Registration Dossier
Registration Dossier
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EC number: 204-399-4 | CAS number: 120-47-8
Endpoint summary
Administrative data
Description of key information
NOAEL (rat, oral, 90 days): 1000 mg/kg bw/d based on read-across
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- See atached read-across justificatiin (chapter 13)
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Based on read-across
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see explanation:
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Critical effects observed:
- no
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018
- 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:
- 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 7-8 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 155 – 190 g (Mean: 172.38 g, ± 20 % = 137.90 – 206.86 g)
females: 127 – 146 g (Mean: 137.30 g, ± 20 % = 109.84 – 164.76 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities.
Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.
Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days)
Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made.
Only healthy animals were used for this study. Before the first administration all animals used for the study were weighed and assigned to the experimental
groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).
- Route of administration:
- oral: gavage
- Vehicle:
- other: 1 % hydroxyethyl-cellulose (viscosity 80-125 cP, 2 % in water at 20 °C).
- Details on oral exposure:
- The test item, as delivered, was weighed into a tared plastic vial on a suitable precision balance and coated with approx. half the target volume with vehicle.
The vehicle 1 % aqueous hydroxyethyl-cellulose was added to give the appropriate final concentration. The formulation was vortexed and/or stirred until visual homogeneity was achieved.
Based on the results of stability testing (Eurofins Munich Study No. 176888), the test item formulations were prepared at least once-weekly
(within stability time frame as given by Eurofins Munich Study No. 176888). The prepared formulation was stored protected from light and at 2-8 °C.
Formulates were kept under magnetic stirring during the daily administration.
The vehicle was also used as control item.
Experimental Groups and Doses
In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).
Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 1000 mg/kg body weight
Administration of Doses
The test item and control formulation were administered at the spezified doses as single application per day to the animals by oral gavage at an application volume of 5 mL/kg bw.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle
at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 176888).
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous according to Eurofins Study No. 176888 (after 30 min without stirring), samples were not collected during the
study for the investigation of homogeneity and only samples were taken for substance concentration in study week 1, 5, 9 and in the last week of treatment (16 samples in total).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 176889)
and until then stored under appropriate conditions based on available stability data. The B-samples will be retained at < -15 °C at BSL Munich (test facility) and discarded after completion of the final study report. - Duration of treatment / exposure:
- Period of 90 days
- Frequency of treatment:
- Once a day; 7 days per week
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
Additionally, 20 animals (5 males and 5 females per group) were used as recovery animals (control and high dose). - Control animals:
- yes, concurrent vehicle
- Details on study design:
- The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 90 days. 10 animals per gender and group were
subjected to necropsy one day after the last administration (end of treatment period). 5 animals per gender of the C and of the HD group were
subjected to necropsy 28 days after the last administration (end of recovery period).
The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and to identify a NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group. - Positive control:
- not applicable
- Observations and examinations performed and frequency:
- Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during
the treatment and recovery period. Food consumption was measured weekly during the treatment and recovery period.
Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days.
The recovery animals were observed for an additional period of 28 days following the last administration.
General clinical observations were made once a day, approximately at the same time each day after dosing. The health condition of the animals
was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were
made once daily. Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea,
asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were
made outside the home cage in a standard arena once before the first administration and weekly thereafter.
Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of the
treatment period as well as at the end of the recovery period in the recovery animals.
Functional Observations
Once before the first exposure and once in the last week of exposure as well as in the last week of the recovery period multiple detailed
behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted in all animals..
Haematology
Haematological parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC),
mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC),
reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos),
basophils (Baso), large unstained cells (Luc).
Blood Coagulation
Coagulation parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.
The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT).
Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes..
The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT),
alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol),
low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TG), glucose (Gluc), sodium (Na), potassium (K).
For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals were retained at the end of treatment (80 animals) and recovery (20 animals) and stored at < -15° C. T3, T4 and TSH serum levels were determined of main study animals (80 animals).
Urinalysis
A urinalysis was performed with samples collected from all animals at necropsy.
The following parameters (specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL),
erythroctes (Ery), leukocytes (Leu)) were measured using qualitative indicators (Heiland Urine Stripes URI 10SL).
Additionally, urine colour / appearance were recorded.
Blood Sampling for potential proof of exposure
For potential proof of exposure after repeated oral administration, blood was sampled from all recovery animals 30 ± 3 minutes after the last treatment (day 90). Approx. 250 µL blood was sampled from the V. Jugularis of slightly anaesthetised rats (Isoflurane) and collected in EDTA- tubes. Samples were placed on ice for potential further processing. Prior to sample analysis an appropriate LC-MS/MS method will be developed for determination of the test item in plasma.
- Sacrifice and pathology:
- Pathology
One day after the last administration (study day 91) all surviving animals of the treatment period and 4 weeks after the last administration all surviving animals
of the recovery period (study day 119) were sacrificed using anesthesia (ketamine and xylazin) and were subjected to a detailed gross necropsy
which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Organ Weight
The wet weight of the following organs (liver, uterus, kidneys, thymus, adrenal glands, thyroid/parathyroid glands, testes, spleen, epididymides,
brain, prostate gland, pituitary gland, seminal vesicles and coagulating glands after ligation, heart, ovaries) of all sacrificed animals was
recorded as soon as possible. Paired organs were weighed individually. Organ weights of animal euthanised for animal welfare reasons were
not recorded.
The wet weight of the organs (liver, uterus with cervix, kidneys, thymus, adrenal glands, thyroid/ parathyroid glands, testes, spleen, epididymides,
brain,prostate, seminal vesicles and coagulating glands, pituitary gland, heart, ovaries) of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together.
Weight of thyroid/parathyroid glands was measured after fixation. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.
The following tissues (adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides,
eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular),
lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole,
rectum, salivary glands (sublingual, submandibular, parotis), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow),
stomach, testes, thymus, thyroid gland including parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina) from all animals were preserved in 4 % neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol.
All animals found dead and/or intercurrently euthanised for animal welfare reasons were also subjected to a gross necropsy and the organs preserved for a histopathological examination.
Histopathology
The afore-listed organs were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the
roups 1 and 4 sacrificed either at the end of the treatment or recovery period and any animal found dead or euthanised before the planned day of sacrifice.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49,
4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH,
AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology). The study phases from test site 1 and 2 was performed in compliance
with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012).
Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study. - Statistics:
- A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ
weights were performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc
Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
Furthermore, statistical comparisons of data acquired during the recovery period were performed with a Dunn’s or Dunnett’s when appropriate.
These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant). - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Slight to moderate salivation was noted in all males and females of the HD group, on several days of treatment and in 2/10 males of the MD group. Furthermore, moving the bedding was regularly observed in all males and all females of the HD group and in few males and females of MD group. The clinical signs salivation and moving the bedding were observed in close timely relation to the dose administration and therefore were considered to be unspecific signs of a local reaction to the test item administration rather than a systemic adverse effect.
Alopecia was observed in one male of the MD group and in single or occasional females of the control, LD, MD and HD groups. Crust/wound was observed in few females of the control and dose groups. These findings are mild common background findings and independent from treatment. Dehydration was observed in 5/10 male animals of the LD group on day 7. This was most probably caused by a block of the drinking bottle and independent of treatment. Single or occasional findings, i.e. piloerection, reduced spontaneous activity, abnormal breathing were observed on single or few days in HD male and female animals and are considered to be an incidental findings and not treatment related.
No clinical signs were observed in HD male and female animals during the recovery period. There were no test item related clinical findings during recovery period. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Female no. 62, dosed at 100 mg/kg was found dead in the cage on study day 39. The cause of morbidity was considered to be most likely incidental and not related to the test item administration due to the fact that no histopathological findings that could be attributed to treatment with the test item were observed in this animal. All remaining animals survived until the end of the treatment or recovery period.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- In males and females of all groups, there was an increase of body weight with the progress of the study. Mean body weight change from day 1 to 90 was comparable between the dose groups and control group. Sporadically observed lower / increased body weight gain in male animals of all groups were whitin the range of historical control data and considered not to be of toxicological relevance.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Mean daily food consumption was comparable between all male and female dose groups and controls during the study treatment and recovery period.
On week 1, there was a slightly but statistically significantly lower food consumption in HD males (approx. 5 % below controls) and on week 7, a statistically significant slightly higher food consumption was observed in HD females (approx. 7 % above controls). However, these slight differences were considered incidental and there was no effect of toxicological relevance on food consumption in any of the dose groups. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No ophthalmologic findings were observed in any of the animals of this study.
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- No toxicologically relevant effects of test item were found on all haematological and coagulation parameters of all male and female animals of the dose groups. A statistical significant slight increase in WBC, in male and female animals of MD and HD groups is not considered toxicologically relevant as individual values were within the historical range. No effect of toxicological relevance on haematological and coagulation parameters was found at the end of the recovery period.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There was no test item related effect on clinical biochemistry parameters measured at the end of the treatment and recovery period.
A slight but statistically significant decreased serum creatinine level observed in male and female animals of the HD group, when compared to controls is of no toxicological relevance. A dose response was not observed and a decrease in this level is usually not associated with toxicity. A slight but statistically significant decrease in total Cholesterol and HDL was observed in male animals of the HD group. These changes were not dose related and fully reversible at the end of the recovery period. Thyroid hormon analysis revealed no statistical significant changes beside an increase T4 level only in HD females. However, due to large natural variation in thyroid hormon levels a toxicological significance of this finding is not seen. This view is supported by the absence of changes in the more relevant TSH levels, the restriction of the finding to just one sex and the absence of any histopathological corrolate in the thyroid, especially in high dose females. In addition, all thyroid hormon values in all dose groups are well whithin physiological ranges given in the literature for this strain of rats.
At the end of recovery, a statistically significant slight increase in sodium was observed in male HD group. As levels were within the range of historical control data this is not considered toxicologically relevant. Besides, the mean values for the remaining parameters are within a normal range of variation. - Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- There were no test item related effects were observed in any of the groups at the end of treatment and recovery period.
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- No test item related effects were observed in any parameter of the functional observation battery at the end of the treatment period when compared to observations before treatment.
Before initiation of treatment (Week 1), there was statistically significantly lower score of urination in male MD males, a higher count of supported rearings in LD females, non-supported rearings in MD females and higher body temperature in LD females when compared to controls, respectively.
The score for moving in cage and the number of supported rearings in the males of the HD group were statistically significantly increased in week 13 when compared to control group, whereas a statistically significantly lower score for sleeping was observed in these animals. Statistical significantly less supported rearings and lower body temperature was observed in HD group at the end of the recovery period, when compared to the respective controls.
As the effects on various functional observation parameters were slight or inconsistent throughout the dose groups before and at the end of treatment and did not coincide with other parameters of activity or motorcoordination, no toxicological significance can be attributed from these findings. - Immunological findings:
- no effects observed
- Description (incidence and severity):
- No indication of adverse immonological reactions and no observations in immunological relevant organs.
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no organ or body weight changes that could be related to treatment with the test item.
A slight but statistically significant higher absolute kidney weight was observed in females of the HD group (10 % above controls), when compared to controls. As no dose response relationship was observed and moreover the changes were not associated with any histopathological findings, this apparent change it is not considered to be toxicologically relevant. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- There were no gross lesions that could be attributed to treatment with the test item. All changes recorded at necropsy are considered to be an incidental finding.
