Oligomerisation products of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane with acrylic acid and fatty acids, C18-unsatd., dimers and nonanoic acid

Endpoint summary

Description of key information

As a conservative approach, the LOAEL of 100 mg/kg bw/day based on the read across study with BADGEDA has been considered further for hazard assessment.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 3, 2011 to November 22, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Principles of method if other than guideline:

Additional guideline: "OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000" (with no deviations).

GLP compliance:

yes

Specific details on test material used for the study:

Specific Gravity / Density: 1.14 g/cm3. pH (1% in water, indicative range): 5.5 – 5.5 (determined at NOTOX). Stability at higher temperatures: yes, maximum temperature 40°C. Stability in vehicle (Polyethylene glycol): at least 6 hours at room temperature over the concentration range 20 to 200 mg/mL.

Species:

rat

Strain:

other: Crl:WI(Han) (outbred, SPF-Quality).

Remarks:

Charles River Deutschland, Sulzfeld, Germany.

Details on species / strain selection:

Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Animals:
Total number of animals: 20 males, 20 females (females were nulliparous and non-pregnant). Age at start of treatment: approximately 6 weeks.
Identification: earmark and tattoo. Randomization: by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean. Acclimatization period: at least 5 days before the start of treatment under laboratory conditions. Health inspection: prior to commencement of treatment to ensure that the animals were in a good state of health.
- Allocation:
5 males and 5 females per group. 4 groups: 0, 100, 300, and 1000 mg/kg bw/day. Dose volume: 5mL/kg bw/day
- Conditions:
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 18.5 – 22.3ºC), a relative humidity of 40-70% (actual range: 41 - 86%) and 12 hour light/12 hour dark cycle. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity (with a maximum of 4 hours). Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
- Accommodation:
Group housing of 5 animals per sex in Makrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet:
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived. During motor activity measurements, animals had no access to food.
Water:
Free access to tap water except during motor activity measurements. Certificates of analysis (performed quarterly) were examined and archived.
Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected the study integrity.

Route of administration:

oral: gavage

Details on route of administration:

Method: oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Vehicle:

polyethylene glycol

Details on oral exposure:

Dose volume 5 mL/kg body weight. Actual dose volumes were adjusted weekly according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analysed in the formulations of Group 2 (100 mg/kg bw/day), Group 3 (300 mg/kg bw/day) and Group 4 (1000 mg/kg bw/day) were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

28 days (All animals were dosed up to the day prior to necropsy).

Frequency of treatment:

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Controls

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

in agreement with target concentrations

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

in agreement with target concentrations

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

in agreement with target concentrations

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Based on the results of a 5-day range finding study (NOTOX Project 497635), the dose levels for this 28-day oral gavage study were selected to be 0, 100, 300 and 1000 mg/kg.
- Study outline: the test substance, formulated in polyethylene glycol 400, was administered daily for at least 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.
- Evaluated parameters: chemical analyses of formulations were conducted once after the study to assess accuracy, homogeneity and stability over 6 hours. The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Positive control:

no

Observations and examinations performed and frequency:

- Mortality / Viability: at least twice daily.
- Clinical signs: at least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. Clinical observations were conducted immediately after dosing, except for those performed in a standard arena which were performed prior to dosing. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades are reported, as well as the percentage of animals affected in summary tables.
- Functional Observations: during week 4 of treatment, the following tests were performed on all animals (abbreviations mentioned in the respective tables indicated between brackets): - hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent), and - motor activity test (recording period: 1 hour under normal laboratory light conditions for individual animals, using a computerized monitoring system (Kinder Scientific LLC, Poway, USA)). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head. Functional observation tests were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
Body weights and food consumption: weekly.
Water consumption: subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Sacrifice and pathology:

- Animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
- The organ weights and terminal body weight were recorded from the animals on the scheduled day of necropsy.
- All organ and tissue samples, as defined under Histopathology (following), were processed, embedded in paraffin wax (Klinipath, Duiven, The Netherlands) cut to slides at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
- The following slides were examined by a pathologist: - all tissues collected at the scheduled sacrifice from all group 1 and 4 animals, - liver of all animals of groups 2 and 3 (males and females), based on (possible) treatment-related changes in this organ in group 4, - all gross lesions.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Histopathology was subjected to a peer review.

Other examinations:

Blood samples were collected under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.. Animals were deprived of food overnight (for a maximum of 20 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin (0.5 mL) for clinical biochemistry parameters. An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids (tubes: Greiner BioOne GmbH, Kremsmünster, Austria).

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one ttest) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. - The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution. - The exact Fisher-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test (Ref. 5) to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Hunched posture was observed for all females at 1000 mg/kg bw/dayduring the second half of the study, starting from Day 19.
- A slight to moderate degree of salivation was shown by females at 100 mg/kg bw/day and all animals at 300 and 1000 mg/kg bw/day. Salivation is often noted in rats of this age and strain following oral gavage and the salivation observed in this study was considered to be a physiological response rather than a sign of systemic toxicity.
- One control female showed scales and scabs in the neck region. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.
- No clinical signs were observed for control males and males at 100 mg/kg bw/day.

