4,4'-Isopropylidenediphenol, polymer with 1-chloro-2,3-epoxypropane, propane-1,2-diol acrylate and succinic anhydride

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Available weight of evidence information from physico-chemical properties, QSAR predictions and other toxicity studies indicates that, the test substance can be overall expected to have a low absorption potential through oral and dermal routes and high to moderate through inhalation routes (if exposed). Further, based on the estimated BCF value and physico-chemical parameters, it is likely to have low bioaccumulation potential and excreted primarily via faeces. Based on metabolism simulators from OECD Toolbox, the constituents of the test substance are likely to undergo ester hydrolysis as the first metabolic reactions leading to the formation of polar metabolites, which would eventually be excreted via urine.

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

50

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

There were no studies available in which the toxicokinetic properties of test substance‘4,4’-Isopropylidenediphenol, polymer with 1-chloro-2,3-epoxypropane, propane-1,2-diol acrylate and succinic anhydride’ were investigated. However, as per REACH guidance document R7.C (2017), information on absorption, distribution, metabolism and excretion may be deduced from the physicochemical properties, QSAR modelling and information from other studies.

ABSORPTION

Oral absorption

Based on physico-chemical properties

According to REACH guidance document R7.C, oral absorption is maximal for substances with molecular weights below 500. Water-soluble substances will readily dissolve into the gastrointestinal fluids. However, absorption of hydrophilic substances via passive diffusion may be limited by the rate at which the substance partitions out of the gastrointestinal fluid. Further, absorption by passive diffusion is higher at moderate log Kow values (between -1 and 4). If signs of systemic toxicity are seen after oral administration (other than those indicative of discomfort or lack of palatability of the test substance), then absorption has occurred.

The test substance is an UVCB, with several constituents having molecular weights (MW) ranging from 130.14 to 1259.37g/mol, with an average MW of 722.43 g/mol. It is a highly viscous liquid with a poor water solubility determined to be <0.007 mg/L and an experimentally determined log Kow ranging from <1.1 to >6.2. Volatility was determined to be low (0.19 Pa).

Based on the R7.C indicative criteria, oral uptake of the test substance is assessed to be low, given their high average molecular weight (exceeding 500), poor water solubility and relatively high log Kow.

 

Based on QSAR predictions:

The Lipinski’s rule OASIS profiler of the OECD QSAR Toolobox v.4.3.1, which describes the molecular properties important for a drug’s pharmacokinetics in the human body, including their absorption, distribution, metabolism, and excretion (”ADME”), predicted‘less bioavailable’for all the constituents except for hydroxypropyl acrylate or tripropylene glycol monoacrylate (i.e., constituent 5).

Based on other toxicity studies:

Some amount of systemic absorption of the test substance via the oral route is confirmed due to presence of clinical signs in an acute toxicity study in rats.

Conclusion:The assessment of oral absorption based on physico-chemical properties as well as Lipinski’s rule of five profiler indicates that, except for the residual constituent 5, which is present at very low amounts, the overall test substance can be expected to have a low absorption potential. However, a maximum of 50% oral absorption has been considered for a conservative risk assessment.

Dermal absorption

Based on physico-chemical properties

According to REACH guidance document R7.C (ECHA, 2017), dermal absorption is maximal for substances having MW below 100 together with log Kow values ranging between 2 and 3 and water solubility in the range of 100-10,000 mg/L. Substances with MW above 500 are considered to be too large to penetrate skin. Further, dermal uptake is likely to be low for substances with log P values <0 or <-1, as they are not likely to be sufficiently lipophilic to cross the stratum corneum (SC). Similarly, substances with water solubility below 1 mg/L are also likely to have low dermal uptake, as the substances must be sufficiently soluble in water to partition from the SC into the epidermis.

The test substance is viscous liquid, with an MW exceeding 100 g/mol, poor water solubility (<1 mg/L) and an experimental log Kow of ≥3 for majority of the constituents. This suggests that the test substance is likely to have a low penetration potential through the skin.

Based on QSAR predictions:

The two well-known parameters often used to characterise percutaneous penetration potential of substances are the dermal permeability coefficient (Kp[1]) and maximum flux (Jmax). Kp reflects the speed with which a chemical penetrates across stratum corneum (SC) and Jmax represents the rate of penetration at steady state of an amount of permeant after application over a given area of SC. Out of the two, although Kp is more widely used in percutaneous absorption studies as a measure of solute penetration into the skin. However, it is not a practical parameter because for a given solute, the value of Kp depends on the vehicle used to deliver the solute. Hence, Jmax i.e., the flux attained at the solubility of the solute in the vehicle is considered as the more useful parameter to assess dermal penetration potential as it is vehicle independent (Robert and Walters, 2007).

