Methyl 4-hydroxybenzoate

Endpoint summary

Description of key information

NOAEL (28 days, OECD 422, gavage, rat): 1000 mg/kg bw/d

NOEL (90 days, OECD 408, gavage rat): 1000 mg/kg bw/d

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-03 to 2019-12-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 29 July 2016

Deviations:

yes

Remarks:

see Overall Remarks

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han) (Full Barrier)

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. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 326 – 398 g (mean: 360.0 g, ± 20 % = 288.0 – 432.0 g)
females: 207 – 258 g (mean: 227.2 g, ± 20 % = 181.8 – 272.6 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)
- Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females (both parental and F1)
and during post-mating period for males (parental and F1) depending on the mating status. During mating period males and females (parental and F1) were housed together in ratio 1:1 (male to female).
After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males (parental and F1) were returned to their original cage.
In each cage Altromin saw fibre was used as bedding.
- Makrolon tunnels were provided for all males and for females until GD 18
- Nesting material were provided latest on GD 18 for all mated females
- 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) under laboratory conditions

Number and Sex of the Animals
80 animals (40 males and 40 females) were included in the study.

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 dosing all females were screened for two weeks for regular oestrous cyclicity and animals (10 females/ group) with regular oestrous cycle (4-5 day cycle)
were used in the study. Before the first administration all animals to be 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).
Each animal was marked with its identification number by individual ear tattoo or tail marking.

Route of administration:

oral: gavage

Vehicle:

other: 1 % hydroxyethyl-cellulose / aqua ad injectionem

Remarks:

hydroxyethyl-cellulose: Manufacturer: Sigma-Aldrich, Batch No.: MKCD0421, Expiry Date: 05 November 2019 aqua ad injectionem: Manufacturer: Deltamedica, Batch No.: 806148, Expiry Date: May 2021 (each bottle was used for up to one week)

Details on oral exposure:

The vehicle has been selected in consultation with the sponsor based on the test item’s characteristics.
Based on the results of stability testing (Eurofins Munich Study No. 187385), the test item formulations were prepared at least every 4 days as given by Eurofins Munich Study No. 187385.
The prepared formulation was stored at room temperature.
The test item, as delivered, was grinded before formulation preparation. Afterwards, the 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).
Formulates were kept under magnetic stirring during the daily administration. The vehicle was also used as control item.

In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating
and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females.
Males were dosed after the mating period until the minimum total dosing period of 28 days is completed.

Control: 0 mg/kg/d
Low Dose: 100 mg/kg/d
Medium Dose: 300 mg/kg/d
High Dose: 1000 mg/kg/d


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.

The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
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. 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 was not shown to be homogenous according to Eurofins Study No. 187385. Therefore, 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 (pre-mating period),
3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) (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. 187386) 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:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males
and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days is completed.

Frequency of treatment:

daily

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.

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. 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.
Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study.
During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with pups.
All animals were weighed directly before termination.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration.
Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Haematology
Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment
prior to or as part of the sacrifice of the animals. 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 from 5 randomly selected males and females (only lactating females were evaluated) of each group
were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
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 from 5 randomly selected males and females (only lactating females were evaluated) of each group were
examined at the end of the treatment prior to or as part of the sacrifice of the animals.
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 bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K)
From 2 female pups per litter on day 4 after birth, from all dams and 2 pups per litter at termination on day 13 and from all adult males at termination, blood samples were collected from the defined site in serum separator tubes. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).
Further assessment of T4 in blood samples from the dams and day 4 pups or other hormones was not deemed necessary as no test item related changes were observed in T4 levels
of adult males and pups on PND 13. Pup blood was pooled by litter for thyroid hormone analysis. Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible
serum hormone assessments. The two pups per litter were female pups to reserve male pups for nipple retention evaluations. No pups were eliminated as litter size was below 8 pups (dam no. 60 of the LD group). As there was only one pup available above a litter size of 8, only one pup was sacrificed from dam numbers 43 and 47 from the control group and dam no. 78 from the HD group.
No pups were available from females number 65 and 67 from the MD group as these females were non-pregnant.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values
of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters
of haematology, blood coagulation and clinical biochemistry were statistically analysed 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. 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):

Moving the bedding was observed in 1/10 females of the control group, 2/10 females of the LD group, 1/10 females of the MD group and 10/10 females and 9/10 males of the HD group mostly in the second half of the treatment period. Increased salivation was noted transiently in 1/10 females of the MD group and in 5/10 males and females of the HD group. Both signs were observed in short timely relation to dose administration or in anticipation thereof and thus were considered to be a sign of discomfort or a local reaction to the test item. These slight signs were not considered as adverse systemic effects.
The clinical sign of piloerection was observed in 4/10 females of the control group, 4/10 females of the LD group, 8/10 females of the MD group, and 7/10 females of the HD group. Due to the absence of dose dependency and the occurrence of piloerection in control animals it was not considered toxicologically relevant. Low incidences of clinical signs without dose dependency (see Details on results P0 ) and thus are not considered test item-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality was observed in p generation. No test item-related effect on mean mortality of pups was observed between PND 0 and PND 4 and between PND 4 and 13 in treatment groups when compared to the control group. Mortality of two pups in the control group was considered incidental.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no statistically significant effect on body weight or body weight change in male animals and body weights of female animals.
Mean body weight gain was slightly but statistically significantly lower in the female LD and female HD group compared to the controls during the first week of the gestation phase. However, without consistency between the groups, without dose dependency and occurrence during only one week of treatment this was not considered adverse. MD females showed no statistically significant differences in body weight change compared to control females. Further slight differences between the groups were within the normal range of variation throughout the treatment period of this study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no toxicologically relevant effect on food consumption in male and female animals of this study.
Slightly but statistically significantly lower food consumption of females of the LD group compared to control females during the second week of gestation was not considered test item related as no differences in food consumption were observed between higher dose groups and the control group. Slightly lower food intake was seen in female animals of the HD group during the premating period. Without achieving statistical significance this is not considered to be an adverse effect.

Food efficiency:

not examined

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

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no statistically significant or biologically relevant effect of the test item on haematological parameters of male animals determined at the end of the treatment period of this study. Differences in haematological parameters between test item-treated groups and the corresponding controls followed no dose dependency and within historical control data and thus were not considered test item-related.
There were no statistically significant effects of the test item on coagulation parameters (PT, aPTT) of female animals and on the parameter aPTT of male animals at the end of the treatment period. PT was statistically significantly but slightly higher in males of the HD group compared to control males (13 % above control). However, without the incidence of other clinical findings or macroscopic findings at the time point of necropsy slightly higher PT value of the HD group was not assumed adverse.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In male animals, mean TBA showed a dose-dependent tendency towards lower TBA levels in test item-treated animals compared to the controls. However, without achieving statistical significance and without a corresponding effect in female animals this difference was not considered adverse. Other parameters of clinical biochemistry showed no statistically significant or biologically relevant differences between males treated with the test item and males of the control group. In female animals there were no statistically significant or biologically relevant differences in any of the parameters of clinical biochemistry when comparing test item treated animals with animals of the control group. The deviation of mean TBA level of HD females from control females (261% deviation from control) was not statistically significant and was mainly based on high TBA levels from two females of the HD group (female numbers 71 and 80) and was not considered adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Slight differences in the mean organ weights between test item-treated groups and the control group are not considered to be caused by treatment with Methyl 4 hydroxybenzoate as no test item related histopathological findings were observed in any of the organs.
Mean relative pituitary gland weight was slightly higher in the male HD group when compared to the control group (deviation from control: 14 %). However, due to the lack of dose dependency this was not considered to be adverse and toxicologically relevant.
Absolute (deviation from control: 21 %) and relative (deviation from control: 18 %) mean adrenal gland weight was statistically significantly lower in females of the MD group. However, this followed no dose dependency and is thereby not considered toxicologically relevant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Only single or occasional macroscopic findings were noted in the groups during necropsy of the animals. These are assumed to be incidental finings without relation to the test item. Findings were enlarged kidneys (bilateral) of LD male no. 13, small testes (bilateral) of MD male no. 27 and small seminal vesicles (left side) of control male no. 7. Without corresponding evidence in histopathological examination these macroscopic findings were not considered of toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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. No treatment-related effects on the testicular histomorphology and interstitial cell structure were noticed. The treatment with Methyl 4 hydroxybenzoate did not induce histomorphological effects in the reproductive organs of the non-pregnant females (animal nos. 65 and 67) and their pairing partners (animal nos. 25 and 27). Therefore, the histopathological NOEL (no observed effect level) may be established at 1000 mg/kg bw/day.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Treatment with the test item had no biologically significant effect on the oestrous cycle analysed during the 2 weeks premating period when comparing test item treated groups to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.
Sperm staging - the testes were checked on completeness of cell populations, completeness of
stages and degenerative changes. No treatment-related effects on the testicular histomorphology
were observed. Further, no treatment-related effect on interstitial cell structure was noticed.
No differences of biological relevance were observed in anogenital distance of male and female pups on PND 0 and in nipple retention of male pups on PND 12 when comparing test item treated pups to pups of the control group.
However, mean male pup nipple retention was shown to be statistically significantly lower in the HD group compared to the control group.
As this value was within the normal range of variation (range of historical control data (±2 fold SD): -1.14-1.50), lower nipple retention in the HD group was not considered test item-related.
Female pups treated with the test item were observed with statistically significantly higher mean pup weight (control: 5.76, MD: 6.04) and mean cube root of pup weight (control: 1.79, MD: 1.82) in the MD group and statistically significantly lower absolute (control: 1.31, LD: 1.07) and relative (control: 0.73, LD: 0.60) anogenital distance in the LD group. Without occurrence of these differences in all groups and dose-dependency this was not considered adverse.

Details on results:

Clinical Observations
Moving the bedding was observed in 1/10 females of the control group, 2/10 females of the LD group, 1/10 females of the MD group and 10/10 females and 9/10 males of the HD group mostly
in the second half of the treatment period. Increased salivation was noted transiently in 1/10 females of the MD group and in 5/10 males and females of the HD group. Both signs were observed
in short timely relation to dose administration or in anticipation thereof and thus were considered to be a sign of discomfort or a local reaction to the test item.
These slight signs were not considered as adverse systemic effects.
The clinical sign of piloerection was observed in 4/10 females of the control group, 4/10 females of the LD group, 8/10 females of the MD group, and 7/10 females of the HD group.
Due to the absence of dose dependency and the occurrence of piloerection in control animals it was not considered toxicologically relevant.
Low incidences of clinical signs like a crust (1/10 males of the HD group and 1/10 females of the MD group), hairless areas (2/10 females of the control group, 3/10 females of the LD group,
6/10 females of the MD group and 1/10 females of the HD group), a scratch/cut (1/10 males of the HD group and 1/10 females of the MD group), an oedema (1/10 males of the control group
and 1/10 females of the MD group), a kinked tail (1/10 females of the LD group), an injured palate (1/10 females of the LD group), hypotonia (1/10 females of the control group),
slow movements (1/10 females of the control group), diarrhoea (1/10 males of the LD group) and aggressiveness (2/10 males of the HD group) were observed in single animals or
without dose dependency and thus are not considered test item-related.

Mortality
No mortality occurred during the treatment period with Methyl 4-hydroxybenzoate in any of the test item-treated groups and the control group. All animals survived the scheduled study period.

Body Weight Development
The test item had no statistically significant effect on body weight or body weight change in male animals and body weights of female animals.
Mean body weight gain was slightly but statistically significantly lower in the female LD and female HD group compared to the controls during the first week of the gestation phase.
However, without consistency between the groups, without dose dependency and occurrence during only one week of treatment this was not considered adverse.
MD females showed no statistically significant differences in body weight change compared to control females.
Further slight differences between the groups were within the normal range of variation throughout the treatment period of this study.

Food Consumption
The test item had no toxicologically relevant effect on food consumption in male and female animals of this study.
Slightly but statistically significantly lower food consumption of females of the LD group compared to control females during the second week of gestation was not considered test item
related as no differences in food consumption were observed between higher dose groups and the control group.
Slightly lower food intake was seen in female animals of the HD group during the premating period. Without achieving statistical significance this is not considered to be an adverse effect.


Haematology and Coagulation
There was no statistically significant or biologically relevant effect of the test item on haematological parameters of male animals determined at the end of the treatment period of this study.
Differences in haematological parameters between test item-treated groups and the corresponding controls followed no dose dependency and thus were not considered test item-related.
There were no statistically significant effects of the test item on coagulation parameters (PT, aPTT) of female animals and on the parameter aPTT of male animals at the end of the treatment period.
PT was statistically significantly but slightly higher in males of the HD group compared to control males (13 % above control). However, without the incidence of other clinical findings
or macroscopic findings at the time point of necropsy slightly higher PT value of the HD group was not assumed adverse.


Clinical Biochemistry
In male animals, mean TBA showed a dose-dependent tendency towards lower TBA levels in test item-treated animals compared to the controls.
However, without achieving statistical significance and without a corresponding effect in female animals this difference was not considered adverse.
Other parameters of clinical biochemistry showed no statistically significant or biologically relevant differences between males treated with the test item and males of the control group.
In female animals there were no statistically significant or biologically relevant differences in any of the parameters of clinical biochemistry when comparing test item treated animals with animals of the control group.
The deviation of mean TBA level of HD females from control females (261% deviation from control) was not statistically significant and was mainly based on high TBA levels from two females of the HD group
(female numbers 71 and 80) and was not considered adverse.

Organ Weights
Slight differences in the mean organ weights between test item-treated groups and the control group are not considered to be caused by treatment with
Methyl 4 hydroxybenzoate as no test item related histopathological findings were observed in any of the organs.
Mean relative pituitary gland weight was slightly higher in the male HD group when compared to the control group (deviation from control: 14 %).
However, due to the lack of dose dependency this was not considered to be adverse and toxicologically relevant.
Absolute (deviation from control: 21 %) and relative (deviation from control: 18 %) mean adrenal gland weight was statistically significantly lower in females of the MD group.
However, this followed no dose dependency and is thereby not considered toxicologically relevant.

Pathology
Only single or occasional macroscopic findings were noted in the groups during necropsy of the animals. These are assumed to be incidental finings without relation to the test item.
Findings were enlarged kidneys (bilateral) of LD male no. 13, small testes (bilateral) of MD male no. 27 and small seminal vesicles (left side) of control male no. 7.
Without corresponding evidence in histopathological examination these macroscopic findings were not considered of toxicological relevance.

