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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-02-16 to 2012-11-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study performed under GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-ETHYLANTHRAQUINONE
- Physical state: yellow solid flakes
- Analytical purity: 98.83%
- Impurities (identity and concentrations): not reported
- Purity test date: not reported
- Lot/batch No.: 110929
- Expiration date of the lot/batch: 31 December 2012
- Stability under test conditions: not reported
- Storage condition of test material: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K.Ltd.; Blackthorn, Bicster, Oxon, UK
- Age at study initiation: (P) 12 wks
- Weight at study initiation: (P) Males: 316-366 g; Females: 189-222 g
- Fasting period before study: none
- Housing: groups of five, during pairing phase one male:one female
- Diet (e.g. ad libitum): ad libitum, Rodent 2018C Tejlad Global Certified Diet, Harlan
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 -23
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 March 2012 To: 5 May 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): arachis oil BP
- Concentration in vehicle: 1.25 mg/mL, 5 mg/mL and 25 mg/mL for low, mid and high dose administration
- Amount of vehicle (if gavage): 4 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the test item formulation was verified by high performance liquid chromatography (HPLC) using an external standard technique. The formulation was extracted with methanol to give a final theoretical concentration of 0.01 mg/mL.
The test item formulations were mixed thoroughly and samples were taken from the bottom, the middle and the top of the container, shaking between sampling. Sampling was performed in triplicates.
Stability, homogeneity and correctness of the prepared concentrations were analysed.
Duration of treatment / exposure:
Exposure period males: 43 days
Exposure period females: 43-45 days
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 20 and 100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of previous toxicity work
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were examined for overt signs of toxicity, ill-health and behavioural changes immediately before dosing, up to 30 minutes after dosing, and one and five hours after dosing, during working days. On weekend and public holidays animals were observed immediately before dosing, soon after dosing, and one hours after dosing (except for females after parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- prior to start of treatment and then weekly. All animals were observed for signs of functional/behavioural toxicity. Functional performance test (motor activity, forelimb/hindlimb grip strength) and assessment of sensory reactivity were performed on 5/sex/dose prior to termination.

BODY WEIGHT: Yes
- Time schedule for examinations: day 1 prior to dosing, and then weekly for males until termination and weekly for females until mating was evident. After this body weight was recorded for females on day 7, 14 and 20 post coitum, and on day 1 and 4 post partum.

FOOD CONSUMPTION :
- Food consumption for each cage determined weekly during pre-mating period and continued for males after mating period. For females after evidence of mating, food consumption was recorded for the periods covering post coitum days 0-7, 7-14 and 14-20. For females with litters, food consumption was recorded on day 1 and 4 pp.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily with exception of pairing phase

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior of termination
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 5/sex/group
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior of termination
- Animals fasted: No
- How many animals: 5/sex/group
- Parameters checked in table 2 were examined.

URINALYSIS: Yes, from 5 males/group
- Time schedule for collection of urine: final week of treatment
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked: under table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: functional/behaviour toxicity in all animals prior treatment and with weekly intervall. Functional performance test was performed in 5/sex/group prior to termination.
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity, grip strength , motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 5)
Statistics:
Bartlett's test was used for analysing homogeneity of variance from mean values. Intergroup variance were assessed using ANOVA or ANCOVA. Williams test for parametric data, Shirley test for non-parametric data, or if no dose response was found data were analysed by stepwise Dunnett's (parametric) or Steel (non-parametric) test. Pair-wise test was performed using Student t-test (parametric) or Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced body weight gain
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
reduced food consumption in females
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
reduced food efficiency
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
increased water consumption in males
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
increased mean cell Hb, cell volume and reticulocytes in females, Hb, erythrocyte count and HK reduced in males
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
increase in bile acid and creatinine in males and total protein in females
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
light amber coloured urine in highest dose
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased spleen and liver weight
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
increased extramedullary hemopoiesis in spleen, hepatocellular hypertrophy, increase incidence of hyaline droplets in kidneys in males.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One female treated with 100 mg/kg bw/day died following bleeding on Day 4 post partum. Animals of both sex treated with 100mg/kg had increased salivation on day 2 (males) and day 10 (females). Episodes of increased salivation were also evident in animals of both sex treated with 20 mg/kg from day 10. One female had generalised fur loss between day 21 and 24. No toxicological significant changes in behaviour could be found. (see Table 3)

BODY WEIGHT AND WEIGHT GAIN
Animals treated with 100 mg/kg showed significant reduction in bw gain during first week of exposure. 5 females showed even bw loss in week 1 and after this reduction in bw gain during maturation. (please see also tables attached)

FOOD CONSUMPTION
Females treated with 100 mg/kg showed reduced food intake in week 1 and during first 2 weeks of gestation.

FOOD EFFICIENCY
food efficiency was reduced in both sex of 100 mg/kg group during first week of treatment.

WATER CONSUMPTION
Males of all treatment groups showed an increase of water consumption throughout the treatment period. Females of treatment group 100 and 20 mg/kg showed also an increase in water consumption during maturation, gestation and lactation. This is commonly observed following oral administration of an unpalatable test item formulation and isolated not considered to be of toxicological importance.

HAEMATOLOGY
Males at 100 mg/kg showed reduced Hb, RBC and HK. Females of this dose group showed increased MCH mean cell volume and reticulocyte count.(please see also tables attached)

CLINICAL CHEMISTRY
Males treated with 100 mg/kg showed increase in bile acid and creatinine. Females of same dose group showed increase in total protein. The effects seen in mid and low dose animals were in the physiological range of these animals (please see also tables attached)

URINALYSIS
Males treated with 100 mg/kg had light amber coloured urine.