In the absence of experimental data, Jmax can be calculated by multiplying the estimated water solubility with the Kp values from DERMWIN v2.01 application of EPI SuiteTMv4.11. The calculated Jmax of the major constituents were found to be very low ranging from 3.1E-14 to 4.4E-06 μg/cm2/h, except for a value of 77 μg/cm2/h calculated for the residual constituent 5 (i.e., hydroxypropyl acrylate), which is present at <3% in the composition. As per Shenet al.2014, the default dermal absorption for substances with Jmax is ≤0.1 μg/cm2/h is less than 10% and for substances with Jmax is >0.1 μg/cm2/h but ≤10 μg/cm2/h, it did not exceed 40%.

The above Jmax based dermal absorption assessment is further supported by the ‘low skin permeability’ categorisation according to the ‘skin permeability (beta)’ profiler from OECD QSAR Toolbox 4.3.1 for all constituents, except for the residual constituent 5.

Therefore, based on these calculations, the test substance is overall predicted to have a low absorption potential except for a moderate potential for the residual constituent 5, via the dermal exposure route.

Based on other toxicity studies:

Some amount of bioavailability of the test substance via the dermal route is confirmed by the positive sensitisation studies indicating that some transfer through the first epidermal layer has taken place.

Further, body weight alterations observed in few animals in the acute dermal toxicity study in rats, indicates some amount of systemic absorption either through dermal route or due to ingestion via grooming.

Conclusion: The assessment of dermal absorption based on physico-chemical properties, QSAR predictions and other toxicity studies indicates that, except for the residual constituent hydroxypropyl acrylate, which is present at very low amounts, the overall test substance can be expected to have a low absorption potential. However, a maximum of 50% dermal absorption has been considered for a conservative risk assessment.

Inhalation absorption

Based on physico-chemical properties:

According to REACH guidance document R7.C, inhalation absorption is maximal for substances with VP >25 KPa, particle size (<100 μm), low water solubility and moderate log Kow values (between -1 and 4). Very hydrophilic substances may be retained within the mucus and not be available for absorption.

The test substance because of its high viscosity and relatively low vapour pressure of 0.19 Pa at 20°C, will not be available as vapour for inhalation under ambient conditions. Also, spray applications (i.e., PROC 7 or 11) are not included in the registration dossier. Therefore, the substance will neither be available for inhalation as vapour nor as aerosols.

Conclusion:Overall based on physico-chemical properties of the test substance, absorption is possible via upper mucosa but is limited by the inhaled amount which is assumed to be low due to the very low vapour pressure. Therefore, a conservative default value of 100% inhalation absorption has been considered for a conservative risk assessment.

METABOLISM:

Based on QSAR predictions:

The predicted metabolism of the test substance was evaluated using the in vivo rat metabolism simulator and the rat liver S9 metabolism simulator of the OECD QSAR Toolbox v.4.3. According to these simulators, the main constituents (present at >5%) are primarily predicted to undergo ester hydrolysis as first metabolic reaction. See table in the CSR for the reaction sites. For further details, refer to the read across justification.

Based on other toxicity studies:

The results of acute, repeated dose and mutagenicity testing suggest that no toxic metabolites are formed when the constituents of test substance are broken down.

DISTRIBUTION:

Based on physico-chemical properties:

The physico-chemical information (high molecular weight, high lipophilicity and low water solubility) indicates that test substance could be distributed to many tissues, once absorbed and bioavailable.

Accumulation potential: Based on the estimated BCF and MW, water solubility values, the bioaccumulation potential of the substance is expected to be low.

EXCRETION:

Based on physico-chemical properties:

Based on the high MW and low water solubility, the test substance as such is expected to be excreted primarily via faeces. Nevertheless, there will be some urinary elimination following formation of water-soluble conjugates via Phase II reactions.

