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

Endpoint summary

Description of key information

Based on the results of an OECD TG 408 compliant 90-day repeated toxicity study with the read across substance BADGEDA, the NOAEL of the test substance for systemic effects is considered to be below the lowest tested dose level of 100 mg/kg bw/day. 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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 test substance.

Executive summary:

A study was conducted to determine the subchronic oral 90-day repeated dose toxicity of the test 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 test 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 test substance to the test animals resulted in no test 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 test substance-related differences in the ophthalmoscopy and no test substance-related effects on urine parameters. Test 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. Test 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 test 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 test 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 test substance-related. The reduction of sperm motility noted at all dose levels was considered to be related to the test 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 test 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 test substance (Braun, 2015).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

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

Adequacy of study:

supporting study

Study period:

From 15 January, 2010 to 20 July, 2010

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:

reference to same study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 9-10 wk
- Weight at study initiation: 310 to 366 g for males and 207 to 244 g for females
- Housing: Up to 5 during pre-mating for all animals and after mating for males, individually with litter for females during gestation and littering.
- Diet (e.g. ad libitum): Standard rodent diet (SDS VRF1 Certified) ad libitum, except overnight before routine blood sampling for toxicity subgroup animals.
- Water (e.g. ad libitum): Potable water taken from the public supply, ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 20 January 2010 To: 13 March 2010

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Approx 50% of the final volume of propylene glycol was added to the test substance. The formulation was warmed in a water bath at approx 50°C and stirred with a spatula until all the test material had dissolved and more propylene glycol was added to make up the required volume. Then, the formulation was returned to the container and mixed using a magnetic stirrer until homogeneous. If necessary, the dose was warmed again to aid homogenisation. Initially all formulations were prepared freshly on the day of use and used within 2 h of completion of preparation. However, following confirmation of the results from a homogeneity and stability study (Huntingdon Life Sciences Study No. RAJ0005) formulations were prepared freshly each week and stored refrigerated (approx 4°C) until required for use.

VEHICLE
- Concentration in vehicle: 20, 60 and 180 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in the first and last weeks of the dosing period were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. Two assays from each group were analysed. The analytical method involved dissolution and dilution using acetonitrile followed by reverse phase high performance liquid chromatographic analysis with ultra-violet detection (230 nm). The mean concentrations in test formulations analysed for the study were within applied limits +10%/-15% of nominal concentrations, confirming accurate formulation, with the exception of one result, Week 1, Day 1, Group 2, 20 mg/mL which was 20.5% below nominal. Additional samples taken from the reformulated Week 1, Day 5, Group 2 formulation were therefore analysed and the achieved concentration was 1.5% above nominal, confirming the accuracy of formulation.

Duration of treatment / exposure:

5 weeks

Frequency of treatment:

Once a day, 7 days a week

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
900 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Toxicity subgroup: 5 males and 5 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: In a preliminary study (Huntingdon Life Sciences Study Number: RAJ0001), dose levels of 80, 250, 750 and 1000 mg/kg bw/day were employed and there was a suggestion of reduced bodyweight gain among males that received 1000 mg/kg bw/day. There was no evidence of an effect of treatment on clinical condition, food consumption, oestrous cycles or organ weights at any dose level investigated, and no treatment-related macroscopic abnormalities were observed. For this study, treatment would continue for approximately 5 weeks and females would be pregnant, therefore in recognition of the potential for reduced bodyweight gain, the high dose level was set at 900 mg/kg bw/day, the low dose of 100 mg/kg bw/day was chosen as an anticipated No Observed Adverse Effect Level (NOAEL), and the intermediate dose of 300 mg/kg bw/day at a logarithmic interval between the low and high doses.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupant(s). During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS:
Detailed observations were recorded in relation to dose administration. For the toxicity subgroup animals and for reproductive subgroup males these were recorded daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and on one occasion during Week 5. For reproductive subgroup females these were recorded daily during the first week of treatment, twice weekly during Week 2 of treatment, on Days 0, 4, 7, 11, 14 and 20 after mating and Day 4 of lactation.

Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal (physical condition and behaviour during handling with particular attention to possible signs of neurotoxicity). For the reproductive subgroup females during the post-mating period, these observations were conducted on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation.

