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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Remarks:
combined repeat dose toxicity test with reproduction/ developmental toxicity screening
Type of information:
experimental study
Adequacy of study:
key study
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol
Cas Number:
1478-61-1
Molecular formula:
C15H10F6O2
IUPAC Name:
4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Sprague-Dawley Crl:CD (SD) IGS BR strain rats were accepted on to the study
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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on 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
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
High Dose
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Intermediate diose
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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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:
no effects observed
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):
As above
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
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.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
for P0 parental female
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
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: 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

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
for F1 offspring
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

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.

The NOEL and NOAEL for first generation offspring was 100 mg/kg/day
Executive summary:

In a combined repeat dose toxicity study with reproductive toxicity screening OECD TG 422 conducted in rats according to GLP, 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. There were no treatment related mortality or effects on behavioural, functional and sensory parameters. No treatment related effects on mating, gestation length, litter size, viability and development.

The key developmental and reproductive toxicity data were lower body weight gain of both sexes at 100 and 300 mg/kg/day. Mating was not affected but there were no pregnancies at 300 mg/kg/day; at 30 and 100 mg/kg/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. Lower epididymides and testis weights at 300 mg/kg/day and Leydig cell atrophy in males at 100 and 300 mg/kg/day, with almost complete regression after 14 days recovery; reduced secretory content in prostate and seminal vesicles with no evidence of recovery

In terms of developmental effects at 30 and 100 mg/kg/day, the numbers of implantations and live young, and the offspring weights did not suggest any developmental effects.  There were no pregnant females at 300 mg/kg/day and therefore developmental effects at this level could not be assessed in this study.

The other key findings in this study were; lower haemoglobin, red cell counts and haematocrit in males at 300 mg/kg/day, lower cholesterol levels in both sexes at all dose levels and lower albumin levels in males at 100 and 300 mg/kg/day.

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