Gross lesion were observed randomly in all the groups at the end of treatment period, like dark lungs, a black left median lobe or dark green spots in the lungs of female animals no. 71 and 77 (MD group) and male animal no. 24 (MD group), liver fused with diaphragm in male animal no. 39 (HD group), a white and hard mass of 1.5 cm at the right kidney of animal no. 34 (HD group), a small right prostate gland in male control animal no. 1, abnormal colored, red spotted thymus in male Control animal no. 4 and male animal no. 24 of the MD group and small, complete thymus of MD group male animal no. 24 and abnormally colored and spotted mesenteric lymph node in 2/10 HD females. Uterus with dilatation on both sides was seen in 2/10 control animals, 2/10 animals of the LD group, 3/10 animals of the MD group and 5/10 animals of the HD group. All these are not considered to be toxicologically relevant.
Gross lesions were also observed at the end of recovery period in HD groups, i.e. a small prostate gland, (1/5 animals), cervix dilatation in 1/5 control animals, small right adrenal gland with abnormal color, in 1/5 HD males (no. 99) and abnormal color spotted mesenteric lymph node in 2/5 control and 2/5 males of the HD group. Based on histopathological evaluation the gross lesions were considered to be incidental and not related to treatment with the test item. - Neuropathological findings:
- no effects observed
- Description (incidence and severity):
- No effects observed in any parameter of the functional observation battery.
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- There were no histopathological findings that could be attributed to treatment with the test item. All recorded findings were considered incidental or
were within the range of background alterations that may be recorded in Wistar rats of this age. - Histopathological findings: neoplastic:
- no effects observed
- Description (incidence and severity):
- No neoplastic findings observed during histopathology.
- Details on results:
- Mortality
Female no. 62, dosed at 100 mg/kg was found dead in the cage on study day 39. A contribution of the macroscopically observed diaphragmatic hernia of the liver cannot be excluded.
However, the cause of morbidity was considered to be most likely incidental and not related to the test item administration. All remaining animals survived until the end of the treatment or recovery period.
Clinical Observations
There were no clinical signs of systemic toxicity in this study.
Slight to moderate salivation was noted in all males and females of the HD group, on several days of treatment and in 2/10 males of the MD group. Furthermore, moving the bedding
was regularly observed in all males and all females of the HD group and in few males and females of MD group. The clinical signs salivation and moving the bedding were observed
in close timely relation to the dose administration and therefore were considered to be signs of a local reaction to the test item rather than a systemic adverse effect.
Alopecia was observed in one male of the MD group and in single or occasional females of the control, LD, MD and HD groups. Crust/wound was observed in few females of the
control and dose groups. These findings are mild common background findings and independent from treatment. Dehydration was observed in 5/10 male animals of the LD group on day 7.
This was most probably caused by a block of the drinking bottle and independent of treatment. Single or occasional findings, i.e. piloerection, reduced spontaneous activity,
abnormal breathing were observed on single or few days in HD male and female animals and are considered to be an incidental findings and not treatment related.
No clinical signs were observed in HD male and female animals during the recovery period.
During the weekly detailed clinical observation, no considerable differences between the groups were found except statistically significant softer faecal consistency
and a lower score for sleeping in the cage in week 1 and a higher score for moving in cages in week 1, higher score of sleeping in the cage and a lower score for moving in cages in week 3.
The statistically significant differences in detailed clinical examination parameters were not considered to be treatment related, as they were observed occasionally without dose dependency.
There were no test item related clinical findings during recovery period.
Functional Observation Battery
No test item related effects were observed in any parameter of the functional observation battery at the end of the treatment period when compared to observations before treatment.
Before initiation of treatment (Week 1), there was statistically significantly lower score of urination in male MD males, a higher count of supported rearings in LD females, non-supported rearings in
MD females and higher body temperature in LD females when compared to controls, respectively.
The score for moving in cage and the number of supported rearings in the males of the HD group were statistically significantly increased in week 13 when compared to control group,
whereas a statistically significantly lower score for sleeping was observed in these animals. Statistical significantly less supported rearings and lower body temperature was observed in
HD group at the end of the recovery period, when compared to the respective controls.
As the effects on various functional observation parameters were slight or inconsistent throughout the dose groups before and at the end of treatment and did not coincide with other
parameters of activity or motorcoordination, no toxicological significance can be attributed from these findings.
Ophthalmoscopic Findings
No ophthalmologic findings were observed in any of the animals of this study.
Body Weight Development
In males and females of all groups, there was an increase of body weight with the progress of the study. Mean body weight change from day 1 to 90 was comparable between the dose groups
and control group. In the HD males, moderate and statistically significantly lower body weight gain was seen in week 11 of treatment when compared to control group (46 % below control)
and in males of LD and MD group moderate and statistically significantly increased body weight gain in week 3 (45% and 24% above control, respectively). In females, no statistically or biologically
significant differences were noted for body weight gain between the dose groups and the control group.
During the recovery period, between day 90 to 118 body weight change was statistically significantly higher in male but not female animals of the HD group, when compared to controls
(88.57% in week 14, 88.6% in week 16 and, 71.42% in week 13). As the body weights were within the range historical control data, this is not assumed to be toxicologically relevant.
Food Consumption
Mean daily food consumption was comparable between all male and female dose groups and controls during the study treatment and recovery period.
On week 1, there was a slightly but statistically significantly lower food consumption in HD males (approx. 5 % below controls) and on week 7, a statistically significant slightly higher
food consumption was observed in HD females (approx. 7 % above controls). However, these slight differences were considered incidental and there was no effect of toxicological relevance
on food consumption in any of the dose groups.
Haematology and Blood Coagulation
No adverse test item related effects were found for all haematological and coagulation parameters for all male and female dose groups at the end of the treatment and recovery period.
A statistically significantly slight increase in WBC in male and females of MD and HD groups is not considered to be adverse as individual values were within the historical control data range.
However, a relation to the test item cannot be excluded. A statistically significant slightly higher Hb and MCHC in females of HD group was observed at the end of the treatment period.
As values were within the range of historical control data this is not considered toxicologically relevant. A tendency towards lower Eos in males and higher Eos in females of the dose
groups is not considered to be toxicologically relevant.
At the end of the recovery period, a slight statistically non-significant increase in WBC in males (33.60 % above control) and a slight but statistical significant slight decrease in WBC in females
of the HD group (36.66 % below control) were observed. As values were within the range of historical control data and due to the inconsistency, these differences are not considered toxicologically relevant.
A statistically significant slightly lower MCH in males of HD group was observed at the end of the recovery period. As values were within the range of historical control data this is not
considered toxicologically relevant.
Clinical Biochemistry
There was no test item related effect on clinical biochemistry parameters measured at the end of the treatment and recovery period.
A slight but statistically significant decreased serum creatinine level observed in male and female animals of the HD group, when compared to controls is of no toxicological relevance. A decrease in this level is usually not associated with toxicity. A slight but statistically significant decrease in total Cholesterol and HDL was observed in male animals of the HD group. A relation to the test item cannot be excluded. Thyroid hormon analysis revealed no statistical significant changes beside an increase T4 level only in HD females. However, due to large natural variation in thyroid hormon levels a toxicological significance of this finding is not seen. This view is supported by the absence of changes in the more relevant TSH levels, the restriction of the finding to just one sex and the absence of any histopathological corrolate in the thyroid, especially in high dose females. In addition, all thyroid hormon values in all dose groups are well whithin physiological ranges given in the literature for this strain of rats.
At the end of recovery, a statistically significant slight increase in sodium was observed in male HD group.
As levels were within the range of historical control data this is not considered toxicologically relevant.
Besides, the mean values for the remaining parameters are within a normal range of variation.
Urinalysis
There were no test item related effects were observed in any of the groups at the end of treatment and recovery period.
Pathology
There were no gross lesions that could be attributed to treatment with the test item. All changes recorded at necropsy are considered to be an incidental finding.
Gross lesion were observed randomly in all the groups at the end of treatment period, like dark lungs, a black left median lobe or dark green spots in the lungs of female animals no. 71 and 77 (MD group) and male animal no. 24 (MD group), liver fused with diaphragm in male animal no. 39 (HD group), a white and hard mass of 1.5 cm at the right kidney of animal no. 34 (HD group), a small right prostate gland in male control animal no. 1, abnormal colored, red spotted thymus in male Control animal no. 4 and male animal no. 24 of the MD group and small, complete thymus of MD group male animal no. 24 and abnormally colored and spotted mesenteric lymph node in 2/10 HD females. Uterus with dilatation on both sides was seen in 2/10 control animals, 2/10 animals of the LD group, 3/10 animals of the MD group and 5/10 animals of the HD group. All these are not considered to be toxicologically relevant. Gross lesions were also observed at the end of recovery period in HD groups, i.e. a small prostate gland, (1/5 animals), cervix dilatation in 1/5 control animals,
small right adrenal gland with abnormal color, in 1/5 HD males (no. 99) and abnormal color spotted mesenteric lymph node in 2/5 control and 2/5 males of the HD group.
Based on histopathological evaluation the gross lesions were considered to be incidental and not related to treatment with the test item.
Organ Weight
There were no organ or body weight changes that could be related to treatment with the test item.
A slight but statistically significant higher absolute kidney weight was observed in females of the HD group (10 % above controls), when compared to controls.
As this was not associated with any histopathological findings, it is not considered to be toxicologically relevant.
Besides, there were no statistically significant differences in weights of any of the other organs between control and dose groups.
A tendency towards lower absolute thyroid/parathyroid weight of male animals of the HD group (25 % below controls) and higher thymus weight in
female animals of the HD group (17 % above controls) were not associated with histopathological findings and are not considered to be toxicologically relevant.
Histopathology
There were no histopathological findings that could be attributed to treatment with the test item. All recorded findings were considered incidental or
were within the range of background alterations that may be recorded in Wistar rats of this age.
Dose Formulation Analysis
Concentration analysis of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL in study weeks 1, 3, 9 and 13 of the study.
The mean recoveries observed for the LD dose group were between 101.6 % and 121.0 % of the nominal value, between 89.7% and 107.8% of the nominal value for the MD dose group and between 86.7 % and 109.2 % of the nominal value for HD dose group. In week 13, the mean recovery observed for the LD was slightly higher (121 %) than the nominal value. However, the overall mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 109.1 %, 97.4 %, and 97.6 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no treatment related signs of toxicity were obseved
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Critical effects observed:
- no
- Conclusions:
- On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with Propyl 4-hydroxybenzoate in male and female Wistar rats, with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made: The no observed adverse effect level (NOAEL) of Propyl 4-hydroxybenzoate in this study is determined at 1000 mg/kg body weight/day.
- Executive summary:
The objective of this study was to assess the possible health hazards which could arise from repeated exposure of Propyl 4-hydroxybenzoate via oral administration to rats over a period of 90 consecutive days.
The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 90 days. Animals of an additional control group were handled identically as the dose groups but received 1 % aqueous hydroxyethyl-cellulose, the vehicle used in this study. The 4 groups comprised of 10 male and 10 female Wistar rats. Control, low, mid and high dose group rats received the dose at 0, 100, 300 and 1000 mg/kg/day respectively, as repeated dose at the dose volume of 5 mL/kg. The test item formulation was prepared at least every 4 days. The test item was suspended in 1 % aqueoushydroxyethyl-cellulose and administered daily during a 90-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements.
During the period of administration, the animals were observed precisely each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. To detect possible delayed occurrence or persistence of, or recovery from toxic effects, animals in the recovery group were observed for a period of 28 days following the last administration.
functional observation battery and ophthalmoscopy examination did not reveal any test item related effects in any of the treatment and recovery groups.
In males and females, there was no test item related effect on body weight during both treatment and recovery period.
There was no effect of toxicological relevance on food consumption in any of the dose groups during both treatment and recovery period.