[1]Log Kp = -2.80 + 0.66 log kow – 0.0056 MW

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from 05-Sep-2014 to 20-Aug-2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the read across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 21 September 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Directive 96/54/EC, 30 September 1996

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: Ca. 7 weeks
- Weight at study initiation: Actual Body Weight Ranges (at Acclimatization) were for the Males 162 - 203 g (mean: 181 g) and for the Females: 125 - 151 g (mean: 138 g)
- Fasting period before study: The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Housing: In groups of up to four in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 20/14 and 46/14) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batches were analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Bacteriological assay, chemical and contaminant analyses of respective samples were performed
- Acclimation period: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

IN-LIFE DATES: From: 11-Sep-2014 (start of 7-day Acclimatization) To: 18/19-Dec-2014 (Necropsy)

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

300

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared with the test item as delivered by the Sponsor.
The vehicle was pre-warmed to a temperature of approximately 40 °C. DGEBADA was weighed into a glass beaker on a tared precision balance and approximately 80 % of the warm vehicle was added (w/v). The mixture was mixed thoroughly using a homogenizer until a clear to colloid-like yellowish solution was obtained. Having obtained a homogeneous mixture, the remaining vehicle was added until the required final volume was reached. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
The dose formulations were stored for up to eight days in glass beakers at room temperature (20 ± 5 °C).

DIET PREPARATION
- Not relevant due to exposure via gavage

VEHICLE
- Justification for use and choice of vehicle: Considered non-toxic to the test animals and ability to form a homogeneous mixture with the test item
- Concentration in vehicle (nominal): 0, 20, 60 and 100 mg/mL (at 0, 100, 300 and 1000 mg/kg bw/day, respectively)
- Amount of vehicle (by gavage): 5 mL/kg body weight
- Lot/batch no.: BCBN0917V (Source: Sigma-Aldrich Chemie GmbH, Steinheim / Germany, Expiry Date: 30-Sep-2016)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At experimental start, on day 1 and during weeks 4, 8 and 13, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 1 g of each concentration were taken to confirm stability (4 hour and 10 days).
These representative samples were dispatched to the analytical laboratories internally (at room temperature and back up samples, on dry ice) and either stored frozen at -20 ± 5 °C until analysis or directly analyzed.
The test item was used as analytical standard.
Stock solutions of DGEBADA in acetonitrile were prepared for external calibration. For example, 20.92 mg of DGEBADA was weighed into a 50 mL volumetric flask and filled to about 75 % of final volume with acetonitrile. The mixture was sonicated for at least five minutes and brought to volume with acetonitrile to yield a solution with a concentration of 418.4 µg/mL. Aliquots of this stock calibration solution were diluted with acetonitrile to obtain calibration solutions with nominal concentrations ranging from 12.55 to 200.8 µg/mL. On each occasion calibration solutions derived from two stock solutions were used for calibration.
Each sample was transferred into an appropriate volumetric flask. The sample vial was successively rinsed with at least two portions of acetonitrile and the rinsings were combined in the volumetric flask. The flask was filled to about 75 % of the target volume with acetonitrile and dissolution was achieved by sonication for at least five minutes. The flask was filled to the mark with acetonitrile. Sample solutions were further diluted with acetonitrile into the calibration range.
Typical HPLC systems from Merck-Hitachi series 7000 were used:
- Computerized System: EZ Chrom Elite; Agilent Technologies
- Column: Kinetex C18; 50 x 4.6 mm, 5 µm
- Pre-Column: Phenomenex 4 x 3 mm
- Column Temperature: Ambient
- Eluent A: Purified water / acetonitrile (50/50, v/v)
- Eluent B: Acetonitrile
- Gradient:
Time [min]; Eluent A [%]; Eluent B [%]
0; 100; 0
4; 100; 0
8; 5; 95
10; 5; 95
10.1; 100; 0
15; 100; 0
- Flow: 1 mL/min
- Wave Length: 230 nm
- Injection Volume: 10 µL
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and calibration solution concentrations. All calibration points used met the acceptance limit of ±20 % variation from the calibration curve derived by linear regression analysis. The coefficients of determination (R²) were higher than 0.99.
The DGEBADA concentrations in the dose formulations ranged from 82.8 to 104.5 % with reference to the nominal and were within the accepted range of ±20 %, except for samples prepared on 11 December 2014 that were in the range from 126.5 to 136.0 %.
The homogeneous distribution of DGEBADA in the preparations was approved because single results found did not deviate more than 4.5 % from the corresponding mean and met the specified acceptance criterion of =15 %. Accordingly the effects were assigned to the nominal concentrations.
In addition, the test item was found to be stable in application formulations when kept up to ten days at room temperature due to recoveries which met the variation limit of 10 % from the time-zero (homogeneity) mean value.