Histopathology
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. No treatment-related effects on the testicular histomorphology and interstitial cell structure were noticed. The treatment with Methyl 4 hydroxybenzoate
did not induce histomorphological effects in the reproductive organs of the non-pregnant females (animal nos. 65 and 67) and their pairing partners (animal nos. 25 and 27).
Therefore, the histopathological NOEL (no observed effect level) may be established at 1000 mg/kg bw/day.


Thyroid Hormone (T4) Analysis
Slightly but statistically lower mean thyroxine hormone (T4) level was not considered adverse without corresponding histopathological findings in the thyroid/parathyroid of male animals.

Oestrous Cycles
Treatment with the test item had no biologically significant effect on the oestrous cycle analysed during the 2 weeks premating period when comparing test item treated groups to the controls.
There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.

Precoital Interval and Duration of Gestation
The pre-coital interval and the duration of gestation were not affected by Methyl 4 hydroxybenzoate.
No statistically significant differences were observed when comparing the test item-treated groups with the control group.

Pre- and Postnatal Data
No considerable test item related effects were noted for mean values of number of corpora lutea, implantations sites, live pups on PND 0, 4 and 13 as well as
pre implantation loss and post implantation loss in all dose groups. Slight differences were within the normal range of variation and were not considered toxicologically relevant.


Reproductive Indices
There were no test item-related effects on the reproductive indices including copulation index, fertility index, delivery index, and viability index.
Slight differences in the reproductive indices followed no dose-dependency and were within the range of biological variation
(range of historical control data (±2 fold SD): copulation index: 88.277-108-086, fertility index: 66.806-118.800, delivery index: 90.130-107.029, viability index: 88.977-110.130).


Dose Formulation Analysis
In this study phase 40 samples in total were measured for determination of Methyl 4-hydroxybenzoate in formulation samples received from Eurofins Munich / BSL Munich Study No. 187381 (main study).
Concentration analysis and homogeneity of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL in study weeks 1, 3, 5 and in the last week of the study. The mean recoveries observed for the LD dose group was between 91.6% and 102.6% of the nominal value, between 92.2% and 98.9% for the MD dose group and between 98.6% and 105.3% 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 97.9%, 96.9%, and 102.2% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15%.
The coefficients of variation of the different sampling locations (top, middle, bottom) was between 3.0% and 4.3% in LD dose group, between 1.8% and 5.7% in MD dose group and between 1.0% and 3.7% in HD dose group. All samples were homogenous, as COV was below or equal 15%.

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: ...

Remarks on result:

other:

Remarks:

No mortality and no clinical signs of systemic toxicity were observed in the animals of this study. Daily oral treatment with Methyl 4-hydroxybenzoate had no effect on body weight and food consumption. At the end of the respective treatment periods of male and female animals there were no changes in clinical pathology results up to 1000 mg/kg bw/day and organ weights were inconspicuous. No relevant macroscopic and no histopathological abnormalities were observed in treated animals. Thus, the NOAEL for general toxicity is determined to be 1000 mg/kg bw/day.

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

other:

Conclusions:

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test 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:
No mortality and no clinical signs of systemic toxicity were observed in the animals of this study. Daily oral treatment with Methyl 4-hydroxybenzoate had no effect on body weight and food consumption. At the end of the respective treatment periods of male and female animals there were no changes in clinical pathology results up to 1000 mg/kg bw/day and organ weights were inconspicuous. No relevant macroscopic and no histopathological abnormalities were observed in treated animals. Thus, the NOAEL for general toxicity is determined to be 1000 mg/kg bw/day.
No test item-related effects on the reproduction and developmental parameters analysed in this study, i.e. oestrous cycle, copulation, fertility and delivery indices, number of corpora lutea, implantation sites and live pups, pre- and post-implantation loss, number of male and female pups, sex ratio, still births, runts, litter weight data, anogenital distance, nipple retention and external abnormalities. Thus, the NOAEL for reproduction/developmental toxicity is determined to be 1000 mg/kg bw/day.

Executive summary:

The aim of this study was to assess the possible effects of Methyl 4-hydroxybenzoateonmale and female fertility and embryo-foetal development after repeated dose administration in Wistar rats.

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 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. 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 10 male and 10 female Wistar rats. Before dosing all females were screened for two weeks for regular oestrous cyclicity and animals (10 females/group) with regular oestrous cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

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

The test item formulation was prepared at least every 4 days. The test item was suspended in 1 % hydroxyethyl-cellulose and administered daily during14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 12 in females. Males were dosed for28 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministrationvolumewas 5 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice fromfive randomly selected males and females from each group.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum period of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natalday 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues was performed on five selected high dose and control animals, in non-pregnant female animals and male mating partners of the LD and MD animals. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in the high dose group. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation ofadditional haematoxylin-PAS (Periodic Acid Schiff)stained slides.All gross lesions macroscopically identified were examined microscopically in all animals.

Summary Results

No test item related mortality or clinical signs of systemic toxicity were observed during daily observations. The clinical signs of moving the bedding and increased salivation observed were observed in short timely relation to dose administration andwere considered to be a sign of discomfort or a local reaction to the test item. Other clinical signs like hairless areas, crusts, scratches/cuts, oedema, kinked tail, hypotonia, slow movements, diarrhoea and aggressiveness were observed without dose dependency in single animals and were not considered adverse.

The test item had no statistically significant or toxicologically relevant effect on body weight or body weight gain and on food consumption in both sexes. However, mean body weight gain was observed to be slightly but statistically significantly lower in the female LD and female HD group compared to the controls during the first week of the gestation phase. However, without consistency between the groups, without dose dependency and occurrence during only one week of treatment this was not considered adverse. Slightly but statistically significantly lower food consumption of females of the LD group during the second week of gestation was not considered test item related as no differences in food consumption were observed between higher dose groups and the control group.

There were no considerable differences in the length or sequence of oestrous cycle stages between the dose groups and the control group. No toxicologically relevant changes in pre-coital interval and duration of gestation were observed in the dose groups when compared to the controls.

No toxicologically relevant effects were noted for corpora lutea, implantations sites, live pups, pre implantation loss and post implantation loss in all dose groups. All values were within the normal range of variation.

There were no test item-related effects on total number of male and female pups, sex ratio and number of still births and runts. Values of these litter parameters were comparable between dose groups and control group.

Litter weight data showed no test item related changes. Slight differences between the groups were within the normal range of variation.

Pre implantation loss and post implantation loss were within the normal range of variation and not relevantly different between dose groups and control group. There was no test item-related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13.

The test item had no relevant effect onreproductive indices (copulation, fertility, viability, and delivery index).

Statistically significantly lower mean male pup nipple retention was observed in the HD group. However, as this value was within the normal range of variation it was not considered test item-related. Female pups in the MD group were observed with statistically significantly higher mean pup weight and mean cube root of pup weight and statistically significantly lower anogenital distance in the LD group. Without occurrence of these differences in all groups and dose-dependency this was not considered adverse.

No test item-related effect was observed on pup thyroid weight and thyroxine hormone (T4) level in males and PND 13 pups of the dose groups when compared to the controls.

Two pups were found dead in the control group and no mortality occurred in any of the test item-treated groups. Mortality in the control group was considered to be within the normal range of background findings.A single finding of an external abnormality (dark tail) in one pup of the control group was considered incidental.

