Formaldehyde, reaction products with bisphenol A and diethanolamine, propoxylated

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(2001)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(Reaction product of 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol, 2-(2-hydroxyethylamino)ethanol and formaldehyde), propoxylated

EC Number:

926-000-9

Cas Number:

1180524-77-9

IUPAC Name:

(Reaction product of 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol, 2-(2-hydroxyethylamino)ethanol and formaldehyde), propoxylated

Specific details on test material used for the study:

- Stability under test conditions: analytically verified
- Solubility and stability of the test substance in the solvent/vehicle: analytically verified

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

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

OTHER SPECIFICS:

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han) (outbred, SPF-Quality)
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at delivery: 10-14 weeks
- Housing: individually in Makrolon cages (MIII type) on sterilized sawdust as bedding material.
- Diet and water: ad libitum
- Acclimation period: at least 5 days prior to treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): approximately 40-70 %
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

Details on exposure:

Administration volume: 5 mL/kg bw; actual dose volumes were calculated according to the latest body weight;
Appearance of formulations: colorless clear solution 80, 100, 300 mg/kg bw); light yellow clear solution (1000 mg/kg bw)
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of formulations were conducted as part of the dose range finding study to confirm stability and homogeneity of the test item in the vehicle prior to the start of the main study:

The concentrations analysed in the formulations of Group Low (4 mg/mL) and Group High (200 mg/mL) were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).
The formulations of Group Low (4 mg/mL) and Group High (200 mg/mL) were homogeneous (i.e. coefficient of variation ≤ 10%).
Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%). The long term storage samples were stable at ≤-70°C for at least 34 days.

Details on mating procedure:

Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating; confirmed by vaginal plug).

Duration of treatment / exposure:

Days 6 - 20 p. c., inclusive

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Duration of test:

from day 0 to necropsy at day 21 p.c.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on results of the dose range finding study.

Four groups of 6 females were exposed to 0, 100, 300 and 1000 mg/kg bw/day for Days 6 to 20 post-coitum inclusive by oral gavage. These dose levels were based on repeated dose oral toxicity study in male and female rats (OECD Test Guideline 407), in which the rats received the test item at dose levels of 0, 100, 500 and 1000 mg/kg bw/day in polyethylene glycol 400. At 1000 mg/kg bw/day ERY, HB and HCT levels were slightly, but significantly reduced in females. The same trend, but not statistically significant, could be seen in high-dosed males. The NOAEL is 500 mg/kg bw/day for male and female rats.

- Maternal findings

No mortality occurred during the study period.
No toxicologically significant clinical signs were noted up to 1000 mg/kg bw/day.
Salivation was noted in all animals at 100, 300 and 1000 mg/kg bw/day from Day 12 post-coitum onwards up to the end of treatment. As there were no correlated findings, the salivation is likely attributed to the taste of the test item.
No treatment related effects were observed on body weights and body weight gain up to 1000 mg/kg bw/day.
Food consumption was unaffected by treatment up to 1000 mg/kg bw/day.
No macroscopic findings were noted at necropsy.
One female in the group treated at 100 mg/kg bw/day delivered early on Day 21 post-coitum.
No treatment related changes for the number of post-implantation loss were observed.

- Fetal findings

Litter sizes were within normal limits for all groups.
The male : female ratios were equal in litters of all groups.
Fetal body weights were unaffected by treatment up to 1000 mg/kg bw/day.
External examination of the fetuses did not show any abnormalities.

Examinations

Maternal examinations:

CLINICAL EXAMINATIONS: Yes
- Time schedule: At least once daily from Day 2 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: liver-, kidney-weight

OTHER: 10 selected females/group were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands). Blood samples were drawn from the aorta for clinical laboratory investigations (for details see below), followed by immediate exsanguination.

Haematology parameters determined:

Red blood cells
Reticulocytes
Red blood cell distribution width (RDW)
Haemoglobin
Haematocrit
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Platelets

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths (early and late resorptions).
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal
inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.