Duration of treatment / exposure:

92/93 days

Frequency of treatment:

Daily, at approximately 24 hour intervals

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor.
- Rationale for animal assignment: Randomly allocated to groups by body weight.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for viability / mortality were recorded twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Daily Observations: The animals were observed for clinical signs once before commencement of administration as well as daily on Days 1 - 92/93 (twice daily during Days 1 - 3) during the treatment period. From Day 34 onwards, symptoms were also recorded immediately after administration.
Weekly Detailed Observations: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 12) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly (on Days 1, 8, 15, 29, 57 and 91)
Body weights were recorded weekly during acclimatization, treatment and recovery periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.
- Body weight observations checked in tables 4 and 5 were included.

FOOD CONSUMPTION: Yes
The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

FOOD EFFICIENCY: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During week 13
- Dose groups that were examined: All animals of the control and high dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood Sampling after 13 Weeks: 18/19-Dec-2014
- Anaesthetic used for blood collection: Yes, light isoflurane anaesthesia
- Animals fasted: Yes. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals
- Parameters examined: Glucose; Urea; Creatinine; Bilirubin, total; Cholesterol, total; Triglycerides; Phospholipids; Aspartate aminotransferase; Alanine aminotransferase; Lactate dehydrogenase; Alkaline phosphatase, Gamma-glutamyl-transferase; Creatine kinase; Sodium; Potassium; Chloride; Calcium; Phosphorus; Protein, total; Albumin; Globulin; Albumin/Globulin ratio; Coagulation Prothrombin time (= Thromboplastin time); Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood Sampling after 13 Weeks: 18/19-Dec-2014
- Animals fasted: Yes. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals
- Parameters examined: Glucose; Urea; Creatinine; Bilirubin, total; Cholesterol, total; Triglycerides; Phospholipids; Aspartate aminotransferase; Alanine aminotransferase; Lactate dehydrogenase; Alkaline phosphatase; Gamma-glutamyl-transferase; Creatine kinase; Sodium; Potassium; Chloride; Calcium; Phosphorus; Protein, total; Albumin; Globulin; Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Urine Sampling after 13 Weeks: 18/19-Dec-2014
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Parameters examined: Urine volume (18 hour); Specific gravity (relative density); Colour; Appearance; pH value; Nitrite; Protein; Glucose; Ketones; Urobilinogen; Bilirubin; Erythrocytes; Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes, relevant parameters from a modified Irwin screen test were evaluated.
- Time schedule for examinations: During week 13
- Dose groups that were examined: All dose groups, all animals
- Battery of functions tested: grip strength / locomotor activity

Sacrifice and pathology:

Sacrifice:
After 13 Weeks: 18/19-Dec-2014
All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

GROSS PATHOLOGY: Yes
Organ weights: The organs were weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight-to-brain weight.

HISTOPATHOLOGY: Yes
Samples of the tissues and organs listed in table 1, below, were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution unless indicated otherwise.

Seminology and Spermatid Count
- Time schedule: After Treatment, 18/19-Dec-2014
- How many animals: Sperm analysis was performed in all males.
Motility: At necropsy of males, a sperm sample from the left caudal epididymis was obtained from each male. The sample was diluted with a pre-warmed (about 37 - 40 °C) physiological medium, and shortly after being obtained, one hundred sperm cells were counted in duplicates (two sperm samples) microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.
Morphology: The sperm cells in the original physiological medium for motility determination were used for morphological assessment after Eosin staining and fixation. The slides were stored at room temperature until analysis. Five hundred (500) sperm cells per sample were evaluated microscopically and classified into the following categories:
Code Description
A Normal, complete sperm
B Normal head, abnormal tail
C Normal head only, tail detached
D Abnormal head only, tail detached
E Abnormal head, normal tail
The percentages of categories A to E were determined. In the absence of treatment-related effects in the control males and high-dose males, the males of the low- and middle-dose were not assessed.
Sperm Count
The left testis and the left cauda epididymis were taken for determination of homogenization resistant sperm cells (HRS) in testes and epididymis. The testes and cauda epididymis were frozen at -20 ± 5 °C pending evaluation. For evaluation the testes and cauda epididymis were thawed and processed separately. Testes and cauda epididymis were weighed and placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm heads were counted microscopically using a modified Neubauer chamber. Dilution factors for each sample were documented in the raw data. These evaluations were performed in the first instance only for group 1 and 4 males. In the absence of a treatment-related effect the remaining frozen tissues were not evaluated.