There were no statistically significant or toxicologically relevant effects of the test item on haematological, clinical biochemistry and coagulation parameters determined at the end of the treatment period of this study.

No test item related macroscopic findings were noted in the groups during necropsy of the animals. Single observed macroscopic findings and slight differences in organ weights between test item treated groups and the controls were considered incidental without the evidence of corresponding histopathological findings.

No test item related changes were observed during the histopathological evaluation. All findings recorded were deemed to be incidental or were within the range of background alterations that may be recorded in Wistar rats.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. No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on interstitial cell structure was noticed.

Conclusion

On the basis ofthiscombined repeated dose oral toxicity and reproduction/ developmental toxicity screening test withMethyl 4-hydroxybenzoatein male and femaleWistarrats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

No mortality and no clinical signs of systemic toxicity were observed in the animals of this study. Daily oral treatment withMethyl 4-hydroxybenzoate had no effect on body weight and food consumption. At the end of the respective treatment periods of male and female animals there were no changes in clinical pathology results up to 1000 mg/kg bw/day and organ weights were inconspicuous. No relevant macroscopic and no histopathological abnormalities were observed in treated animals. Thus, the NOAEL for general toxicity is determined to be 1000 mg/kg bw/day.

No test item-related effects on the reproduction and developmental parameters analysed in this study, i.e. oestrous cycle, copulation, fertility and delivery indices, number of corpora lutea, implantation sites and live pups, pre- and post-implantation loss, number of male and female pups, sex ratio, still births, runts, litter weight data, anogenital distance, nipple retention and external abnormalities. Thus, the NOAEL forreproduction/developmental toxicity isdetermined to be1000 mg/kg bw/day.

Reference 2

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

Summary Body Weight - Treatment Phase

Sex: Male - Phase: In-life

		C / M	LD / M	MD / M	HD / M
day 1	Mean	245.67 a	244.20	245.10	242.40
	S.d.	12.46	10.01	8.89	8.24
	N	15	10	10	15
	Deviation Vs Control [%]		-0.60	-0.23	-1.33
day 8	Mean	275.73 a	273.10	273.00	267.93
	S.d.	14.41	9.43	7.62	10.91
	N	15	10	10	15
	Deviation Vs Control [%]		-0.96	-0.99	-2.83
day 15	Mean	299.67 a	294.70	298.90	289.27
	S.d.	18.32	13.24	8.80	15.12
	N	15	10	10	15
	Deviation Vs Control [%]		-1.66	-0.26	-3.47
day 22	Mean	319.67 a	313.80	317.80	306.40
	S.d.	19.82	15.06	7.89	15.27
	N	15	10	10	15
	Deviation Vs Control [%]		-1.84	-0.58	-4.15
day 29	Mean	334.87 a	326.40	334.70	318.73
	S.d.	23.97	16.22	8.92	18.55
	N	15	10	10	15
	Deviation Vs Control [%]		-2.53	-0.05	-4.82
day 36	Mean	348.07 a	338.60	347.90	329.67
	S.d.	25.07	18.60	8.12	19.97
	N	15	10	10	15
	Deviation Vs Control [%]		-2.72	-0.05	-5.29
day 43	Mean	356.53 ad	347.00	357.20	334.47 *
	S.d.	25.45	18.74	9.10	20.40
	N	15	10	10	15
	Deviation Vs Control [%]		-2.67	0.19	-6.19

		C / M	LD / M	MD / M	HD / M
day 50	Mean	366.87 a	361.90	371.30	347.87
	S.d.	28.37	23.16	9.27	23.53
	N	15	10	10	15
	Deviation Vs Control [%]		-1.35	1.21	-5.18
day 57	Mean	377.40 ad	365.50	375.20	352.87 *
	S.d.	31.29	23.78	11.39	22.87
	N	15	10	10	15
	Deviation Vs Control [%]		-3.15	-0.58	-6.50
day 64	Mean	386.07 ad	370.80	384.80	357.40 **
	S.d.	30.88	22.93	12.36	23.48
	N	15	10	10	15
	Deviation Vs Control [%]		-3.95	-0.33	-7.43
day 71	Mean	390.33 ad	376.80	389.30	360.33 **
	S.d.	32.02	24.42	14.79	23.20
	N	15	10	10	15
	Deviation Vs Control [%]		-3.47	-0.26	-7.69
day 78	Mean	396.53 ad	383.80	396.90	365.80 **
	S.d.	34.50	23.20	15.63	24.86
	N	15	10	10	15
	Deviation Vs Control [%]		-3.21	0.09	-7.75
day 85	Mean	401.80 u	389.70	406.60	369.87 *
	S.d.	34.94	24.50	16.65	26.40
	N	15	10	10	15
	Deviation Vs Control [%]		-3.01	1.19	-7.95
day 90	Mean	406.53 u	393.10	410.40	376.47 *
	S.d.	35.44	22.39	16.95	26.33
	N	15	10	10	15
	Deviation Vs Control [%]		-3.30	0.95	-7.40

Conslusion of CRO:

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 (Historical control Data are summerized in full study report page 869- Annex III).
Overall, treatment with test item did not show any adverse effects on body weight
development at the end of treatment and recovery period.

Sex: Female - Phase: In-life

		C / F	LD / F	MD / F	HD / F
day 1	Mean	171.67 a	171.40	172.60	169.13
	S.d.	9.17	5.95	8.49	7.14
	N	15	10	10	15
	Deviation Vs Control [%]		-0.16	0.54	-1.48
day 8	Mean	184.00 k	181.70	184.00	179.40
	S.d.	8.90	8.78	9.32	8.73
	N	15	10	10	15
	Deviation Vs Control [%]		-1.25	0.00	-2.50
day 15	Mean	192.13 a	191.90	192.80	187.87
	S.d.	11.66	7.78	6.07	9.05
	N	15	10	10	15
	Deviation Vs Control [%]		-0.12	0.35	-2.22
day 22	Mean	200.27 k	201.80	201.10	196.93
	S.d.	11.20	5.53	5.49	10.51
	N	15	10	10	15
	Deviation Vs Control [%]		0.77	0.42	-1.66
day 29	Mean	205.93 a	206.00	205.50	203.53
	S.d.	12.57	7.04	7.26	11.13
	N	15	10	10	15
	Deviation Vs Control [%]		0.03	-0.21	-1.17
day 36	Mean	211.67 a	212.50	209.60	209.53
	S.d.	12.91	8.53	8.11	10.91
	N	15	10	10	15
	Deviation Vs Control [%]		0.39	-0.98	-1.01
day 43	Mean	216.13 a	216.60	216.00	215.33
	S.d.	12.78	7.89	8.04	13.41
	N	15	10	10	15
	Deviation Vs Control [%]		0.22	-0.06	-0.37

