Registration Dossier

Administrative data

Description of key information

Subacute repeat oral dose toxicity study (OECD 407): NOAEL 10 mg/kg bw/day (Umano 2012)

Combined repeat dose toxicity study with reproductive toxicity screening (OECD 422): NOAEL 30 mg/kg/day; Anon (2011)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
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: Not reported
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Confirmed
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not reported
- Preliminary purification step (if any): Not reported
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: Not reported

FORM AS APPLIED IN THE TEST (if different from that of starting material): Not reported

OTHER SPECIFICS: n/a
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Crj:CD (SD) rats were purchased from Charles River, Japan, Inc (Shiga, Japan)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Crj:CD (SD) rats were purchased from Charles River, Japan, Inc (Shiga, Japan)
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 8 weeks
- Weight at study initiation: ca. 310 - 340 g (males) and ca. 203 - 226 g (females)
- Fasting period before study: Not reported
- Housing: Housed individually in stainless stell wire-mesh cages.
- Diet (e.g. ad libitum): Ad libitum, commercial diet (MF, Oriental Yeast Co., Tokyo, Japan)
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Not reported

DETAILS OF FOOD AND WATER QUALITY: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 55 ± 20 %
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12: 12 (light: dark)

IN-LIFE DATES: Not reported
Route of administration:
oral: gavage
Details on route of administration:
The volume of the olive oil that contained the chemical for gavage was 5 mL/kg.
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): olive oil - OECD recommended to aid solubility of test item.
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 mL/kg/bw
- Lot/batch no. (if required): Not reported
- Purity: Not reported
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Chosen after completition of a range-finder test. Concentrations did not induce death or severe suffering.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No
- Post-exposure recovery period in satellite groups: n/a
- Section schedule rationale (if not random): Males were necropsied on Day 29. Females were necropsied after having been dosed for at least 29 days and killed ondays 30–33 to allow them to be killed in the diestrous stage or in the continuous same stage.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in Table 2 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): n/a
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: n/a
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: n/a

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): n/a
- Time schedule for examinations: n/a

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (chemical name not reported)
- Animals fasted: Not specified
- How many animals: 20 animals per treatment group (10 males and 10 females, 80 animals in total)
- Parameters checked in Table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: Not specified
- How many animals: 20 animals per treatment group (10 males and 10 females, 80 animals in total)
- Parameters checked in Table 3 were examined.

URINALYSIS: No
- Time schedule for collection of urine: n/a
- Metabolism cages used for collection of urine: n/a
- Animals fasted: n/a

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 4th week after test initialtion
- Dose groups that were examined: Control, 10, 30 and 100 mg/kg bw/day
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a

OTHER: Hormone analysis (in serum), estrous cycle, spermatology and histopathology observations were also undertaken.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes - Table 4

HISTOPATHOLOGY: Yes - Table 4
Statistics:
Bartlett’s variance test was performed for the parametric data (body weight, food consumption, hematological data, clinical biochemical data, hormonal data, organ weight, sperm count, grip strength, and locomotor activity). Bartlett’s test revealed a homogeneous variance, so one-way analysis of variance was conducted and if the result of the one-way analysis was significant, Dunnett’s test was performed to compare the treated and the control groups. Data with an inhomogeneous variance shown by Bartlett’s test or nonparametric data (the number of stools, the number of urinary pools, and incidence rate of abnormal spermatozoa) were subjected to the Kruskal–Wallis rank test, and if a significant divergence was observed, a Dunnett’s approach was carried out. Incidence rates of abnormal estrous cycles, gross pathological findings, and histopathological findings were analyzed by Fisher’s “exact” probability test. In the evaluation of the examination results, when a divergence from the control was found at a significance level of 1 or 5%, it was regarded as a significant change.

A male rat in the control group was diagnosed as being the subject of an administration error on gross and histopathological examination. The error was thought to have happened just before day 22, based on changes in body weights and food consumption, so data that contained body weights at days 26 and 28, food consumption at days 22 and 28, and all organ weights of this rat were excluded from the statistical analysis.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Several male and female rats in the 100 mg/kg group showed salivation from the first week, and this sign disappeared within 90 min of administration. No other abnormal general findings were observed in any of the groups.

Salivation in the 100 mg/kg group was not found to be a sign of toxicity because this sign disappeared soon after administration and no other related changes were observed.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A decrease in body weight gains was found in the male rat 100 mg/kg group and the female rat 30 and 100 mg/kg groups from the first week after administration and was accompanied by decreased food consumption.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A decrease in body weight gains was found in the male rat 100 mg/kg group and the female rat 30 and 100 mg/kg groups from the first week after administration and was accompanied by decreased food consumption.
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):
In male rats, white blood cell counts, total cholesterol, and albumin values decreased in the 100 mg/kg group. In female rats, cholinesterase and total cholesterol values decreased and total bilirubin values increased in the 100 mg/kg group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In male rats, white blood cell counts, total cholesterol, and albumin values decreased in the 100 mg/kg group. In female rats, cholinesterase and total cholesterol values decreased and total bilirubin values increased in the 100 mg/kg group.

Serum T4 values increased in the 100 mg/kg groups of both sexes, but no changes in TSH were detected in any treated groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males, the relative weights of kidney, adrenal, and brain increased signifcantly in the 100 mg/kg group and the absolute weights of prostate, ventral prostate, seminal vesicle, liver, heart, and spleen decreased in this group. In female rats, the relative brain weights increased significantly in the 30 and 100 mg/kg groups, and the absolute heart weights decreased in the 100 mg/kg group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Dilatation of the large intestinal lumen was observed in 9 male and female rats in the 100 mg/kg groups, respectively.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the male rats, the incidence of changes, such as atrophy of testicular Leydig cells, hypertrophy of the adrenal zona fasciculata, and decreased hepatocytic glycogen, was higher in the 100 mg/kg group than in the control group. In addition, decreased hematopoiesis in the bone marrow and spleen, atrophy of the mammary glands, and atrophy of pituitary basophilic cells were also observed in the 100 mg/kg group. In female rats, hypertrophy of the adrenal zona fasciculata and decreased hepatocytic glycogen were detected in several rats given the chemical.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Sperm analysis - no effects observed.

Estrous cycle - effects observed, treatment-related. Irregular estrous cycles were observed in the 30 and 100 mg/kg groups, and the diestrous stage continued in some animals. The mean duration of estrous cycles was prolonged in the 100 mg/kg group without statistical diverence (0 mg/kg group = 4.2 ± 0.4 days, 100 mg/kg group = 4.9 ± 0.9 days), and estrous cycling days were not measured in one and three of 10 rats in the 30 and 100 mg/kg groups, respectively, due to the irregularity of their estrous cycles.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other:
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
seminal vesicle
other: Prostate weights
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
endocrine system
Organ:
adrenal glands
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 5       Mean body weight changes following 28 days of treatment

Dose Group

Mean Initial Body Weight (g)

Mean Terminal Body Weight (g)

Male

Female

Male

Female

Control

324 ± 14

211 ± 7

449 ± 27

274 ± 18

10 mg/kg/day

324 ± 13

215 ± 11

451 ± 26

277 ± 18

30 mg/kg/day

325 ± 15

213 ± 10

450 ± 33

255 ± 18*

100 mg/kg/day

326 ± 13

214 ± 11

396 ± 29**

253 ± 15*

* statistically different from control group (p < 0.05)

** statistically different from control group (p < 0.01)

Table 6:         Selected haematology, clinical chemistry and pathology findings

 

Daily Dose

(mg/kg bw/day)

Male

Female

Control

10

30

100

Control

10

30

100

No. of animals

10

10

10

10

10

10

10

10

Haematology

- WBC (x103/mm3)

12.28 ± 1.05

11.83 ± 2.66

11.17 ± 2.67

9.17 ± 2.46*

8.59 ± 2.49

9.06 ± 2.15

9.28 ± 1.77

8.90 ± 1.84

Blood chemistry

- Cholinesterase (IU/L)

54 ±18

56 ± 19

48 ± 11

50 ± 10

525 ± 163

537 ± 98

465 ± 236

300 ± 127*

- Total cholesterol (mg/dL)

69 ± 8

60 ± 12

60 ± 9

49 ± 7**

69 ± 12

65 ± 7

60 ± 11

47 ± 5**

- Total bilirubin (mg/dL)

0.03 ± 0.02

0.02 ± 0.01

0.02 ± 0.01

0.03 ± 0.02

0.02 ± 0.01

0.01 ± 0.01

0.02 ± 0.01

0.04 ± 0.02**

- Albumin (g/dL)

3.00 ± 0.28

3.05 ± 0.12

3.05 ± 0.09

2.84 ± 0.11**

3.51 ± 0.45

3.56 ±0.16

3.35 ± 0.33

3.30 ± 0.27

- T4 (ng/dL)

3.704 ± 0.452

4.158 ± 0.826

4.061 ± 0.846

4.754 ± 0.762**

2.397 ± 0.297

2.376 ± 0.443

2.826 ± 0.950

3.671 ± 0.479**

Histopathology

- Bone marrow,decreased hematopoiesis

0

0

0

4

0

NE

NE

0

- Spleen,decreased extramedullary

0

0

0

2

0

NE

NE

0

- Testis,atrophy of Leydig cells

0

0

0

5*

 

 

 

 

- Mammary gland,atrophy of glands

0

0

0

3

0

NE

NE

0

- Adrenal gland,hypertrophy of zona fasiculata

1

1

0

8**

0

1

1

2

- Pituitary gland,atrophy of basophilic cells

0

0

0

1

0

NE

NE

0

- Liver,decreased hepatocytic glycogen

1

0

1

8**

0

0

1

2

- Organs in thoracic cavity,inflammation and granuloma

1

NE

NE

0

0

NE

NE

0

NE not examined

* significant difference from control (p < 0.05)