Histotechnique: All organ and tissue samples were processed, embedded and cut at an approximate thickness of 4 micrometers and stained with haematoxylin and eosin.
Histopathology: Slides of all organs and tissues collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. In addition, test item-related findings noted in the livers and mesenteric lymph nodes of high-dose males and females and in the pituitary glands of high-dose males trigger the evaluation of these organs in the middle- and low-dose groups. Attempts were made to correlate gross observations with microscopic findings. The stage of oestrus was evaluated, as the stage of spermatogenesis and histopathology of the interstitial cell structure. A peer review was performed by W. Henderson.
Immunohistochemistry: Slides of the kidney from all male animals of the control and high-dose groups were immunostained with an anti-alpha-2-microglobulin-antibody. The slides were examined by the principal investigator for histopathology. Slides of the kidney from all male animals of the control and high-dose groups were shipped to: Stewart Jones, Propath UK Limited, Willow Court, Netherwood Road, Hereford / United Kingdom

Other examinations:

no

Statistics:

The following statistical methods were used to analyse body weight, food consumption, grip strength, locomotor activity, clinical laboratory data, ophthalmoscopy, sperm analysis, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test

Clinical signs:

no effects observed

Description (incidence and severity):

No fatalities occurred. Increased salivation in males and females treated in groups 3 and 4 from week 5 onwards were considered to be incidental.

Mortality:

no mortality observed

Description (incidence):

No fatalities occurred. Increased salivation in males and females treated in groups 3 and 4 from week 5 onwards were considered to be incidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group 4 the males had test item related lower mean body weight from day 15 of treatment. Differences in mean body weights of both sexes in group 3 & 4 were considered to be within the range of typical variation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption was not affected in males and females.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The mean daily food consumption was not affected, but in group 4 the males had test item related lower mean body weight gain from day 15 of treatment.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test item-related ophthalmoscopic changes.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Group 3 & 4, both sexes: Lower mean platelet counts. Group 4 males: higher relative neutrophil & monocyte and lower mean relative lymphocyte counts; females: elevated mean relative monocyte and mean absolute neutrophil counts

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Group 2-4 males, group 3&4 females: Increased cholesterol and phospholipid levels. Group 4 males: reduced glucose, potassium, protein and globulin levels & hepatic enzyme activation. Group 4 females: Aminotransferase activities increased

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related differences in the urinalysis parameters of males and females at all dose levels.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

No effects in the functional observation battery. Mean fore- and hind limb grip strength values increased. Locomotor Activity reductions in males of group 2 and both sexes of groups 3&4

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group 2-4: Lower mean absolute and relative prostate weights; Group 3&4 males: Higher mean relative kidney weights; Group 4 females: Lower mean absolute thymus weight, mean thymus-to body weight ratio and mean thymus-to-brain weight ratio

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related adverse macroscopical findings at any dose level.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Group 4: Moderate hepatocellular hypertrophy/vacuolation in one females, Minimal multifocal hepatocellular necrosis in the liver, minimal decreased lymphocytes in the mesenteric lymph nodes

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no test item-related deaths at any dose level. Increased salivation was noted during daily observations in males and females treated with 300 mg/kg bw/day and 1000 mg/kg bw/day from week 5 onwards or in detailed weekly observations. Rats treated with 100 mg/kg bw/day were unaffected. All other findings were considered to be incidental. There were no test item-related findings noted during the weekly detailed observations.

BODY WEIGHT AND WEIGHT GAIN
At 1000 mg/kg bw/day, the males had lower mean body weight and lower mean body weight gain from day 15 of treatment. These were considered to be test item related. The differences in the mean body weights of both sexes treated with 100 and 300 mg/kg bw/day and in females treated with 1000 mg/kg bw/day were considered to be within the range of typical variation.

FOOD CONSUMPTION
The mean daily food consumption of the test item-treated rats was not affected.

FOOD EFFICIENCY
The mean daily food consumption was not affected, but in group 4 the males had test item related lower mean body weight gain from day 15 of treatment.

