Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-02-16 until 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

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 exposure:
PREPARATION OF DOSING SOLUTIONS:

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
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max 14 days
- Proof of pregnancy: vaginal plug or presence of sperm in vagina referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
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 technic. The formulation were 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: base on the results of previous toxicity work
Positive control:
no

Examinations

Parental animals: Observations and examinations:
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 8motor 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
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.


Postmortem examinations (offspring):
SACRIFICE
- all offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations
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).
Reproductive indices:
pre-coital indices; fertility indices (mating index and pregnancy index), gestation length, parturition index, implantation losses (pre-implantation and post-implantation losses)
Offspring viability indices:
live birth index, viability index, sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced body weight gain, reduced food consumption in females, increased water consumption in males
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
reduced body weight gain, reduced food consumption in females, increased water consumption in males
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
extramedullary hemopoiesis in spleen, hepatocellular hypertrophy, increase incidence of hyaline droplets in kidneys in males
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

See repeated dose toxicity, Dunster and Watson, 2012

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
repeated dose toxicity
Effect level:
>= 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related findings in the fertility of parents

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
teratogenicity
Generation:
F1
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related findings in offspring

Overall reproductive toxicity

Reproductive 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 reproduction/ developmental toxicity was considered to be 100 mg/kg/day for parent and offspring.
The NOAEL for repreated dose toxicity is considered to be 20 mg/kg bw for the rat under the conditions of this study.
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.

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.