** significant difference from control (p <0.01)

Table 7:         Absolute and relative organ weights

 

Daily Dose

(mg/kg/day)

Male

Female

Control

10

30

100

Control

10

30

100

No. of animals

9

10

10

10

10

10

10

10

Organ Weights

Prostate (ventral and dorsolateral)

mg

1068 ± 188

1134 ± 179

1061 ± 189

827 ± 183*

-

-

-

-

g/100 g

0.237 ± 0.045

0.251 ± 0.031

0.236 ± 0.041

0.208 ± 0.039

-

-

-

-

Ventral prostate

mg

631 ± 166

741 ± 110

676 ± 150

474 ± 112*

-

-

-

-

g/100 g

0.139 ± 0.034

0.164 ± 0.021

0.150 ± 0.030

0.120 ± 0.027

-

-

-

-

Seminal vesicle

g

1.41 ± 0.19

1.43 ± 0.30

1.38 ± 0.24

1.02 ± 0.36*

-

-

-

-

g/100 g

0.312 ± 0.045

0.317 ± 0.069

0.307 ± 0.045

0.254 ± 0.079

-

-

-

-

Liver

g

17.13 ± 2.18

16.90 ± 1.37

16.38 ± 2.85

14.10 ± 1.31**

-

-

-

-

g/100 g

3.778 ± 0.322

3.747 ± 0.179

3.623 ± 0.399

3.564 ± 0.271

-

-

-

-

Kidney

g

3.02 ± 0.23

3.01 ± 0.18

3.00 ± 0.37

2.89 ± 0.30

-

-

-

-

g/100 g

0.667 ± 0.021

0.671 ± 0.058

0.666 ± 0.064

0.729 ± 0.043*

-

-

-

-

Heart

g

1.28 ± 0.09

1.32 ± 0.08

1.28 ± 0.09

1.13 ± 0.13**

0.87 ± 0.08

0.88 ± 0.05

0.82 ± 0.11

0.78 ± 0.06*

g/100 g

0.283 ± 0.013

0.293 ± 0.018

0.285 ± 0.011

0.286 ± 0.017

0.316 ± 0.015

0.319 ± 0.021

0.321 ± 0.024

0.308 ± 0.020

Spleen

g

0.71 ± 0.08

0.70 ± 0.08

0.71 ± 0.10

0.59 ± 0.09*

-

-

-

-

g/100 g

0.158 ± 0.018

0.155 ± 0.015

0.158 ± 0.020

0.149 ± 0.016

-

-

-

-

Adrenals

mg

58 ± 8

58 ± 11

56 ± 4

63 ± 9

-

-

-

-

g/100 g

0.013 ± 0.002

0.013 ± 0.002

0.012 ± 0.001

0.016 ± 0.003**

-

-

-

-

Brain

g

2.17 ± 0.05

2.21 ± 0.09

2.23 ± 0.06

2.19 ± 0.07

2.03 ± 0.05

2.00 ± 0.06

2.05 ± 0.10

2.02 ± 0.08

g/100 g

0.482 ± 0.034

0491 ± 0.032

0.498 ± 0.036

0.554 ± 0.035**

0.742 ± 0.042

0.725 ± 0.044

0.809 ± 0.070*

0.799 ± 0.039*

* significantly different from control (p < 0.05)

** significantly different from control (p < 0.01)

Conclusions:
The no adverse effect level (NOAEL) for the test item was determined to be 10 mg/kg bw/day, due to findings of reduced body weight gain and abnormal estrous cycles in the female rat 30 mg/kg bw/day group.
Executive summary:

In a subacute repeat dose toxicity study (OECD 407), Bisphenol AF was administered to 30 male and 30 female Sprague-Dawley rats by oral gavage at dose levels of 10, 30 and 100 mg/kg bw/day for up to 28 consecutive days. In addition, control rats were dosed with vehicle (olive oil) alone for each test group.

A summary of adult responses to Bisphenol AF is described below;

Mortality - no treatment-related deaths were detected.

Clinical signs - clinical signs were generally confined to post-dose increased salvation and staining around the mouth for animals treated at 100 mg/kg/day. Regression of all signs was evident 90 minutes after treatment.

Behavioural assessments - open field observations did not reveal any treatment-related effects.

Functional performance - no treatment-related effects were evident for grip or motor activity.

Sensory reactivity assessments - no treatment-related effects were observed.

Bodyweight - significant (p< 0.01) reduced bodyweight gains were evident for males in the 100 mg/kg/day group during the treatment period. For females, bodyweight gain was significantly (p <0.05) reduced in the 30 and 100 mg/kg/day groups.

Haematology and blood chemistry - for males, a significant reduction in white blood cell count (p < 0.05), cholinesterase and albumin (p < 0.01) was observed in the 100 mg/kg bw/day treatment group. Further, a significant (p < 0.01) increase in T4 hormone was also observed in the same treatment group. For females, a significant decrease in cholinesterase (p < 0.05) and total cholesterol (p < 0.01) was observed in the 100 mg/kg bw/day treatment group. Further, a significant (p < 0.01) increase in total bilirubin and T4 hormone was also observed in the same treatment group.

Oestrous cycle assessment - irregular estrous cycles were observed in the 30 and 100 mg/kg groups, and the diestrous stage continued in some animals. The mean duration of estrous cycles was prolonged in the 100 mg/kg group without statistical difference.

Necropsy - dilatation of the large intestinal lumen was observed in 9 male and female rats in the 100 mg/kg groups, respectively.

Organ weights - in males, the relative weights of kidney, adrenal, and brain increased significantly in the 100 mg/kg group and the absolute weights of prostate, ventral prostate, seminal vesicle, liver, heart, and spleen decreased in the same group. In female rats, the relative brain weights increased significantly in the 30 and 100 mg/kg groups, and the absolute heart weights decreased in the 100 mg/kg group.

Histopathological changes - in the male rats, the incidence of changes, such as atrophy of testicular Leydig cells, hypertrophy of the adrenal zona fasciculata, and decreased hepatocytic glycogen, was statistically significantly higher in the 100 mg/kg group than in the control group. In addition, decreased hematopoiesis in the bone marrow and spleen, atrophy of the mammary glands, and atrophy of pituitary basophilic cells were also observed in the 100 mg/kg group. In female rats, hypertrophy of the adrenal zona fasciculata and decreased hepatocytic glycogen were detected in several rats at each dose level, although these changes were not statistically significantly.

In conclusion, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 10 mg/kg bw/day due to findings of reduced body weight gain and abnormal estrous cycles in the female rat treated at 30 mg/kg bw/day.

This subacute repeat dose toxicity study in the rat is acceptable and satisfies the guideline requirements for an OECD 417 in the rat.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined repeat dose toxicity test with reproduction/ developmental toxicity screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27 October - 22 December 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study was conducted in acordance with International guidelines and in accordance with GLP. Exposure duration in males was not consistent with guideline requirements for repeated dose toxicity, which restricts the reliability of the endpoint for males only.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, dark
- Stability under test conditions: Stable- confirmed by IR spectrums pre and post testing
- Solubility and stability of the test substance in the solvent/vehicle: Confirmed in a previous study (stable for at least 21 days)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Suspended in Arachis oil BP
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 7.5, 25 and 75 mg/mL suspensions prepared
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) Liquid suspension

OTHER SPECIFICS:

Formultions were prepared every 2 weeks and stored at ca. 4 ºc in the dark.
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley Crl:CD (SD) IGS BR strain rats were accepted on to the study
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Females (if applicable) nulliparous and non-pregnant: Yes- at study inititation
- Age at study initiation: ca. 9 weeks old
- Weight at study initiation: 301 - 375 g
- Fasting period before study: No
- Housing:
Initially, animals housed in groups of 4 or 5 in solid floor polypropylene cages (22 x 52 x 33 cm) with stainless steel mesh lids with softwood bedding.

During the mating phase, the non-recovery dose group animals were transferred to polypropylene grid floor cages (20 x 41.5 x 24 cm) suspended over trays lined with absorbent paper on a 1 male: 1 female basis within each dose group. Males were returned to original cages after successful mating.

Mated females were housed individually during gestation and lactation in solid floor polypropylene cages (20 x 41.5 x 24 cm) with stainless stell mesh lids and softwood flakes.

Recovery animals were housed in groups of 4 or 5 in solid florr polypropylene cages (22 x 52 x 33 cm) with stainless steel mesh lids and softwood flake bedding.

- Diet (e.g. ad libitum): free aces to pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories, Oxon, UK)
- Water (e.g. ad libitum): free acess to tap water
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: Diet certified (with CoA's). Neither diet or water considered to contain contaminants at a level that might have affected the integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 55 ± 15 %
- Air changes (per hr): minimum of 15
- Photoperiod (hrs dark / hrs light): 12:12 light: dark

IN-LIFE DATES: From: 27 October 2009 To: 22 December 2009
Route of administration:
oral: gavage
Details on route of administration:
Test material administered by gavage using a stainless steel cunnula attahed to a disposable plastic syringe.
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

Test material was prepared at the appropriate concentration as a suspension in Arachis oil BP. Stability and homogeneity of formulations was verified in a previous study. Fresh formulations were prepared every 2 weeks and stored at ca. +4 ºC in the dark. Subsamples were taken from each formualtion to verify concentration using a validated HPLC method. Measured concentrations were within ± 9 % of the nominal concentration throughout the study.