BODY WEIGHT:
Toxicity subgroup males and females and reproductive subgroup males were weighed weekly throughout the study. Reproductive subgroup females were weighed weekly until mating was detected, on Days 0, 7, 14 and 20 after mating and on Days 1, 4, 6 and 7 of lactation.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded weekly for all animals for the first two weeks of treatment. Food consumption continued to be recorded weekly for the remaining three weeks of treatment for toxicity subgroup females. Food consumption was not recorded during Week 3 of treatment for toxicity subgroup males or reproductive subgroup males as the animals were in pairing with the reproductive subgroup females during this period. Food consumption was recorded, however, during Weeks 4 and 5 of treatment for these males.
For each reproductive subgroup female, the weight of food supplied, that remaining and an estimate of any spilled was recorded for the periods Days 0-6, 7-13 and 14-19 after mating and Days 1-3 and 4-6 of lactation.

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation.

OTHER:
- SENSORY REACTIVITY:
Sensory reactivity and grip strength assessments were performed (before dosing) on all toxicity subgroup animals during Week 5 of treatment. The following measurements, reflexes and responses were recorded: approach response, touch response, auditory startle reflex, tail pinch response and grip strength.

- MOTOR ACTIVITY:
During Week 5 of treatment (before dosing), the motor activity of each toxicity subgroup animal was measured using a Rodent Activity Monitoring System (Version 2.0.3). Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-min intervals (one hour total).

- HAEMATOLOGY:
During Week 5 of treatment (after sensory reactivity, grip strength and motor activity assessments), blood samples were obtained from all toxicity subgroup animals after overnight withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein. The following were measured using a Bayer Advia 120 haematology analyser: haematocrit (Hct), haemoglobin concentration (Hb), erythrocyte count (RBC), reticulocyte count (Retic), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV), total leucocyte count (WBC), differential leucocyte count (neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B)
monocytes (M), large unstained cells (LUC)) and platelet count (Plt). The most common morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were recorded. Prothrombin time (PT) (using an ACL 3000 Plus analyser and IL PT-Fibrinogen reagent) and activated partial thromboplastin time (APTT) (using an ACL 3000 Plus Analyser and IL APTT reagent) were also measured.

- BLOOD CHEMISTRY:
At the same time and using the same animals as for peripheral haematology, the plasma was examined using a Roche P Modular Analyser for: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile Acids (BIAC), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb) (by chemical assay).

Sacrifice and pathology:

SACRIFICE:
Animals were killed in Week 6 after completion of the Week 5 investigations.

GROSS NECROPSY:
All animals were subject to a detailed necropsy. For reproductive subgroup females, the number of uterine implantation sites was also recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The tissues indicated in Table 1 were weighed.
In the toxicity subgroup, the following organs were fixed for histopathology: adrenal glands, peyer’s patch, brain, pituitary, caecum, prostate, colon, rectum, duodenum, sciatic nerves, epididymides (L&R), seminal vesicles and coagulation gland, heart, spinal cord, ileum, spleen, jejunum, sternum with marrow, kidneys, stomach, liver, testes (L&R), lungs, thymus, lymph nodes (left axillary and mesenteric), thyroid with parathyroids, trachea, mammary area (caudal), urinary bladder, oesophagus, uterus with cervix and oviducts, ovaries (L&R) and vagina.
In the reproductive subgroup, the following organs were fixed for histopathology: mammary area (caudal), testes (L&R), ovaries (L&R), uterus with cervix and oviducts, pituitary, vagina and prostate. Samples of any abnormal tissues were also retained and processed for examination.
All tissues preserved for examination were examined for all toxicity subgroup and reproductive subgroup animals of control group and 900 mg/kg bw/day group. In addition, based on the results of the initial assessment, the lungs and kidneys were examined from all toxicity subgroup males in the 100 and 300 mg/kg bw/day groups.