No toxicologically relevant effects of test item were found on all haematological and coagulation parameters of all male and female animals of the dose groups. A statistical significant slight increase in WBC, in male and female animals of MD and HD groups is not considered toxicologically relevant as individual values were within the historical range. No effect of toxicological relevance on haematological and coagulation parameters was found at the end of the recovery period. There was no adverse effect on clinical biochemistry parameters measured at the end of the treatment and recovery period. Changes in some clinical biochemistry parameter were in most cases not dose related and not considered to be of toxicological relevance. There was no adverse effect on urinary parameters measured at the end of the treatment and recovery period. Based on histopathological evaluation, none of the occasional gross lesions observed at necropsy were considered to be related to treatment with test iteml. No statistically significant difference in organ weight was found in male and female animals at any of the dose levels tested when compared to control. At histopathological evaluation, there were no abnormalities that could be attributed to treatment with the test items.
Conclusion
On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with Propyl 4-hydroxybenzoate in male and femaleWistarrats, with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:
The no observed adverse effect level (NOAEL) of Propyl 4-hydroxybenzoate in this study is determined at 1000 mg/kg body weight/day.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-01-16 to 2020-01-23
- Reliability:
- 1 (reliable without restriction)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- adopted 25 June 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 7-8 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 183 – 224 g (mean: 205.9 g, ± 20 % = 164.7 – 247.0 g)
females: 131 – 158 g (mean: 145.5 g, ± 20 % = 116.4 – 174.6 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.
Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days)
Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Only healthy animals were used for the study Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).
- Route of administration:
- oral: gavage
- Vehicle:
- other: 1 % hydroxyethyl-cellulose (viscosity 80-125 cP, 2 % in water at 20 °C).
- Details on oral exposure:
- The vehicle has been selected in consultation with the sponsor based on the test item’s characteristics.
The test item, as delivered, was grinded before formulation preparation. Afterwards, test item was weighed into a tared plastic vial on a suitable precision balance and coated with approx. 1/3 of the target volume with 1 % aqueous hydroxyethyl-cellulose, the vehicle used in this study. After producing slurry with the glass rod for 1 minute, the rest of the vehicle was added to give the appropriate final concentration. The formulation was then stirred until visual homogeneity was achieved (at least 30 min).
Based on the results of stability testing (Eurofins Munich Study No. 187385), the test item formulations were prepared once in 4 days. The prepared formulation was stored at room temperature.
Formulates were kept under magnetic stirring during the daily administration.
The vehicle was also used as control item.
Experimental Groups and Doses
In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).
Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 1000 mg/kg body weight
Administration of Doses
The test item and control formulation were administered at a single dose to the animals by oral gavage at an application volume of 5 mL/kg bw.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Study No. 187385).
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
The test item is shown to be suspension according to Eurofins Study No. 187385, so the samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1, 5, 9 and in the last week of treatment (40 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 190461) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at below 15 °C at BSL Munich (test facility) and discarded after completion of the final study report. - Duration of treatment / exposure:
- Period of 90 days
- Frequency of treatment:
- Once a day; 7 days per week
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
Additionally, 20 animals (5 males and 5 females per group) were used as recovery animals. - Control animals:
- yes, concurrent vehicle
- Details on study design:
- The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 90 days. 10 animals per gender and group were
subjected to necropsy one day after the last administration (end of treatment period). 5 animals per gender of the C and of the HD group were
subjected to necropsy 28 days after the last administration (end of recovery period).
The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group. - Positive control:
- not applicable
- Observations and examinations performed and frequency:
- Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during
the treatment and recovery period. Food consumption was measured weekly during the treatment and recovery period.
Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days. The recovery animals were observed for an additional period of 28 days following the last administration.
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examination using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period as well as at the end of the recovery period in the recovery animals.
Functional Observations
Once before the first exposure and once in the last week of exposure as well as in the last week of the recovery period multiple detailed
behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted in all animals..
Haematology
Haematological parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value (HCT), haemoglobin content (HGB), red blood cell count (RBC),
mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC),
reticulocytes (Ret), platelet count (PLT), white blood cells (WBC), neutrophils (Neut), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos),
basophils (Baso), large unstained cells (Luc).
Blood Coagulation
Coagulation parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.
The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT).
Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes..
The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT),
alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TG), glucose (Gluc), sodium (Na), potassium (K).
For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals were retained at the end of treatment (80 animals) and recovery (20 animals) and stored at < -15° C. T3, T4 and TSH serum levels were determined of main study animals (80 animals).
Urinalysis
A urinalysis was performed with samples collected from all animals at necropsy.
The following parameters (specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL),
erythrocytes (Ery), leukocytes (Leuc)) were measured using qualitative indicators (Heiland Urine Stripes URI 10SL).
Additionally, urine colour / appearance were recorded.
Blood Sampling for Proof of Exposure
For proof of exposure of test item after repeated oral administration, blood was sampled from the control (1 rat/sex), HD (1 male, 2 females) main group and recovery groups (4 5 animals / group) on treatment week 12.
Approx. 250 µL blood (2 time points / rat) were sampled from the V. Jugularis of slightly anaesthetised rats (Isoflurane) and collected in K3EDTA-tubes. Samples were placed on ice. They were then centrifuged for 10 min at 4 °C at approx. 1000 g. After centrifugation, 100 µL of plasma were transferred into a tube containing 5 µL of 1 M citric acid solution. The results of the main study animals (control - 10, 60; HD - 40, 89, 90) were evaluated carefully at the end of the study, as these animals were not handled fully as the other main study animals.
All samples were vortexed for 5-10 seconds and were immediately stored at < -70 °C until further analyses. In case, less than 100 µL plasma, the volume difference was recorded and the dilution factor was adjusted and considered during the sample measurement. The samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 187389).
The Principal investigator for the determination of plasma levels of the Methyl 4-hydroxybenzoate and 4-hydroxybenzoic acid for proof of exposure provided the analytical results to the study director of this study via email and sent the analytical report to the study director upon the completion of the study.
Prior to sample analysis appropriate LC-MS/MS Methods were developed for determination of the test item in plasma. The Validation of the bioanalytical method was described in individual phase plans under Eurofins Munich Study No. 187388.
Sperm Analysis
At terminal sacrifice, left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters (Motility, cauda epididymal sperm count or testicular sperm head count and sperm morphology) from all males sacrificed at the end of treatment period were performed by using Hamilton Thorn Sperm Analyser (TOX IVOS Version 13.0C) except for sperm morphology, which was addressed manually by manual method. Sperm motility evaluation was not performed for the recovery animals at the end of recovery due to logistic reasons.
Sperm morphology slides were prepared from all males. Initially, evaluation was made in male animals of the groups 1 (control) and 4 (HD group) sacrificed at the end of the treatment period. Sperm morphology examinations were not extended to male animals of all other dosage groups and recovery groups, as there were no treatment-related changes observed in the high dose group. For morphology evaluation, sperm from left vas deferens was transferred to 0.1 % bovine serum albumin solution. For staining, two drops of 1 % aqueous Eosin-Y solution was mixed with six drops of the sperm-suspension. The stained sperm suspension was used to prepare smears on slides. After complete drying, the slides were dipped into 0.1 % acetic acid for approximately 30 seconds to intensify the colouring.
- Sacrifice and pathology:
- Pathology
One day after the last administration (study day 91) all surviving animals of the treatment period and 4 weeks after the last administration all surviving animals of the recovery period (study day 119) were sacrificed using anaesthesia (ketamine and xylazin) and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle.
Organ Weight
The wet weight of the following organs (liver, uterus with cervix, kidneys, thymus, prostate, seminal vesicles and coagulating glands, thyroid/ parathyroid glands (were weighed after fixation), testes, spleen, epididymides, brain, adrenals, pituitary gland, ovaries, heart) of all sacrificed animals was recorded as soon as possible.
Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.
The following tissues (adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides (right), eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys,
liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular, parotis), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes (right), thymus, thyroid gland including parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina) from all animals were preserved in 4 % neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol.
All animals found dead and/or intercurrently euthanised for animal welfare reasons were also subjected to a gross necropsy and the organs preserved for a histopathological examination.
Histopathology
The afore-listed organs were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the groups 1 and 4 sacrificed either at the end of the treatment or recovery period and animal euthanised before the planned day of sacrifice.
Tissue samples of all main animals from the control and the high dose group, the animal intercurrently euthanized (animal no. 98) and all organs with macroscopic findings (main and recovery animals) were processed. All resulting tissue sections were embedded in paraffin and cut at an approximated thickness of 2 – 4 µm and stained with haematoxylin and eosin (H&E).
Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology). The study phases from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites. The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director. The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study. - Statistics:
- A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
Furthermore, statistical comparisons of data acquired during the recovery period may be performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant). - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no clinical signs of systemic toxicity in this study.
Slight to moderate/severe salivation was noted in males and females of the HD group, on several days of treatment. Furthermore, moving the bedding was regularly observed in all males and all females of the HD group and in one male at MD group. The clinical signs salivation and moving the bedding were observed in close timely relation to the dose administration and therefore were considered to be unspecific signs of a local reaction to test item administration rather than a systemic adverse effect. For more information see the section details on results - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- HD recovery female no. 98, dosed at 1000 mg/kg was moribund sacrificed on day 56 due to animal welfare reasons. Predominant clinical signs observed on the day of moribund sacrifice were abnormal breathing and reduced spontaneous activity. The abnormal dark red colour in the lungs at necropsy observation correlated with the multifocal alveolar haemorrhages during the histopathology evaluation. The alveolar haemorrhages were considered to be most likely incidental and not related to treatment with test item. Due to the signle incidance of mortality this effect is not considered to be test item related. All remaining animals survived until the end of the treatment or recovery period.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In males and females of all groups, there was an increase of body weight with the progress of the study. Mean body weight from day 1 to 90 was comparable between the dose groups and control group. Some changes in body weight/body weight gain are observed is some animals which are mainly due to higher body weight gain of control animals are not considered to be test item related. For more information see the section details on results.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Beside some minor changes in food consumption the mean daily food consumption was comparable between all male and female dose groups and controls during the study treatment and recovery period. For more information see the section details on results.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No adverse or test item related effects were found for any haematological and coagulation parameters in male and female dose groups at the end of the treatment and recovery period.
A slight increase in mean WBC in MD males and in LD, MD females groups are not considered to be adverse as individual values were within the historical control data range. A tendency towards lower or higher percent differential leucocytes counts in males and females of the dose groups is not considered to be toxicologically relevant. At the end of the recovery period, a slight but statistically significant increase in percent reticulocytes in HD males (112 % above control) was observed. Slight and non-statistically significant decrease or increase in WBC in male and females of the HD group were observed.
A slight but statistically significant increase in aPTT in HD females (34 % above control) was observed when compared to control. This is considered to be an incidental finding. No test item related effects on coagulation parameters were observed in both main and recovery groups.
Treatment with the test item had no dose-dependent effect on haematology parameters of males and females in any of the test item-treated groups. Differences between test item-treated males or females and their respective controls showed no dose-dependency or consistency and thus were not considered toxicologically relevant. All the values were found to be within the normal range of historical values in this strain. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- There were no test item related effects observed on various urine parameters in any of the groups at the end of treatment and recovery periods.
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item related effects were observed in any parameter of the functional observation battery at the end of the treatment period when compared to observations before treatment.
In males, before initiation of treatment (Week 1), there was a slight but statistically significant higher mean score for grooming and body temperature was observed in the MD group. On week 13, there was a slight but statistically significant lower score was observed for animal sleeps, rearing supported, defecation, righting reflex ground, air righting reflex and a higher mean score for animal moving in cage, startle response, equilibrium reflex, positional passivity, visual placing, grip strength, pinching the tail, toe pinch reflex, hind limb reflex, pupil response and limb tone in the MD group.
