Formaldehyde, reaction products with bisphenol A and diethanolamine, propoxylated

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In a repeated dose oral toxicity study in rats (Wistar, OECD TG 407), the substance was administered via gavage to 5 rats/sex/dose at 0, 100, 500, and 1000 mg/kg bw and day in PEG 400 for 4 weeks. Neither gross pathological nor histopathological changes of the male and female reproductive organs (testes, epididymides, prostate, seminal vesicles including coagulation glands, ovaries with oviducts, uterus with cervix, vagina, and mammary glands) were reported at any exposure concentration, including the highest dose revealing slight systemic toxicity. The NOAEL for adverse effects on reproductive organs or tissues can therefore be determined with 1000 mg/kg bw/day. Overall, based on the results of the subacute oral study there is evidence for a very mild systemic toxicity and not any indication of a reproductive toxicity potential of the test material in doses up to and including the limit dose. Although studies on fertility, respectively extended one- or multigeneration- studies are not available for the test material further testing is considered to be of low priority. In accordance to REACH Annex XI, section 1.2. there is sufficient weight of evidence to conclude that Desmorapid 01 is not a reproductive toxicant and therefore further testing on vertebrate animals for that property shall be omitted (for details see waiver for fertility/multigeneration study). In line with REACH Annex XI, section 1.1.2 the available information on the reproductive toxicity potential of the test material is adequate for the purpose of classification and labeling and/or risk assessment.

Link to relevant study records

Reference

Endpoint:

reproductive toxicity, other

Remarks:

subacute study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD TG 407

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: PEG 400

Details on mating procedure:

not applicable because it is a subacute study

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

once a day

Remarks:

Doses / Concentrations:
0, 100, 500, 1000 mg/kg bw and day
Basis:
other: actual dose received

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: adverse effects on reproductive organs or tissues

Remarks on result:

other: Generation: not applicable because it is a subacute study (migrated information)

Reproductive effects observed:

not specified

Conclusions:

No adverse effects on reproductive organs or tissues were observed in a subacute oral study with rats at 1000 mg/kg and bw.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

No extended one- or multigeneration- studies are required because the 28-day study indicates no adverse effects on reproductive organs or tissue. Data waiver is claimed.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a repeated dose oral toxicity study in rats (Wistar, OECD TG 407), the substance was adimistered via gavage to 5 rats/sex/dose at 0, 100, 500, 1000 mg/kg bw in PEG 400 for 4 weeks. No adverse effects on reproductive organs or tissue were observed up to and including 1000 mg/kg bw

Effects on developmental toxicity

Description of key information

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.

A GLP-compliant OECD 414 gudieline study in time-mated female New Zealand White rabbits was conducted in order to examine the prenatal developmental toxicity of (Reaction product of 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol, 2-(2 -hydroxyethylamino)ethanol and formaldehyde), propoxylated. The dose levels were set at 720, 300 and 100 mg/kg bw/day. The maternal NOAEL was established as being 300 mg/kg/day based on the preterm sacrifice observed at 720 mg/kg/day. No adverse effects regarding developmental toxicity were observed at the highest dose level tested. The developmental NOAEL was established as being at least 720 mg/kg/day.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(2001)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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:

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

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.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

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.

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

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

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

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).

Abnormalities:

no effects observed

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

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

Abnormalities:

no effects observed

Developmental effects observed:

no

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-09-14 to 2022-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted August 1998

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: at room temperature
- Stability and homogeneity of the test material in the vehicle under test conditions and during storage: stable, maximum temperature 40°C; Stability for 4 days in the refrigerator protected from light (followed by at least 12 hours at room temperature, protected from light) and for 20 days in the freezer, protected from light in Eppendorf tubes is confirmed over the concentration range 1 to 800 mg/mL

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 17-19 weeks (age at mating)
- Weight at study initiation: 2977 - 4180 g
- Fasting period before study:
- Housing: individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm). For psychological/environmental enrichment, animals were provided with shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm) and wooden sticks (Swedish aspen wood, Bioservices, Schaijk, The Netherlands).
- Diet: restricted access; KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding pellets(Granovit AG, Kaiseraugst, Swizerland); on arrival animals received approximately 25 grams pelleted diet and on subsequent days 140-160 grams were supplied.
- Water: ad libitum, municipal tap water
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-19
- Humidity (%): 47-62
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2021-12-15 To: 2022-01-14

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Daily, within 24 hours prior to dosing.
- Storage Conditions set to maintain: 15-25 °C
- Formulations (w/w) homogenized to visually acceptable levels. Formulations were heated up to 40 °C.
- Formulations and vehicle were continuously stirred until and during dosing.