	C / F	LD / F	MD / F	HD / F
day 50       Mean	218.00 a	221.00	219.50	219.20
S.d.	12.59	7.56	7.04	12.34
N	15	10	10	15
Deviation Vs Control [%]		1.38	0.69	0.55
day 57       Mean	222.33 a	223.30	219.60	222.14
S.d.	13.75	8.34	5.38	12.27
N	15	10	10	14
Deviation Vs Control [%]		0.43	-1.23	-0.09
day 64       Mean	223.93 a	227.50	222.30	224.21
S.d.	14.82	10.87	9.02	12.47
N	15	10	10	14
Deviation Vs Control [%]		1.59	-0.73	0.13
day 71       Mean	232.33 a	229.90	227.50	226.43
S.d.	13.63	13.05	6.36	13.00
N	15	10	10	14
Deviation Vs Control [%]		-1.05	-2.08	-2.54
day 78       Mean	231.67 a	231.70	228.70	228.14
S.d.	14.50	11.34	7.09	14.41
N	15	10	10	14
Deviation Vs Control [%]		0.01	-1.28	-1.52
day 85       Mean	234.40 a	234.20	230.50	230.36
S.d.	14.74	11.52	7.41	13.42
N	15	10	10	14
Deviation Vs Control [%]		-0.09	-1.66	-1.72
day 90       Mean	232.87 a	234.00	230.40	231.71
S.d.	14.68	13.46	7.23	14.09
N	15	10	10	14
Deviation Vs Control [%]		0.49	-1.06	-0.49
a=ANOVA

Summary Body Weight (g) - Recovery Phase		187383 - 1
Ascentos™ 1.3		26-Nov-2019 12:16
Sex: Male - Phase: In-life
	C / M	HD / M
day 90                          Mean	410.6 n	369.0
S.d.	41.37	15.48
N	5	5
Deviation Vs Control [%]		-10.1
day 97                          Mean	422.8 n	388.8
S.d.	41.54	13.74
N	5	5
Deviation Vs Control [%]		-8.0
day 104                        Mean	426.8 n	381.0
S.d.	42.48	16.14
N	5	5
Deviation Vs Control [%]		-10.7
day 118                        Mean	384.0 n	366.6
S.d.	39.96	15.01
N	5	5
Deviation Vs Control [%]		-4.5
n=DUNNETT

Summary Body Weight (g) - Recovery Phase		187383 - 2
Ascentos™ 1.3		26-Nov-2019 12:16
Sex: Female - Phase: In-life
	C / F	HD / F
day 90                          Mean	224.0 n	234.2
S.d.	11.55	19.52
N	5	4
Deviation Vs Control [%]		4.6
day 97                          Mean	231.0 n	234.2
S.d.	15.70	19.35
N	5	4
Deviation Vs Control [%]		1.4
day 104                        Mean	230.8 n	232.0
S.d.	11.01	18.24
N	5	4
Deviation Vs Control [%]		0.5
day 118                        Mean	236.4 s	237.8
S.d.	11.84	19.67
N	5	4
Deviation Vs Control [%]		0.6

 

Summary Body Weight Change (g) - Treatment Phase

Sex: Male - Phase: In-life
		C / M	LD / M	MD / M	HD / M
d 1 -> 8	Mean	30.1 k	28.9	27.9	25.5
	S.d.	6.86	6.47	5.86	7.51
	N	15	10	10	15
d 8 -> 15	Mean	23.9 k	21.6	25.9	21.3
	S.d.	6.50	4.74	3.18	5.72
	N	15	10	10	15
d 15 -> 22	Mean	20.0 k	19.1	18.9	17.1
	S.d.	3.98	4.77	5.51	4.64
	N	15	10	10	15
d 22 -> 29	Mean	15.2 a	12.6	16.9	12.3
	S.d.	6.12	3.60	6.38	4.59
	N	15	10	10	15
d 29 -> 36	Mean	13.2 a	12.2	13.2	10.9
	S.d.	4.16	5.65	4.10	3.65
	N	15	10	10	15
d 36 -> 43	Mean	8.5 a	8.4	9.3	4.8
	S.d.	3.91	2.27	6.18	6.65
	N	15	10	10	15
d 43 -> 50	Mean	10.3 k	14.9	14.1	13.4
	S.d.	4.56	6.74	6.26	7.67
	N	15	10	10	15
d 50 -> 57	Mean	10.5 ad                          3.6 **                              3.9 **                             5.0 *
	S.d.	4.78                             3.47                                4.43                               6.29
	N	15	10	10	15
d 57 -> 64	Mean	8.7 u	5.3	9.6	4.5 *
	S.d.	4.64	3.83	3.53	5.87
	N	15	10	10	15
d 64 -> 71	Mean	4.3 k	6.0	4.5	2.9
	S.d.	4.03	4.08	4.81	4.23
	N	15	10	10	15
d 71 -> 78	Mean	6.2 k	7.0	7.6	5.5
	S.d.	4.89	3.50	5.93	5.74
	N	15	10	10	15
d 78 -> 85	Mean	5.3 k	5.9	9.7	4.1
	S.d.	2.46	5.69	20.01	2.94
	N	15	10	10	15
d 85 -> 90	Mean	4.7 a	3.4	3.8	6.6
	S.d.	5.86	4.03	3.12	2.23
	N	15	10	10	15
d 1 -> 90	Mean	160.9 ad	148.9	165.3	134.1 **
	S.d.	28.68	19.52	18.92	23.26
	N	15	10	10	15

d = day; k=KRUSKALL-WALLIS; a=ANOVA; ad=ANOVA-DUNNETT; ** = p < 0.01; * = p < 0.05; u=KRUSKALL-WALLIS-DUNN

Summary Body Weight Change (g) - Treatment Phase

Sex: Female - Phase: In-life

C / F                          LD / F				MD / F	HD / F
d 1 -> 8	Mean	12.3 k	10.3	11.4	10.3
	S.d.	3.11	4.85	6.06	5.70
	N	15	10	10	15
d 8 -> 15	Mean	8.1 k	10.2	8.8	8.5
	S.d.	4.19	4.59	4.85	4.96
	N	15	10	10	15
d 15 -> 22	Mean	8.1 a	9.9	8.3	9.1
	S.d.	3.98	4.20	3.74	6.02
	N	15	10	10	15
d 22 -> 29	Mean	5.7 k	4.2	4.4	6.6
	S.d.	4.76	3.68	3.37	4.95
	N	15	10	10	15
d 29 -> 36	Mean	5.7 k	6.5	4.1	6.0
	S.d.	4.48	4.65	6.28	4.28
	N	15	10	10	15
d 36 -> 43	Mean	4.5 a	4.1	6.4	5.8
	S.d.	5.07	3.60	3.92	7.80
	N	15	10	10	15
d 43 -> 50	Mean	1.9 a	4.4	3.5	3.9
	S.d.	4.07	4.72	6.84	6.75
	N	15	10	10	15
d 50 -> 57	Mean	4.3 a	2.3	0.1	1.6
	S.d.	3.66	2.79	3.38	4.77
	N	15	10	10	14
d 57 -> 64	Mean	1.6 a	4.2	2.7	2.1
	S.d.	6.16	3.12	4.79	5.14
	N	15	10	10	14
d 64 -> 71	Mean	8.4 k	2.4	5.2	2.2
	S.d.	8.73	3.60	5.59	4.15
	N	15	10	10	14
d 71 -> 78	Mean	-0.7 a	1.8	1.2	1.7
	S.d.	4.32	4.32	3.79	5.37
	N	15	10	10	14
d 78 -> 85	Mean	2.7 k	2.5	1.8	2.2
	S.d.	8.34	3.63	3.58	3.04
	N	15	10	10	14
d 85 -> 90	Mean	-1.5 k	-0.2	-0.1	1.4
	S.d.	8.43	3.61	3.28	4.20
	N	15	10	10	14
d 1 -> 90	Mean	61.2 a	62.6	57.8	61.6
	S.d.	8.68	13.44	6.37	11.02
	N	15	10	10	14