In LD males on week 13, slight but statistically significant higher mean score was observed for head touch, startle response (includes MD), equilibrium reflex, positional passivity, visual placing, grip strength, pinching the tail, toe pinch reflex, hind limb reflex, pupil response and limb tone and lower mean score for defecation, righting reflex ground and air righting reflex.
In females, on week 13, there was a slight but statistically significant higher mean score observed for startle response, equilibrium reflex, positional passivity (LD group), visual placing (LD group), grip strength, pinching the tail, toe pinch reflex, hind limb reflex, pupil response, and limb tone and lower score for righting reflex ground and air righting reflex in LD and MD groups. Higher score for HD group was observed for pupil response.
During the recovery period, on week 17, the recovery HD males showed slightly higher score for defecation and in females, slightly higher score for body temperature.
As the effects on various functional observation parameters were slight or inconsistent throughout the dose groups before and at the end of treatment/recovery. These findings are not considered to be a toxicological significance. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no organ or body weight changes that could be related to treatment with the test item.
A slight but statistically significant lower absolute mean heart weight was observed in males of the HD group (9 % below) when compared to controls. A slight but statistically significant higher relative mean liver weight was observed in males of the HD group (10 % above), when compared to the controls.
A moderate but statistically significant higher relative mean thymus weight was observed in males of the HD recovery group (47 % above), when compared to controls. A slight but statistically significant lower absolute mean pituitary gland weight was observed in males of the HD recovery group (13 % below), when compared to controls. A slight but statistically significant lower absolute mean liver weight was observed in males of the HD recovery group (10 % below), when compared to controls. Moderate increase in mean absolute and relative weight of epididymis (right & left) was observed in HD recovery male at the end of recovery when compared to control. A slight increase in absolute and relative mean pituitary gland weight was observed in females of the HD recovery group, when compared to controls. As these were not associated with any histopathological findings, it is not considered to be toxicologically relevant.
Moderate increase in absolute and relative mean uterus with cervix weight was observed in females of the HD recovery group, when compared to the controls. The differences between test item-treated females and control animals in mean weight of uterus and ovaries were not considered toxicologically relevant as female reproductive organs undergo variable changes depending on the stage of the oestrous cycle. In the absence of corresponding microscopic changes, all observed body and organ weight changes were considered of no toxicological relevance. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Gross lesions like displaced red jejunum (animal no. 35, HD), dark red colour caecum (animal no. 13, LD), mass at papillary process in liver (animal no. 23, MD), small testes on both sides (animal no. 39, HD), red colour thymus with focus (animal no. 32, 37, HD) and in females, mass at diaphragm (animal no. 74, MD), uterus dilatation [animal no. 55 (control), 61 (LD), 78, 79 (MD), 83, 85 (HD)], black focus in the adrenal glands (animal no. 58, control), red colour thymus with focus [animal no. 58 (control), 64 (LD), 76, 77 (MD) and 87 (HD)], red colour and enlarged (both sides) mandibular lymph node (animal no. 64, LD) and dark red mesenteric lymph node (animal no. 75, MD) were observed randomly in all the groups of males and females at the end of treatment period,
Gross lesions were also observed at the end of recovery period in HD groups, i.e. in animal no. 98 abnormal foamy content in trachea, dark red abnormal colour in lungs, liver herniation in animal no. 100 and in animal no. 43 (control) red focus at right epididymides. Further, in the decedent animal no. 98 the abnormal dark red colour in the lungs observed at necropsy correlated with the multifocal alveolar haemorrhages during the histopathology evaluation. The alveolar haemorrhages were considered most likely to be incidental.
All gross lesions observed in the decedent and survivors animals were deemed to be incidental and without toxicological relevance and considered not to be induced by the treatment with the test item. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no histopathological findings that could be attributed to treatment with the test item. All recorded microscopic findings were considered incidental or were within the range of background alterations that may be recorded in Wistar rats of this age.
There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. In addition, in the investigated testes, no treatment-related effects on the testicular histomorphology were observed and the histological appearance reflected the animal physiology. - Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- Mortality
HD recovery female no. 98, dosed at 1000 mg/kg was moribund sacrificed on day 56 due to animal welfare reasons. Predominant clinical signs observed on the day of moribund sacrifice were abnormal breathing and reduced spontaneous activity. The abnormal dark red colour in the lungs at necropsy observation correlated with the multifocal alveolar haemorrhages during the histopathology evaluation. The alveolar haemorrhages were considered to be most likely incidental and not related to treatment with test item.
All remaining animals survived until the end of the treatment or recovery period.
Clinical Observations
There were no clinical signs of systemic toxicity in this study.
Slight to moderate/severe salivation was noted in males and females of the HD group, on several days of treatment. Furthermore, moving the bedding was regularly observed in all males and all females of the HD group and in one male at MD group. The clinical signs salivation and moving the bedding were observed in close timely relation to the dose administration and therefore were considered to be unspecific signs of a local reaction to test item administration rather than a systemic adverse effect.
Low incidences of clinical signs like overgrown teeth, cough/sneezing, crust at skin/fur, diarrhoea, scratch/cut at head and hairless are at right neck are considered to be an incidental findings and not related to the treatment. Animal no. 98 (HD recovery group) showed clinical signs including apathy, prone position, spontaneous reduced activity (moderate), both eyes closed and abnormal breathing immediately after treatment with test item on day 56.
Moving the bedding in HD recovery males and moving the bedding and salivations (slight) were observed in HD recovery females. No clinical signs were observed in HD male and female animals during the recovery period. No ophthalmologic findings were observed in any of the animals of this study. There were no treatment related findings during the detailed weekly clinical examination in any of the groups. Statistical significance and the intermittent clinical findings during the detailed clinical examinations; including animal sleeps, moving in the cage, salivation and response to handing were observed during the week 1, 3, 5, 6, 10, 11 and 12 in the treated groups are considered to be incidental and are not considered to be a toxicological significance.
Functional Observation Battery
No test item related effects were observed in any parameter of the functional observation battery at the end of the treatment period when compared to observations before treatment.
In males, before initiation of treatment (Week 1), there was a slight but statistically significant higher mean score for grooming and body temperature was observed in the MD group. On week 13, there was a slight but statistically significant lower score was observed for animal sleeps, rearing supported, defecation, righting reflex ground, air righting reflex and a higher mean score for animal moving in cage, startle response, equilibrium reflex, positional passivity, visual placing, grip strength, pinching the tail, toe pinch reflex, hind limb reflex, pupil response and limb tone in the MD group.
In LD males on week 13, slight but statistically significant higher mean score was observed for head touch, startle response (includes MD), equilibrium reflex, positional passivity, visual placing, grip strength, pinching the tail, toe pinch reflex, hind limb reflex, pupil response and limb tone and lower mean score for defecation, righting reflex ground and air righting reflex.
In females, on week 13, there was a slight but statistically significant higher mean score observed for startle response, equilibrium reflex, positional passivity (LD group), visual placing (LD group), grip strength, pinching the tail, toe pinch reflex, hind limb reflex, pupil response, and limb tone and lower score for righting reflex ground and air righting reflex in LD and MD groups. Higher score for HD group was observed for pupil response.
During the recovery period, on week 17, the recovery HD males showed slightly higher score for defecation and in females, slightly higher score for body temperature.
As the effects on various functional observation parameters were slight or inconsistent throughout the dose groups before and at the end of treatment/recovery. These findings are not considered to be a toxicological significance.
Body Weight Development
In males and females of all groups, there was an increase of body weight with the progress of the study. Mean body weight from day 1 to 90 was comparable between the dose groups and control group.
In males, LD, MD and HD groups showed moderate but statistically significant lower body weight gain on week 8 of treatment when compared to control group (53-66 % below control). This was mainly due to higher body weight gain of control animals. In LD and HD groups, a moderate but statistically significant (HD only) lower body weight gain was seen in week 9 (39 % and 49 % below control, respectively) when compared to the controls. In females of LD, MD and HD groups, slight to moderate decrease in mean body weight gain was observed on weeks 8, 10, 11, 12 and 13 and this was mainly due to the higher body weight gain of control animals. These changes are considered to be an apparent statistical significances and not treatment related effects.
All above-mentioned differences in body weight gain did have a statistically significant effect on mean body weight in males of HD group from day 50-57 and 57-64. In females, no statistically or biologically significant differences were noted for mean body weight between the dose groups and the control group. During the recovery period, between day 104 to 118 and 90 to 118 body weight gain was statistically significantly higher in male but not female animals of the HD group, when compared to controls. All the values of control and treated groups were found to be within the normal range of historical values in this strain.
Overall, treatment with test item did not show any adverse effects on body weight development at the end of treatment and recovery period.
Food Consumption
Mean daily food consumption was comparable between all male and female dose groups and controls during the study treatment and recovery period.
In week 1 and 2, there was a slight but statistically significantly lower food consumption in HD males (approx. 6 % below controls) and on week 6, 8 and 9, a slight but statistically significant lower food consumption was observed in HD males (approx. 6-8 % below controls). In week 10, higher food consumption (52-61 % above control) was observed at all dose groups. In females, higher food consumption was observed at all dose groups on week 10 and 11.
However, these differences were considered incidental and there was no treatment related effects on food consumption in any of the dose groups in males and females.
Haematology and Blood Coagulation
No adverse or test item related effects were found for any haematological and coagulation parameters in male and female dose groups at the end of the treatment and recovery period.
A slight increase in mean WBC in MD males and in LD, MD females groups are not considered to be adverse as individual values were within the historical control data range. A tendency towards lower or higher percent differential leucocytes counts in males and females of the dose groups is not considered to be toxicologically relevant. At the end of the recovery period, a slight but statistically significant increase in percent reticulocytes in HD males (112 % above control) was observed. Slight and non-statistically significant decrease or increase in WBC in male and females of the HD group were observed.
A slight but statistically significant increase in aPTT in HD females (34 % above control) was observed when compared to control. This is considered to be an incidental finding. No test item related effects on coagulation parameters were observed in both main and recovery groups.
Treatment with the test item had no dose-dependent effect on haematology parameters of males and females in any of the test item-treated groups. Differences between test item-treated males or females and their respective controls showed no dose-dependency or consistency and thus were not considered toxicologically relevant. All the values were found to be within the normal range of historical values in this strain.
Clinical Biochemistry
There was no test item related effect observed on clinical biochemistry parameters measured at the end of the treatment and recovery period.
A slight but statistically significant decrease in mean sodium level in MD males (3.7 % below control) and statistically significant increase in mean potassium levels in MD and HD males (26.7 % and 32.9 % above control) and females (17.1 % and 37.4 % above control) were observed when compared to control and considered to be of no toxicological relevance. A slight but statistically significant decrease in mean albumin level in MD males (5.4 % below control) was observed when compared to the control. A Higher and statistically significant (HD only) increase in mean TBA level in LD and HD females (44.3 % and 160.5 % above control) were observed when compared to the control.
A slight but statistically insignificant increase in HDL was observed in female of the MD and HD groups (22.7% and 16.7% above control respectively). The total cholesterol levels were slightly increased (26.1 % and 22.5 % above control in MD and HD respectively) to a similar extent. The LDL (calculated) levels were moderately elevated (47 % and 55 % above controls in MD and HD respectively), this increase is not considered to be of biological relevance. In males, there were no changes in mean HDL or LDL levels when compared to the control as well as mean total cholesterol. Slightly higher mean HDL and LDL levels (23.1 % and 59.4 % respectively, above controls) were observed in recovery HD females at the end of recovery.
