squamous cell hyperplasia in the forestomach of all animals at 500 mg/kg bw/day and also in the forestomach for 3 (male)/5 (female) animals at 100 mg/kg bw/day. In female animals, very slight-slight submucosal forestomach edema in 1/1/5/3 animals (0/20/100/500 mg/kg bw/day). Single female (controls) and 2 females (100 mg/kg bw/day) with very slight-slight lymphocytic infiltration in the glandular stomach mucosa. One male (20 mg/kg bw/day) with very slight lymphocytic and neutrophile infiltrations in the muscular layer.

- Liver: all males/3 females with very slight - slight centrilobular hypertrophy of hepatocytes (500 mg/kg bw/day).

Incidental findings affecting only single treated animals or noted in the control group:
Almost all animals in control and treated groups with very slight-moderate periportal fatty changes (13/13/13/12 males and 12/11/13/13 females at 0/20/100/500 mg/kg bw/day, respectively).

- Testes: single animal findings (very slight granulation tissue in the seminiferous tubule interstitium in 1 animal at 500 mg/kg bw/day; 1 animal of control and 1 animal of 100 mg/kg bw/d showed very slight atrophy in the seminiferous tubule). None of these abnormalities was deemed to cause a failure of conception.

- Epididymes: single animal findings (slight unilateral sperm granuloma) at 100 mg/kg bw/day.
High dose and control group animals showed comparable interstitial lymphocytic infiltration in the prostate. No abnormalities were seen in the seminal vesicle.

- Kidney: 2 females and 1 male (500 mg/kg bw/d) with very slight -moderate basophilic tubules in the cortex (vs. 4 males and 1 female in controls); 1 of these females with slight vacuolar degeneration in the proximal renal tubule.

- Heart (only males): very slight - slight myocardial degeneration/fibrosis (9/10/8/12 males at 0/20/100/500 mg/kg bw/day, respectively). There were no differences in the onset frequency and severity between the control group and each test substance-treated group.

- Spleen: All male and female animals (control; 500 mg/kg bw/day) with very slight - slight extramedullary hematopoiesis and brown pigmentation. No differences in the frequency and severity between the two groups observed.

- Pancreas: Single animal finding (male 500 mg/kg bw/day) with very slight focal acinar cell atrophy.

- Vagina, Ovary and Uterus: 1 animal (control) with cyst in the lamina propria; no other abnormalities observed.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
During pre-mating no significant differences in mean cycle length were observed. Mean times of vaginal estrous during mating period were not significantly different between test groups.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw/day, a statistically significant decrease in the number of pregnant dams (6 vs 12 in the control) was noted. The respective fertility index was also reduced (46.2% vs 92.3% in the control). No test substance related effects were observed at 100 or 20 mg/kg bw/day.

All animals mated and the copulation index and time to copulation was not significantly different in the testing groups.
There were no statistically significant differences in the number of corpora lutea, number of implantation sites, implantation index, gestation index and gestation length between the control group and the test substance-treated groups.
No abnormalities were observed during parturition and lactation in any group.
Key result
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no systemic adverse effects noted up to and including the high dose of 500 mg/kg bw/day
Remarks on result:
other:
Remarks:
adaptive, non-adverse effects noted in the liver at 500 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
the findings reported for the rat are considered not to be relevant for humans
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw/day, when calculated for pregnant dams (n = 6), the number of pups born per litter was statistically significantly decreased (9.3 vs 14 in the control). When calculated for dams with live pups (n = 5), the number of liveborn pups (day 0 of lactation) per litter (10.6 vs 13.8 in the control) and the number of live pups (day 4 of lactation) per litter (10.2 vs. 14.8 in the control) was statistically significantly decreased.

The respective delivery index was statistically significantly decreased (66.5% vs 94.7% in the control), and the birth index showed a statistically non-significant decrease (76.0% vs 85.3%). No statistically significant changes in the live birth index and the viability index were observed.

No test substance effects were observed at 20 and 100 mg/kg bw/day.

The effects noted at 500 mg/kg bw/day were considered secondary to fertility effects on the dams, because during the 4 day lactation phase the viability index was not decreased.

Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No external malformations were found in all pups born; and also there were no abnormalities in internal organs at autopsy on day 4 of lactation. In addition, no abnormalities were observed at autopsy of dead pups.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noted up to and including the high dose of 500 mg/kg bw/day
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Summary of pregnancy data

Parameter

Vehicle

(0 mg/kg bw/day)

Low dose

(20 mg/kg bw/day)

Mid dose

(100 mg/kg bw/day)

High dose

(500 mg/kg bw/day)

Copulation index (%)

100

100.0

100.0

100.0

Fertility index (%)

92.3

92.3

92.3

46.2*

Number of corpora lutea

17.8

18.0

16.3

13.3P

Number of implantation sites

14.8

15.6

13.0

12.0P

Implantation index (%)

82.3

87.0

79.1

86.4P

Number of pregnant dams

12

12

12

6*

Number of dams delivering liveborns

12

12

12

5

Gestation index (%)

100.0

100.0

100.0

83.3

Gestation length

22.8

22.3

22.8

23.0

*: Significantly decreased relative to control (p < 0.05)

P: calculated for pregnant females (n = 6)

 

Table 2: Summary of litter data

Parameter

Vehicle

(0 mg/kg bw/day)

Low dose

(20 mg/kg bw/day)

Mid dose

(100 mg/kg bw/day)

High dose

(500 mg/kg bw/day)

Number of Pups Born

14.0

14.3

11.8

9.3* P

Delivery Index (%)

94.7

92.2

90.9

66.5*P

Number of Liveborns (Day 0 of Lactation)

13.8

14.2

11.8

10.6*

Birth Index (%)

85.3

91.6

90.9

76.0#L

Live Birth Index (%)

90.5

99.3

100.0

95.4L

Sex Ratio (♂/♀)

0.7

1.0

1.0

1.0

Number of Live Pups (Day 4 of Lactation)

14.8

14.2

11.8

10.2*L

Viability Index of Newborns on Day 4 after

Birth (%)

98.7

100.0

100.0

96.0L

Pup Weight (g) (Day 0 of Lactation)           Males

Females

7.0

7.0

7.4

6.6

6.5

6.6

7.0

6.1

Pup Weight (g) (Day 4 of Lactation)            Males

Females

11.3

11.3

12.7

11.3

10.8

10.9

12.3

11.1

External Findings

0

0

0

0

Visceral Findings

0

0

0

0

*: Significantly decreased relative to control; #: tendency for decrease compared to control 

P: calculated for pregnant females (n = 6)

L: calculated for females with live pups (n = 5)

Conclusions:
In the present study, the NOAEL with respect to systemic toxicity was set at 500 mg/kg bw/day, the highest dose level tested; findings in liver seen at this dose level were considered as adaptative rather than degenerative effects under the test conditions used. A local NOAEL was set at 20 mg/kg bw/day, which was the lowest dose tested. The local NOAEL refers to irritation effects seen at the mid and high dose levels (100 and 500 mg/kg bw/day) in the forestomach of the rats; these effects however are considered of no relevance for humans, due to their localisation. With respect to reproduction toxicity, the NOAEL for the parental animals was set at 100 mg/kg bw/day, referring to reduced fertility indices noted at 500 mg/kg bw/day. These effects were considered to be secondary to a metabolic acidosis affecting reproduction performance. No adverse effects were noted on live born pups, therefore, with respect to developmental toxicity and under the conditions of the present screening test, the NOAEL is set at 500 mg/kg bw/day.


Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 1996
Deviations:
yes
Remarks:
behaviour was not examined
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Anisaldehyde
EC Number:
204-602-6
EC Name:
Anisaldehyde
Cas Number:
123-11-5
Molecular formula:
C8H8O2
IUPAC Name:
4-methoxybenzaldehyde
Details on test material:
Purity: 99.9%
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the closed bottle at the room temperature
- Stability under test conditions: verified at the beginning and at the end of the test

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crj: CD (SD) IGS
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Tsukuba Breeding Center of Charles River Laboratories Japan, Inc.
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 297.7 – 427.7 g after acclimatization and 403.0 – 356.5 g after grouping; Females: 205.5 – 297.8 g after acclimatization and 254.9 – 218.6 g after grouping for females
- Fasting period before study: no
- Housing: animals were single-housed in metal wire-mesh flooring cages (220 x 270 x 190 mm)
From day 14 post coitum females were housed in a plastic reproduction cage for rats (350 x 400 x 180 mm), to which paper pulp chips (ALPHA-dri manufactured by Kasho Company Limited) were supplied appropriately as bedding. Moreover, the temperature and humidity of the breeding room was set at 23.5-25.5 °C and 45-70 %, respectively during the breeding period.
- Diet: pellets (CE-2 manufactured by Clea Japan, Inc.), ad libitum
- Water: tap water (Hatano City Waterworks Bureau), ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 50 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test article was weighed, dissolved in corn oil and administered within 8 days after the preparation.