Fetal examinations:

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural
anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
External:
Each viable fetus was examined in detail, weighed and sexed. All live fetuses were euthanized by administration of approximately 0.05 mL (=10mg) of sodium pentobarbital into the oral cavity using a small flexible plastic or metal feeding tube.
Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination performed (if possible).
Visceral (Internal):
Approximately one-half of the fetuses (live and dead) in each litter (all groups)were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (Ref. 2). This
examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (Ref. 3). The sex of all fetuses was confirmed by internal examination.
The heads were removed from these fetuses and placed in Bouin's solution. Tissues were then transferred to a 70% aqueous ethanol solution for subsequent processing and soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues were stored in 10% buffered formalin. Any remaining tissues (from the fetuses used for fresh visceral examination) was discarded. The carcasses were processed and stained with Alizarin Red S (as described below), but not examined in first instance.
Skeletal:
From the other one-half of the fetuses (live and dead) in each litter (all groups), the sex was confirmed by internal examination. All fetuses were eviscerated, fixed in 96% aqueous ethanol, macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson (Ref. 5). Skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). The specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

For each litter the following calculations were performed:
Percent Pre-implantation loss, percent Post-implantation loss
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a
mean litter proportion on a total group basis, i.e. percent Viable fetuses affected/litter
The following definitions were applicable for implantation data:
- Fetal (late) resorptions were defined as a dead fetus with external degenerative changes and presence of distinguishable features such as head or limbs.
- Embryonic (early) resorptions were defined as evidence of implantation without presence of distinguishable features such as head or limbs.
- Dead fetus was defined as a non-viable fetus without external degenerative changes and presence of distinguishable features such as head or limbs.
- Post-implantation loss included embryonic (early) resorptions, fetal (late) resorptions and dead fetuses.

Historical control data:

Yes - are reported in the Appendix "Historical Data Fetal Morphology" of the study report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation was observed in 9/22 and 19/22 females treated at 300 and 1000 mg/kg bw/day, respectively, for two to several days at the end of treatment. As there were no correlated findings noted, the salivation was likely attributed to the taste of the test item.
The incidence of scabs and alopecia remained within the range of background findings to be expected for rats this strain and age.
No other treatment related clinical signs were noted in any of the groups.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was slightly, but significantly, reduced from from Day 6-9 post-coitum at 300 mg/kg bw/day and from Day 6-12 at 1000 mg/kg bw/day. As the effect was only minor, recovered fully from Day 12 post-coitum onwards, and there was no concurrent effect on body weight or body weight gain, the reduced food consumption was not considered to be adverse.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

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):

Kidney weights were unaffected by treatment up to 1000 mg/kg bw/day.

The weight of the liver (absolute and relative) showed a dose related increase. The increase of the absolute liver weights of approximately 11% at 1000 mg/kg bw/day was statistically significant. Relative liver weights were significantly increased with 7% and 11% in females treated at 300 and 1000 mg/kg bw/day. Although the effect at 300 mg/kg bw/day was likely the result of treatment, it is only minor (<10%) and therefore not considered adverse in isolation. However, as the increase of the absolute and relative liver weights at 1000 mg/kg bw/day was marginally above more than 10%, % (11%) this was considered to be an adverse effect of treatment because either data on clinical chemistry or histopathology were collected within the scope of this prenatal developmental toxicity study.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day and 1000 mg/kg bw/day, two females (No. 51 and 73, respectively) had a visibly enlarged liver. Although incidental, this finding corresponds with the increased liver weights observed in these groups.

In female No. 30, treated at 100 mg/kg bw/day, many reddish/black foci were observed in the glandular mucosa of the stomach, which was also thickened and had an irregular surface. Additionaly, clotted blood was noted in the thoracic cavity at the heart for this animal. In female No. 35, which was non-pregnant , the uterus was filled with fluid. The presence of scab formation and alopecia were confirmed at necropsy for female No. 12 (Control) and 52 (300 mg/kg bw/day), respectively. These findings were considered to be incidental and not related to treatment.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

2

Dose descriptor:

LOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

The effect on liver weight is treatment related - whether it may be adverse cannot be decided from this study (see chapter 7.5).