ORGAN WEIGHTS
increased in absolute and relative spleen and liver weight at 100 mg/kg in both sex. Males at the same dose group showed also increase in absolute and relative kidney weight.

GROSS PATHOLOGY
Animals of both sex had at 100 mg/kg reddened lungs at necropsy (incidental, no histological findings). One female at 5 mg/kg bw had malformed median lobe of the liver and mass in the mid-thoracic region. One female at 20 mg/kg had also malformed left liver lobe (incidental occurrence). One male hare increased pelvic space in the right kidney which is considered as congenital abnormality. One other male of high dose group has a small left epididymis and left testis. (see Table 4)

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver: central to diffuse hepatocellular hypertrophy was at minimal severity in both sex at 100 mg/kg. In teh spleen at 100 mg/kg extramedullary hemopiesis mainly erythropoiesis was seen in males and females. In the kidneys increased incidence of hyaline droplets in the epithelium and tubular basophilia was observed in males at 100 mg/kg. (see Table 5)

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 3: Summary Incidence of Daily Clinical Observation

Findings

Group 1

0 mg/kg bw/d

Group 2

5 mg/kg bw/d

Group 3

20 mg/kg bw/d

Group 4

100 mg/kg bw/d

Treatment/No of animals

10 M

10 F

10 M

10 F

10 M

10 F

10 M

10 F

Scheduled kill

10

10

10

10

10

10

10

9

found dead

0

0

 0

0

1 (after bleeding)

increased salivations

0

0

 0

 0

 4

10

10

 Generalised fur loss

 0  0  0  0  0  0  0  1

 M: Males; F: Females

Table 4: Necropsy findings

Findings

Group 1

0 mg/kg bw/d

Group 2

5 mg/kg bw/d

Group 3

20 mg/kg bw/d

Group 4

100 mg/kg bw/d

No of findings/No of animals

10 M

10 F

10 M

10 F

10 M

10 F

10 M

9 F

Epididymides, small, left

0/10

-

0/10

-

0/10

-

1/10

-

Kidneys, increased pelvic space, right

0/10

-

0/10 

 -

0/10 

1/10

-

Testes, small, left

0/10

-

 0/10

 -

0/10 

 -

1/10

-

 Liver, malformed left lobe

-  0/10  -  0/10  -  1/10 - 0/9

 Liver, malformed median lobe

 -  0/10  -  1/10  -  0710  -  0/9

 Lungs, reddened

 -  1/10  -  0/10  -    -  1/9

 Spinal cord, mass mid-thoracic

 -  1/10  -  0/10  -  0/10  -  0/10

 M: Males; F: Females

Table 5: Histopathology findings

Findings

Group 1

0 mg/kg bw/d

Group 2

5 mg/kg bw/d

Group 3

20 mg/kg bw/d

Group 4

100 mg/kg bw/d

Incidence/Mean Severity

5 M

5 F

5 M

5 F

5 M

6F

5 M

5 F

Liver: Hepathocellular hypertrophy

-

-

-

-

-

-

3/1.0

2/1.0

Spleen: Extramedullary hemopoiesis

5/1.0

5/2.0

5/1.0 

5/2.0

5/1.4

5/2.2 

5/1.8

5/2.8

Kidney: Hyaline droplets

1/1.0

-

 2/1.0

 -

2/1.0 

 -

1/10

-

 Kidne: Tubular basophilia  1/1.0  -  2/1.0  -  1/1.0  2/1.0  5/1.0  1/1.0

 M: Males; F: Females

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the NOAEL for repeated dose toxicity is considered to be 20 mg/kg bw for the rat.
Executive summary:

2 -ethylanthraquinone was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats with application via the oral route according to OECD Guideline 422 and GLP. The test substance was administered as a solution in Arachis oil BP orally by gavage to 3 groups of 10 male and 10 female Wistar rats each, once a day on 7 days per week for 43 - 45 consecutive days.

Doses used:

•         Group 2 (low dose): 5 mg per kg body weight and day,

•         Group 3 (mid dose): 20 mg per kg body weight and day,

•         Group 4 (high dose): 100 mg per kg body weight and day.

An equally sized negative control group (group 1) received Arachis oil BP, the vehicle for the test substance. The duration of treatment covered a 2 -week pre-mating period in both sexes, the mating period, the gestation period as well as 4 days of lactation in females and treatment up to day 43 for males.

One unscheduled death occurred in the high dose female group on day of termination after bleeding. All other animals survived until termination. No treatment related effects could be observed on reproductive/developmental parameters. Therefore the NOAEL for reproductive toxicity was considered to be 100 mg/kg bw/day.

The NOAEL for general, systemic parental toxicity of the test substance was determined to be 20 mg/kg bw/day based on adverse clinical pathological findings at next higher dose (reduced body weight gain, reduced food consumption in females, changes in haematology, increased extramedullary hemopoiesis in spleen, increased spleen weight, increase bile acid in males and total protein in females). Changes in liver weight connected with hepatocellular hypertrophy is commonly observed in rat liver following administration of xenobiotics and does not represent an adverse effect. Increase incidence of hyaline droplets in kidneys of males is a well known response to treatment with some hydrocarbons and known to represent limited relevance to human.