VEHICLE
- Concentration in vehicle: 0.4, 2 and 10%(w/v), respectively in the low, mid and high dose groups
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: V8P7069
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
It was confirmed in a previous acute oral study that the prepared test article formulation (0.40, 2.00, 10.0 % (w/v)) contained the test article at the predetermined concentration, respectively. Each 2 mL of the prepared samples were volume-adjusted with n-propanol and measured by high performance liquid chromatography (HPLC; analytical column: Inertsil ODS-2 [4.6 mm i.d. x 150 mm, particle size 5 μm]; mobile phase: water/acetonitrile [60 : 40]). Their concentrations were obtained by using a calibration curve constructed from the measured values (MBA: 10-50 μg/mL).
Duration of treatment / exposure:
- Males: at least 42 days (2 weeks premating, 2 weeks during mating, 2 weeks after the completion of mating period)
- Females: at least 40 days (2 weeks premating, throughout mating periods up to 2 weeks, then throughout pregnancy up to day 4 of lactation after parturition (females copulated without parturition were dosed until the day corresponding to day 24 of gestation).
Frequency of treatment:
daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a 14-day repeated-dose toxicity study, 5 animals per sex and per dose group were orally dosed at 0, 250, 500 and 1000 mg/kg bw/day. Males and females showed salivation after administration from 500 mg/kg bw/day. High dose males and females exhibited significantly depressed food consumption (there was also a tendency toward retarded body weight gain in males). Hematological examination revealed a significant decrease in platelet count for high dose females. Blood biochemical examination showed significant increases in alkaline phosphatase activity for males and GPT activity for males and females as well as a tendency toward an increase in the GOT activities for males and females, in the high dose group.
At autopsy, thickening, edema, and reddish or dark spots of the surface of gastric mucosa were found in high dose males and females, in mid dose females and in low dose males. Additionally, an increase in the absolute liver weight was seen for males and females from 500 mg/kg bw/day onwards, as well as in relative liver weights in males from 500 mg/kg bw/day onwards and females from the 250 mg/kg bw/day onwards.

The high dose for the present study was therefore determined to be 500 mg/kg bw/day, to induce apparent toxicity but no mortality; the medium dose was set at 100 mg/kg bw/day and the low dose was 20 mg/kg bw/day (obtained by dividing the medium dose by a common ratio of 5).
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: More than once a day before and after administration during the dosing period.

BODY WEIGHT: Yes
- Time schedule for examinations: on days 1 (starting day of administration), 7, 14, 21, 28, 35 and 42 of administration as well as on day of autopsy for all males, and on days 1, 7 and 14 of administration for all females. In addition, females that required some time to mate were weighed also on days 21 and 28 of administration. Body weights were also determined on days 0, 7, 14 and 20 of gestation for females copulated, and on days 0 and 4 of lactation as well as on day of autopsy for females with parturition. Females without parturition, that were confirmed to have copulated, were weighed on the day corresponding to day 25 of gestation (day of autopsy).

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to autopsy on the day after day 42 of administration for all males, on the day after day 4 of lactation for females with parturition, and on the day corresponding to day 25 of gestation for females copulated without parturition
- Anaesthetic used for blood collection: Yes (pentobarbital sodium)
- Animals fasted: Yes; 18 - 24 hours
- How many animals: all
- Parameters examined: reticulocyte percentage (control and high dose), red blood cell count (RBC), white blood cell count (WBC), platelet count, hemoglobin content (Hb), mean corpuscular volume (MCV), hematocrit (Ht), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), differential white blood count, prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: subsequent to the blood sampling for the purpose of hematological examination; blood samples were collected again from the abdominal caudal vena cava by using heparin as an anticoagulant.
- Animals fasted: Yes
- How many animals: all
- Parameters examined: total protein, albumin, total cholesterol, glucose, blood urea nitrogen (BUN), creatinine, alkaline phosphatase activity (ALP), GOT activity, GPT activity, γ-GTP activity, triglyceride, inorganic phosphorus(Inorg. phos.), total bilirubin, calcium, A/G ratio, sodium, potassium, chlorine.