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no treatment related effects on: litter size, sex ratio, fetal body weights, fetal mophological examination, external malformations and variations, visceral malformations and variations, sceletal malformations and variations

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: itter size, sex ratio, fetal body weights, fetal mophological examination, external malformations and variations, visceral malformations and variations, sceletal malformations and variations

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

An a prenatal developmental toxicity study performed according to OECD TG 414 mated female Wistar rats were assigned to four dose groups, each containing twenty-two animals. The test item was administered once daily by gavage from Day 6 to 20 post-coitum at doses of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, Polyethylene glycol 400, alone.
No treatment related clinical signs were noted in any dam of the groups. Body weights and body weight gain remained within the same range for all groups. Food consumption was slightly, but significantly, reduced from from Day 6-9 post-coitum at 300 mg/kg bw/day and from Day 6-12 at 1000 mg/kg bw/day. As the effect was only minor, recovered fully from Day 12 post-coitum onwards, and there was no concurrent effect on body weight or body weight gain, the reduced food consumption was not considered to be adverse. All haematology parameters were unaffected by treatment up to 1000 mg/kg bw/day.No treatment related macroscopic findings were noted. Kidney weights were unaffected by treatment up to 1000 mg/kg bw/day.The weight of the liver (absolute and relative) showed a dose related increase. The increase of the absolute liver weights of approximately 11% at 1000 mg/kg bw/day was statistically significant. Relative liver weights were significantly increased with 7% and 11% in females treated at 300 and 1000 mg/kg bw/day. Although the effect at 300 mg/kg bw/day was likely the result of treatment, it is only minor (<10%) and therefore not considered adverse in isolation. However, as the increase of the absolute and relative liver weights at 1000 mg/kg bw/daywas marginally above 10% (11%) this was considered to be an adverse effect of treatment because either data on clinical chemistry or histopathology were collected within the scope of this prenatal developmental toxicity study
No treatment-related effects on the fetal development were observed in all test item treated groups. In detail, litter size, sex-ratio, fetal body weight, and fetal morphological examinations (external, visceral and skeletal) were not affected by the treatment.
In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for the test item was established as being 300 mg/kg bw/day. The developmental NOAEL was established as being at least 1000 mg/kg bw/day.

Executive summary:

An a prenatal developmental toxicity study performed according to OECD TG 414 mated female Wistar rats were assigned to four dose groups, each containing twenty-two animals. The test material was administered once daily by gavage from Day 6 to 20 post-coitum at doses of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, Polyethylene glycol 400, alone. No treatment related clinical signs were noted in any dam of the groups. Body weights and body weight gain remained within the same range for all groups. Food consumption was slightly, but significantly, reduced from from Day 6-9 post-coitum at 300 mg/kg bw/day and from Day 6-12 at 1000 mg/kg bw/day. As the effect was only minor, recovered fully from Day 12 post-coitum onwards, and there was no concurrent effect on body weight or body weight gain, the reduced food consumption was not considered to be adverse. All haematology parameters were unaffected by treatment up to 1000 mg/kg bw/day.No treatment related macroscopic findings were noted. Kidney weights were unaffected by treatment up to 1000 mg/kg bw/day.The weight of the liver (absolute and relative) showed a dose related increase. The increase of the absolute liver weights of approximately 11% at 1000 mg/kg bw/day was statistically significant. Relative liver weights were significantly increased with 7% and 11% in females treated at 300 and 1000 mg/kg bw/day. Although the effect at 300 mg/kg bw/day was likely the result of treatment, it is only minor (< 10%) and therefore not considered adverse in isolation. However, as the increase of the absolute and relative liver weights at 1000 mg/kg bw/day was marginally above 10% (11%) this was considered to be an adverse effect of treatment because either data on clinical chemistry or histopathology were collected within the scope of this prenatal developmental toxicity study. No treatment-related effects on the fetal development were observed in all test material treated groups. In detail, litter size, sex-ratio, fetal body weight, and fetal morphological examinations (external, visceral and skeletal) were not affected by the treatment. In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for the test material was established as being 300 mg/kg bw/day. The developmental NOAEL was established as being at least 1000 mg/kg bw/day.