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 7.5, 25 and 75 mg/mL prepared for 30, 100 and 300 mg/kg/day test groups
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): not reported
- Purity: not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Aliquots of each formulation were analysed using a validated HPLC method. Measured concentrations were within ± 9 % of the nominal concentration throughout the study.
Duration of treatment / exposure:
Test groups and controls: 55 consecutive days
Recovery groups: 42 consecutive days followed by a recovery phase of 14 days where no test item was administered.
Frequency of treatment:
Once, daily
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
High dose
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Intermediate dose
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Recovery high dose
No. of animals per sex per dose:
Treatment groups: 12 males + 12 females per treatment group
Recovery groups: 5 males + 5 females per treatment group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose concentrations were based on the findings of a preliminary study conducted at 1000, 400 and 100 mg/kg bw.
- Rationale for animal assignment (if not random): not specified
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: yes, 14 days
- Section schedule rationale (if not random): not specified
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to 30 mins after dosing, one and 5 hours after dosing, during the working week. Animals were obsered immediately before dosing, soon after dosing and 1 hour after dosing, at weekends. During the treatment-free period, recovery animals were observed twice daily (once at weekends).
- Cage side observations checked in table ## were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: behavioural assessment: weekly
functional performance tests: prior to termination (in 5 males and 5 females per non-recovery treatment group)

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to dosing then weekly (males until termination, females until mating was evident). Female bodyweights were then recorded on Day 0, 7, 14 and 20 post coitum, and on Day 0 and 4 post partum. During the mating phase, females were weighed daily untl mating was confirmed. Reovery animals were weighed before test initiation and weekly thereafter until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Non-recovery males: Day 14 and 42; recovery males: Day 56; non-recovery females: Day 14 and Day 4 post-partum; recovery females Day 56.
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Not specified
- How many animals: 5 per sex and test group
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as above
- Animals fasted: Not specified
- How many animals: 5 per sex and test group
- Parameters checked in table 2 were examined.

URINALYSIS: Yes (males only)
- Time schedule for collection of urine: week 6
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: not specified
- Dose groups that were examined: non-recovery animals (control, 30, 100 and 300 mg/kg/day)
- Battery of functions tested: grip strength, motor activity

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)

HISTOPATHOLOGY: Yes (see table 3)
Statistics:
The following paameters were subjected to statistical analysis;

quantitative functional performance data
bodyweight and bodyweight change
food consumption
water consumption
haematology, blood chemistry and urinalysis
pre-coital intervals
gestation lengths
litter data- litter size, corpora lutea, implantation sites, litter weight
sex ratio
implantation losses, live birth index, viability indices, implantation index and delivery index
offspring bodyweight and bodyweight change
offspring surface righting
adult absolute and bodyweight relative organ weights

The following statistical procedures were used;

Data was assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparissons were conducted using Dunett's test. In case of recovery group data, the analysis used was a two-tailed t-test incorporating Levene's test for homogeneity of variance. Where Levene's test showed unequal variances the data was analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test.

Non parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

Probability values (p) are presented as follows:
P<0.001***
P<0.01**
P<0.05*
P≥0.05 (not significant)

Histapathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes;

1. Chi-squared analysis for differences in the incidence of lesions occuring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Probability values (p) were calculated as follows:

P<0.001 +++ --- ***
P<0.01 ++ -- **
P<0.05 + - *
P<0.1 (+) (-) (*)
P≥0.1 (n.s.)

+/- difference vs. control
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Dehydration and staining around the ano-genital region was evident for one female treted at 300 mg/kg/day on Days 6 and 7.
A second female showed dehydration on Day 7 and was hunched from Day 8-10.
A third female showed staining of the ano-genital region on Day 7.
Regresion of these signs was evident thereafter.

Other signs in the 300 mg/kg/day consisted of increased salivation after dosing and up to one hour after dosing on occasion of animals of either sex during the treatment period. Staining around the mouth were recorded and instances of noisy respiration noted in 5 males and 1 recovery female. Regression of these signs was evident followign cessation of treatment in recovery animals.

Increased salivation was observed in the 100 mg/kg/day group (both sexes) from week 3 with red/brown staining around the mouth observed. The incidence of signs was less in this group vs. the 300 mg/kg/day group. Increased salivation were detected up to 1 hour after dosing in the 30 mg/kg/day group (both sexes).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
300 mg/kg/day female displayed signs of hunched posture, lethargy, laboured and gasping breathing and tiptoe gait. Termination on Day 6 and resulting pathology of this individual concluded that the death was not material toxicity related but rather the result of an inappropriate dosing technique.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males;

300 mg/kg/day - significant reduction through the test period vs. controls. Bodyweight gains were statistically higher in the recovery group during the treatment free period. Reduced bodyweight increases throught the treatment period inevitably resulted in significantly lower mean bodyweights from Day 15 onwards.
100 mg/kg/day - significantly reduced bodyweight gain during the first 3 weeks of treatment.
30 mg/kd/day - no adverse effects noted.

Females;

300 mg/kg/day - 1 individual showed substancial weight loss (28 g) in week 1. Four other individuals showed slight bodyweight losses, resulting in a significant reduction in mean bodyweight gain vs. controls in week 1.
100 mg/kg/day - significantly lower bodyweight gains in the first week of treatment vs. controls. Bodyweight gain during gestation was comparable to controls. Mean bodyweights on Day 0 and 4 of lactation were significantly lower vs. controls.
30 mg/kg/day - significant reduction in bodyweight on Day 0 and 4 post partum.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males;

300 mg/kg/day - significant reduction in consumption during week 1 in non recovery (-22 %) and recovery (-28 %) animals vs. controls. Reduced consumption was also noted in both groups in week 2. Intake ws not measured during mating period however reduced intake was evident during this time. Reductions were evident in recovery animals through the remaining treatment period, although no difference was observed in the non-recovery group vs. controls. During the treatment-free period, treated animals intake was comparable to the controls.
100 mg/kg/day - significant reductions noted pre-mating when compared to controls. Intake improved and was comparable to controls after mating.
30 mg/kg/day - no adverse effects noted.

Females;
300 mg/kg/day - 25 % and 31 % reduction in intake during week 1 compared to controls in non-recovery and recovery groups. Recovery animals showed further reductions in intake up to week 5. Regression was evident during the treatment-free period.
100 mg/kg/day - 19 % reduction in week 1 and significant reductions through weeks 2 and 3 of the gestation period. Intake was comparable to controls during lactation.
30 mg/kg/day - Reduced intake during week 2 of gestation vs. controls.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males;

300 mg/kg/day - reduced efficieny vs control through weeks 1 - 7. Increased in efficiency (vs control) during treatment-free period
100 mg/kg/day - reduced efficiency vs control in week 1, although comparable to control though remainder of test
30 mg/kg/day - no difference vs controls

Females;

300 mg/kg/day - reduction in efficiency in week 1, comparable to controls through remainder of the test
100 mg/kg/day - reduction in week 1, comparable to controls through remainder of the test
30 mg/kg/day - reduction in week 1, comparable to controls through remainder of the test
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males;

300 mg/kg/day - increased water intake noted in non-recovery individuals throught the whole test period versus control. Regression occuring in recovery males during treatment-free period.
100 mg/kg/day - increased water intake pre-mating (statistically) and post-mating (statistically, only in week 5) versus control.
30 mg/kd/day - increased water intake pre-mating (statistically) and post-mating (statistically, only in week 5) versus control.

Females

300 mg/kg/day - significant increase during week 1 and 2 in the recovery females (week 1-3, for non-recovery females) versus controls. Recovery evident during the treatment-free period.
100 mg/kg/day - significant increase during week 1versus controls. Increase during gestation and early lactation , although not statistically significant.
30 mg/kg/day - not significantly different vs controls.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males;

300 mg/kg/day - lower (not statistically significant) haemoglobin and erythrocyte values observed on Day 14. Significant (slight) reduction in reticulocyte counts versus controls on Day 14. At Day 42, significant reduction in haemoglobin and erythrocytes counts were oberved versus controls. Hematocrit counts also lower (although not significantly) at this timepoint. Regression of these findings was observed in the recovery males during the treatment-free period with the exception of erythrocyte counts. Significant increase in mean cell volume and mean cell haemoglobinwas observed in the recovery males versus controls.
30 and 100 mg/kg/day - No changes versus control.

Females;

300 mg/kg/day - no significant differences versus controls.
100 mg/kg/day - significant reduction in mean cell haemoglobin compared to controls on Day 14 (slight and in the absence of a dose-related response and other haemotological changes at this level, this finding was considered to have been incidental).
30 mg/kg/day - no significant change versus controls
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males;

300 mg/kg/day (pre-mating) - significant reduction in blood albumin on Day 14 (pre-mating). Lower A/G ratios and increased alanine aminotransferase levels were also evident, although not significantly so. Significant increase in blood urea levels versus controls. Significantly, blood cholesterol was reduced during pre-mating, versus control values.
300 mg/kg/day (pre-termination) - significant increase in blood urea levels versus controls with reduction in blood albumin levels evident also. Reduction of blood cholesterol and increase in alanine aminotrasferase continued at significant levels.

Regression of changes observed during the treatment-free period in recovery males with slight reduction in plasma bilirubin noted, versus controls. Slight increases in A/G ratio and plasma chloride levels noted.