URINALYSIS: Yes
- Time schedule for collection of urine: week 6 of administration
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: by collecting approximately 4-hour urines or freshly voided urines; pH, occult blood, protein, sugar, urobilinogen, ketone bodies, and bilirubin were examined through the test paper method, as well as color tone and turbidity by visual inspection, respectively.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Male animals: All surviving animals on the day after day 42
- Maternal animals: All surviving animals on the day after day 4 of lactation
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

ORGAN WEIGHTS: YES
- Brain, heart, thymus, liver, kidneys, spleen and adrenal glands (in addition, testes and epididymes for males) were weighed, and the respective organ weights to body weight (relative weights) were obtained. In females, ovaries and uterus were weighed.

HISTOPATHOLOGY: Yes
- Brain, pituitary gland, spinal cord, digestive tract, liver, kidneys, adrenal glands, spleen, pancreas, heart, thymus, thyroid gland, trachea, lungs, urinary bladder, mesenteric lymph nodes, mandibular lymph nodes, sciatic nerve and femur marrow, in addition, seminal vesicle and prostate ventral lobe, as well as vagina, ovaries and uterus were fixed in 10 % phosphate-buffered formalin. Lungs were fixed by infusion. Testes and epididymes were fixed and preserved in Bouin's fluid, and then moved into 10 % phosphate-buffered formalin for long-term storage. The fixed organs were paraffin sectioned in the usual manner and stained with hematoxylin and eosin, in order to conduct histopathological examination.

Uterus and vagina for 8 females having failed to conceive or having total embryonic resorption in the high-dose group and seminal vesicle and prostate for male mating partners of such females were additionally paraffin-sectioned in the usual manner and stained with hematoxylin and eosin, in order to conduct histopathological examination.
Other examinations:
Reproduction / developmental effects were also evaluated (see 7.8.1-1)
Statistics:
Histopathological graded data and the sum of positive grades were subjected to significance test respectively by using Mann-Whitney U test (level of significance: 5 %) and Fisher’s one-sided exact test (level of significance: 5 %), in comparison with the control group.
Other data, i. e. body weights, food consumptions, hematological and blood biochemical values in the cases subjected to regular autopsy, and organ weights underwent first a test for uniformity of variance (level of significance: 5 %) through Bartlett’s method, by defining the values obtained from individual rat or the average values per litter as one sample. If the variance was uniform, one-way analysis of variance (level of significance: 5 %) was conducted. And if any significance was found between the groups, multiple comparisons were performed through Dunnett’s method (level of significance: 5 %). In contrast, in the cases where the variance was 0 in any group and where the variance was not uniform, Kruskal-Wallis rank test (level of significance: 5 %) was carried out. If any significance was seen between the groups, multiple comparisons were performed through Dunnett’s test (level of significance: 5 %).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
12/13 males and 11/13 females of the high dose group showed transient salivation.
Mortality:
no mortality observed
Description (incidence):
No dead or moribund animals were seen in any sex and treatment group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Males: slightly increased (104%) at both 100, 500 mg/kg bw/day compared to the vehicle control values (not statistically significant) at the end of the study;
- Females:
(pre-)mating: slightly increased body weights (107% at day 14) at 500 mg/kg bw/day compared to the vehicle control values (not statistically significant);
gestation: significantly increased body weights on days 0 and 7 of gestation (113% and 108% of control respectively) at 500 mg/kg bw/day; marked individual differences not reaching statisitcal significance after day 14 of gestation in this dose group;
lactation: significantly increased body weights on day 0 of lactation at 100 and 500 mg/kg bw/day (108% and 115% of control respectively), slight increase on day 4 of lactation (104% of control) at 500 mg/kg bw/day (not statistically significant).
See Tables 1 and 2 for data on male and female body weight development.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Males: significantly increased only on day 35 at 100 mg/kg bw/day; significantly increased from day 7 onward at 500 mg/kg bw/day
- Females: significantly increased during the premating period (days 7 and 13) at 500 mg/kg bw/day
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
platelet count: significant decrease in males at 500 mg/kg bw/day and in females at 100 and 500 mg/kg bw/day (no effects were noted on prothrombin time or activated partial thromboplastin time)
red blood cell count: significant decrease in males at 100 and 500 mg/kg bw/day
mean corpuscular volume: significant increases in females at 100 mg/kg bw/day
mean corpuscular hemoglobin: significant increases in females at 100 mg/kg bw/day