VEHICLE
- Justification for use and choice of vehicle: polyethylene glycol 400 was used as vehicle for solubility reasons
- Concentration in vehicle: 66.7 mg/mL, 200 mg/mL, 480 mg/mL
- Amount of vehicle: 1.5 mL/kg
- Supplier: Merck, Darmstadt, Germany
- Specific gravity: 1.125

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity and were within acceptance criteria. Analytical verification was performed via HPLC/UV.

Details on mating procedure:

- Impregnation procedure: purchased time-mated females

Duration of treatment / exposure:

day 7 to day 28 post-coitum

Frequency of treatment:

once daily

Duration of test:

21 days (dosing)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

720 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on dose range finding study: At 720 mg/ kg bw/day, one female was sacrificed in extremis on Day 24 post-coitum due body weight loss (8%) and absent food consumption for ten consecutive days. The female was observed with erected fur
on Days 23 and 24 post-coitum and at necropsy, multiple red foci were found on the liver. The surviving females were observed with erected fur (2/5 females), body weight loss (4% between Days 12-21 post-coitum, 1/5 females) and severely reduced to absent food consumption for several days. No macroscopic findings were observed for the females sacrificed according to study schedule. Therefore, 720 mg/kg/day was considered as maximum dose level to be tolerated by pregnant rabbits.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency will be at least once daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily; starting on Day 7 post-coitum up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 3, 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 29 post-coitum
- Organs examined:
All animals were subjected to an external, thoracic and abdominal examination,with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in the appropriate fixative, together with the animal identification; Each ovary and uterine horn of all animals was dissected and examined as quickly as possible

OTHER: Food consumption observe daily from Day 3 post-coitum onwards. Water consumption observed on a regular basis throughout the study

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of late resorptions: Yes
- Other: number and distribution of live and dead fetuses

Blood sampling:

No data

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]

Statistics:

Parametric/Non-Parametric
Levene’s test will be used to assess the homogeneity of group variances.
The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons will be conducted using Dunnett’s or Dunn’s test, respectively.

Non-Parametric
The groups will be compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons will be conducted using Dunn’s test.

ANCOVA
The data corresponding to a response variable of interest and to a related covariate will be submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons will be conducted using Dunnett’s test.

Indices:

Male Fetuses (%): (No. of male fetuses x 100) / No. of fetuses
Female Fetuses (%): (No. of female fetuses x 100) / No. of fetuses
Pre-Implantation Loss (%): (No. of corpora lutea – No. of implantations) / No. of corpora lutea x 100
Post-Implantation Loss (%): (No. of implantations – No. of live fetuses) / No. of implantations x 100
Litter % of Fetuses with Abnormalities: No. of fetuses in litter with a given finding / No. of fetuses in litter examined x 100

Historical control data:

Historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source are available at the laboratory.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: one female (1/22, No. 37) was observed with non-treatment related clinical signs: erected fur, decreased activity, animal felt cold to touch

Mortality:

mortality observed, treatment-related

Description (incidence):

Control group:
- one female (1/22) found dead on Day 25 post-coitum due to gavage-related incident
100 mg/kg bw/day:
- one female (1/22, No. 37) found dead on Day 18 post-coitum. Mortaility was considered unrelated to treatment with test material
300 mg/kg bw/day:
- all animals survived until scheduled sacrifice
720 mg/kg bw/day:
- two females (2/22) were sacrificed on Day 20 post-coitum based on prolonged and absent food consumption.
- Mortality is considered treatment-related with the test material.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day:
- no effects observed
300 and 720 mg/kg bw/day:
- mean adjusted body weight loss over the dosing period (something that is usually seen in rabbits) was less pronounced compared to control (-112.7 g and -102.2 g, respectively vs -152.9 g in the control group). The apparent higher mean weight gain was attributed to the control group having a slightly lower mean starting weight on Day 7 post-coitum with subsequent higher mean weight gain.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

100 and 300 mg/kg bw/day:
- no effects observed
720 mg/kg bw/day:
- lower mean food consumption (up to 22%) compared to control between Days 9-21 post-coitum, recovered to control levels as dosing progressed
- Overall mean food intake was in the same range as the concurrent control
- As the effect was reversible and occurred in the absence of effects on body weight, this was considered to be non-adverse