100 mg/kg/day - significant reduction in blood albumin on Day 14 (pre-mating) and Day 42. Reduction in blood choloesterol on Day 42 versus controls with an increse in alanine aminotransferase also noted.
30 mg/kg/day - reduced blood cholesterol pre-mating

Females;

300 mg/kg/day (pre-mating) - significant reduction in blood albumin and A/G ratios on Day 14 (pre-mating). Significant increase in alanine aminotransferase levels and reduction in plasma chloride levels compared to controls observed. Significantly, blood cholesterol was reduced during pre-mating, versus control values although a dose response curve was not apparent.
300 mg/kg/day (pre-termination) - Day 4 post-partum blood levels were not significantly changed versus the controls.
30 and 100 mg/kg/day - reduced blood cholesterol pre-mating (significant)
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant changes versus controls observed in any of the treated males
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Behavioural assessment - Noisy respiration noted in one female of 300 mg/kg/day group- also noted in clinical observations.

Functional observations - no significant changes in treated animals versus controls

Functional performance - no significant changes in treated animals versus controls

Sensory reactivity assessment - no significant changes in treated animals versus controls
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males;

Individuals treated at 300 mg/kg/day had significant reductions in absolute epididymis weights versus controls, which reflected in lower bodyweight-relative epididymis weights. Lower absolute and bodyweight-relative testis weights were also evident at 300 mg/kg/day in comparison to the controls, althought only statistically significant for absolute weight. Elevated absolute adrenal weights versus controls, with statistically significant increase in bodyweight-relative adrenal weights observed in comparison to the controls. Elevation in bodyweight-relative liver weights were observed versus controls. Organ weight data for recovery males after the 14 treatment-free days still showed elevated bodyweight-relative adrenal weights when compared to controls. Bodyweight-relative spleen and thymus weights were also elevated when compared to controls. Bodyweight-relative brain weights were elevated compared to the controls. In the absence of histopathological correlates, these increases were not considered to represent delayed systemic toxicity.

No effects were noted in the 100 or 30 mg/kg/day treated individuals.

Females;

No effects were detected in treated post partum females compared to controls.

Slight but statistically significant organ weight changes were evident for females treated at 100 and 30 mg/kg/day. These consisted of slight reduction in absolute heart weights (100 and 30 mg/kg/day). Higher bodyweight-relative brain weights were also observed for females treated at 100 and 30 mg/kg/day. A dose-related response was not evident and in the absence of histopathological changes in these organs, these findings were not considered to represent a true effect of treatment.

No significant organ weight changes were noted in the recovery females during the treatment-free period.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mammary gland

Tubuloalveolar differentiation of mamary tissue was seen in males treated at all three concentrations, although only statistically significant data was observed at 300 mg/kg/day. There was no evidence of regression of the observation in the recovery males after the treatment-free period. Minimal glandular hyperplasia of the mammary tissue was seen for four non-pregnant females treated at 300 mg/kg/day. This may have been an effect of treatment. Hyperplasia was not seen in recovery control or 300 mg/kg/day females following completion of the treatment free period.

Ovaries

Follicular cysts were seen among non-pregnant females in the 300 mg/kg/day group, this may have been an effect in the absence of directly comparable controls. Follicular cysts were seen in the recovery females versus controls suggesting that the effect had not regressed.

Testes

Leydig cell atrophy was seen in relation to treatment for males treated with 300 mg/kg/day and at 100 mg/kg/day, although not statistically significant at this level. The condition regressed among the recovery males after the treatment free period. Moderate or severe testicular atrophy was seen for two recovery males. This condition does occur spontaneously among laboratory maintained rats and there was no evidence to suggest this was a treatment related consequence.

Seminal vesicles/ coagulating gland

Reduced secretory content as indicated by smaller organ size was seen in relation to treatment for males treated with 300 mg/kg/day and 100 mg/kg/day compared to control, but not statistically significant at 30 mg/kg/day. There was no evidence of regression of the condition among 300 mg/kg/day males after the treatment-free period has elapsed.

Prostate

Reduced secretory content as indicated by smaller organ size was seen in relation to treatment for males treated with 300 mg/kg/day and 100 mg/kg/day, but not at 30 mg/kg/day. There was no convincing regression observed in the recovery males.

Liver

Centrilobular hepatocyte enlargement was seen in relation to treatment for males treated with 300 and 100 mg/kg/day with the effects also evident at 30 mg/kg/day (statistically significant). Females were also affected at 300 mg/kg/day at (not statistically significant) and 100 mg/kg/day (statistically significant). Regression was observed in both sexes in the recovery animals.

Kidneys

A greater incidence of higher grades of severity of groups of basophillic tubules and tubular dilatation were seen as a consequence of treatment for males with 300 mg/kg/day compared to controls (statistically significant)) but not at other dose levels. Not observed (convincingly) for females. Both conditions regressed in the recovery group.

Adrenal glands

Cortical vacuolation is relatively common in lab-maintained rats and is especially prevalent among males and more rarely seen among females. The condition was significantly less prevalent among males treated at 300 mg/kg/day and 100 mg/kg/day. Although this may be a spurious group distribution of incidence and severity grades and effect of treatment on the adrenal cortex cannot be excluded. A similar effect was not seen in the females. A group differential was maintained among recovery animals suggesting that any effec was not fully regressed.

Lungs

Groups of alveolar macrophages were prevalent among control animals of either sex and grades of severity ranged from minimal to moderate. Such a macrophage response was rather greater than might normally be seen in te control animals of this age. The incidence and severity of alveolar macrophage populations wa significantly lower for males and femlaes treated at 300 mg/kg/day (stat. analysis not performed on females) and 100 mg/kg/day. Although such incidnece and severity could be fortuitous, an effect of treatment cannot be excluded. No evidence of alveolar macrophage accumulation after the treatment-free period had elapsed, suggesting regression of any effect.

Pituitary

Vacuolation of pars anterior cells is commonly seen among male rats but it is rarely present in female rats of this age. The prevalence and severity grades of vacuolation were normal or slightly above normal for control males but significantly lower for males treated at 300 mg/kg/day, indicating a dose-related effect. This effect was not seen in females or males treated at other concentrations. There was no evidence of regression in the recovery males.

Uterus/ cervix

Dilation of the uterine horn, with or without keratinisation in the cervix was found in one female treated with 30 mg/kg/day and one female treated with 100 mg/kg/day which displayed in utero total litter losse and one non-pregnant female treatde with 30 mg/kg/day, 2 non-pregnant females treated with 100 mg/kg/day and 2 nonpregnant females from the 300 mg/kg/day dose groups. This simply represents normal cyclical changes in the female rat. In addition, necrotic contenets were present in the uterus from one female treated at 100 mg/kg/day. This female showedd a corpus luteum and implantation site during the post mortum procedure, therefore this was considered to represent resorption of the foetuses.

Vagina

Hyperplasia of the vaginal epithelium was sen for 4 non-pregnant females at 300 mg/kg/day, but allowing for cyclical changes, there was insufficient evidence to suggest an effect of treatment. Similarly, higher grades of severity of vascuolar degeneration of the post partum vaginal epithelium as normal conversion from mucinous to non-mucinous morpholgy were seen among intermediate dose females compared to controls. There was no convincing effect of the treatment in this study. Keratinisation of the vaginal epithelium is a normal cyclical change in the female rat.

Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See above
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
for P0 parental male
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
not determinable
Remarks:
Microscopic changes in the mammary gland observed at all tested concentrations. Effects did not recover following cessation of treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
for P0 parental female
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
food efficiency
gross pathology
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
urinalysis
water consumption and compound intake
Remarks on result:
other:
Remarks:
NOAEL for systemic toxicity was achieved at 30 mg/kg/day because effects at 30 mg/kg/day were not considered to represent an adverse health effect for systemic toxicity.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
Leydig cells
seminal vesicle
testes
other: epididymides, prostate
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 4:       Average body weights and body weight gains during 55 days of treatment

Dose rate (mg/kg/day)

Male Body Weights at Day (g)

Total Weight Gain (g)

1

8

15

22

29

36

43

50

57

Male

Control

340

370

390

412

436

456

461

-

-

120

Low

342

370

393

411

437

455

466

-

-

124

Mid

340

359

375

395

421

439

449

-

-

108

High

343

356

371

384

404

418

425

-

-

83*

Recovery control

346

387

422

455

484

507

528

548

556

210

Recovery high

344

352

367*

382*

395*

406*

412*

445*

463*

120**

Female Body Weights (Weight Gain) During (g);

Dose rate (mg/kg/day)

Maturation (at day)

Gestation (at day)

Lactation (at day)

Total Weight Gain (g)

1

8

15

0

7

14

20

0

4

Control

241

250

(9)

256

(6)

271

304

(33)

340

(36)

421

(81)

324

332

(8)

-

Low

238

241

(3)

246

(5)

253

284

(31)

314

(30)

377*

(63)

301*

300**

(-1)

-

Mid

233

233

(1**)

239

(6*)

248

282

(34)

312

(31)

382

(70)

292*

301*

(9)

-

High

242

240

(-2**)

250

(9)

-

-

-

-

-

-

-

Recovery control

236

245

254

262

271

280

283

290

295

59

Recovery high

236

235

242

249

257

263

267

279

279

43

*  Significantly different (p <0.05) from the control.