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:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- A prolonged prothrombin time (PT) was observed for males at 1000 mg/kg bw/day when compared to control males. The following statistically significant differences between controls and animals at 1000 mg/kg bw/day were considered not to represent a change of toxicological significance, because the differences were slight in nature and the values of the animals at 1000 mg/kg bw/day remained well within the range considered normal for rats of this age and strain: increased relative monocyte counts in males and lower haemoglobin levels and lower haematocrit in females.
- Lower relative neutrophil counts in females at 100 mg/kg bw/day and lower red blood cell counts in females at 300 mg/kg bw/day were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher total bilirubin levels in males and females at 1000 mg/kg bw/day,
- Higher cholesterol levels in males at 300 mg/kg bw/day and males and females at 1000 mg/kg bw/day,
- Higher bile acids levels in females at 1000 mg/kg bw/day and (not statistically significant) in males at 300 and 1000 mg/kg bw/day.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional observations:
- No toxicologically significant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.
- Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic examination:
Necropsy did not reveal any toxicologically relevant alterations. The incidence of the macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included: reduced size of the right seminal vesicle (one control male and one male at 1000 mg/kg bw/day), foci on the thymus (one control female, one male at 100 mg/kg bw/day and one female at 300 mg/kg bw/day), diaphragmatic hernia of the right medial lobe of the liver (one male at 300 mg/kg bw/day), a yellowish, hard nodule in the body of the left epididymis (one male at 300 mg/kg bw/day), pelvic dilation of the right kidney (two males at 1000 mg/kg bw/day), presence of fluid in the uterus (one or two females of all groups), scab formation on the skin of the back of the neck (one control female), dark red discolouration of the left mandibular lymph node (one female at 100 mg/kg bw/day), tan foci on the right side of the clitoral gland (one female at 300 mg/kg bw/day and one female at 1000 mg/kg bw/day) and an enlarged left mandibular lymph node (one female at 1000 mg/kg bw/day).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A minor treatment-related microscopic finding, vacuolation of Kupffer cells, was present in the livers of rats at 300 and 1000 mg/kg bw/day. All males and females at 1000 mg/kg bw/day had minimal vacuolation of Kupffer cells. Four of five males and two of five females at 300 mg/kg bw/day also had minimal vacuolation of Kupffer cells. There were no incidences at 100 mg/kg bw/day. Vacuolation was minimal in the sense that the observation was barely noticeable.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat. (dissolved fraction)

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

clinical biochemistry

clinical signs

gross pathology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

other: changes in bile acids

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Results summary:

At 1000 mg/kg bw/day, minimal vacuolation of Kupffer cells (barely noticeable) was present in the livers of all males and females and was accompanied with a prolonged prothrombin time in males, higher total bilirubin levels in males and females and higher bile acids levels in males and females. Although most levels were only slightly above the range considered normal for rats of this age and strain, the change in bile acids levels in females was remarkable. In addition, hunched posture was observed for females at 1000 mg/kg bw/day during the second half of the study. Taking all findings into account, the combination of effects in animals at 1000 mg/kg bw/day was considered to be adverse in nature.

At 300 mg/kg bw/day, minimal vacuolation of Kupffer cells (barely noticeable) was observed among several animals and was accompanied with slightly higher cholesterol levels and higher bile acids levels. These findings were considered not to be adverse in nature based on the slight nature of these findings and the absence of any other toxicologically relevant findings.

Animals at 100 mg/kg bw/day did not show toxicologically significant changes in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

From the results presented in this report, a No Observed Adverse Effect Level (NOAEL) for the test substance of 300 mg/kg bw/day was established.

Conclusions:

Wistar rats were treated with the test substance for 28 consecutive days by daily oral gavage at dose levels up to 1000 mg/kg. Under the study conditions, a NOAEL of 300 mg/kg was established.

Executive summary:

A study was conducted to determine the chronic toxicity of the test substance according to OECD Guideline 407, EU Method B.7 and EPA OPPTS 870.3050. Groups of 5 male and 5 female Wistar rats were treated with the test substance (dissolved in polyethylene glycol) for 28 consecutive days by oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Chemical analyses of formulations were conducted once after the study and confirmed accuracy, homogeneity and stability over 6 h. The following parameters were evaluated: clinical signs daily, functional observation tests in Week 4, bodyweight and food consumption weekly, clinical pathology and macroscopy at termination, organ weights and histopathology on a selection of tissues. At 1000 mg/kg bw/day, minimal vacuolation of Kupffer cells (barely noticeable) was present in the livers of all males and females and was accompanied with a prolonged prothrombin time in males, higher total bilirubin levels in males and females and higher bile acids levels in males and females. Although most levels were only slightly above the range considered normal for rats of this age and strain, the change in bile acids levels in females was remarkable. In addition, hunched posture was observed for females at 1000 mg/kg bw/day during the second half of the study. Taking all findings into account, the combination of effects in animals at 1000 mg/kg bw/day was considered to be adverse. At 300 mg/kg bw/day, minimal vacuolation of Kupffer cells (barely noticeable) was observed among several animals and was accompanied with slightly higher cholesterol levels and higher bile acids levels. These findings were not considered not to be adverse due to their slight nature and the absence of any other toxicologically relevant effects. Animals at 100 mg/kg bw/day did not show toxicologically significant changes in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). Under the study conditions, the NOAEL was determined to be 300 mg/kg bw/day (Stitzinger, 2011).

