4,4'-isopropylidenediphenol

Immunotoxicity

Description of key information

In the course of the CLARITY BPA core study various endpoints regarding immunotoxicity were analyzed. Overall, this study indicated no consistent effect up to the highest dose tested on any parameter. This is in line with recently published Grantee studies, in which no persistent and reasonable change in immune cell composition, lymphoproliferative and immune effector responses by splenic leukocytes was observed. 

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

immunotoxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study.

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study.
Core Study was conducted under GLP conditions. For further information and details see Clarity Core Study.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

This study is part of the CLARITY program and belongs to the Grantee Studies. For further information and details see Clarity Core Study.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study. For further information and details see Clarity Core Study.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study. For further information and details see Clarity Core Study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study. For further information and details see Clarity Core Study.

Duration of treatment / exposure:

thymus: PND21; spleen: PND 21, PND 90, PND 183, PND 365

Frequency of treatment:

daily

Dose / conc.:

0.003 mg/kg bw/day (nominal)

Dose / conc.:

0.025 mg/kg bw/day (nominal)

Dose / conc.:

0.25 mg/kg bw/day (nominal)

Dose / conc.:

2.5 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

No. of animals per sex per dose:

2-10

Control animals:

yes, concurrent vehicle

Details on study design:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study. For further information and details see Clarity Core Study.

Specific cell-mediated immunity:

SPLEEN: Yes
- Method: Cell Counting, Flow Cytometry
- Dose groups: Vehicle; 0.05 & 0.5 µg/kg bw/d EE2; 2.5-25.000 µg/kg bw/d
- No. of animals: 2-10

Analysis of
- BPA-induced alteration of spleen cellularity by calculating the number of splenocytes per milligram of spleen at PND 21, PND 90, PND 183, PND 365.
- of lymphoid cell populations in splenocytes (B cells, CD3 T cells, CD4 T cells, CD8 T cells, natural killer cells (NK), natural killer T cells (NKT)) after BPA treatment at PND 21, PND 90, PND 183, PND 365.
- of BPA induced alterations of spleen associated myeloid cell populations (Monocytes, Macrophages, antigen presenting cells (APC), classic dendritic cells (cDC) and mature cDC) at PND 21, PND 90, PND 183 and PND 365.

THYMUS: Yes
- Method: Flow Cytometry
- Dose groups: Vehicle; 0.05 & 0.5 µg/kg bw/d EE2; 2.5-25.000 µg/kg bw/d
- No. of animals: 3-10

Analysis of BPA-induced changes on thymic cellularity by determination of the proportion of CD3+, CD4+/CD8+ double positive T cells, CD4+ helper cells and CD8+ cytotoxic T cells at PND 21.

Positive control:

Ethinyl estradiol (0.05 & 0.5 µg/kg bw/d)

Statistics:

Two-way ANOVA with Dunnett’s posttest was used.
Vehicle-treated animals, which were housed in the same room as those dosed with 25 mg/kg bw/d were excluded in this study. An exception is the 6-month timepoint. Here, all vehicle-treated animals were housed in the same room as the high-dose group.

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

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:

effects observed, non-treatment-related

Description (incidence and severity):

Within 530 measurements, 10 significant, BPA-related alterations.
No BPA-induced effects on the proportion of T cells in the thymus (PND 21).
Decreases in the percentage of macrophage and dendritic cells in BPA-treated male rats after 6 months and 1 year without persistent trend or dose response.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Cell viabilities:

not examined

Humoral immunity examinations:

not examined

Specific cell-mediated immunity:

effects observed, non-treatment-related

Description (incidence and severity):

Within 530 measurements, 10 significant, BPA-related alterations.
No BPA-induced effects on the proportion of T cells in the thymus (PND 21).
Decreases in the percentage of macrophage and dendritic cells in BPA-treated male rats after 6 months and 1 year without persistent trend or dose response.

Non-specific cell-mediated immunity:

not examined

Other functional activity assays:

not examined

Other findings:

not examined

Dose descriptor:

LOEL

Effect level:

0.003 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

immunology

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Lowest effective dose / conc.:

0.003 mg/kg bw/day (nominal)

System:

immune system

Organ:

spleen

Conclusions:

Authors concluded that most, if not all, of BPA-treatment related effects were found to be transient with no persistent trend.
This study shows that chronic BPA treatment in Sprague Dawley rats beginning on gestation day 6 continuing up to 1 year of age produced no persistent and reasonable changes in immune cell composition.

Executive summary:

This study is part of the BPA-Clarity Program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study.