OPHTHALMOSCOPIC EXAMINATION
There were no test item-related ophthalmoscopic changes.

HAEMATOLOGY
In males treated with 1000 mg/kg bw/day, higher relative neutrophil and monocyte counts, lower mean relative lymphocyte count, lower platelet count were noted when compared with the controls. Females treated with 1000 mg/kg bw/day had elevated mean relative monocyte counts, reduced platelets and increased mean absolute neutrophil count. Males and females treated with 300 mg/kg bw/day had only lower mean platelet counts when compared with the controls. Males and females treated with 100 mg/kg bw/day were unaffected.

CLINICAL CHEMISTRY
Test item-related differences in clinical biochemistry included increased mean cholesterol and mean phospholipid levels in males at all dose levels. At 1000 mg/kg bw/day, parameters that were reduced in males included glucose levels, potassium levels, protein and globulin levels. Males also showed differences in several hepatic enzymes indicative of increased activation, including aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase. Increased activities of the aspartate and alanine aminotransferases were noted in females at 1000 mg/kg bw/day. These values are generally associated with increased metabolism in the liver and represent an adaptive response. Hence, these changes are considered to be test item related. Higher cholesterol and phospholipid levels seen in females at 1000 mg/kg/day and 300 mg/kg bw/day were also considered to be related to the test item. Increased potassium levels were noted in males treated with 300 mg/kg bw/day.

URINALYSIS
There were no test item-related differences in the urinalysis parameters of males and females at all dose levels.

NEUROBEHAVIOUR
There were no test item-related findings in the functional observation battery at week 13. The mean fore- and hind limb grip strength values of the test item-treated rats compared favourably with those of the respective control rats. Test item related reductions of locomotor activity was noted in males treated with 100 mg/kg/day and 300 mg/kg bw/day and both sexes treated with 1000 mg/kg bw/day.

ORGAN WEIGHTS
Lower mean absolute and relative prostate weights noted at 100, 300 and 1000 mg/kg/day were dose-related and considered to be related to the treatment with the test item. Other test item related differences in organ weights included higher mean relative kidney weights in males at 300 mg/kg/day and 1000 mg/kg/day, and lower mean absolute thymus weight, mean thymus-to body weight ratio and mean thymus-to-brain weight ratio in females at 1000 mg/kg bw/day.

GROSS PATHOLOGY
There were no test item-related adverse macroscopical findings at any dose level.

HISTOPATHOLOGY: NON-NEOPLASTIC
At 1000 mg/kg/day, moderate hepatocellular hypertrophy/vacuolation in one of ten females was noted and considered to be associated with changes in kidneys, lymphoid organs and ovaries consistent with stress/altered metabolic status.
Further changes seen at 1000 mg/kg/day included
• Minimal multifocal hepatocellular necrosis in the liver, minimal decreased lymphocytes in the mesenteric lymph nodes, and minimal multifocal cell hypertrophy in the pituitary gland (pars distalis) of males.
• Minimal multifocal hepatocellular necrosis the liver of a few rats (in two of ten males and one of ten females)
• Minimal to slight decreased lymphocytes in the mesenteric lymph nodes of four of ten males and four of ten females,
• Minimal multifocal cell hypertrophy in the pituitary gland (pars distalis) of seven of nine males.
In addition, minimal decreased lymphocytes in the mesenteric lymph nodes was observed in one of ten females at 300 mg/kg bw/day.
There were no test item-related effects in the testis, the integrity of the various cell types present within the different stages of the sperm cycle (spermatogenesis), the quantification of the different stages (quantitative staging) and the integrity of interstitial cells being unaffected.

- Seminology
The evaluation of sperm motility showed that the number of progressive sperm cells was lower in males at all dose levels. The mean number of stationary sperm cells was according higher at all dose levels when compared with control sperm. The reduction of sperm motility in males was considered to be a test item-related change.
There were no test item-related differences in the sperm morphology and sperm counts of the control group and rats treated with 1000 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

< 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

clinical biochemistry

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Locomotor Activity

Reductions seen in the mean locomotor activity of the males treated with 100 mg/kg/day and 300 mg/kg bw/day and both sexes treated with 1000 mg/kg bw/day were considered to be test item-related. The mean locomotor activity of the females treated with 100 or 300 mg/ kg bw/day were considered to be unaffected.