** Significantly different (p <0.01) from the control.

*** Significantly different (p <0.001) from the control.

Table 5:       Selected haematology, clinical chemistry and pathology findings

Doses (mg/kg/day)

Control

30

100

300

Control

30

100

300

male

female

Number of animals/group

5

5

5

5

5

5

5

5 (day 56 recovery)

Haematology(day 42 for males and day 4 post-partum for females)

- Hb (g/dl)

16.6

16.2

16.1

15.2**

13.3

12.7

12.4

15.5

- RBC (1012/L)

8.89

8.78

8.46

8.09**

6.91

6.62

6.53

8.29

- Hct (%)

47.5

46.9

46.9

44.9

39.6

37.4

36.9

45.4

- MCH (pg)

18.7

18.5

19.0

18.7

19.4

19.1

19.0

18.7

- MCV (fl)

53

53

55

55

57

56

57

55

- MCHC (g/df)

34.9

34.7

34.2

33.8

33.7

33.8

33.6

34.1

- WBC (109/L)

10.8

9.2

11.3

8.0

9.0

7.5

12.1

7.3

Blood chemistry(day 42 and day 4 post-partum for females)

- Urea (mg/dl)

29

32

29

37**

35

40

50

44

- Glucose (mg/dl)

147

159

140

153

118

122

128

163

- Tot. Prot. (g/dl)

6.57

6.45

6.34

6.42

5.65

5.74

5.57

7.58

- Albumin (g/dl)

3.6

3.5

3.3*

3.3**

3.2

3.2

3.1

4.2

- A/G ratio

1.21

1.17

1.14

1.09

1.34

1.30

1.20

1.26

- Na+ (mmol/L)

150

150

150

150

151

149

152

152

- K+ (mmol/L)

4.58

4.32

4.46

4.16

4.80

4.08

4.59

4.20

- Cl- (mmol/L)

104

103

105

104

106

105

104

107

Pathology

male

female

Number of animals/group

12

12

12

12

12

12

12

11a

- External,mass under right forelimb

0

0

1

0

0

0

0

0

- Internal,epididymides: small

0

0

1

0

0

0

0

0

- Internal,right kidney: hydronephrosis

0

0

1

0

0

0

0

0

- Internal,lungs: mottled appearance

0

0

1

0

0

0

0

0

- Internal,mandibular lymph nodes: enlarged

0

0

1

0

0

0

0

0

- Internal,mass: approx.. 2 cm, spherical, containing green/yellow substance

0

0

1

0

0

0

0

0

- Internal,seminal vesicles: small

0

0

0

4

-

-

-

-

- Internal,prostate: small

0

0

0

4

-

-

-

-

- Internal,testes: small

0

0

1

0

-

-

-

-

- Internal,adrenals: pale

0

0

0

0

0

1

0

0

- Internal,cervical lymph nodes: enlarged

0

0

0

0

0

0

0

1

- Internal,lungs: reddened

0

0

0

0

1

0

0

1

- Internal,mass: approx. 1.5 cm, containing white coloured viscous liquid

0

0

0

0

0

0

0

1

- Internal,ovaries: dark red discolouration

-

-

-

-

0

0

0

1

- Internal,stomach: sloughing- glandular region

0

0

0

0

0

1

0

0

- Internal,Uterus: 1 dead foetus in each horn

-

-

-

-

0

1

0

0

- No abnormalities

12

12

9

8

11

11

12

9

Statistics: Anova + Dunnetts tests (two sided): * P,0.05   ** P,0.01

aone female killed in extremis on Day 6 exhibited the following pathological signs; adrenals (dark and enlarged), gastro-intestinal tract (gaseous distention), liver (dark), lungs (reddened), stomach (sloughing- non-glandular region), thoracic cavity (containing liquid), mass under left forelimb (containing foods and liquid).

Table 6:       Absolute and relative organ weights

 

Males (non-recovery)

Females (non-recovery)

Females (recovery)

DAILY DOSE

(mg/kg bw/day)

Control

30

100

300

Control

30

100

Control

300

NUMBER OF ANIMALS

12

12

12

12

7-11

7-11

7-11

5

4-5

BODY WEIGHT (g)a

461

466

449

425

332

301

304

295

279

BRAIN

Absolute Weighta

g

2.0179

2.0543

2.0266

2.0070

1.9028

1.8502

1.9078

1.9121

1.8922

Per Body Weighta

%

0.4423

0.4419

0.4535

0.4754

0.5755

0.6164*

0.6282*

0.6530

0.6810

ADRENALS

Absolute Weighta

g

0.0562

0.0562

0.0527

0.0636

0.0760

0.0705

0.0700

0.0628

0.0571

Per Body Weighta

%

0.0122

0.0121

0.0118

0.0150**

0.0229

0.0234

0.0229

0.0214

0.0202

EPIDIDYMIDES

Absolute Weighta

g

1.2917

1.3495

1.2100

1.0385***

n.a.b

n.a.b

n.a.b

n.a.b

n.a.b

Per Body Weighta

%

0.2824

0.2900

0.2695

0.2448**

n.a.b

n.a.b

n.a.b

n.a.b

n.a.b

HEART

Absolute Weighta

g

1.7237

1.6602

1.5566

1.5537

1.3490

1.1218*

1.1404*

1.0731

1.1116

Per Body Weighta

%

0.3738

0.3563

0.3477

0.3666

0.4053

0.3726

0.3762

0.3661

0.3994

KIDNEYS

Absolute Weighta

g

3.1949

3.2058

3.0922

3.1681

1.9178

1.8878

1.9243

1.9781

1.8940

Per Body Weighta

%

0.6952

0.6877

0.6910

0.7431

0.5778

0.6261

0.6316

0.6713

0.6802

LIVER

Absolute Weighta

g

15.6764

15.5205

14.9837

15.8695

12.7095

11.6779

11.9166

9.3369

9.3088

Per Body Weighta

%

3.3934

3.3273

3.3404

3.7250*

3.8193

3.8884

3.9040

3.1752

3.3435

SPLEEN

Absolute Weighta

g

0.7653

0.7845

0.7309

0.6956

0.6289

0.5669

0.5916

0.5168

0.4972

Per Body Weighta

%

0.1659

0.1688

0.1634

0.1638

0.1888

0.1890

0.1941

0.1746

0.1784

TESTES

Absolute Weighta

g

3.4920

3.5732

3.2223

3.1171*

n.a.b

n.a.b

n.a.b

n.a.b

n.a.b

Per Body Weighta

%

0.7641

0.7701

0.7177

0.7350

n.a.b

n.a.b

n.a.b

n.a.b

n.a.b

THYROID

Absolute Weighta

g

0.0178

0.0189

0.0196

0.0170

0.0149

0.0118

0.0135

0.0141

0.0127

Per Body Weighta

%

0.0039

0.0041

0.0043

0.0040

0.0045

0.0040

0.0044

0.0049

0.0046

THYMUS

Absolute Weighta

g

0.3756

0.3925

0.3732

0.3393

0.2620

0.2118

0.2171

0.3301

0.2999

Per Body Weighta

%

0.0823

0.0838

0.0840

0.0801

0.0790

0.0706

0.0715

0.1118

0.1072

OVARIES

Absolute Weighta

g

n.a.b

n.a.b

n.a.b

n.a.b

0.2076

0.0935

0.0958

0.0851

0.0712

Per Body Weighta

%

n.a.b

n.a.b

n.a.b

n.a.b

0.0594

0.0312

0.0313

0.0289

0.0256

UTERUS

Absolute Weighta

 

n.a.b

n.a.b

n.a.b

n.a.b

0.8779

0.7427

0.8500

0.9308

0.6539

Per Body Weighta

 

n.a.b

n.a.b

n.a.b

n.a.b

0.2642

0.2484

0.2787

0.3140

0.2356

aGroup means at the end of terminal necropsy are shown.

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity and reproduction in male rats could not be established as treatment related effects were observed at the lowest tested concentration.

The NOAEL for systemic toxicity in female rate was 30 mg/kg/day. The NOAEL for reproduction in female rats could not be established as pregnancy rates were reduced at the lowest tested concentration compared to the controls.
Executive summary:

In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422) Bisphenol AF was administered to 36 male and 36 female Sprague-Dawley rats by gavage at dose levels of 30, 100 and 300 mg/kg bw/day for up to 55 consecutive days. Two recovery groups, each of 5 males and 5 females, were also treated at 300 mg/kg/day for 42 days consecutive days followed by a 14 day treatment-free period. In addition, control rats were dosed with vehicle (Arachis oil BP) alone for each test group.

A summary of adult responses to Bisphenol AF is described below;

Mortality - no treatment-related deaths were detected.

Clinical signs - clinical signs were generally confined to post-dose increased salvation and staining around the mouth for animals treated at 300 mg/kg/day. These signs were also observed at lower concetrations, but to a lesser extent. Dehydration and staining around the ano-genital region was evident for one treated female at 300 mg/kg/day on Day 6 and Day 7. Another female showed signs of hunched posture and dehydration between Day 7 and Day 10. Staining around the ano-genital region was also evident for another female treated at 300 mg/kg/day. Regression of all signs was evident following cessation of treatment.

Behavioural assessments - weekly open field observations did not reveal any treatment-related effects.

Functional performance - no treatment-related effects were evident for grip or motor activity.

Sensory reactivity assessments - no treatment-related effects were observed.

Bodyweight - Reduced bodyweight gains were evident for males in the 300 mg/kg/day group during the treatment period, although regression was observed during the treatment-free period. Slight bodyweight gain reduction was also observed in the 100 mg/kg/day group. For females, actual bodyweight loss was observed in the first week of treatment at 300 mg/kg/day. Bodyweight gain reduction was evident at 100 mg/kg/day in the first week. No effects were noted for pregnant females during gestation however bodyweights of females in both the 100 and 30 mg/kg/day groups were reduced during early lactation.

Food consumption and efficiency - reduction in dietary intake and food efficiency was observed in males at 100 and 300 mg/kg/day during the first 2 weeks. For females, dietary intake was reduced for females at 300 mg/kg/day during the pre-mating phase and for 100 mg/kg/day in week 1. Reduced intake was also observed during gestation in the 30 and 100 mg/kg/day groups and a slight reduction with 30 mg/kg/day during lactation.