For further information and details regarding housing conditions, dosing, study design, etc. see Clarity Core Study. Within this study the relative number and proportion of leukocyte populations in the spleen (PND 21, PND 90, PND 183, PND 365) and thymus (PND 21) was analyzed. Within 530 measurements, 10 significant, BPA-related alterations were found. No BPA-induced effects on the proportion of T cells in the thymus (PND 21) was observed. Analysis of leukocyte population in the spleen revealed decreased percentage of macrophage and dendritic cells in BPA-treated male rats after 6 months and 1 year without persistent trend or dose response. The authors concluded that most, if not all, of BPA-treatment related effects were found to be transient with no persistent trend.

In conclusion, this study shows that chronic BPA treatment in Sprague Dawley rats beginning on gestation day 6 continuing up to 1 year of age produced no persistent and reasonable changes in immune cell composition.

Reference 2

Endpoint:

immunotoxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study.

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study.
Core Study was conducted under GLP conditions. For further information and details see Clarity Core Study.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

This study is part of the CLARITY program and belongs to the Grantee Studies. For further information and details see Clarity Core Study.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study. For further information and details see Clarity Core Study.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study. For further information and details see Clarity Core Study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study. For further information and details see Clarity Core Study.

Duration of treatment / exposure:

PND 90, PND 183, PND 365

Frequency of treatment:

daily

Dose / conc.:

0.003 mg/kg bw/day (nominal)

Dose / conc.:

0.025 mg/kg bw/day (nominal)

Dose / conc.:

0.25 mg/kg bw/day (nominal)

Dose / conc.:

2.5 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

No. of animals per sex per dose:

1-10

Control animals:

yes, concurrent no treatment

Details on study design:

This study is part of the CLARITY program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study. For further information and details see Clarity Core Study.

Specific cell-mediated immunity:

SPLEEN: Yes
- Method: Cell Culture/Activation, ELISA, Proliferation Assay, Flow Cytometry
- Dose groups: Vehicle; 0.05 & 0.5 µg/kg bw/d EE2; 2.5-25.000 µg/kg bw/d
- No. of animals: 1-10

Analysis of
- Lipopolysaccharide (LPS)-, Pokeweed-mitogen (PWM)- and anti-CD3/28-induced spleenocyte proliferation after BPA-treatment at PND 90, PND 183 and PND 365.
- the effect of BPA on IgM responses at PND 90, PND 183, PND 365 - determination of intracellular IgM and secreted IgM after LPS or PWM activation.
- CD3/CD28-induced T cell activation after BPA treatment at PND 90, PND 183, PND 365.
- LPS-induced natural killer cell activation after BPA treatment at PND 183, PND 365.
- LPS-induced activation of monocytes/macrophages/granulocytes after BPA treatment at PND 90, PND 183, PND 365.
-LPS-induced activation macrophages/dendritic cells after BPA treatment at PND 90, PND 183, PND 365.

Positive control:

Ethinyl estradiol (EE2): 0.05 mg/kg bw/d; 0.5 mg/kg bw/d

Statistics:

Two-way ANOVA with Dunnett’s posttest was used. Vehicle-treated animals, which were housed in the same room as those dosed with 25 mg/kg bw/d were excluded in this study. An exception is the 6-month timepoint. Here, all vehicle-treated animals were housed in the same room as the high-dose group.

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

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:

effects observed, non-treatment-related

Description (incidence and severity):

Within 630 measurements, 35 significant BPA related alterations.
BPA-associated, augmented cell proliferation in respone to LPS or PWM in 1-year old male rats.
Most statistically significant changes associated with BPA treatment were sporadic and not dose-dependent with only one out of five BPA dose groups showing a significant difference.
Observed alterations were mostly moderate and showed no persistent trend over time.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Cell viabilities:

not examined

Humoral immunity examinations:

not examined

Specific cell-mediated immunity:

effects observed, non-treatment-related

Description (incidence and severity):

Within 630 measurements, 35 significant BPA related alterations.
BPA-associated, augmented cell proliferation in respone to LPS or PWM in 1-year old male rats.
Most statistically significant changes associated with BPA treatment were sporadic and not dose-dependent with only one out of five BPA dose groups showing a significant difference.
Observed alterations were mostly moderate and showed no persistent trend over time.

Non-specific cell-mediated immunity:

not examined

Other functional activity assays:

not examined

Other findings:

not examined

Dose descriptor:

LOEL

Effect level:

<= 0.003 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

immunology

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Within 630 measurements, 35 significant BPA related alterations. Several BPA-related changes at 0.0025 mg/kg bw/d, without persistent trend over time or dose response.