Table 2: Locomotor activity of male test animals

Allocation and Dose Levels mg/kg bw/day	Group 1 Control Males 0	Group 2 Males 100	Group 3 Males 300	Group 4 Males 1000
6 minutes	330	315 (-4.5 %)	292 (-11.5 %)	293 (-11.2 %)
12 minutes	206	150 (-27.2 %)	189 (-8.3 %)	181 (-12.1 %)
18 minutes	165	116 (-29.7 %)	156 (-5.5 %)	87 (-47.3 %)
24 minutes	152	116 (-23.7 %)	65 (-57.2 %)	60 (-60.5 %)
30 minutes	144	74 (-48.6 %)	28 (-80.6 %)	22 (-84.7 %)
Total	997	771 (-22.7 %)	730 (-26.8 %)	643 (-35.5 %)

Bold values denote statistically significant changes.

Table 3: Locomotor activity of female test animals

Allocation and Dose Levels mg/kg bw/day	Group 1 Control Females 0	Group 2 Females 100	Group 3 Females 300	Group 4 Females 1000
6 minutes	349	371 (+6.3 %)	290 (-16.9 %)	316 (-9.5 %)
12 minutes	198	208 (+5.1 %)	158 (-20.2 %)	147 (-25.8 %)
18 minutes	113	116 (+2.7 %)	85 (-24.8 %)	51 (-54.9 %)
24 minutes	24	67 (+179.2 %)	42 (+75.0 %)	11 (-54.2 %)
30 minutes	31	72 (+132.3 %)	43 (+38.7 %)	14 (-54.8 %)
Total	714	835 (+16.9 %)	617 (-13.6 %)	538 (-24.6 %)

Bold values denote statistically significant changes.

Body Weights

At 1000 mg/kg bw/day, the males had lower mean body weight and lower mean body weight gain from day 15 of treatment; the differences to the control males attained statistical significance (p<0.05 or p<0.01) from days 57 and 36 of treatment, respectively. These were considered to be test item-related.

At 300 mg/kg bw/day, the mean body weight and mean body weight gain of males were reduced from day 57 and day 15 of treatment, respectively. These differences were not statistically significant. Males at 100 mg/kg bw/day were not affected.

Table 4: Male Body Weights (g) and Difference to Control (%)

Allocation and Dose Levels mg/kg bw/day	Group 1 Control* 0	Group 2 100	Group 3 300	Group 4 1000
Day 1	216	214 (-0.9 %)	217 (+0.5 %)	217 (+0.5 %)
Day 8	261	261 (0.0 %)	264 (+1.1 %)	260 (-0.4 %)
Day 15	283	284 (+0.4 %)	287 (+1.4 %)	275 (-2.8 %)
Day 29	335	338 (-0.9 %)	336 (+0.3 %)	319 (-4.8 %)
Day 57	394	384 (-2.5 %)	379 (-3.8 %)	358 (-9.1 %)
Day 91	431	420 (-2.6 %)	412 (-4.4 %)	386 (-10.4 %)

Bold values denote statistically significant changes.

There were no test item-related differences in the mean body weights or mean body weight gain of females at any dose level.

Table 5: Female Body Weights (g) and Difference to Control (%)

Allocation and Dose Levels mg/kg bw/day	Group 1 Control* 0	Group 2 100	Group 3 300	Group 4 1000
Day 1	153	155 (+1.3 %)	153 (0.0 %)	154 (+0.7 %)
Day 8	175	175 (0.0 %)	177 (+1.1 %)	173 (-1.1 %)
Day 15	181	182 (+0.6 %)	182 (+0.6 %)	177 (-2.2 %)
Day 29	201	196 (-2.5 %)	203 (+0.5 %)	192 (-4.5 %)
Day 57	226	220 (-2.7 %)	223 (-1.3 %)	211 (-6.6 %)
Day 91	239	235 (-1.7 %)	239 (0.0 %)	220 (-7.9 %)

Bold values denote statistically significant changes.

Conclusions:

Based on the results of the read across study, NOAEL for systemic effects is considered to be <100 mg/kg bw/day.