Treatment with the test item had no effects on parameters of clinical biochemistry of males and females in any of the test item-treated groups which were considered toxicologically relevant. Differences between test item-treated males or females and their respective controls showed no dose-dependency or consistency. All the values were found to be within the normal range of historical values in this strain and without toxicological relevance.
Determination of Thyroxine (T3, T4 and TSH)
No adverse test item related effect was observed on T3, T4 and TSH measurement after the end of treatment and recovery period.
A slight but statistically significant lower TSH level was observed in males of the HD and HD recovery group when compared to controls. In the light of absence of effect on T3, T4, thyroid weight and no histopathological findings, this statistically significant effect on TSH was not considered to be toxicologically relevant or adverse.
Urinalysis
There were no test item related effects observed on various urine parameters in any of the groups at the end of treatment and recovery periods.
Sperm Analysis
There were no statistical significances for mean testicular sperm count and mean sperm motility for all dose groups in the treatment groups. No treatment-related effects on the mean testis weight, mean sperm motility and mean testicular sperm count in the treatment groups were observed. Mean total number of abnormal and normal sperms/findings (sperm morphology) in HD group showed statistical significances at the end of treatment period when compared to control. This statistical significance is considered to be very minimal change; however all the values were found to be within the normal range of historical values in this strain. In addition, there were no treatment-related effects on the testicular histomorphology were observed during histopathological evaluations.
Slight but statistically significant lower mean calculated weight of parenchyma in HD groups was observed, when compared to control at the end of treatment of main groups. No treatment related effects on the mean testis weight and mean testicular sperm counts in the recovery periods were observed.
Pathology
Gross lesions like displaced red jejunum (animal no. 35, HD), dark red colour caecum (animal no. 13, LD), mass at papillary process in liver (animal no. 23, MD), small testes on both sides (animal no. 39, HD), red colour thymus with focus (animal no. 32, 37, HD) and in females, mass at diaphragm (animal no. 74, MD), uterus dilatation [animal no. 55 (control), 61 (LD), 78, 79 (MD), 83, 85 (HD)], black focus in the adrenal glands (animal no. 58, control), red colour thymus with focus [animal no. 58 (control), 64 (LD), 76, 77 (MD) and 87 (HD)], red colour and enlarged (both sides) mandibular lymph node (animal no. 64, LD) and dark red mesenteric lymph node (animal no. 75, MD) were observed randomly in all the groups of males and females at the end of treatment period,
Gross lesions were also observed at the end of recovery period in HD groups, i.e. in animal no. 98 abnormal foamy content in trachea, dark red abnormal colour in lungs, liver herniation in animal no. 100 and in animal no. 43 (control) red focus at right epididymides. Further, in the decedent animal no. 98 the abnormal dark red colour in the lungs observed at necropsy correlated with the multifocal alveolar haemorrhages during the histopathology evaluation. The alveolar haemorrhages were considered most likely to be incidental.
All gross lesions observed in the decedent and survivors animals were deemed to be incidental and considered not to be induced by the treatment with the test item.
Organ Weight
There were no organ or body weight changes that could be related to treatment with the test item.
A slight but statistically significant lower absolute mean heart weight was observed in males of the HD group (9 % below) when compared to controls. A slight but statistically significant higher relative mean liver weight was observed in males of the HD group (10 % above), when compared to the controls.
A moderate but statistically significant higher relative mean thymus weight was observed in males of the HD recovery group (47 % above), when compared to controls. A slight but statistically significant lower absolute mean pituitary gland weight was observed in males of the HD recovery group (13 % below), when compared to controls. A slight but statistically significant lower absolute mean liver weight was observed in males of the HD recovery group (10 % below), when compared to controls. Moderate increase in mean absolute and relative weight of epididymis (right & left) was observed in HD recovery male at the end of recovery when compared to control. A slight increase in absolute and relative mean pituitary gland weight was observed in females of the HD recovery group, when compared to controls. As these were not associated with any histopathological findings, it is not considered to be toxicologically relevant.
Moderate increase in absolute and relative mean uterus with cervix weight was observed in females of the HD recovery group, when compared to the controls. The differences between test item-treated females and control animals in mean weight of uterus and ovaries were not considered toxicologically relevant as female reproductive organs undergo variable changes depending on the stage of the oestrous cycle.
In the absence of corresponding microscopic changes, all observed body and organ weight changes were considered of no toxicological relevance.
Histopathology
There were no histopathological findings that could be attributed to treatment with the test item. All recorded findings were considered incidental or were within the range of background alterations that may be recorded in Wistar rats of this age.
There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. In addition, in the investigated testes, no treatment-related effects on the testicular histomorphology were observed and the histological appearance reflected the animal physiology.
Dose Formulation Analysis
Concentration analysis of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL in study weeks 1, 5, 9 and in the last week of the study. The mean recoveries observed for the LD dose group was between 94.8% and 105.3% of the nominal value, between 95.2% and 106.1% for the MD dose group and between 98.0% and 102.0% of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 100.8%, 99.8%, and 100.3% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15%. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Remarks:
- No test item related effects were observed up to 1000 mg/kg bw/d
- Critical effects observed:
- not specified
- Conclusions:
- On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with Methyl 4-hydroxybenzoate in male and female Wistar rats, with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:
There was no test item-related effect observed on mortality, clinical signs, body weight development, food consumption, functional observation battery, weekly detailed clinical observations, haematology and blood coagulation, clinical biochemistry, urinalysis, sperm analysis, gross pathological findings, organ weight and histopathology in males and females sacrificed at the end of treatment period or recovery period up to the highest dose tested of 1000 mg/kg bw/day.
The no observed adverse effect level (NOEL) of Methyl 4-hydroxybenzoate in this study is determined at 1000 mg/kg body weight/day. - Executive summary:
The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Methyl 4-hydroxybenzoate via oral administration to rats over a period of 90 days.
The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 90 days. Animals of an additional control group were handled identically as the dose groups but received 1 % hydroxyethyl-cellulose (viscosity 80-125 cP, 2 % in water at 20 °C), the vehicle used in this study. The 4 groups comprised of 10 male and 10 femaleWistar rats.
In addition, two groups of recovery animals comprised of 5 male and 5 female Wistar rats, respectively, were treated identically to the control and high dose group of the main study, followed by a 28 day recovery period.
The following doses were evaluated:
Control (C): 0 mg/kg body weight/day
Low Dose (LD): 100 mg/kg body weight/day
Medium Dose (MD): 300 mg/kg body weight/day
High Dose (HD): 1000 mg/kg body weight/day
The test item formulation was prepared at least once in 4 days. The test item was suspended in 1 % hydroxyethyl-cellulose (viscosity 80-125 cP, 2 % in water at 20 °C) and administered daily during a 90 day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements.
During the period of administration, the animals were observed precisely each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. To detect possible delayed occurrence or persistence of or recovery from toxic effects, animals in the recovery group were observed for a period of 28 days following the last administration.
Body weight and food consumption were measured weekly.
In addition to weekly detailed cage side observations, once before the first exposure, once in the last week of exposure as well as in the last week of the recovery period, multiple detailed behavioural observations were made outside the home cage using a battery of functional observational tests.
Haematological, coagulation and clinical biochemical parameters were determined with blood samples obtained from overnight fasted animals at their terminal sacrifice.A urinalysis was performed on samples collected from all animals prior to or as part of their terminal sacrifice.
To evaluate possible toxic effects on male fertility, sperm motility and testicular sperm head count at the end of the treatment period and only testicular sperm head count at the end of the recovery period was performed. Sperm morphology from vas deferens was evaluated at the end of the treatment period from control and high dose males. It was not evaluated from intermediate groups and recovery groups as no test item related effect was observed in HD group males sacrificed at the end of treatment period.
For an evaluation of endocrine effects, thyroid hormones (T3, T4 and TSH) and serum total cholesterol, HDL, LDL levels were determined from the main study and recovery group animals.
At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved.
A full histopathological evaluation of the tissues was performed on high dose and control animals. These examinations were not extended to animals of the other dosage groups as no treatment-related changes were observed in the HD group. Any gross lesion macroscopically identified was examined microscopically in all animals including moribund sacrificed rat.
Summary Results:
One female (animal no. 98) of the HD recovery group was moribund sacrificed due to the clinical signs observation immediately after dosing including apathy, prone position, reduced spontaneous activity and abnormal breathing on treatment day 56. Macroscopically, the abnormal dark red colour in the lungs observed at necropsy correlated with the multifocal alveolar haemorrhages during the histopathology evaluation. The alveolar haemorrhages were considered to be most likely incidental and not related to treatment with test item. All remaining animals survived until the end of the treatment or recovery period.
There were no test item related clinical signs of systemic toxicity observed during the treatment and recovery period in any of the animals. In addition, detailed clinical examinations, functional observation battery (FOB) and ophthalmoscopy examination did not reveal any test item related effects in any of the treatment and recovery groups.
In males and females, there was no test item related effect on body weight during both treatment and recovery period.
There was no effect of toxicological relevance on food consumption in any of the dose groups during both treatment and recovery period.
No toxicologically relevant effects of test item were found on all haematological and coagulation parameters of all male and female animals of the dose groups. A statistical significant slight increase or decrease in WBC, in male and female animals of dose groups is not considered to be toxicologically relevant as individual values were within the historical range. No effect of toxicological relevance on haematological and coagulation parameters was found at the end of the recovery period.
There was no adverse effect onclinical biochemistry parametersmeasured at the end of the treatment and recovery period. Slightly but statistically significant either increase or decrease in sodium, potassium, TBA, HDL level observed in male or female dose groups are considered to be without biological or toxicological relevant as individual values were within the historical range. No adverse test item related effect was observed on T3, T4 and TSH measurement after the end of treatment and recovery period. There was no adverse effect onurinary parametersmeasured at the end of the treatment and recovery period.
No treatment-related effects on the mean testis weight, mean sperm motility and mean testicular sperm count was observed at the end of treatment. No treatment related effects on the mean testis weight and mean testicular sperm count was observed at the end of recovery. The sperm morphology of the HD treatment group was comparable to the control group and no specific findings were observed.
All gross lesions observed in the decedent and survivors animals were deemed to be incidental, and considered not to be induced by the treatment with the test item.
There were no organ or body weight changes that could be related to treatment with the test item.
There were no histopathological findings that could be attributed to treatment with the test item. There was no histological evidence of toxicity in the reproductive organs and tissues examined.In addition,in the investigated testesno treatment-related effects on the testicular histomorphology were observed and the histological appearance reflected the animal physiology.
At dose formulation analysis nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %.
Exposure to Methyl 4-hydroxybenzoate and the metabolite 4-hydroxybenzoic acid assessed on week 12 at 4 time points (10 min, 30 min, 1 and 4 h post dose) after the dose administration was demonstrated at the HD level of 1000 mg/kg/day. The mean values of measured Methyl 4-hydroxybenzoateand4-hydroxybenzoic acid revealed a systemic exposure of the animals to test item.
Conclusion:
On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with Methyl 4-hydroxybenzoate in male and femaleWistarrats, with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:
There was no test item-related effect observed on mortality, clinical signs, body weight development, food consumption, functional observation battery, weekly detailed clinical observations, haematology and blood coagulation, clinical biochemistry, urinalysis, sperm analysis, gross pathological findings, organ weight and histopathology in males and females sacrificed at the end of treatment period or recovery period up to the highest dose tested of 1000 mg/kg bw/day.
The no observed adverse effect level (NOEL) of Methyl 4-hydroxybenzoate in this study is determined at 1000 mg/kg body weight/day.