No significant changes in hematocrit value and hemoglobin content (males/females), reticulocyte count and the histopathological examination of bone marrow; spleen did not show any changes related to hematological changes. Therefore, all the changes in the erythrocyte were considered by the authors to occur accidentally. See Table 3 for selected haematological parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A/G ratio: significant increases in males at 500 mg/kg bw/day
A/G ratio: significant decreases in females at 100 mg/kg bw/day
GOT activity: significant increases in males at 500 mg/kg bw/day
Inorganic phosphorus: significant increases in males at 500 mg/kg bw/day
Albumin: significant decreases in females at 100 mg/kg bw/day
Chloride: significant increases in females at 100 mg/kg bw/day
Glucose: significant increase in females at 500 mg/kg bw/day
Calcium: significant decrease in females at 500 mg/kg bw/day

Females changes (A/G ratio, albumin, chloride) were not dose-dependent and therefore considered not to be test substance related.
Male changes (A/G ratio, inorganic phosphorus) were - although statistically significant - considered to be of no toxicological relevance because their were only marginal in magnitude, not observed in females or changes noted in females were reciprocal in nature.
See Table 4 for selected clinical biochemistry parameters.
Urinalysis findings:
no effects observed
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):
Liver: increase in absolute (113%; statistically non-significant) and relative (by 111%; statistically significant) weight in males at 500 mg/kg bw/day
Liver: increase in absolute (117%; statistically significant) and relative (109%; statistically non-significant) weight in females at 500 mg/kg bw/day
Epididymis: statistically significant decreases in absolute/relative (91%/88%) weight in males at 500 mg/kg bw/day
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related findings were noted at 500 and 100 mg/kg bw/day (dose-dependent incidence):
500 mg/kg bw/day
Males: 1 animal with black spots in the glandular stomach mucosa
Females: 3 animals with dark or black spots/areas in glandular stomach; 1 animal with black deposits in glandular stomach

Females in 100 mg/kg bw/day
2 animals with thickening of the forestomach mucosa, one of which was accompanied by edema, the other had black spots in the glandular stomach mucosa.

Incidental findings affecting only single treated animals or noted in the control group:
500 mg/kg bw/day
Males: 1 animal with epididymal nodule
Females: 1 animal with cystic ovarian bursa

100 mg/kg bw/day
Single animal findings (dark-red spot in the lung; ovarian cyst on the right side; accumulated white gelatinous content in the vagina cavity)

Females in 20 mg/kg bw/day
Single animal findings (darkening of the caudate lobe of the liver, diverticulum in the ileum, abnormal formation of kidney)

Males and females in the control group:
Single animal findings (1 female with small thymus; 1 male with dark-red spot in the lung)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related findings were noted at 500 and 100 mg/kg bw/day (dose-dependent incidence):
- Stomach: squamous cell hyperplasia in the forestomach of all animals at 500 mg/kg bw/day and also in the forestomach for 3 (male)/5 (female) animals at 100 mg/kg bw/day.

- Liver: all males/3 females with very slight - slight centrilobular hypertrophy of hepatocytes (500 mg/kg bw/day).

Incidental findings affecting only single treated animals or noted in the control group:

Stomach: in female animals, very slight-slight submucosal forestomach edema in 1/1/5/3 animals (0/20/100/500 mg/kg bw/day). Single female (controls) and 2 females (100 mg/kg bw/day) with very slight-slight lymphocytic infiltration in the glandular stomach mucosa. One male (20 mg/kg bw/day) with very slight lymphocytic and neutrophile infiltrations in the muscular layer.

Liver: almost all animals in control and treated groups with very slight-moderate periportal fatty changes (13/13/13/12 males and 12/11/13/13 females at 0/20/100/500 mg/kg bw/day, respectively).