Statistics:

The following sequence of statistical tests was used for grip strength, motor activity, bodyweight, food consumption, organ weight and survival indices and clinical pathology data: 1) a parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. 2) a non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.
For organ weight data, analysis of covariance was performed. Sex ratio were analysed by Wald chi-square test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
One reproductive subgroup male receiving 100 mg/kg bw/day was found dead prior to dose administration on Day 34 of the study. Ante mortem signs for this animal were restricted to noisy respiration (rales) on the previous day and there were no macroscopic abnormalities detected. In the absence of any other mortalities in this or higher dose groups, it was considered incidental and unrelated to treatment.

BODY WEIGHT AND FOOD CONSUMPTION:
At 900 mg/kg bw/day, mean bodyweight gain was satistically significantly lower than control during the first week of treatment (67% of control). During the second week of treatment, the weight gain of these males remained slightly (but not significantly) lower than control. Thereafter, mean weight gain was essentially similar to control and overall mean gain during the treatment period (Week 0 to Week 5) was 81% of Control. The mean bodyweight gain of males receiving 300 or 100 mg/kg bw/day was considered unaffected by treatment. Mean bodyweight gain of females in all dose groups was considered unaffected by treatment throughout the study. It was noted that during the first two weeks of treatment, mean weight gain in all treated groups was lower than control, and between Weeks 2 and 5 the weight gain of toxicity subgroup females was higher than control, with all differences attaining statistical significance. There was, however, no dose response apparent, and therefore these differences were considered to be of no toxicological importance.

TEST SUBSTANCE INTAKE:
The mean food intake of males and females in all dose groups was considered unaffected by treatment throughout the study.

ORGAN WEIGHTS:
At scheduled termination, toxicity subgroup females in all treated groups showed slightly low mean unadjusted adrenal weights compared with control, with a dose-related trend apparent and differences attaining statistical significance. All values were, however, within the 5-95% confidence limit of general HCD for rats of this age and strain (0.053-0.093 g). All other differences in unadjusted and adjusted organ weights for toxicity and reproductive subgroup animals were small, and no clear relationship to treatment.

GROSS PATHOLOGY:
There were no macroscopic abnormalities detected at scheduled termination of the toxicity or reproductive subgroup animals that were considered to represent an adverse effect of treatment.

HISTOPATHOLOGY:
No test substance related changes were observed in any of the organs microscopically examined from toxicity and reproductive subgroup animals.
Seminiferous tubules of all males of both the subgroups were evaluated with respect to their stage in the spermatogenic cycle and integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted. During initial microscopical examination, lungs from three toxicity subgroup males receiving 900 mg/kg bw/day had minimal aggregations of alveolar macrophages. In addition, the kidney from one male in this group had minimal multiple cortical scarring, minimal focal cortical tubular basophilia and cortical tubular dilatation. To assess the toxicological significance of this finding, a trackdown was carried out on lungs and kidney from both the subgroup males given 100 or 300 mg/kg bw/day. Subsequent microscopical examination showed no dose dependent trend in severity or incidence level for these findings and hence they were considered as incidental. The incidence and distribution of all the other findings were consistent with the common background for animals of this age at these laboratories.

SIGNS AND ARENA OBSERVATIONS:
No signs were observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment.

SENSORY REACTIVITY OBSERVATIONS AND GRIP STRENGTH:
Sensory reactivity observations and grip strength values for toxicity subgroup animals were similar to those for controls, and considered unaffected by treatment.

MOTOR ACTIVITY:
No dose response was apparent and no association with treatment was identified.

HAEMATOLOGY:
Assessment of haematological parameters for toxicity subgroup animals during Week 5 of treatment revealed slightly prolonged prothrombin time among males and females receiving 900 or 300 mg/kg bw/day, with a dose-related trend apparent and statistical significance attained. The time for males receiving 900 mg/kg bw/day was marginally outside the 5-95% confidence limits of general historical control data (HCD) for rats of this age and strain (13.2-16.4 sec) but males in the 300 mg/kg bw/day were within the range, as were the females in the 300 or 900 mg/kg bw/day groups (13.4-17.6 seconds).
At 900 or 300 mg/kg bw/day, group mean activated partial thromboplastin time was slightly shortened compared with the concurrent controls. A dose dependent trend was apparent, however statistical significance was not attained, and at 900 mg/kg bw/day the group mean time for males was influenced by one atypical animal with a particularly short clotting time (8.1 sec). With this animal excluded from the group mean, the mean time for this group would be 14.6 sec, and all group mean times for these groups would be within the 5-95% confidence limits of general (HCD) for rats of this age and strain (14.1-24.4 sec for males and 10.0-19.0 sec for females).
Some other minor inter-group differences in haematological parameters attained statistical significance, but no clear evidence of an adverse effect of treatment. Among all groups of treated males, the mean cell haemoglobin concentration was higher than control, with a dose-response apparent. In the absence of any other changes in erythrocytic parameters, or a similar effect among females, these differences were considered likely to be fortuitous and unrelated to treatment.