Referenceopen allclose all
Mortality
Sex:Male- Phase:In-life |
|
|
|
|
|
|
|
|
|
C/M |
LD/M |
MD/M |
HD/M |
|
Animals examined |
N |
15 |
10 |
10 |
15 |
day1->118 |
Alive |
N |
15 |
10 |
10 |
15 |
|
Alive |
% |
100.0 |
100.0 |
100.0 |
100.0 |
Mortality
Sex:Female-Phase:In-life |
|
|
|
|
|
|
|
|
|
C/M |
LD/M |
MD/M |
HD/M |
|
Animals examined |
N |
15 |
10 |
10 |
15 |
day1->118 |
Alive |
N |
15 |
9 |
10 |
15 |
|
Alive |
% |
100.0 |
90.00 |
100.0 |
100.0 |
Clinical Biochemistry
Sex:Male- Phase:In-life
|
C / M |
LD / M |
MD / M |
HD / M |
|
ALAT |
Mean |
36.21 |
36.82 |
34.78 |
36.24 |
[U/L] |
S.d. |
5.935 |
5.677 |
4.268 |
6.667 |
day 91 |
N |
10 |
10 |
10 |
9 |
Gluc |
Mean |
11.243 |
12.370 |
13.107 |
12.109 |
[mmol/L] |
S.d. |
2.6142 |
2.1722 |
3.0591 |
1.8560 |
day 91 |
N |
10 |
10 |
10 |
9 |
Crea |
Mean |
21.3 |
19.5 |
22.4 |
17.7 |
[µmol/L] |
S.d. |
2.71 |
3.84 |
7.65 |
1.66 |
day 91 |
N |
10 |
10 |
10 |
9 |
ASAT |
Mean |
116.15 |
103.21 |
99.23 |
92.31 |
[U/L] |
S.d. |
29.435 |
14.777 |
19.046 |
17.059 |
day 91 |
N |
10 |
10 |
10 |
9 |
AP |
Mean |
122.795 |
125.367 |
113.942 |
118.826 |
[U/L] |
S.d. |
24.8284 |
36.0696 |
35.6057 |
44.7871 |
day 91 |
N |
10 |
10 |
10 |
9 |
Na |
Mean |
142.3 |
141.2 |
140.7 |
142.0 |
[mmol/L] |
S.d. |
2.75 |
3.08 |
2.75 |
1.66 |
day 91 |
N |
10 |
10 |
10 |
9 |
K |
Mean |
4.949 |
4.742 |
4.798 |
5.006 |
[mmol/L] |
S.d. |
0.6791 |
0.7578 |
0.8119 |
0.6875 |
day 91 |
N |
10 |
10 |
10 |
9 |
TP |
Mean |
59.84 |
61.12 |
59.23 |
57.98 |
[g/L] |
S.d. |
2.991 |
3.556 |
3.165 |
1.731 |
day 91 |
N |
10 |
10 |
10 |
9 |
ALB |
Mean |
32.702 |
33.676 |
32.144 |
32.378 |
[g/L] |
S.d. |
1.8167 |
1.8430 |
1.4392 |
0.9289 |
day 91 |
N |
10 |
10 |
10 |
9 |
|
|
C / M |
LD / M |
MD / M |
HD / M |
TBIL |
Mean |
2.01 |
2.08 |
1.91 |
2.07 |
[µmol/L] |
S.d. |
0.285 |
0.466 |
0.335 |
0.250 |
day 91 |
N |
10 |
10 |
10 |
9 |
CHOL |
Mean |
2.014 |
1.957 |
2.020 |
1.661 |
[mmol/L] |
S.d. |
0.2878 |
0.1357 |
0.1890 |
0.2318 |
day 91 |
N |
10 |
10 |
10 |
9 |
TBA |
Mean |
17.878 |
22.622 |
22.256 |
18.074 |
[µmol/L] |
S.d. |
5.9091 |
10.0550 |
7.2659 |
6.8755 |
day 91 |
N |
9 |
10 |
10 |
9 |
TG |
Mean |
0.97 |
1.09 |
1.26 |
1.46 |
[mmol/L] |
S.d. |
0.352 |
0.405 |
0.371 |
0.431 |
day 91 |
N |
8 |
10 |
9 |
9 |
Urea |
Mean |
7.261 |
7.216 |
7.556 |
6.232 |
[mmol/L] |
S.d. |
1.1827 |
0.7894 |
1.0914 |
0.7559 |
day 91 |
N |
10 |
10 |
10 |
9 |
HDL |
Mean |
1.284 |
1.296 |
1.311 |
1.076 |
[mmol/L] |
S.d. |
0.1361 |
0.1059 |
0.1073 |
0.1680 |
day 91 |
N |
10 |
10 |
10 |
9 |
LDL (calculated) |
Mean |
0.528 |
0.443 |
0.458 |
0.294 |
[mmol/L] |
S.d. |
0.1402 |
0.0696 |
0.1156 |
0.1328 |
day 91 |
N |
8 |
10 |
9 |
9 |
ALAT |
Mean |
34.62 |
|
|
26.95 |
[U/L] |
S.d. |
5.798 |
|
|
4.268 |
day 119 |
N |
5 |
0 |
0 |
4 |
Gluc |
Mean |
15.076 |
|
|
12.480 |
[mmol/L] |
S.d. |
2.7719 |
|
|
2.1391 |
day 119 |
N |
5 |
0 |
0 |
4 |
|
C / M |
LD / M |
MD / M |
HD / M |
|
Crea |
Mean |
28.4 |
|
|
27.0 |
[µmol/L] |
S.d. |
9.07 |
|
|
3.46 |
day 119 |
N |
5 |
0 |
0 |
4 |
ASAT |
Mean |
94.34 |
|
|
96.32 |
[U/L] |
S.d. |
20.240 |
|
|
16.872 |
day 119 |
N |
5 |
0 |
0 |
4 |
AP |
Mean |
93.424 |
|
|
81.502 |
[U/L] |
S.d. |
23.9905 |
|
|
34.2293 |
day 119 |
N |
5 |
0 |
0 |
4 |
Na |
Mean |
140.6 |
|
|
143.5 |
[mmol/L] |
S.d. |
1.14 |
|
|
1.29 |
day 119 |
N |
5 |
0 |
0 |
4 |
K |
Mean |
4.590 |
|
|
4.110 |
[mmol/L] |
S.d. |
0.4584 |
|
|
0.4334 |
day 119 |
N |
5 |
0 |
0 |
4 |
TP |
Mean |
53.36 |
|
|
55.52 |
[g/L] |
S.d. |
3.304 |
|
|
1.936 |
day 119 |
N |
5 |
0 |
0 |
4 |
ALB |
Mean |
31.116 |
|
|
32.168 |
[g/L] |
S.d. |
2.0129 |
|
|
1.1706 |
day 119 |
N |
5 |
0 |
0 |
4 |
TBIL |
Mean |
1.84 |
|
|
1.82 |
[µmol/L] |
S.d. |
0.251 |
|
|
0.206 |
day 119 |
N |
5 |
0 |
0 |
4 |
CHOL |
Mean |
1.666 |
|
|
1.852 |
[mmol/L] |
S.d. |
0.1941 |
|
|
0.1179 |
day 119 |
N |
5 |
0 |
0 |
4 |
|
|
C / M |
LD / M |
MD / M |
HD / M |
TBA |
Mean |
21.266 |
|
|
19.305 |
[µmol/L] |
S.d. |
7.6762 |
|
|
7.8503 |
day 119 |
N |
5 |
0 |
0 |
4 |
TG |
Mean |
0.90 |
|
|
1.00 |
[mmol/L] |
S.d. |
0.254 |
|
|
0.160 |
day 119 |
N |
5 |
0 |
0 |
4 |
Urea |
Mean |
8.392 |
|
|
6.300 |
[mmol/L] |
S.d. |
1.7579 |
|
|
1.0450 |
day 119 |
N |
5 |
0 |
0 |
4 |
HDL |
Mean |
1.110 |
|
|
1.218 |
[mmol/L] |
S.d. |
0.0970 |
|
|
0.0981 |
day 119 |
N |
5 |
0 |
0 |
4 |
LDL (calculated) |
Mean |
0.376 |
|
|
0.436 |
[mmol/L] |
S.d. |
0.1349 |
|
|
0.0322 |
day 119 |
N |
5 |
0 |
0 |
4 |
Sex:Female- Phase:In-life
|
C / F |
LD / F |
MD / F |
HD / F |
|
ALAT |
Mean |
32.96 |
31.92 |
32.43 |
30.57 |
[U/L] |
S.d. |
6.598 |
5.738 |
4.585 |
9.530 |
day 91 |
N |
10 |
9 |
10 |
10 |
Gluc |
Mean |
10.500 |
9.811 |
10.836 |
9.920 |
[mmol/L] |
S.d. |
1.9542 |
1.6568 |
1.7542 |
2.2668 |
day 91 |
N |
10 |
9 |
10 |
10 |
Crea |
Mean |
26.0 |
22.3 |
22.9 |
21.3 |
[mmol/L] |
S.d. |
3.59 |
2.83 |
3.75 |
3.09 |
day 91 |
N |
10 |
9 |
10 |
10 |
ASAT |
Mean |
80.37 |
80.93 |
75.79 |
73.90 |
[U/L] |
S.d. |
5.358 |
13.419 |
10.277 |
14.983 |
day 91 |
N |
10 |
9 |
10 |
10 |
AP |
Mean |
70.535 |
79.556 |
69.461 |
60.147 |
[U/L] |
S.d. |
37.3140 |
37.5844 |
20.6289 |
16.6869 |
day 91 |
N |
10 |
9 |
10 |
10 |
Na |
Mean |
143.7 |
143.3 |
142.7 |
143.3 |
[mmol/L] |
S.d. |
1.77 |
2.50 |
1.77 |
2.54 |
day 91 |
N |
10 |
9 |
10 |
10 |
K |
Mean |
3.664 |
3.690 |
3.873 |
3.627 |
[mmol/L] |
S.d. |
0.2393 |
0.2533 |
0.3836 |
0.1601 |
day 91 |
N |
10 |
9 |
10 |
10 |
TP |
Mean |
59.77 |
61.06 |
60.39 |
61.06 |
[g/L] |
S.d. |
3.540 |
2.905 |
3.213 |
3.737 |
day 91 |
N |
10 |
9 |
10 |
10 |
ALB |
Mean |
33.824 |
34.467 |
34.195 |
34.611 |
[g/L] |
S.d. |
2.1958 |
1.4768 |
1.8785 |
1.9025 |
day 91 |
N |
10 |
9 |
10 |
10 |
|
|
C / F |
LD / F |
MD / F |
HD / F |
TBIL |
Mean |
2.30 |
2.36 |
2.70 |
2.77 |
[µmol/L] |
S.d. |
0.598 |
0.340 |
0.478 |
0.521 |
day 91 |
N |
10 |
9 |
10 |
10 |
CHOL |
Mean |
1.362 |
1.344 |
1.311 |
1.276 |
[mmol/L] |
S.d. |
0.3601 |
0.2532 |
0.3015 |
0.3420 |
day 91 |
N |
10 |
9 |
10 |
10 |
TBA |
Mean |
14.322 |
13.810 |
14.748 |
15.200 |
[µmol/L] |
S.d. |
7.7652 |
4.3278 |
6.1424 |
13.3457 |
day 91 |
N |
10 |
9 |
10 |
10 |
TG |
Mean |
0.82 |
0.91 |
0.91 |
0.94 |
[mmol/L] |
S.d. |
0.329 |
0.322 |
0.349 |
0.380 |
day 91 |
N |
10 |
8 |
10 |
10 |
Urea |
Mean |
6.909 |
6.532 |
6.004 |
6.652 |
[mmol/L] |
S.d. |
0.7241 |
0.6999 |
0.5171 |
1.0934 |
day 91 |
N |
10 |
9 |
10 |
10 |
HDL |
Mean |
0.948 |
0.929 |
0.885 |
0.828 |
[mmol/L] |
S.d. |
0.2581 |
0.2037 |
0.2218 |
0.3040 |
day 91 |
N |
10 |
9 |
10 |
10 |
LDL (calculated) |
Mean |
0.250 |
0.233 |
0.244 |
0.261 |
[mmol/L] |
S.d. |
0.1115 |
0.1055 |
0.1220 |
0.0964 |
day 91 |
N |
10 |
8 |
10 |
10 |
ALAT |
Mean |
30.98 |
|
|
22.54 |
[U/L] |
S.d. |
8.708 |
|
|
3.868 |
day 119 |
N |
5 |
0 |
0 |
5 |
Gluc |
Mean |
9.988 |
|
|
8.180 |
[mmol/L] |
S.d. |
1.7758 |
|
|
0.9301 |
day 119 |
N |
5 |
0 |
0 |
5 |
|
C / F |
LD / F |
MD / F |
HD / F |
|
Crea |
Mean |
36.6 |
|
|
25.6 |
[mmol/L] |