 

Summary Body Weight Change (g) - Recovery Phase		187383 - 1
Ascentos™ 1.3		26-Nov-2019 12:20
Sex: Male - Phase: In-life
	C / M	HD / M
d 90 -> 97                                                                   Mean	12.2 s	19.8
S.d.	2.59	21.64
N	5	5
d 97 -> 104                                                                 Mean	4.0 n	-7.8
S.d.	4.80	20.08
N	5	5
d 104 -> 118                                                               Mean	-42.8 n	-14.4 ***
S.d.	3.27	8.02
N	5	5
d 90 -> 118                                                                 Mean	-26.6 n	-2.4 ***
S.d.	3.91	3.65
N	5	5
d = day; s=DUNN; n=DUNNETT; *** = p < 0.001

Summary Body Weight Change (g) - Recovery	Phase		187383 - 2
Ascentos™ 1.3			26-Nov-2019 12:20
Sex: Female - Phase: In-life
		C / F	HD / F
d 90 -> 97	Mean	7.0 s	0.0
	S.d.	5.96	2.94
	N	5	4
d 97 -> 104	Mean	-0.2 n	-2.2
	S.d.	4.87	2.99
	N	5	4
d 104 -> 118	Mean	5.6 n	5.8
	S.d.	4.28	11.87
	N	5	4
d 90 -> 118	Mean	12.4 n	3.5
	S.d.	1.34	12.77
	N	5	4
d = day; s=DUNN; n=DUNNETT

 

Summary Food Consumption (g/day/animal) - Treatment Phase   Ascentos™ 1.3					187383- 1   09-Jul-2019 09:24
Sex: Male - Phase: In-life
C / M			LD / M	MD / M	HD / M
d 1 -> 8	Mean [g]	23.09 n	22.90 X	22.73 X		21.70 **
	S.d.	0.15	0.63	0.14		0.26
	N	3	2	2		3
	Deviation Vs Control [%]	0.0	-0.8	-1.5		-6.0
d 8 -> 15	Mean [g]	23.92 s	23.04 X	23.74 X		22.50 *
	S.d.	0.07	0.71	0.16		0.68
	N	3	2	2		3
	Deviation Vs Control [%]	0.0	-3.7	-0.8		-5.9
d 15 -> 22	Mean [g]	23.90 n	22.64 X	23.41 X		22.78
	S.d.	0.36	0.22	0.51		0.72
	N	3	2	2		3
	Deviation Vs Control [%]	0.0	-5.3	-2.1		-4.7
d 22 -> 29	Mean [g]	23.99 s	23.11 X	23.93 X		23.24
	S.d.	0.31	0.12	0.14		0.49
	N	3	2	2		3
	Deviation Vs Control [%]	0.0	-3.7	-0.3		-3.1
d 29 -> 36	Mean [g]	22.89 n	22.20 X	22.80 X		22.09
	S.d.	0.37	0.93	0.24		0.73
	N	3	2	2		3
	Deviation Vs Control [%]	0.0	-3.0	-0.4		-3.5
d 36 -> 43	Mean [g]	22.48 n	21.36 X	22.40 X		21.23 *
	S.d.	0.47	1.23	0.20		0.11
	N	3	2	2		3
	Deviation Vs Control [%]	0.0	-5.0	-0.3		-5.6
d 43 -> 50	Mean [g]	21.64 n	21.01 X	21.94 X		21.03
	S.d.	2.12	0.51	0.28		0.25
	N	3	2	2		3
	Deviation Vs Control [%]	0.0	-2.9	1.4		-2.8

		C / M	LD / M	MD / M	HD / M
d 50 -> 57	Mean [g]	22.61 n	21.33 X	21.33 X	21.02 *
	S.d.	0.72	1.56	0.71	0.21
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	-5.7	-5.7	-7.0
d 57 -> 64	Mean [g]	22.16 n	20.73 X	21.37 X	20.30 *
	S.d.	0.90	1.52	0.28	0.27
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	-6.5	-3.6	-8.4
d 64 -> 71	Mean [g]	13.84 n	22.26 X	22.09 X	21.09
	S.d.	9.41	1.54	0.24	0.20
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	60.8	59.6	52.4
d 71 -> 78	Mean [g]	20.76 n	21.11 X	21.51 X	19.03
	S.d.	2.95	1.37	0.44	0.98
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	1.7	3.6	-8.3
d 78 -> 85	Mean [g]	20.27 n	20.87 X	20.93 X	19.87
	S.d.	1.07	0.99	0.63	0.59
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	3.0	3.3	-2.0
d 85 -> 90	Mean [g]	27.28 n	21.50 X	21.22 X	20.44
	S.d.	8.04	0.99	0.37	1.02
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	-21.2	-22.2	-25.1

Summary Food Consumption (g/day/animal) - Treatment Phase

Sex: Female -	Phase: In-life
		C / F	LD / F	MD / F	HD / F
d 1 -> 8	Mean [g]	15.08 s	15.34 X	15.51 X	14.50
	S.d.	0.31	0.32	0.24	1.51
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	1.8	2.9	-3.9
d 8 -> 15	Mean [g]	15.74 s	15.94 X	15.70 X	15.10
	S.d.	0.34	0.24	0.10	0.73
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	1.3	-0.3	-4.1
d 15 -> 22	Mean [g]	15.55 n	16.36 X	16.19 X	14.67
	S.d.	0.66	0.71	0.55	1.06
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	5.2	4.1	-5.7
d 22 -> 29	Mean [g]	15.63 n	15.79 X	15.60 X	15.38
	S.d.	0.62	0.02	0.24	1.03
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	1.0	-0.2	-1.6
d 29 -> 36	Mean [g]	15.28 n	15.99 X	15.79 X	15.53
	S.d.	0.81	0.51	0.79	0.98
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	4.6	3.3	1.7
d 36 -> 43	Mean [g]	14.91 n	15.61 X	15.13 X	15.19
	S.d.	0.51	0.06	0.06	1.20
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	4.7	1.4	1.9
d 43 -> 50	Mean [g]	15.00 n	15.43 X	15.14 X	15.76
	S.d.	0.67	0.08	0.08	0.89
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	2.9	1.0	5.1

Sex: Female -	Phase: In-life
		C / F	LD / F	MD / F	HD / F
d 50 -> 57	Mean [g]	15.69 n	15.97 X	15.37 X	15.23
	S.d.	1.50	0.44	0.53	0.61
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	1.8	-2.0	-2.9
d 57 -> 64	Mean [g]	15.04 n	15.94 X	15.19 X	15.27
	S.d.	0.91	0.24	0.46	0.69
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	6.0	1.0	1.6
d 64 -> 71	Mean [g]	12.76 n	15.83 X	15.37 X	15.57
	S.d.	5.06	0.32	0.16	1.20
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	24.0	20.4	22.0
d 71 -> 78	Mean [g]	13.32 n	15.53 X	15.26 X	15.30
	S.d.	4.16	0.10	0.85	0.84
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	16.5	14.5	14.9
d 78 -> 85	Mean [g]	15.43 n	15.76 X	15.09 X	14.38
	S.d.	1.39	0.55	0.48	1.27
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	2.1	-2.2	-6.8
d 85 -> 90	Mean [g]	15.53 n	15.32 X	14.78 X	15.25
	S.d.	0.40	0.51	0.03	1.01
	N	3	2	2	3
	Deviation Vs Control [%]	0.0	-1.4	-4.8	-1.8