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

300 mg/kg bw/day:
- Constriction of the spleen or right caudal lobe of the lung in combination with an enlarged
spleen was observed in (1/22)
720 mg/kg bw/day:
- Constriction of the spleen or right caudal lobe of the lung in combination with an enlarged
spleen was observed in (1/22)

These findings occurred at similar or lower incidences as in the concurrent control group and were without a dose response and remained within the range of biological variation for rabbits of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/day:
- higher mean percentage of pre-implantation loss observed in absence of a dose-response
- higher mean percentage of postimplantation loss (8.72%) compared to control considered unrelated to treatment with the test material (caused by the high number of resorptions observed for Female No. 56 with a postimplantation loss of 66.7%).

720 mg/kg bw/day:
- higher mean percentage of postimplantation loss compared to control (11.28% vs 5.82%, respectively), caused by the high number of early resorptions observed for Female No. 68 (postimplantation loss of 75%). However, when excluding this female, the mean percentage of postimplantation loss was still slightly higher than the control value (mean of 7.9% after correction vs 5.82% in the control group)
- At the small magnitude, these changes were considered non-adverse

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

720 mg/kg bw/day: high number of early resorptions observed for Female No. 68 (i.e., six, resulting in a postimplantation loss of 75%)

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

mortality

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/day:
-slightly lower mean number of fetuses (i.e., 9.1 vs 9.6 for concurrent control), caused by one female (No. 56) with only one live fetus. The remaining females at this dose group were observed with litter sizes comparable to control. As this only concerned a single female, this was considered unrelated to treatment with the test material.
720 mg/kg bw/day:
- lower mean number of fetuses compared to control (8.4 vs 9.6 for concurrent control), mainly caused by the low number of fetuses observed for Female Nos. 68 and 78 (2 and 3 live fetuses, respectively) but was still below the control value after excluding these two females from the mean calculation (9.1 after correction). At the small magnitude, these changes were considered non-adverse.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Control group:
- one fetus (1/172) was observed with multiple severe malformations (i.e., absent head, hyperflexed hindlimb, malrotated hindpaw, ectrodactyly, short tail and thoracogastroschisis).

300 mg/kg bw/day:
- 2 fetuses (2/180) presented with hyperflexion of the forepaw and omphalocele, respectively
- no dose-response observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Control group:
- one fetus (1/172) with skeletal malformations observed
720 mg/kg bw/day:
- five fetuses (5/168) with visceral malformations observed
100 mg/kg bw/day:
- two fetuses (2/179) with visceral malformations observed

skeletal variations were either scored infrequently and/or in instances comparable to the control group or were within historical control data

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Control group:
- five fetuses (5/172) with visceral malformations observed
720 mg/kg bw/day:
- one fetus (1/168) with visceral malformations observed
300 mg/kg bw/day:
- three fetuses (3/180) with visceral malformations observed
100 mg/kg bw/day:
- two fetuses (2/179) with visceral malformations observed

Visceral variations and incidental findings were observed in a diverse range of visceral structures across all groups. However, in all cases these observations were either scored infrequently, in instances comparable to the control group or in the absence of a dose-relationship

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

720 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects obsereved at highest dose level tested not resulting in maternal toxicity

Abnormalities:

no effects observed

Description (incidence and severity):

no adverse effects observed related to treatment with the test-material. All abnormalities that were observed are within control and/or historical control data

Developmental effects observed:

yes

Lowest effective dose / conc.:

720 mg/kg bw/day (actual dose received)

Treatment related:

not specified

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Conclusions:

In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female New Zealand White rabbits, the maternal NOAEL was established as being 300 mg/kg/day based on the preterm sacrifice observed at 720 mg/kg/day. No adverse effects regarding developmental toxicity were observed at the highest dose level tested. Thus, the developmental NOAEL was established as being at least 720 mg/kg/day.

Executive summary:

A GLP-compliant OECD 414 gudieline study in time-mated female New Zealand White rabbits was conducted in order to examine the prenatal developmental toxicity of (Reaction product of 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol, 2-(2 -hydroxyethylamino)ethanol and formaldehyde), propoxylated. The dose levels were set at 720, 300 and 100 mg/kg bw/day. The maternal NOAEL was established as being 300 mg/kg/day based on the preterm sacrifice observed at 720 mg/kg/day. The developmental NOAEL was established as being at least 720 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

720 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

guideline study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available study results a classification according to Regulation (EC) No 1272/2008, Annex I is not warranted.