Critical effects observed:

no

Lowest effective dose / conc.:

0.003 mg/kg bw/day (nominal)

System:

immune system

Organ:

spleen

Conclusions:

Authors concluded that most observed effects were sporadic, moderate in magnitude, showed no persistent trend, were not dose-dependent and only one out of five BPA dose groups showed statistically significant difference.
This study shows that chronic BPA treatment in Sprague Dawley rats beginning on gestation day 6 continuing up to 1 year of age leads to no persistent and reasonable changes of lymphoproliferative and immune effector responses by splenic leukocytes.

Executive summary:

This study is part of the BPA-Clarity Program and belongs to the Grantee Studies. These Grantee studies used animals raised in the same condition and exposed to the same doses of BPA as the Core study.

For further information and details regarding housing conditions, dosing, study design, etc. see Clarity Core Study. Within this study i) Lipopolysaccharide (LPS)-, Pokeweed-mitogen (PWM)- and anti-CD3/28-induced spleenocyte proliferation after BPA-treatment at PND 90, PND 183 and PND 365, ii) the effect of BPA on IgM responses at PND 90, PND 183, PND 365 - determination of intracellular IgM and secreted IgM after LPS or PWM activation, iii) CD3/CD28-induced T cell activation after BPA treatment at PND 90, PND 183, PND 365, iv) LPS-induced natural killer cell activation after BPA treatment at PND 183, PND 365, v) LPS-induced activation of monocytes/macrophages/granulocytes after BPA treatment at PND 90, PND 183, PND 365 and vi) LPS-induced activation macrophages/dendritic cells after BPA treatment at PND 90, PND 183, PND 365 were analyzed. Within 630 measurements, 35 significant, BPA-related alterations were found. Except of a BPA-associated, augmented cell proliferation in respone to LPS or PWM in 1 -year old male rats, only sporadic and not dose-dependent (only one out of five dose groups showing a significant difference) BPA mediated effects were found. These BPA-induced alterations were mostly moderate and showed no persistent trend over time. The authors concluded that these BPA related changes are unlikely to compromise immune competence in adult rats. In conclusion, this study shows that chronic BPA treatment in Sprague Dawley rats beginning on gestation day 6 continuing up to 1 year of age produced no persistent and reasonable changes in lymphoproliferative and immune effector responses by splenic leukocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on immunotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Recent information taken into account for the dossier update:

 

There are two recent studies in the context of the US NTP CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) study (Li et al., 2018a & Li et al., 2018b). To address potential immunotoxicological effects, the present studies were conducted as part of the CLARITY-Bisphenol A program. The impact of Bisphenol A exposure (2.5, 25, 250, 2500 & 25000 µg/kg bw/d) on immune cell composition, lymphoproliferative and immune effector responses by splenic leukocytes was assessed in juvenile and adult NCTR Sprague-Dawley rats of both sexes. Ethinyl estradiol (EE; 0.5 µg/kg bw/day) was used as a reference estrogen. Exposure spanned gestation and lactation with dams gavaged from gestational day 6 until birth, and then the offspring gavaged directly through weaning until either PND 21, PND 90, PND 183 or PND 365. 

Immune cell composition was analyzed by cell counting and flow cytometry in spleen and thymus of various immune cell populations. Overall, no consistent effects of Bisphenol A were observed for any endpoint, in either sex, at either age compared to vehicle controls (Li et al., 2018a). Splenocyte proliferation, determination of intercellular and secreted IgM as well as activation of various immune cell populations was analyzed by ELISA, [3H]-Thymidine proliferation assay and flow cytometry. Also here Bisphenol-A treatment resulted only in sporadic findings, which were mostly moderate in magnitude and therefore, it is unlikely that BPA compromises immune competence in rats (Li et al., 2018b). Of note, vehicle-treated animals, which were housed in the same room as those dosed with 25000 µg BPA/kg bw/d were excluded in these studies due to the hypothesized potential for unintentional exposure.  

 

EFSA Opinion 2015 Conclusions on immune effects: 

"Based on recent human studies, there are indications that Bisphenol A may be linked to immunological outcomes in humans, although these studies had limitations and confounding factors may have been present. A causal link between Bisphenol A exposure during pregnancy or in childhood and immune effects in humans cannot be established. 

Studies in animals lend support to the possibility of immunological effects of Bisphenol A. Most of these studies suffered from shortcomings in experimental design and reporting. Although dose-responses could not be confidently established in most studies, a dose-related effect was observed in allergic lung inflammation. 

Using a WoE approach, the CEF Panel assigned a likelihood level of “-as likely as not- to likely” to immunotoxic effects of Bisphenol A. Since the likelihood level for this endpoint is less than “likely” (see Appendix A), this endpoint was not taken forward for assessing the toxicological reference point, but was taken into account in the evaluation of uncertainty for hazard characterisation and risk characterisation." 

Justification for classification or non-classification

Bisphenol A is included in Annex VI of Regulation (EC) No 1272/2008.