Water consumption - increase in water intake was observed for males in all treatment groups, with regression during the treatment-free period. For females, increases were observed at 100 and 300 mg/kg/day pre-mating with slight increases observed during gestation and early lactation in the 100 mg/kg/day group. Regression of effect was also evident in the females during the treatment-free period.

Haematology - reduction of haemoglobin, erythrocyte and haemacrit counts were observed in males at 300 mg/kg/day, with partial regression.

Blood chemistry - reduction of albumin, A/G, cholesterol and increase in blood urea was evident in males at 300 mg/kg/day. Reduction in albumin and cholesterol noted at 100 mg/kg/day and cholesterol at 30 mg/kg/day were also noted. ALAT levels were elevated for males at 100 and 300 mg/kg/day, with regression observed after cessation of treatment. Similar effects were noted for the females, although no significant effects were evident prior to termination on Day 4 post partum.

Urinalysis - no adverse effects noted.

Oestrous cycle assessment - 1 female at 300 mg/kg/day showed continuous anestrus and failed to mate. A second female showed extended oestrous, mated, but did not achieve pregnancy.

Mating - no adverse effects noted.

Fertility - no pregnancies observed at 300 mg/kg/day. Three females at 100 mg/kg/day mated, but did not achieve pregnancy. There were 2 non-pregnant females at 30 mg/kg/day.

Gestation length - no adverse effects noted.

Litter size, viability, development - no adverse effects noted.

Necropsy - small seminal vessels and prostates observed at 300 mg/kg/day. Small testes and epididymides observed at 100 mg/kg/day. No effects were noted in males at 30 mg/kg/day or females at any treatment level. No macroscopic abnormalities were detected in offspring.

Organ weights - reduced absolute and bodyweight-relative epididymis and testes were evident in males at 300 mg/kg/day, together with increased adrenal and liver weights. No regression of increased adrenal weights was observed after cessation of treatment. No organ weight changes were evident in post partum females or males treated at 30 or 100 mg/kg/day.

Histopathological changes;

Mammary gland - tubuloaveolar differentiation of mammary tissue seen in males treated at all doses with no evidence of regression. Minimal glandular hyperplasia was seen in 4 non-pregnant females at 300 mg/kg/day however no such effects were observed in females following cessation of treatment.

Ovaries - follicular cysts were observed in non-pregnant females in the 300 mg/kg/day group, with no regression observed following cessation of treatment.

Testes - leydig cell atrophy was seen in 100 and 300 mg/kg/day groups. Almost complete regression followed after cessation of treatment.

Seminal vesicles/ coagulating gland - reduced secretory content in the 100 and 300 mg/kg/day groups with no evidence of regression.

Prostate - reduced secretory content in 100 and 300 mg/kg/day groups with no evidence of regression.

Liver - centrilobular hepatocyte enlargement was observed in males in all treatment groups and in females treated at 100 and 300 mg/kg/day. Regression was observed following cessation of treatment.

Kidneys - an increased incidence of higher grades of severity of groups of basophilic tubules and tubular dilation were seen in males treated at 300 mg/kg/day. Regression of both conditions was observed following the recovery period.

In conclusion, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity and reproduction in male rats could not be established as treatment related effects were observed at the lowest tested concentration. The NOAEL for systemic toxicity in female rate was 30 mg/kg/day.  The NOAEL for reproduction in female rats could not be established as pregnancy rates were reduced at the lowest tested concentration compared to the controls. The No Observed Effect Limit and NOAEL for first generation offspring was 100 mg/kg/day under these test conditions.

This combined toxicity study with reproduction screening in the rat is acceptable and satisfies the guideline requirements for an OECD 422 in the rat.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
Study duration is only 14 days
GLP compliance:
no
Remarks:
Published study not conducted to GLP
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not reported
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Dissolved in corn oil and 4 % ethanol.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test time dissolved in corn oil with 4 % ethanol.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Test time dissolved in corn oil with 4 % ethanol.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as a liquid.

OTHER SPECIFICS: n/a
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Academy of Military Medical Sciences, Beijing, China.
- Females (if applicable) nulliparous and non-pregnant: n/a
- Age at study initiation: 7 weeks
- Weight at study initiation: Not reported
- Fasting period before study: Not reported
- Housing: 1 animal per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

DETAILS OF FOOD AND WATER QUALITY: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12:12, light:dark

IN-LIFE DATES: Not reported
Route of administration:
oral: gavage
Details on route of administration:
BPAF was administered orally via gavage to rats in the treatment group for 14 days at doses of 2, 10, 50 and 200 mg/kg body weight/day in a volume of 5 ml/kg of body weight.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

BPAF was dissolved in corn oil vehicle including 4% ethanol, which was prepared fresh each day.

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): No justification reported however, corn oil is OECD guideline recommended.
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 mL/kg of body weight
- Lot/batch no. (if required): Not reported
- Purity: Not reported
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
n/a
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily
Dose / conc.:
2 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The chosen doses were based on a study in which the oral 50% lethal dose (LD50) in rats exposed acutely was up to 3400 mg BPAF/kg.
- Rationale for animal assignment (if not random): Not reported
- Rationale for selecting satellite groups: n/a
- Post-exposure recovery period in satellite groups: n/a
- Section schedule rationale (if not random): Not reported
Positive control:
n/a
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No
- Time schedule: n/a

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule: n/a

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): n/a
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: n/a
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: n/a

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: n/a

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): n/a
- Time schedule for examinations: n/a

OPHTHALMOSCOPIC EXAMINATION: n/a
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 14
- Anaesthetic used for blood collection: No
- Animals fasted: Not specified
- How many animals: 6 per treatment group (30 in total)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 14
- Animals fasted: Not specified
- How many animals: 6 per treatment group (30 in total)

URINALYSIS: No
- Time schedule for collection of urine: n/a
- Metabolism cages used for collection of urine: n/a
- Animals fasted: n/a

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a
- Battery of functions tested: n/a

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a

OTHER: n/a
Sacrifice and pathology:
GROSS PATHOLOGY: No

HISTOPATHOLOGY: Yes- testes weighed
Statistics:
Data were analyzed using SPSS for Windows 13.0 Software and presented as the mean with standard error (mean ± SEM). Differences between the control and the treatment groups were determined using a one-way ANOVA followed by the LSD multiple range test. A p < 0.05 was considered statistically significant.
Clinical signs:
not examined
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Compared with the control group, no significant difference in body weights was observed in the 2 and 10 mg/kg/d groups. However, the body weights were markedly decreased in the rats dosed with 50 and 200 mg/kg/d BPAF, by 12.3% and 16.2%, respectively (p < 0.01).
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:
effects observed, treatment-related
Description (incidence and severity):
No significant difference in cholesterol levels was observed following exposure to 2 and 10 mg BPAF/kg/d compared with the control group. However, in the 50 and 200 mg BPAF/kg/d groups, the total cholesterol concentrations in serum were significantly reduced by 26.9% and 36.8%, respectively (p < 0.01).
Description (incidence and severity):
In rats receiving 200 mg/kg/d of BPAF, testosterone levels significantly decreased by 90.6% of the control (p < 0.01). No significant difference was observed between control and other exposure groups, though testosterone levels exhibited a degressive trend at 50 mg/kg/d group. In rats receiving 50 and 200 mg/kg/d BPAF, the levels of luteinizing hormone (LH) in serum were dramatically increased by 65.2% and 90.2%, respectively (p < 0.05). Additionally, the levels of follicle-stimulating hormone (FSH) in rat serum exposed to 10, 50 and 200 mg/kg/d BPAF were markedly increased by 33.3%, 35.1% and 34.5%, respectively (p < 0.05).

The mRNA levels of scavenger receptor class B type 1 (SR-B1), which is responsible for transporting plasma cholesterol to steroidogenic tissues, were significantly reduced at the dose of 200 mg/kg/d BPAF (p < 0.05). The expression of steroidogenic acute regulatory protein (StAR), a gene involved in cholesterol transport to the inner mitochondrial membrane, was also dramatically reduced in the 200 mg BPAF/kg/d group compared with control group (p < 0.01). Consistent with the induction of StAR, the rats dosed with 200 mg BPAF/kg/d also exhibited reduced levels (p < 0.01) of cholesterol side-chain cleavage enzyme (P450scc) mRNA, which is translated into a rate-limiting enzyme that catalyzes cholesterol side-chain cleavage to form pregnenolone. Similarly, 17-beta-hydroxysteroid dehydrogenase (17-HSD), which has the ability to convert androstenedione to testosterone, was notably down-regulated in testes from rats dosed with 200 mg/kg/d BPAF (p < 0.05). No significant difference between the control and the BPAF treatment groups was detected for 3-betahydroxysteroid dehydrogenase (3-HSD) and cytochrome P450c17 subfamily a (CYP17) mRNA. Compared to the control group, the mRNA expression of HMGCoA reductase (HMGR), which is the rate-limiting enzyme of cholesterol synthesis, was unchanged in testes from rats exposed to BPAF. Conversely, sterol regulatory element binding protein 1c (SREBP-1c), which plays an important role in the regulation of cholesterol biosynthesis in somatic cells, was reduced in rats dosed with 200 mg/kg/d BPAF (p < 0.05). Inhibin B, which is used as a biomarker of testicular toxicity, was markedly reduced in the 200 mg/kg/d group (p < 0.05). Similarly, the mRNA expression of luteinizing hormone receptor (LHR) and ER in the testes of rats receiving 200 mg/kg/d BPAF were statistically decreased compared with the control group (p < 0.05). However, no difference in mRNA expression levels of AR, ER, and Mullerian inhibiting substance (MIS) were detected between the BPAF treated groups and the control group.