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Rationale for reliability incl. deficiencies:

other:

Justification for type of information:

This endpoint is read across from 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid; Refer to section 13 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 provided endpoint summary.

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

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.

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

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:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Lower mean absolute and relative prostate weights, increased cholesterol and phospholipid levels, Locomotor Activity reductions

Key result

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:

In conclusion based on the results of this study with the read across substance, no-observed adverse effect levels (NOAEL) for systemic effects were below the lowest dose level of 100 mg/kg bw/day.

Executive summary:

The subchronic oral (gavage) 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 (CAS 55818-57-0, BADGEDA), to Wistar rats of both sexes was investigated in a GLP-compliant dose-effect study according to the OECD TG 408 (1998) and EU B.29 (1996) protocols.

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 body weight. 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.

In conclusion based on the results of this read across study, no-observed adverse effect levels (NOAEL) for systemic effects were below the lowest dose level of 100 mg/kg bwday.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: reliable guideline study under GLP

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): DGEBADA
- Storage condition of test material: At room temperature (20 ± 5 °C)

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

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day (considered groups 1, 2, 3 and 4, respectively), corresponding nominal concentrations is vehicle were 0, 20, 60 and 200 mg/mL PEG 300, respectively
Basis:
actual ingested

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

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.

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

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:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Lower mean absolute and relative prostate weights, increased cholesterol and phospholipid levels, Locomotor Activity reductions

Key result

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:

In conclusion based on the results of this study, no-observed adverse effect levels (NOAEL) for systemic effects were below the lowest dose level of 100 mg/kg body weight/day of BADGEDA.

Executive summary:

The subchronic oral (gavage) 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 (CAS 55818-57-0, BADGEDA) to Wistar rats of both sexes was investigated in a GLP-compliant dose-effect study according to the OECD TG 408 (1998) and EU B.29 (1996) protocols.

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 body weight. 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.

In conclusion based on the results of this study, no-observed adverse effect levels (NOAEL) for systemic effects were below the lowest dose level of 100 mg/kg body weight/day of BADGEDA.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Well-conducted guideline compliant study

Repeated dose toxicity: inhalation - systemic effects

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine the chronic toxicity of the test substance according to OECD Guideline 407, EU Method B.7 and EPA OPPTS 870.3050. Groups of 5 male and 5 female Wistar rats were treated with the test substance (dissolved in polyethylene glycol) for 28 consecutive days by oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Chemical analyses of formulations were conducted once after the study and confirmed accuracy, homogeneity and stability over 6 h. The following parameters were evaluated: clinical signs daily, functional observation tests in Week 4, bodyweight and food consumption weekly, clinical pathology and macroscopy at termination, organ weights and histopathology on a selection of tissues. At 1000 mg/kg bw/day, minimal vacuolation of Kupffer cells (barely noticeable) was present in the livers of all males and females and was accompanied with a prolonged prothrombin time in males, higher total bilirubin levels in males and females and higher bile acids levels in males and females. Although most levels were only slightly above the range considered normal for rats of this age and strain, the change in bile acids levels in females was remarkable. In addition, hunched posture was observed for females at 1000 mg/kg bw/day during the second half of the study. Taking all findings into account, the combination of effects in animals at 1000 mg/kg bw/day was considered to be adverse. At 300 mg/kg bw/day, minimal vacuolation of Kupffer cells (barely noticeable) was observed among several animals and was accompanied with slightly higher cholesterol levels and higher bile acids levels. These findings were not considered not to be adverse due to their slight nature and the absence of any other toxicologically relevant effects. Animals at 100 mg/kg bw/day did not show toxicologically significant changes in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). Under the study conditions, the NOAEL was determined to be 300 mg/kg bw/day (Stitzinger, 2011).

Further, as 90-day study with a read across substance, BADGEDA, has been additionally discussed here, as its effects on gonads has been used as read across for the reproductive toxicity endpoint.

The subchronic oral (gavage) 90-day repeated dose toxicity of the read across substance, BADGEDA (CAS 55818-57-0), to Wistar rats of both sexes was investigated in a GLP-compliant dose-effect study according to the OECD TG 408 (1998) and EU B.29 (1996) protocols.

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 body weight. 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.

In conclusion based on the results of this read across study, no-observed adverse effect levels (NOAEL) for systemic effects were below the lowest dose level of 100 mg/kg bwday (Braun, 2015).

Justification for classification or non-classification

Based on the results from a 28 d or 90-day repeated dose toxicity studies in rats, the test substance does not require classification for this endpoint according to CLP (1272/2008) criteria.