Executive summary:

A study was conducted to determine the subchronic oral 90-day repeated dose toxicity of the read across substance '4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid (BADGEDA)' to Wistar rats of both sexes according to the OECD Guideline 408 and EU Method B.29, in compliance with GLP. The read across substance was administered daily by oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day (named groups 1 to 4, respectively) using a volume of 5 mL/kg bw. The control group was treated with the vehicle, PEG 300, only. The groups comprised 10 animals per sex which were sacrificed after 92/93 days of treatment. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization and treatment periods. Functional observational battery, locomotor activity and grip strength were performed during week 13. At the end of the dosing period, blood samples were withdrawn for haematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Sperm samples were collected at necropsy to assess count, motility and morphology. Histological examinations were performed on organs and tissues from all control and high dose animals and all gross lesions from all animals. The administration of the read across substance to the test animals resulted in no read across substance-related deaths, no differences in mean food consumption, weekly observations (weeks 1 - 13) or functional observational battery (week 13), no differences of toxicological relevance in the fore- and hind limb grip strength values, no read across substance-related differences in the ophthalmoscopy and no read across substance-related effects on urine parameters. Read across substance-related clinical signs included salivation in both sexes treated with 300 mg/kg bw/day and 1000 mg/kg bw/day. This finding was first noted in week 5 and was frequently seen throughout the rest of the study. This finding is generally associated with a bitter taste of the dosing solutions and is a secondary, non-adverse effect. Clearly lower mean body weight development was noted from day 15 of treatment in males treated with 1000 mg/kg bw/day. Reduced locomotor activity was noted in males at all dose levels and in females treated at 1000 mg/kg bw/day. In males treated with 1000 mg/kg bw/day, higher relative neutrophil and monocyte counts, lower mean relative lymphocyte count and lower platelet count were noted when compared with the controls. Females treated with 1000 mg/kg bw/day had elevated mean relative monocyte counts, reduced platelets and an increased mean absolute neutrophil count. Males and females treated with 300 mg/kg bw/day had only lower mean platelet counts when compared with the controls. Males and females treated with 100 mg/kg bw/day were unaffected. Read across substance-related differences in clinical biochemistry parameters were noted in males at all dose levels. Increased mean cholesterol and mean phospholipid levels were noted in males at all dose levels, and is often associated with changes in lipid metabolism. Males also showed increased enzyme activation of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase. Similar findings were seen in females treated with 1000 mg/kg bw/day: increased activities of the aspartate and alanine aminotransferases. These differences, while read across substance related, are generally considered to be metabolic adaptation and of no toxicological relevance. However, parameters that were reduced in males at 1000 mg/kg bw/day included glucose levels, potassium levels, protein and globulin levels. Although these values remained within the limits of the historical control values, they were considered to be related to the treatment with the read across substance. In females, significantly higher cholesterol and phospholipid levels (both p<0.01) were seen at 1000 mg/kg/day and 300 mg/kg/day, exceeded the upper limits of the historical control data and were considered to be read across substance-related. The reduction of sperm motility noted at all dose levels was considered to be related to the read across substance. Lower mean absolute and relative prostate weights noted at 100, 300 and 1000 mg/kg/day, and higher mean relative kidney weights in males at 300 mg/kg/day and 1000 mg/kg/day were considered to be read across substance-related effects. Lower mean absolute thymus weight, mean thymus-to body weight ratio and mean thymus-to-brain weight ratio in females at 1000 mg/kg bw/day were considered to be a stress reaction. The test-substance induced microscopic findings in the liver of a few rats at 1000 mg/kg bw/day (minimal hepatocellular necrosis, moderate hepatocellular hypertrophy/vacuolation); they were considered to be the histological correlates of the increased liver enzymes recorded in clinical biochemistry. The most prominent liver microscopic findings were observed in the female rat no. 72 (1000 mg/kg bw/day) and were associated with changes in kidneys, lymphoid organs and ovaries consistent with stress/altered metabolic status. The pathogenesis of the multifocal cell hypertrophy observed in the pars distalis of the pituitary gland of males cannot be ascertained. However, the pituitary findings and the decreased prostate/seminal vesicles weights may represent an indicator of mild disruption of testosterone production/levels. The pathogenesis underlying the decreased lymphocytes in the mesenteric lymph nodes is unknown in the absence of changes in the other lymphoid organs. Under the study conditions, no-observed adverse effect levels (NOAEL) for systemic effects were below the lowest dose level of 100 mg/kg bw/day of the read across substance (Braun, 2015). Based on the results of the read across study, the NOAEL of test substance for systemic effects is considered to be <100 mg/kg bw/day.

Results and discussion

Target system / organ toxicity

Critical effects observed:

not specified