Due to the significant changes in their mRNA expression levels and their important roles in steroidogenesis, SR-B1, StAR, and P450scc were also evaluated at the protein level. Protein expression of SR-B1, StAR, and P450scc in testes was sharply reduced only in the 200 mg/kg/d BPAF group, by 70.9%, 42.2%, and 36.7%, respectively (p < 0.05). However, in the rats given 2, 10, and 50 mg/kg/day of BPAF, the expression levels of these proteins were unchanged compared with the control group.
Urinalysis findings:
not examined
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):
The absolute testis weight was unchanged in the BPAF treatment groups compared to the control group. The relative testis weight significantly increased (by 9.9%, p < 0.05) only in the highest dose group.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
NOAEL
Effect level:
< 10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
The NOEAL for bisphenol-AF for male Sprague-Dawley rats was < 10 mg/kg/bw, where the most sensitive endpoint was the concentration of follicle-stimulating hormone (FSH) in blood serum which was significantly (p < 0.05) increased in all tested groups versus the control.

The endocrine disrupting activity of bisphenol-AF in the male rat was investigated. The data demonstrated that BPAF has the potential to impair pituitary–gonadal function at different levels by increasing LH and FSH concentrations and decreasing testosterone levels in serum. Additionally, The dramatic decrease in testosterone concentration, reduced levels of genes and proteins involved in steroidogenesis and the reduced expression of inhibin B suggested that the testes may be a primary target organ of BPAF exposure.
Executive summary:

In a subchronic toxicity study bisphenol-AF (99 % purity) was administered to 30 Sprague-Dawley male rats by gavage at dose levels of 2, 10, 50 and 100 mg/kg bw/day along with a vehicle control group for 14 days.

Total cholesterol levels in serum were decreased in rats given a dose of 50 and 200 mg/kg/d. BPAF concentration in the testes increased with increasing doses of BPAF. Reduced serum testosterone and increased luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were observed in rats in the higher dose groups. Furthermore, BPAF exposure resulted in a dramatic decline in genes and protein involved in cholesterol biosynthesis, transport and steroid biosynthesis. Similarly, the testicular mRNA levels of inhibin B, estrogen receptor (ER) and luteinizing hormone receptor (LHR) also decreased in rats given a dosage of 200 mg/kg/d BPAF. Together, these data demonstrate that BPAF-induced inhibition of testosterone production primarily resulted from the alteration of genes and proteins in the testosterone biosynthesis pathway.

The NOEAL for bisphenol-AF for male Sprague-Dawley rats was < 10 mg/kg/bw, where the most sensitive endpoint was the concentration of follicle-stimulating hormone (FSH) in blood serum which was significantly (p < 0.05) increased in all tested groups versus the control.

This subchronic toxicity study in the Sprague-Dawley rat is acceptable based on the guideline requirements for a subchronic oral study (OECD 407) where the purpose of the testing guideline is to investigate a broad variety of potential targets of toxicity. In this investigation, the effects on the endocrine system indicated that further longer-term studies would need to be conducted to conclude on the endocrine disrupting potential of bisphenol-AF.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2002
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
other: US EPA OCSPP 890.1400
Qualifier:
equivalent or similar to guideline
Guideline:
other: US EPA OCSPP 890.1600
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: Not reported
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Dissolved in olive oil
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in olive oil
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Dissolved in olive oil
- Final preparation of a solid: n/

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as a liquid

OTHER SPECIFICS: n/a
Species:
rat
Strain:
other: Females = Sprague-Dawley, Males = Wistar
Details on species / strain selection:
Females = Crj:CD (SD)
Males = Brl Han: WIT Jcl (GALAS)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan (females) and Clea Japan (males)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 19 days for uterophic assay (females) and 7 weeks old for Hershberger assay (males)
- Weight at study initiation: Not reported
- Fasting period before study: Not reported
- Housing: Housed individually (females) and in groups of 3 (males)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Not reported for females and 14 days post-op for males (castration).

DETAILS OF FOOD AND WATER QUALITY: Commercial diet

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 ºC
- Humidity (%): 55 ± 5 %
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): Not reported

IN-LIFE DATES: Not reported
Route of administration:
other: Females = subcutaneous injection, males = oral gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dissolved in olive oil (ratio not reported).

DIET PREPARATION
- Rate of preparation of diet (frequency): Not reported
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): Not reported
- Purity: Not reported
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Females = 3 days
Males = 10 days
Frequency of treatment:
Daily
Dose / conc.:
8 mg/kg bw/day (nominal)
Remarks:
Female
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Female
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Female
Dose / conc.:
8 mg/kg bw/day (nominal)
Remarks:
Female + ethynyl estradiol (EE) at a rate of 0.6 µg/kg bw
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Female + ethynyl estradiol (EE) at a rate of 0.6 µg/kg bw
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Female + ethynyl estradiol (EE) at a rate of 0.6 µg/kg bw
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Male
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Male
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
Male
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Male + testosterone propionate at a rate of 0.2 mg/kg bw
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Male + testosterone propionate at a rate of 0.2 mg/kg bw
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
Male + testosterone propionate at a rate of 0.2 mg/kg bw
No. of animals per sex per dose:
6 individuals per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not reported
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: n/a
- Post-exposure recovery period in satellite groups: n/a
- Section schedule rationale (if not random): n/a
Positive control:
Females = tamoxifen + estradiol
Males = flutamide + testosterone
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: At end of incubation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): n/a
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: n/a
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: n/a

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No
- Time schedule for collection of blood: n/a
- Anaesthetic used for blood collection: n/a
- Animals fasted: n/a
- How many animals: n/a

CLINICAL CHEMISTRY: No
- Time schedule for collection of blood: n/a
- Animals fasted: n/a
- How many animals: n/a

URINALYSIS: No
- Time schedule for collection of urine: n/a
- Metabolism cages used for collection of urine: n/a
- Animals fasted: n/a

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a
- Battery of functions tested: n/a

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a

OTHER: n/a
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (See Table 1 (Uterotrophic assay) and Table 2 (Hershberger assay))
Statistics:
Uterotrophic assay - Differences in body weight and organ weight between the vehicle group and each of the chemical groups and between the vehicle-plus-EE group and each of the chemical plus EE groups were assessed for statistical significance by the two-tailed Student’s t -test.

Hershberger assay - As above.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Uterotrophic assay - No abnormalities observed in any of the test groups.

Hershberger assay: Decreased spontaneous locomotion was seen in rats given 200 and 600 mg/kg bw.
Mortality:
mortality observed, treatment-related
Description (incidence):
Uterotrophic assay - No mortality observed.

Hershberger assay - Mortality observed (2) in the 600 mg/kg bw + TP group
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Uterotrophic assay - Body weight increased normally in all test groups.

Hershberger assay - Significant decrease in weight gain observed in the 200 and 600 mg/kg bw test groups.
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
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):
Uterotrophic assay - Uterine blotted weights increased significantly in the 8, 40 and 200 mg/kg bw test groups (See Table 1).

Hershberger assay - Significant decrease in relative bulbocavernosus/ levator ani muscle (BC/LA) weight in the 200 mg/kg bw test group. Significant increase in relative glans penis weight in the 600 mg/kg bw test group. (See Table 2).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See above.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
< 8 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
not determinable
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
8 mg/kg bw/day (nominal)
System:
female reproductive system
Organ:
uterus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
glans penis
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1:       Summary of uterotrophic assay results

Dose group

(mg/kg per day)

Body weight

(g)

Uterus blotted weight

Absolute

(mg)

Relative

(mg)

Vehicle

56.1 ± 4.3

28.6 ± 4.9

50.9 ± 7.4

8

55.0 ± 4.5

47.2 ± 9.9**

85.1 ± 11.9**

40

56.6 ± 4.0

65.9 ± 9.8**

116.0 ± 11.7**

100

54.7 ± 4.2

96.4 ± 9.0 **

177.2 ± 22.2

** significantly different from vehicle control ( <0.01)

Table 2:       Summary of Hershberger assay results

Dose group

(mg/kg per day)

Body weight

(g)

Ventral prostate

(mg/100 g bw)

Semical vesicle

(mg/100 g bw)

BC/LA (mg/100 g bw)

Glans penis

(mg/100 g bw)

Cowper’s gland

(mg/100 g bw)

Vehicle control

275.1 ± 9.7

5.8 ± 0.9

11.3 ± 2.1

53.2 ± 6.3

12.6 ± 1.0

1.5 ± 0.5

50

257.0 ± 18.2

5.5 ± 1.5

12.0 ± 1.8

55.9 ± 8.4

12.8 ± 1.1

1.7 ± 0.5

200

259.9 ± 12.8*

5.7 ± 1.4

13.2 ± 2.3

44.2 ± 4.7*

12.2 ± 2.0

1.3 ± 0.4

600 (400)a

219.6 ± 30.9*

6.2 ± 1.6

13.6 ± 1.2

48.9 ± 3.5

15.1 ± 1.8*

1.7 ± 0.3

* significantly different from vehicle control ( <0.05)

Conclusions:
The NOAEL for body weight increase in female and male rats was 100 and 50 mg/kg bw per day, respectively. The NOAEL for mortality in male rats was 200 mg/kg bw per day, where mortality was observed in the top concentration (600 mg/kg bw per day).

In the uterotrophic test, a significant increase in relative uterus weight (p < 0.01) was observed in female rats treated at 8 and 40 mg/kg bw per day.