S.d. |
23.41 |
|
|
1.14 |
day 119 |
N |
5 |
0 |
0 |
5 |
ASAT |
Mean |
81.82 |
|
|
68.90 |
[U/L] |
S.d. |
19.355 |
|
|
15.175 |
day 119 |
N |
5 |
0 |
0 |
5 |
AP |
Mean |
51.558 |
|
|
35.658 |
[U/L] |
S.d. |
10.2563 |
|
|
13.7959 |
day 119 |
N |
5 |
0 |
0 |
5 |
Na |
Mean |
139.8 |
|
|
141.8 |
[mmol/L] |
S.d. |
3.49 |
|
|
1.48 |
day 119 |
N |
5 |
0 |
0 |
5 |
K |
Mean |
3.822 |
|
|
3.756 |
[mmol/L] |
S.d. |
0.4126 |
|
|
0.2085 |
day 119 |
N |
5 |
0 |
0 |
5 |
TP |
Mean |
59.14 |
|
|
61.20 |
[g/L] |
S.d. |
2.827 |
|
|
3.798 |
day 119 |
N |
5 |
0 |
0 |
5 |
ALB |
Mean |
34.914 |
|
|
36.208 |
[g/L] |
S.d. |
1.6809 |
|
|
2.6896 |
day 119 |
N |
5 |
0 |
0 |
5 |
TBIL |
Mean |
2.84 |
|
|
2.64 |
[µmol/L] |
S.d. |
0.699 |
|
|
0.706 |
day 119 |
N |
5 |
0 |
0 |
5 |
CHOL |
Mean |
1.118 |
|
|
1.302 |
[mmol/L] |
S.d. |
0.2750 |
|
|
0.0887 |
day 119 |
N |
5 |
0 |
0 |
5 |
|
|
C / F |
LD / F |
MD / F |
HD / F |
TBA |
Mean |
22.076 |
|
|
17.520 |
[µmol/L] |
S.d. |
9.8022 |
|
|
7.3873 |
day 119 |
N |
5 |
0 |
0 |
5 |
TG |
Mean |
0.55 |
|
|
0.60 |
[mmol/L] |
S.d. |
0.182 |
|
|
0.142 |
day 119 |
N |
5 |
0 |
0 |
5 |
Urea |
Mean |
7.480 |
|
|
5.984 |
[mmol/L] |
S.d. |
2.6890 |
|
|
0.7884 |
day 119 |
N |
5 |
0 |
0 |
5 |
HDL |
Mean |
0.756 |
|
|
0.892 |
[mmol/L] |
S.d. |
0.2341 |
|
|
0.0826 |
day 119 |
N |
5 |
0 |
0 |
5 |
LDL (calculated) |
Mean |
0.252 |
|
|
0.290 |
[mmol/L] |
S.d. |
0.0611 |
|
|
0.0229 |
day 119 |
N |
5 |
0 |
0 |
5 |
Summary Body weight (g)- male - Phase: in -life
|
C/M |
LD/M |
MD/M |
HD/M |
|
day1 |
Mean |
220.47a |
219.10 |
224.70 |
222.93 |
|
S.d. |
10.38 |
12.27 |
9.67 |
13.40 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
-0.62 |
1.92 |
1.12 |
day8 |
Mean |
255.60k |
231.80 |
257.90 |
255.87 |
|
S.d. |
13.41 |
24.53 |
10.80 |
15.70 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
-9.31 |
0.90 |
0.10 |
day15 |
Mean |
284.73a |
271.20 |
286.70 |
285.13 |
|
S.d. |
18.05 |
16.81 |
12.63 |
16.58 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
-4.75 |
0.69 |
0.14 |
day22 |
Mean |
307.13a |
303.60 |
314.50 |
309.40 |
|
S.d. |
21.46 |
17.03 |
14.44 |
17.88 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
-1.15 |
2.40 |
0.74 |
day29 |
Mean |
321.93a |
321.80 |
328.20 |
325.07 |
|
S.d. |
24.02 |
17.83 |
17.69 |
19.59 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
-0.04 |
1.95 |
0.97 |
day36 |
Mean |
335.13a |
337.00 |
345.30 |
341.33 |
|
S.d. |
25.39 |
19.74 |
18.52 |
18.80 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
0.56 |
3.03 |
1.85 |
day43 |
Mean |
346.47a |
347.70 |
357.30 |
352.93 |
|
S.d. |
27.14 |
21.03 |
19.26 |
21.40 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
0.36 |
3.13 |
1.87 |
day50 |
Mean |
356.00a |
359.90 |
364.10 |
362.93 |
|
S.d. |
27.38 |
23.96 |
19.81 |
24.52 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
1.10 |
2.28 |
1.95 |
day57 |
Mean |
364.33a |
367.00 |
375.00 |
369.40 |
|
S.d. |
29.41 |
23.63 |
20.78 |
25.56 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
0.73 |
2.93 |
1.39 |
day64 |
Mean |
371.27a |
377.00 |
383.30 |
377.07 |
|
S.d. |
31.47 |
24.24 |
24.98 |
26.65 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
1.54 |
3.24 |
1.56 |
day71 |
Mean |
376.67a |
382.40 |
391.00 |
384.87 |
|
S.d. |
30.98 |
24.13 |
28.49 |
26.99 |
|
N |
15 |
10 |
10 |
15 |
|
DeviationVsControl[%] |
|
1.52 |
3.81 |
2.18 |
|
C/M |
LD/M |
MD/M |
HD/M |
|
day78 |
Mean |
387.33a |
393.30 |
399.10 |
390.67 |
|
S.d. |
31.68 |
24.18 |
27.79 |
27.86 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
1.54 |
3.04 |
0.86 |
day85 |
Mean |
390.33a |
397.30 |
403.80 |
394.93 |
|
S.d. |
32.16 |
24.53 |
29.17 |
31.04 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
1.78 |
3.45 |
1.18 |
day90 |
Mean |
397.47a |
410.80 |
410.00 |
400.27 |
|
S.d. |
35.08 |
25.53 |
30.76 |
33.19 |
|
N |
15 |
10 |
10 |
15 |
|
DeviationVsControl[%] |
|
3.35 |
3.15 |
0.70 |
|
C/F |
LD/F |
MD/F |
HD/F |
|
day1 |
Mean |
161.80k |
163.90 |
166.20 |
162.38 |
|
S.d. |
7.04 |
8.84 |
11.04 |
6.15 |
|
N |
15 |
10 |
10 |
16 |
|
DeviationVsControl[%] |
|
1.30 |
2.72 |
0.36 |
day8 |
Mean |
176.93a |
176.60 |
176.90 |
177.07 |
|
S.d. |
8.58 |
9.97 |
11.35 |
9.66 |
|
N |
15 |
10 |
10 |
15 |
|
DeviationVsControl[%] |
|
-0.19 |
-0.02 |
0.08 |
day15 |
Mean |
189.00a |
189.20 |
191.10 |
189.40 |
|
S.d. |
10.60 |
11.04 |
13.46 |
10.20 |
N 15 10 10 15 |
|||||
|
DeviationVsControl[%] |
|
0.11 |
1.11 |
0.21 |
day22 |
Mean |
197.60a |
199.20 |
200.10 |
196.67 |
|
S.d. |
9.49 |
8.82 |
13.83 |
12.83 |
|
N |
15 |
10 |
10 |
15 |
|
DeviationVsControl[%] |
|
0.81 |
1.27 |
-0.47 |
day29 |
Mean |
206.53a |
209.80 |
209.20 |
209.00 |
|
S.d. |
11.09 |
9.67 |
15.17 |
9.96 |
|
N |
15 |
10 |
10 |
15 |
|
DeviationVsControl[%] |
|
1.58 |
1.29 |
1.19 |
day36 Mean 213.93a 217.60 214.60 214.67 |
|||||
|
S.d. |
9.48 |
11.17 |
13.47 |
11.78 |
|
N |
15 |
10 |
10 |
15 |
|
DeviationVsControl[%] |
|
1.71 |
0.31 |
0.34 |
day43 |
Mean |
215.27k |
220.22 |
219.80 |
217.13 |
|
S.d. |
10.44 |
13.24 |
16.22 |
11.97 |
N 15 9 10 15 |
|||||
|
DeviationVsControl[%] |
|
2.30 |
2.11 |
0.87 |
day50 |
Mean |
218.67k |
222.44 |
220.90 |
220.40 |
|
S.d. |
11.90 |
9.76 |
14.12 |
10.36 |
N 15 9 10 15 |
|||||
|
DeviationVsControl[%] |
|
1.73 |
1.02 |
0.79 |
day57 |
Mean |
221.47k |
225.78 |
225.10 |
224.00 |
|
S.d. |
10.97 |
10.95 |
14.72 |
9.11 |
N 15 9 10 15 |
|||||
|
DeviationVsControl[%] |
|
1.95 |
1.64 |
1.14 |
day64 |
Mean |
224.80a |
231.78 |
228.50 |
228.53 |
|
S.d. |
11.83 |
13.92 |
16.08 |
14.15 |
N 15 9 10 15 |
|||||
|
DeviationVsControl[%] |
|
3.10 |
1.65 |
1.66 |
day71 |
Mean |
229.80k |
233.11 |
233.80 |
232.00 |
|
S.d. |
13.11 |
10.73 |
14.11 |
13.93 |
|
N |
15 |
9 |
10 |
15 |
|
DeviationVsControl[%] |
|
1.44 |
1.74 |
0.96 |
|
C/F |
LD/F |
MD/F |
HD/F |
|
day78 |
Mean |
230.27a |
235.00 |
235.60 |
234.40 |
|
S.d. |
13.77 |
10.61 |
16.75 |
13.39 |
N 15 9 10 15 |
|||||
|
DeviationVsControl[%] |
|
2.06 |
2.32 |
1.80 |
day85 |
Mean |
230.73a |
238.00 |
238.70 |
238.00 |
|
S.d. |
12.87 |
12.50 |
17.27 |
12.42 |
N 15 9 10 15 |
|||||
|
DeviationVsControl[%] |
|
3.15 |
3.45 |
3.15 |
day90 |
Mean |
232.87a |
238.44 |
237.30 |
236.93 |
|
S.d. |
12.78 |
12.10 |
15.85 |
15.61 |
|
N |
15 |
9 |
10 |
15 |
|
DeviationVsControl[%] |
|
2.40 |
1.90 |
1.75 |
Summary of Hematology
Male- Phase: in-life
|
C/M |
LD/M |
MD/M |
HD/M |
|
WBC |
Mean |
4.1450ad |
4.9411 |
5.7090* |
6.3489** |
[1E9/L] |
S.d. |
1.0948 |
0.9088 |
1.0546 |
2.0651 |
day91 |
N |
10 |
9 |
10 |
9 |
|
DeviationVsControl[%] |
|
19.2065 |
37.7322 |
53.1698 |
RBC Mean 9.6040k 9.6878 9.9930 9.9444 |
|||||
[1E12/L] |
S.d. |
0.4133 |
0.2589 |
0.3748 |
0.3792 |
day91 |
N |
10 |
9 |
10 |
9 |
|
DeviationVsControl[%] |
|
0.8723 |
4.0504 |
3.5448 |
HGB Mean 16.350k 16.556 16.800 16.989 |
|||||
[g/dL] |
S.d. |
0.688 |
0.579 |
0.855 |
0.280 |
day91 |
N |
10 |
9 |
10 |
9 |
|