 

Summary Food Consumption (g/day/animal) - Recovery Phase		187383 - 1
Ascentos™ 1.3		26-Nov-2019 12:22
Sex: Male - Phase: In-life
	C / M	HD / M
d 90 -> 97                                    Mean [g]	21.3	28.8
N	1	1
Deviation Vs Control [%]		34.8
d 97 -> 104                                  Mean [g]	22.9	24.5
N	1	1
Deviation Vs Control [%]		6.7
d 104 -> 118                                Mean [g]	16.2	16.9
N	1	1
Deviation Vs Control [%]		4.1
d = day

Summary Food Consumption (g/day/animal) - Recovery Phase		187383 - 2
Ascentos™ 1.3		26-Nov-2019 12:22
Sex: Female - Phase: In-life
	C / F	HD / F
d 90 -> 97                                     Mean [g]	15.3	14.5
N	1	1
Deviation Vs Control [%]		-5.6
d 97 -> 104                                   Mean [g]	15.8	15.0
N	1	1
Deviation Vs Control [%]		-4.9
d 104 -> 118                                 Mean [g]	15.0	14.5
N	1	1
Deviation Vs Control [%]		-3.3
d = day

 

Summary Clinical Signs - Treatment Phase

Summary Clinical Signs - Treatment Phase   Ascentos™ 1.3						187383- 1   08-Jul-2019 11:26
Sex: Male - Phase: In-life
			C / M	LD / M	MD / M	HD / M
	Animals examined	N	15	10	10	15
	Animals with signs	N	1	0	3	15
		%	6.7	0.0	30.0	100.0
	Digestive Tract	N	0	0	1	7
		%	0.0	0.0	10.0	46.7
	Salivation increased	N	0	0	0	7
		%	0.0	0.0	0.0	46.7
day 1 -> 90	Overgrown teeth	N	0	0	1	0

		%	0.0	0.0	10.0	0.0
	Central Nervous System	N	0	0	1	15
	Moving the bedding	%	0.0	0.0	10.0	100.0
	Skin and Fur	N	0	0	0	1
	Crust	%	0.0	0.0	0.0	6.7
	Respiratory / Pulmonary Tract	N	1	0	1	0
	Cough/Sneezing	%	6.7	0.0	10.0	0.0

 

Ascentos™ 1.3						08-Jul-2019 11:27
Sex: Female - Phase: In-life
			C / F	LD / F	MD / F	HD / F
	Animals examined	N	15	10	10	15
	Animals with signs	N	2	0	0	15
		%	13.3	0.0	0.0	100.0
	Digestive Tract	N	1	0	0	10
		%	6.7	0.0	0.0	66.7
	Salivation increased	N	0	0	0	10
		%	0.0	0.0	0.0	66.7
	Diarrhoea	N	1	0	0	0
		%	6.7	0.0	0.0	0.0
	Central Nervous System	N	0	0	0	15
		%	0.0	0.0	0.0	100.0
	Moving the bedding	N	0	0	0	14
		%	0.0	0.0	0.0	93.3
	Apathy	N	0	0	0	1
day 1 -> 90		%	0.0	0.0	0.0	6.7
	Prone Position	N	0	0	0	1
		%	0.0	0.0	0.0	6.7
	Spontaneous activity reduced	N	0	0	0	1
		%	0.0	0.0	0.0	6.7
	Eyes / Mucous Membranes	N	0	0	0	1
	Eyes closed	%	0.0	0.0	0.0	6.7
	Skin and Fur	N	1	0	0	1
		%	6.7	0.0	0.0	6.7
	Hairless area	N	0	0	0	1
		%	0.0	0.0	0.0	6.7
	Scratch/ Cut	N	1	0	0	0
		%	6.7	0.0	0.0	0.0
	Respiratory / Pulmonary Tract	N	0	0	0	1
	Abnormal breathing	%	0.0	0.0	0.0	6.7

Summary Clinical Signs - Recovery			187383- 1
Ascentos™ 1.3			09-Jul-2019 09:09
Sex: Male - Phase: In-life
		C / M	HD / M
Animals examined	N	5	5
Animals with signs	N	0	5
day 90->118	%	0.0	100
Central nervous system Moving the bedding	N	0	5
	%	0.0	100

 

Summary Haematology - Treatment Phase

Sex: Male -	Phase: In-life
		C / M	LD / M	MD / M	HD / M
WBC	Mean	4.531 a	4.663	5.106	4.594
[1E9/L]	S.d.	1.120	0.888	1.261	2.046
day 91	N	8	9	9	9
	Deviation Vs Control [%]		2.915	12.674	1.395
RBC	Mean	9.430 k	9.282	9.388	9.387
[1E12/L]	S.d.	0.186	0.438	0.529	0.487
day 91	N	8	9	9	9
	Deviation Vs Control [%]		-1.567	-0.448	-0.460
HGB	Mean	16.51 k	16.48	17.31	17.09
[g/dL]	S.d.	1.65	0.66	0.53	0.66
day 91	N	8	9	9	9
	Deviation Vs Control [%]		-0.21	4.84	3.49
HCT	Mean	49.32 k	48.29	49.67	49.51
[%]	S.d.	0.84	1.89	3.26	2.43
day 91	N	8	9	9	9
	Deviation Vs Control [%]		-2.10	0.69	0.38
MCV	Mean	52.32 a	52.04	52.90	52.77
[fL]	S.d.	1.16	1.55	1.41	0.86
day 91	N	8	9	9	9
	Deviation Vs Control [%]		-0.54	1.10	0.84
MCH	Mean	17.50 k	17.77	18.47	18.21
[pg]	S.d.	1.66	0.34	0.85	0.44
day 91	N	8	9	9	9
	Deviation Vs Control [%]		1.52	5.52	4.06
MCHC	Mean	33.51 k	34.12	34.94	34.54
[g/dL]	S.d.	3.63	0.84	1.64	0.72
day 91	N	8	9	9	9
	Deviation Vs Control [%]		1.82	4.27	3.08
RET%	Mean	1.641 a	1.456	1.598	1.516
[%]	S.d.	0.109	0.204	0.215	0.262
day 91	N	8	9	9	9
	Deviation Vs Control [%]		-11.314	-2.649	-7.658

		C / M	LD / M	MD / M	HD / M
PLT	Mean	666.2 a	642.1	671.3	652.9
[1E9/L]	S.d.	92.2	87.2	90.1	95.0
day 91	N	8	9	9	9
	Deviation Vs Control [%]		-3.6	0.8	-2.0
EOS%	Mean	0.562 k	0.522	0.589	0.856
[%]	S.d.	0.297	0.387	0.376	0.590
day 91	N	8	9	9	9
	Deviation Vs Control [%]		-7.160	4.691	52.099
LYM%	Mean	73.74 a	75.20	76.39	76.94
[%]	S.d.	7.78	3.73	4.97	5.95
day 91	N	8	9	9	9
	Deviation Vs Control [%]		1.98	3.60	4.35
NEUT%	Mean	22.92 a	21.21	19.93	19.63
[%]	S.d.	7.87	2.75	3.82	5.60
day 91	N	8	9	9	9
	Deviation Vs Control [%]		-7.48	-13.05	-14.36
MONO%	Mean	2.40 a	2.61	2.62	2.07
[%]	S.d.	0.34	1.17	1.14	0.49
day 91	N	8	9	9	9
	Deviation Vs Control [%]		8.80	9.26	-13.89
BASO%	Mean	0.150 k	0.133	0.156	0.200
[%]	S.d.	0.093	0.071	0.088	0.122
day 91	N	8	9	9	9
	Deviation Vs Control [%]		-11.111	3.704	33.333
LUC%	Mean	0.225 k	0.356	0.322	0.322
[%]	S.d.	0.158	0.159	0.199	0.148
day 91	N	8	9	9	9
	Deviation Vs Control [%]		58.025	43.210	43.210