In the Hershberger test, a significant reduction (p < 0.05) in BC/LA weight and a significant increase (p < 0.05) in glans penis weight was observed in male rats treated at 200 and 600 mg/kg bw per day, respectively.
Executive summary:

In a chronic toxicity study bisphenol-AF (98.8 % purity) was administered to male and female rats in an uterotrophic and Hershberger repeat dose test. Female rats were Sprague-Dawley (Crj:CD) and of 17 days of age whereas the males were castrated Wistar (Brl Han: WIST Jcl (GALAS)) of 56 days of age. Test item was applied to female individuals in the uterotrophic assay by subcutaneous injection (in olive oil) on three consecutive days at dose levels of 8, 40 and 100 mg/kg bw/day. Males, in the Hershberger assay, were treated by oral gavage on ten consecutive days at dose levels of 50, 200 and 600 mg/kg bw/day. The top concentration in the ersberger assay was reduced to 400 mg/kg bw/day midway throught the testing period due to observed mortality.

Test item related effects were noted in both female and male individuals in their respective tests at the tested concentrations. No reduction in body weight increase was observed in the female uterotrophic assay however, a significant reduction in weight increase (p < 0.05) was observed in the 200 and 600 mg/kg bw/day male treatment groups. Mortality of two individuals was also observed in the male Hershberger test at the highest tested dose concentration. Assay specific responses included significant increase (p < 0.01) in relative uterus weight in the 8 and 40 mg/kg bw/day treatment groups in comparison to the controls. A significant decrease (p < 0.05) in BC/LA weight and a significant increase (p < 0.05) in glans penis weight was observed in the 200 and 600 mg/kg bw/day treatment groups, versus the controls, respectively.

The NOAEL for bisphenol-AF to male and female rats was < 8 mg/kg bw/day based on histopathology results, where a significant increase in organ weight (uterus) was observed in female individuals- indicating that the test item has estrogen agonistic properties.

This chronic study is acceptable and satisfies the guideline requirement for a chronic study (OPPTS 890.1600 and OPPTS 890.1400), Hersberger and uterotrophic assays in male and female rats, respectively.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The conclusion for this endpoint is based on a weight of evidence, consisting of 2 reliable studies (Klimisch 1 and 2) and 2 unreliable studies that provide supporting information (Klimisch 3).
System:
other: The NOAEL was based on the reduced body weight gain and abnormal estrous cycles in the female rat in the intermediate dose group

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

During the treatment period, a dose-dependent decrease in overall mean body weight gain was apparent in all groups of toxicity and recovery phase males and females exposed to the test item when compared to controls; this decrease in overall weight gain was largely attributable to lower body weight gain and body weight losses. The reduction in body weight performance was evident among the reproductive phase females in the gestation and lactation period and was considered to be an adverse effect. Another finding is the possible endocrine effects as demonstrated by the Leydig cell atrophy and irregular estrous cycles noted in both the OECD 407 and OECD 422. This effect correlate with the reduced secretory content in prostate and seminal vesicles with no evidence of recovery. Effect on estrous cycles also correlate with the failure in mating and inability to achieve pregnancy

Additional information

In a subacute toxicity study conducted on rats in accordance with OECD 407 under GLP (Umano, 2012), The substance was administered to 30 male and 30 female Sprague-Dawley rats by oral gavage at dose levels of 10, 30 and 100 mg/kg bw/day for up to 28 consecutive days.  Although there was no recovery phase, this is not a guideline requirement.  The key findings in this study were lower body weight gain at 100 mg/kg/day for males and at 30 and 100 mg/kg/day for females; lower white cell counts in males at 100 mg/kg/day; lower cholesterol levels in both sexes at 100 mg/kg/day; lower cholinesterase and higher bilirubin levels in females at 100 mg/kg/day; longer oestrous cycles in females at 100 mg/kg/day; lower absolute and relative prostate and seminal vesicles weights at 100 mg/kg/day, possibly reflecting the lower body weight; higher adrenal weight in males at 100 mg/kg/day; Leydig cell atrophy at 100 mg/kg/day in 5/10 males; hypertrophy of the adrenal zona fasciculata at 100 mg/kg/day in 8/10 males, 2/10 females; atrophy of mammary glands in 3/10 males at 100 mg/kg/day; decreased haematopoiesis in bone marrow and extramedullary haematopoiesis in spleen at 100 mg/kg/day in 4/10 and 2/10 males, respectively.  Many findings were related to endocrine effects, but had not considered that some may have been secondary to the lower body weights.  Without having the individual data, it is not possible to comment further.  In conclusion, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 10 mg/kg bw/day due to findings of reduced body weight gain and abnormal estrous cycles in the female rat treated at 30 mg/kg bw/day.

In a combined repeated dose toxicity study with reproductive toxicity screening OECD TG 422 conducted in rats according to GLP, dose levels of 30, 100 and 300 mg/kg bw/day and generally followed OECD Guideline 422, but the males were dosed for a total of 6 weeks, with a recovery group maintained for a further 2 weeks.  All non-recovery females were mated after 2 weeks dosing, then reared until around Day 4 post partum.  Recovery females were not mated, were dosed for a total of 6 weeks followed by 2 weeks recovery.  Although mating was not obviously affected, there were no pregnancies at 300 mg/kg bw/day.  At 30 and 100 mg/kg bw/day, there were 10/12 and 8/12 females pregnant, respectively, although only 9 and 7 females had live offspring compared with 11 control females.  Because of these findings, inter-group comparisons for females were confounded by the different pregnancy statuses of the females.  Reproductive findings will be discussed under developmental/ reproductive toxicology.  The other key findings were lower body weight gain of both sexes at 100 and 300 mg/kg bw/day; lower haemoglobin, red cell counts and haematocrit in males at 300 mg/kg bw/day; lower cholesterol levels in both sexes at all dose levels; lower albumin levels in males at 100 and 300 mg/kg bw/day; lower epididymides and testis weights at 300 mg/kg bw/day; higher liver and adrenal weights in males at 300 mg/kg bw/day; lower heart weights in females at 30 and 100 mg/kg bw/day possibly reflecting lower body weights; Leydig cell atrophy in males at 100 and 300 mg/kg bw/day, with almost complete regression after 14 days recovery; reduced secretory content in prostate and seminal vesicles with no evidence of recovery; centrilobular hepatocyte enlargement in males at all levels and in females at 100 and 300 mg/kg bw/day, with evidence of recovery.

Based on the 2 studies, the key findings were lower weight gain in males at ≥100 mg/kg/day and in females at ≥ 30 mg/kg/day; lower cholesterol levels in both sexes at ≥100 mg/kg/day and possibly at 30 mg/kg/day; lower epididymides and testis weights at 300 mg/kg/day; higher liver and adrenal weights in males at 300 mg/kg/day, with possible increase in adrenal weights at 100 mg/kg/day; Leydig cell atrophy at ≥100 mg/kg/day with regression during recovery period; reduced secretory content in prostate and seminal vesicles with no evidence of recovery at ≥100 mg/kg/day; centrilobular hepatocyte enlargement in males at ≥30 mg/kg/day and in females at ≥100 mg/kg/day, with evidence of recovery.

The key value for chemical safety assessment used was NOAEL = 10 mg/kg bw/day (Umano, 2012).

In another repeated dose toxicity study (Klimisch 3 - unreliable study; Feng 2012), the substance was administered to 30 Sprague-Dawley male rats by gavage at dose levels of 2, 10, 50 and 100 mg/kg bw/day along with a vehicle control group for 14 days.  Key finding included, decreased total serum cholesterol at dose of 50 and 200 mg/kg/day,   Reduced serum testosterone and increased luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were observed in rats in the higher dose groups. Dose related accumulation of the testate in the testes, decline in genes and protein involved in cholesterol biosynthesis, transport and steroid biosynthesis. Similarly, the testicular mRNA levels of inhibin B, estrogen receptor (ER) and luteinizing hormone receptor (LHR) also decreased in rats given a dosage of 200 mg/kg/d. The NOEAL for male Sprague-Dawley rats was < 10 mg/kg/bw.

In an uterotrophic and Hershberger repeated dose test (Klimisch 3 - unreliable study; Yamasaki 2003), the test item was administered to rats via subcutaneous injections and oral garage at dose levels of 8, 40 and 100 mg/kg bw/day and 50, 200 and 600 mg/kg bw/day respectively. Test item related effects were noted in both female and male individuals in their respective tests at the tested concentrations, significant reduction in weight increase and significant increase glans penis weight observed in the 200 and 600 mg/kg bw/day male treatment groups. Mortality of two individuals was also observed in the male Hershberger test at the highest dose group.  There was also significant increase in relative uterus weight in the 8 and 40 mg/kg bw/day dose groups. The NOAEL for male and female rats was < 8 mg/kg bw/day based on histopathology results, where a significant increase in organ weight (uterus) was observed in female individuals, there is an implication that the test item has estrogen agonistic properties.

Justification for classification or non-classification

The substance meets the criteria for classification as STOT-RE Category 2 in accordance with Regulation (EC) No 1272/2008 (CLP). This classification is based on the following:

- Lower absolute and relative prostate and seminal vesicle weights ar 100 mg/kg bw/day (Umano et al., 2012)

- Higher adrenal weights in males at 100 mg/kg bw/day (Umano et al., 2012)

- Leydig cell atrophy at 100 mg/kg bw/day (Umano et al., 2012)

- Lower epididymide & testes weights at 300 mg/kg bw/day (OECD 422, Anon., 2011)

- Higher liver & adrenal weights in males at 300 mg/kg bw/day (OECD 422, Anon., 2011)

- Leydig cell atrophy at 100 mg/kg bw/day (OECD 422, Anon., 2011)

- Reduced secretory content of prostate and seminal vesicle at 100 mg/kg bw/day (OECD 422, Anon., 2011)