DeviationVsControl[%] |
|
1.257 |
2.752 |
3.908 |
HCT Mean 52.610k 52.711 53.620 54.100 |
|||||
[%] |
S.d. |
2.416 |
1.437 |
2.711 |
1.672 |
day91 |
N |
10 |
9 |
10 |
9 |
|
DeviationVsControl[%] |
|
0.192 |
1.920 |
2.832 |
MCV Mean 54.770a 54.422 53.650 54.433 |
|||||
[fL] |
S.d. |
0.910 |
1.861 |
1.377 |
1.713 |
day91 |
N |
10 |
9 |
10 |
9 |
|
DeviationVsControl[%] |
|
-0.635 |
-2.045 |
-0.615 |
MCH Mean 17.000a 17.100 16.820 17.100 |
|||||
[pg] |
S.d. |
0.170 |
0.696 |
0.421 |
0.574 |
day91 |
N |
10 |
9 |
10 |
9 |
|
DeviationVsControl[%] |
|
0.588 |
-1.059 |
0.588 |
MCHC Mean 31.110a 31.422 31.340 31.411 |
|||||
[g/dL] |
S.d. |
0.615 |
0.800 |
0.723 |
0.717 |
day91 |
N |
10 |
9 |
10 |
9 |
|
DeviationVsControl[%] |
|
1.004 |
0.739 |
0.968 |
RET% Mean 1.6520k 1.6744 1.7510 1.6256 |
|||||
[%] |
S.d. |
0.4626 |
0.2351 |
0.2942 |
0.2815 |
day91 |
N |
10 |
9 |
10 |
9 |
|
DeviationVsControl[%] |
|
1.3586 |
5.9927 |
-1.6008 |
|
C/M |
LD/M |
MD/M |
HD/M |
|
PLT |
Mean |
635.80a |
622.78 |
659.70 |
624.22 |
[1E9/L] |
S.d. |
69.24 |
126.33 |
81.55 |
68.87 |
day91 N 10 9 10 9 |
|||||
|
DeviationVsControl[%] |
|
-2.05 |
3.76 |
-1.82 |
EOS% |
Mean |
0.6300k |
0.3333 |
0.3600 |
0.2778 |
[%] |
S.d. |
0.6273 |
0.2449 |
0.2989 |
0.1856 |
day91 N 10 9 10 9 |
|||||
|
DeviationVsControl[%] |
|
-47.0899 |
-42.8571 |
-55.9083 |
LYM% |
Mean |
77.870a |
76.667 |
79.230 |
78.322 |
[%] |
S.d. |
5.447 |
8.829 |
3.746 |
4.614 |
day91 N 10 9 10 9 |
|||||
|
DeviationVsControl[%] |
|
-1.545 |
1.747 |
0.581 |
NEUT% |
Mean |
18.990a |
20.478 |
16.950 |
19.078 |
[%] |
S.d. |
5.007 |
8.037 |
2.668 |
4.135 |
day91 |
N |
10 |
9 |
8 |
9 |
|
DeviationVsControl[%] |
|
7.835 |
-10.742 |
0.462 |
MONO% |
Mean |
2.220a |
2.189 |
2.012 |
1.944 |
[%] |
S.d. |
0.603 |
0.779 |
0.617 |
0.498 |
day91 |
N |
10 |
9 |
8 |
9 |
|
DeviationVsControl[%] |
|
-1.401 |
-9.347 |
-12.412 |
BASO% |
Mean |
0.1100k |
0.1000 |
0.1000 |
0.1222 |
[%] |
S.d. |
0.0876 |
0.0866 |
0.0667 |
0.0667 |
day91 |
N |
10 |
9 |
10 |
9 |
|
DeviationVsControl[%] |
|
-9.0909 |
-9.0909 |
11.1111 |
LUC% Mean 0.1700k 0.2111 0.2250 0.2333 |
|||||
[%] |
S.d. |
0.1059 |
0.1269 |
0.1035 |
0.0866 |
day91 |
N |
10 |
9 |
8 |
9 |
|
DeviationVsControl[%] |
|
24.1830 |
32.3529 |
37.2549 |
Sex: female
Phase: Life-in
|
C/F |
LD/F |
MD/F |
HD/F |
|
WBC |
Mean |
2.7910ad |
2.8133 |
3.8510* |
3.7720* |
[1E9/L] |
S.d. |
0.3375 |
0.7408 |
0.9480 |
0.9162 |
day91 |
N |
10 |
9 |
10 |
10 |
|
DeviationVsControl[%] |
|
0.8002 |
37.9792 |
35.1487 |
RBC Mean 8.3350a 8.5889 8.2960 8.6410 |
|||||
[1E12/L] |
S.d. |
0.3947 |
0.4125 |
0.4556 |
0.4314 |
day91 |
N |
10 |
9 |
10 |
10 |
|
DeviationVsControl[%] |
|
3.0461 |
-0.4679 |
3.6713 |
HGB Mean 14.840ad 15.411 15.060 15.530* |
|||||
[g/dL] |
S.d. |
0.544 |
0.542 |
0.597 |
0.587 |
day91 |
N |
10 |
9 |
10 |
10 |
|
DeviationVsControl[%] |
|
3.848 |
1.482 |
4.650 |
HCT Mean 47.620a 48.867 47.560 48.490 |
|||||
[%] |
S.d. |
1.997 |
2.037 |
2.166 |
2.155 |
day91 |
N |
10 |
9 |
10 |
10 |
|
DeviationVsControl[%] |
|
2.618 |
-0.126 |
1.827 |
MCV Mean 57.160k 56.933 57.370 56.130 |
|||||
[fL] |
S.d. |
1.512 |
1.546 |
1.569 |
1.431 |
day91 |
N |
10 |
9 |
10 |
10 |
|
DeviationVsControl[%] |
|
-0.397 |
0.367 |
-1.802 |
MCH Mean 17.830a 17.956 18.170 18.000 |
|||||
[pg] |
S.d. |
0.558 |
0.546 |
0.611 |
0.618 |
day91 |
N |
10 |
9 |
10 |
10 |
|
DeviationVsControl[%] |
|
0.704 |
1.907 |
0.953 |
MCHC Mean 31.170ad 31.556 31.670 32.070** |
|||||
[g/dL] |
S.d. |
0.435 |
0.553 |
0.501 |
0.653 |
day91 |
N |
10 |
9 |
10 |
10 |
|
DeviationVsControl[%] |
|
1.237 |
1.604 |
2.887 |
RET% Mean 1.9380a 1.8556 1.8470 1.8300 |
|||||
[%] |
S.d. |
0.3466 |
0.2912 |
0.4640 |
0.2517 |
day91 |
N |
10 |
9 |
10 |
10 |
|
DeviationVsControl[%] |
|
-4.2541 |
-4.6956 |
-5.5728 |
Sex:Fe |
male-Phase:In-life |
|
||||
|
|
C/F |
LD/F |
MD/F |
HD/F |
|
PLT[1E9/L] |
Mean S.d. |
644.80k 60.97 |
548.33 185.07 |
639.00 100.47 |
625.40 118.12 |
|
day91 |
N |
10 |
9 |
10 |
10 |
|
|
DeviationVsControl[%] |
|
-14.96 |
-0.90 |
-3.01 |
|
EOS%[%] |
Mean S.d. |
0.2200k 0.1476 |
0.2667 0.2062 |
0.3200 0.2486 |
0.4600 0.5929 |
|
day91 |
N |
10 |
9 |
10 |
10 |
|
|
DeviationVsControl[%] |
|
21.2121 |
45.4545 |
109.0909 |
|
LYM%[%] |
Mean S.d. |
83.180k 4.415 |
81.478 6.798 |
81.990 6.503 |
81.990 7.776 |
|
day91 |
N |
10 |
9 |
10 |
10 |
|
|
DeviationVsControl[%] |
|
-2.046 |
-1.431 |
-1.431 |
|
NEUT[%] |
% Mean S.d. |
14.630a 3.984 |
15.889 6.402 |
15.520 6.026 |
15.030 7.338 |
|
day91 |
N |
10 |
9 |
10 |
10 |
|
|
DeviationVsControl[%] |
|
8.605 |
6.083 |
2.734 |
|
MONO% |
Mean |
1.760a |
2.011 |
1.770 |
2.200 |
|
[%] |
S.d. |
0.508 |
0.499 |
0.365 |
0.455 |
|
day91 |
N |
10 |
9 |
10 |
10 |
|
|
DeviationVsControl[%] |
|
14.268 |
0.568 |
25.000 |
|
BASO% |
Mean |
0.0700k |
0.1000 |
0.0500 |
0.0600 |
|
[%] |
S.d. |
0.0675 |
0.1118 |
0.0850 |
0.0516 |
|
day91 |
N |
10 |
9 |
10 |
10 |
|
|
DeviationVsControl[%] |
|
42.8571 |
-28.5714 |
-14.2857 |
|
LUC% Mean 0.1500a 0.2111 0.3000 0.2800 |
||||||
[%] |
|
S.d. |
0.1080 |
0.1167 |
0.1414 |
0.1687 |
day91 |
|
N |
10 |
9 |
10 |
10 |
|
|
DeviationVsControl[%] |
|
40.7407 |
100.0000 |
86.6667 |
Histopathological data summary attached as pdf
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- The available information comprises adequate, reliable (Klimisch score 1) and consistent studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on the identified similarities in structure and intrinsic properties between source and target substance (see attached read-aross justification). The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
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
For the endpoint repeated dose toxicity no OECD conform experimental studies on ethyl 4-hydroxybenzoate are conducted because of the close structural and functional similarities and metabolism to methyl 4-hydroxybenzoate and propyl 4-hydroxybenzoate. Sub-chronic (90-day) oral application of methyl 4-hydroxybenzoate or propyl 4-hydroxybenzoate to rats according to OECD TG 408 did not induce any signs of systemic toxicity. With regard to parameters indicating potential for endocrine disruption, no effects on reproductive organs (organ weight and histopathological changes), sperm parameters, oestrous cyclicity and serum thyroid hormone levels were observed in either the male or female rats. For both
substances, the no-observed adverse effect level (NOAEL) was set at 1000 mg/kg bw/day, i.e. the highest dose tested as per OECD TG 408 (BSL Bioservices 2018, BSL Bioservices 2019).
Justification for classification or non-classification
The available data on the repeated dose toxicity of the test substance and on surrogate substances does not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and is therefore conclusive but not sufficient for classification.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