BLOOD CHEMISTRY:
During Week 5, total bilirubin and total bile acid concentrations were statistically significantly high in males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day. Total bilirubin concentrations for animals in the 900 mg/kg/day group were outside the 5-95% confidence limits of general HCD for rats of this age and strain (1-3 μmol/L for males and females); no HCD are available for total bile acid concentrations. In addition, cholesterol concentrations were statistically significantly high in males and females in all treated groups with a dose related trend apparent; concentrations for males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day were outside the 5-95% confidence limits of general HCD (1.07-2.07 mmol/L for males and 1.25-2.97 mmol/L for females).

Key result

Dose descriptor:

NOAEL

Effect level:

> 900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects were observed

Critical effects observed:

not specified

 Table 2: Main statistically significant changes in haematology and blood chemistry parameters:

Dose (mg/kg bw/day)	0	100	300	900
	Males
MCHC (g/dL)	35.3 ± 0.44 (n=5)	35.8 ± 0.44 * (n=5)	36.4 ± 0.25 ** (n=3)	36.8 ± 0.29 ** (n=5)
PT (sec)	14.7 ± 0.29 (n=5)	14.3 ± 0.48 (n=5)	15.8 ± 0.57 ** (n=5)	16.5 ± 0.38 ** (n=5)
Bili (µmol/L)	2± 0.5 (n=5)	2± 0.5 (n=5)	3± 0.4 ** (n=5)	5± 0.8 ** (n=5)
BIAC (umol/L)	30.4± 16.71 (n=5)	38.8± 21.33 (n=5)	68.6± 26.54 * (n=5)	151.7± 27.29 ** (n=5)
Chol (mmol/L)	1.32± 0.270 (n=5)	1.99± 0.227 ** (n=5)	2.91± 0.407 ** (n=5)	3.55± 0.178 ** (n=5)
	Females
PT (sec)	14.7 ± 0.59 (n=5)	15.0 ± 0.37 (n=5)	15.7 ± 0.34 ** (n=4)	15.6 ± 0.37 ** (n=5)
Bili (µmol/L)	2± 0.4 (n=5)	2± 0.0 (n=5)	3± 0.9 (n=5)	4± 0.8 ** (n=5)
BIAC (umol/L)	16.0± 3.55 (n=5)	23.2± 16.24 (n=5)	67.4± 96.49 (n=5)	165.5± 77.74 ** (n=5)
Chol (mmol/L)	1.42± 0.308 (n=5)	2.47± 0.413 ** (n=5)	2.70± 0.524 ** (n=5)	3.91± 0.598 ** (n=5)

Conclusions:

Based on the results of the read across study, the NOAEL for the test substrance for systemic toxicity is considered > 900 mg/kg bw/day, the highest dose tested.

Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the read across substance '4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid (BADGEDA)' according to the OECD Guideline 422, in compliance with GLP. Three groups each comprising five male and five female rats received the read across substance at doses of 100, 300 or 900 mg/kg bw/day for five consecutive weeks. A similarly constituted control group received the vehicle, propylene glycol, at the same volume-dose. During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology and blood chemistry. Organ weight, macroscopic and microscopic pathology investigations were undertaken. There was only one mortality during the course of the study, considered unrelated to treatment. There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. There was no effect of treatment on food consumption, sensory reactivity findings, grip strength values and motor activity in males and females throughout the study. At 900 mg/kg bw/day, mean bodyweight gain of males was slightly lower than controls during the first week of treatment. During the second week, weight gain for these males remained slightly (but not significantly) lower than controls, but thereafter, mean weight gain was essentially similar to control in all treated groups and the bodyweight gain of females was unaffected by treatment at all dose levels investigated. Haematological assessment revealed slightly prolonged prothrombin time among males and females receiving 900 or 300 mg/kg bw/day. Biochemical changes in the blood plasma comprised high total bilirubin and total bile acid concentrations in males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day, and high cholesterol concentrations in males and females in all treated groups. A dose-related trend was evident for all of these haematological and biochemical parameters. Upon detailed examination, these changes were not considered to be adverse. There was no clear evidence of an adverse effect of treatment on mean organ weights among all animals at scheduled termination. There were no macroscopic abnormalities and no read across substance-related lesions at microscopic examination. Based on the results of the read across study, the NOAEL for systemic toxicity was determined to be >900 mg/kg bw/day (Stannard, 2010).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The information requirements for this tonnage band is sufficiently met with the available data.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

A study was conducted to determine the repeated dose oral toxicity of the read across substance BADGEDA according to the OECD Guideline 422, in compliance with GLP. Three groups each comprising five male and five female rats received the read across substance at doses of 100, 300 or 900 mg/kg bw/day for five consecutive weeks. A similarly constituted control group received the vehicle, propylene glycol, at the same volume-dose. During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology and blood chemistry. Organ weight, macroscopic and microscopic pathology investigations were undertaken. There was only one mortality during the course of the study, considered unrelated to treatment. There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. There was no effect of treatment on food consumption, sensory reactivity findings, grip strength values and motor activity in males and females throughout the study. At 900 mg/kg bw/day, mean bodyweight gain of males was slightly lower than controls during the first week of treatment. During the second week, weight gain for these males remained slightly (but not significantly) lower than controls, but thereafter, mean weight gain was essentially similar to control in all treated groups and the bodyweight gain of females was unaffected by treatment at all dose levels investigated. Haematological assessment revealed slightly prolonged prothrombin time among males and females receiving 900 or 300 mg/kg bw/day. Biochemical changes in the blood plasma comprised high total bilirubin and total bile acid concentrations in males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day, and high cholesterol concentrations in males and females in all treated groups. A dose-related trend was evident for all of these haematological and biochemical parameters. Upon detailed examination, these changes were not considered to be adverse. There was no clear evidence of an adverse effect of treatment on mean organ weights among all animals at scheduled termination. There were no macroscopic abnormalities and no read across substance-related lesions at microscopic examination. Based on the results of the read across study, the NOAEL for systemic toxicity was determined to be >900 mg/kg bw/day (Stannard, 2010).

A study was conducted to determine the subchronic oral 90-day repeated dose toxicity of the read across substance 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.

Inhalation:

The test substance has low vapour pressure (0.19 Pa at 20°C) so that normal processing and use conditions will not generate inhalation exposure. Furthermore, there are no spray applications of the substance. Repeated inhalation exposure is therefore not expected to pose an issue for human health and no further testing is required for this endpoint, in accordance with Annex IX, Section 8.6, Column 2 of the REACH legislation. Nevertheless, the risk assessment for this route has been carried out based on a 90-day repeated dose oral study with the read across substance, using appropriate route-to-route extrapolation assessment factors as per the ECHA Guidance R.8.

 

Dermal:

A repeated dose dermal toxicity study is not required because an OECD 422 as well as a 90-day repeated dose oral studies are available with the read across substance. Further, given the physico-chemical properties of the substance, dermal absorption is not expected to be higher than via the oral route. Hence, testing via dermal route will less likely result in any additional hazard identification and further testing involving vertebrate animals may be omitted, in accordance with Annex XI (1.2) of the REACH regulation. Nevertheless, the risk assessment for this route has been carried out based on the 90-day oral study available with the read across substance, using appropriate route-to-route extrapolation assessment factors as per the ECHA Guidance R.8.

Justification for classification or non-classification

Based on the available data from read across studies with BADGEDA, no adverse effect and no target organ was identified, therefore the test substance is not required to be classified for repeated dose toxicity or STOT RE according to EU CLP (EC 1272/2008) criteria.