- Testes: single animal findings (very slight granulation tissue in the seminiferous tubule interstitium in 1 animal at 500 mg/kg bw/day; 1 animal of control and 1 animal of 100 mg/kg bw/d showed very slight atrophy in the seminiferous tubule). None of these abnormalities was deemed to cause a failure of conception.

- Epididymes: single animal findings (slight unilateral sperm granuloma) at 100 mg/kg bw/day.
High dose and control group animals showed comparable interstitial lymphocytic infiltration in the prostate. No abnormalities were seen in the seminal vesicle.

- Kidney: 2 females and 1 male (500 mg/kg bw/d) with very slight -moderate basophilic tubules in the cortex (vs. 4 males and 1 female in controls); 1 of these females with slight vacuolar degeneration in the proximal renal tubule.

- Heart (only males): very slight - slight myocardial degeneration/fibrosis (9/10/8/12 males at 0/20/100/500 mg/kg bw/day, respectively). There were no differences in the onset frequency and severity between the control group and each test substance-treated group.

- Spleen: All male and female animals (control; 500 mg/kg bw/day) with very slight - slight extramedullary hematopoiesis and brown pigmentation. No differences in the frequency and severity between the two groups observed.

- Pancreas: Single animal finding (male 500 mg/kg bw/day) with very slight focal acinar cell atrophy.

- Vagina, Ovary and Uterus: 1 animal (control) with cyst in the lamina propria; no other abnormalities observed.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
the findings as reported for the rat are considered not to be relevant for humans
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no systemic adverse effects noted up to and including the high dose of 500 mg/kg bw/day
Remarks on result:
other:
Remarks:
adaptative, non-adverse effects seen in the liver at 500 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Table 1. Body weight changes in males

 

Body weight gain (g, mean ± S.D.)

Dose level

(mg/kg bw/day)

0

20

100

500

N

13

13

13

13

Day of administration

 

1 (initial body weight)

381.8 ± 12.0

385.1± 13.2

383.2± 13.5

384.4± 13.4

7

406.3 ± 17.1

407.9± 20.3

413.8± 21 1

413.1± 21.3

14

431.7 ± 24.1

434.7 ± 20.3

440.4 ± 28.3

442.9 ± 27.4

21

451.8 ± 27.2

458.0 ± 21.8

467.8 ± 30.9

469.1 ± 32.5

28

480.4 ± 33.8

490.2 ± 25.6

498.9 ± 33.9

499.5 ± 35.6

35

407.9 ± 38.5

518.7 ± 31.6

527.6 ± 39.4

529.0 ± 42.2

42

525.8 ± 44.6

537.5 ± 38.7

547.4 ± 44.1

549.2 ± 45.8

Table 2. Body weight changes in females

 

Body weight gain (g, mean ± S.D. (N)

Dose level

(mg/kg bw/day)

0

20

100

500

Day of administration

 

1

236.5 ± 8.4 (13)

237.4 ± 10.8 (13)

237.0 ± 9.6 (13)

237.9 ± 9.8 (13)

7

247.1 ± 10.9 (13)

251.2 ± 12.4 (13)

252.1 ± 13.1 (13)

257.5 ± 16.2 (13)

14

254.5 ± 13.0 (13)

259.6 ± 15.4 (13)

260.4 ± 16.4 (13)

271.4 ± 19.4 (13)

21

 

 

 

303.3 (1)

Day of pregnancy

 

 

 

 

0

262.1 ± 14.3 (12)

269.2 ± 16.0 (12)

271.6 ± 13.0 (12)

249.9 ± 15.2 **(6)

7

302.4 ± 11.7 (12)

307.7 ± 16.5 (12)

309.7 ± 16.3 (12)

325.8 ± 19.3 * (6)

14

340.7 ± 14.8 (12)

346.8 ± 15.8 (12)

349.0 ± 18.9 (12)

359.9 ± 29.6 (6)

20

412.2 ± 31.7 (12)

420.4 ± 15.5 (12)

414.1 ± 30.3 (12)

416.6 ± 61.9 (6)

Day of lactation

 

 

 

 

0

306.7 ± 14.7

317.7 ± 20.2

331.5 ± 21.9 **

352.3 ± 14.1 * (5)

4

337.4 ± 16.9

335.7 ± 12.5

335.7 ± 15.9

352.1 ± 11.7 (5)

*  significantly different from control, p < 0.05

** significantly different from control, p < 0.01

Table 3. Selected haematological findings

Males (N = 13)

Dose level

(mg/kg bw/day)

RBC

(x104/mm3)

Hemoglobin

(g/dL)

Hematocrit

(%)

MCV

(µm3)

MCH
(pg)

Platelet

(x104/mm3)

PT

(sec)

APTT

(sec)

0

811 ± 35

15.2 ± 0.6

44.6 ± 1.8

55.0 ± 1.9

18.8 ± 0.6

91.6 ± 7.4

14.6 ± 1.5

20.9 ± 1.6

20

801 ± 33

15.2 ± 0.5

44.3 ± 2.1

55.3 ± 1.3

19.0 ± 0.5

94.7 ± 11.5

14.5 ± 1.4

21.4 ± 2.3

100

771 ± 33**

14.8 ± 0.8

43.1 ± 1.9

55.9 ± 1.9

19.2 ± 0.6

86.2 ± 7.7

14.4 ± 1.1

21.4 ± 1.5

500

779 ± 23*

14.9 ± 0.4

43.3 ± 1.5

55.6 ± 1.7

19.1 ± 0.6

82.4 ± 8.6*

13.8 ± 1.0

19.7 ± 1.9

Females (N = 13)

0

684 ± 48

13.3 ±0.8

39.8 ± 2.4

58.2 ± 1.5

19.4 ± 0.6

108.6 ± 11.5

13.0 ± 0.5

17.9 ± 1.2

20

649 ± 44

13.0± 0.8

38.8 ± 2.0

58.8 ± 2.2

20.0 ± 0.6

111.2 ± 9.3

12.7 ± 0.6

17.1 ± 1.5

100

649 ± 67

13.2 ± 1.3

39.1 ± 4.0

60.3 ± 2.3*

20.3 ± 0.7**

96.6 ± 11.8*

12.5 ± 0.6

16.9 ± 0.9

500

712 ± 69

14.0 ± 1.1

41.5 ± 3.5

58.4 ± 1.9

19.7 ±0.7

87.3 ± 8.9**

12.9 ± 0.7

16.8 ± 0.8

*  significantly different from control, p < 0.05

** significantly different from control, p < 0.01

Table 4. Selected clinical biochemistry findings

Males (N = 13)

Dose level

(mg/kg bw/day)

Total protein

(g/dL)

Albumin

(g/dL)

A/G

ALP

(U/L)

GPT
(U/L)

GOT

(U/L)

Inorg. Phosph.
(mg/dL)

0

5.5 ± 0.3

3.0 ± 0.2

1.23 ± 0.19

211 ± 68

30 ± 4

62 ± 7

6.3 ± 0.3

20

5.5± 0.2

3.1 ± 0.2

1.37 ± 0.12

243 ± 47

27 ± 4

57 ± 5

6.5 ± 0.5

100

5.3± 0.3

3.0 ± 0.2

1.30 ± 0.14

210 ± 44

31 ± 5

34 ± 5

6.2 ± 0.4

500

5.2± 0.2

3.1 ± 0.2

1.42 ± 0.16**

248 ± 86

39 ± 16

84 ± 46*

6.7 ± 0.7*

Females (N = 13)

0

5.5 ± 0.4

3.2 ± 0.3

1.44 ± 0.16

113 ± 22

42 ± 15

77 ± 18

6.3 ± 0.9

20

5.5 ± 0.4

3.1 ± 0.2

1.37 ± 0.18

123 ± 98

38 ± 8

68 ± 9

6.1 ± 0.6

100

5.3 ± 0.3

2.9 ± 0.2*

1.24 ± 0.08**

125 ± 34

40 ± 12

74 ± 16

6.0 ± 0.8

500

5.2 ± 0.2

3.1 ± 0.2

1.54 ± 0.20

124 ± 35

67 ± 26

67 ± 26

6.1 ± 0.6

*  significantly different from control, p < 0.05

** significantly different from control, p < 0.01

Applicant's summary and conclusion

Conclusions:
No adverse systemic findings were noted in this OECD 422 study. The findings on local irritation of the forestomach are not considered relevant for humans. Accordingly, the data on the repeated dose toxicity of p-anisic acid are conclusive but not sufficient for classification.