 

Summary Haematology - Treatment Phase   Ascentos™ 1.3				187383 - 3   16-Jan-2020 10:14
Sex: Female - Phase: In-life
	C / F	LD / F	MD / F	HD / F
WBC                   Mean	1.692 k	2.166	1.976	1.707
[1E9/L]                S.d.	0.301	0.851	0.609	0.661
day 91                 N	10	10	10	10
Deviation Vs Control [%]		28.014	16.785	0.887
RBC                    Mean	8.267 a	8.337	8.154	8.133
[1E12/L]              S.d.	0.404	0.358	0.494	0.392
day 91                 N	10	10	10	10
Deviation Vs Control [%]		0.847	-1.367	-1.621
HGB                    Mean	14.97 a	15.02	14.99	14.97
[g/dL]                   S.d.	0.56	0.50	0.70	0.69
day 91                 N	10	10	10	10
Deviation Vs Control [%]		0.33	0.13	0.00
HCT                    Mean	44.29 a	44.42	43.96	43.11
[%]                       S.d.	1.75	1.78	2.25	2.36
day 91                 N	10	10	10	10
Deviation Vs Control [%]		0.29	-0.75	-2.66
MCV                    Mean	53.60 a	53.31	53.95	53.01
[fL]                       S.d.	1.31	1.70	1.37	1.42
day 91                 N	10	10	10	10
Deviation Vs Control [%]		-0.54	0.65	-1.10
MCH                   Mean	18.13 a	18.03	18.41	18.34
[pg]                      S.d.	0.67	0.69	0.74	0.40
day 91                 N	10	10	10	10
Deviation Vs Control [%]		-0.55	1.54	1.16
MCHC                Mean	33.81 a	33.84	34.11	34.62
[g/dL]                   S.d.	0.71	0.75	0.77	0.59
day 91                 N	10	10	10	10
Deviation Vs Control [%]		0.09	0.89	2.40
RET%                 Mean	1.773 a	1.855	1.889	2.018
[%]                       S.d.	0.200	0.325	0.374	0.526
day 91                 N	10	10	10	10
Deviation Vs Control [%]		4.625	6.543	13.818

Summary Haematology - Treatment Phase   Ascentos™ 1.3				187383 - 4   16-Jan-2020 10:14
Sex: Female - Phase: In-life
	C / F	LD / F	MD / F	HD / F
PLT                      Mean	727.6 a	718.8	766.4	764.7
[1E9/L]                S.d.	99.4	66.4	119.1	80.3
day 91                 N	10	10	10	10
Deviation Vs Control [%]		-1.2	5.3	5.1
EOS%                 Mean	0.250 k	0.410	0.330	0.520
[%]                       S.d.	0.212	0.202	0.313	0.509
day 91                 N	10	10	10	10
Deviation Vs Control [%]		64.000	32.000	108.000
LYM%                 Mean	84.36 k	80.07	85.20	80.01
[%]                       S.d.	4.92	6.77	4.46	9.90
day 91                 N	10	10	10	10
Deviation Vs Control [%]		-5.09	1.00	-5.16
NEUT%              Mean	13.35 k	17.08	12.18	17.04
[%]                       S.d.	4.99	6.51	4.02	9.53
day 91                 N	10	10	10	10
Deviation Vs Control [%]		27.94	-8.76	27.64
MONO%             Mean	1.65 a	1.99	1.95	2.04
[%]                       S.d.	0.50	0.71	0.69	0.45
day 91                 N	10	10	10	10
Deviation Vs Control [%]		20.61	18.18	23.64
BASO%              Mean	0.080 k	0.070	0.070	0.110
[%]                       S.d.	0.063	0.095	0.048	0.099
day 91                 N	10	10	10	10
Deviation Vs Control [%]		-12.500	-12.500	37.500
LUC%                 Mean	0.320 a	0.350	0.300	0.270
[%]                       S.d.	0.148	0.196	0.211	0.250
day 91                 N	10	10	10	10
Deviation Vs Control [%]		9.375	-6.250	-15.625

Summary Heamatology - End of Recovery		187383- 1
Ascentos™ 1.3		27-Jun-2019 11:03
Sex: Male - Phase: In-life
	C / M	HD / M
WBC                            Mean	3.902 n	2.710
[1E9/L]                         S.d.	0.738	1.042
day 119                       N	4	4
Deviation Vs Control [%]		-30.557
RBC                             Mean	10.368 n	9.582
[1E12/L]                      S.d.	0.372	0.604
day 119                       N	4	4
Deviation Vs Control [%]		-7.572
HGB                             Mean	17.60 s	16.58
[g/dL]                           S.d.	0.53	1.04
day 119                       N	4	4
Deviation Vs Control [%]		-5.82
HCT                             Mean	54.05 s	51.28
[%]                                S.d.	0.37	3.12
day 119                       N	4	4
Deviation Vs Control [%]		-5.13
MCV                            Mean	52.18 n	53.52
[fL]                                S.d.	2.14	1.52
day 119                       N	4	4
Deviation Vs Control [%]		2.59
MCH                            Mean	17.00 s	17.30
[pg]                               S.d.	0.79	0.16
day 119                       N	4	4
Deviation Vs Control [%]		1.76
MCHC                         Mean	32.58 n	32.28
[g/dL]                           S.d.	0.85	0.78
day 119                       N	4	4
Deviation Vs Control [%]		-0.92
RET%                          Mean	0.475 n	1.005 **
[%]                                S.d.	0.206	0.113
day 119                       N	4	4
Deviation Vs Control [%]		111.579

 

		C / M	HD / M
PLT	Mean	623.5 n	651.5
[1E9/L]	S.d.	51.5	56.3
day 119	N	4	4
	Deviation Vs Control [%]		4.5
EOS%	Mean	0.575 n	1.175
[%]	S.d.	0.250	1.014
day 119	N	4	4
	Deviation Vs Control [%]		104.348
LYM%	Mean	83.28 s	82.82
[%]	S.d.	8.32	1.23
day 119	N	4	4
	Deviation Vs Control [%]		-0.54
NEUT%	Mean	12.75 s	13.82
[%]	S.d.	7.61	1.52
day 119	N	4	4
	Deviation Vs Control [%]		8.43
MONO%	Mean	2.62 s	1.65
[%]	S.d.	1.16	0.19
day 119	N	4	4
	Deviation Vs Control [%]		-37.14
BASO%	Mean	0.225 n	0.100
[%]	S.d.	0.126	0.082
day 119	N	4	4
	Deviation Vs Control [%]		-55.556
LUC%	Mean	0.575 n	0.425
[%]	S.d.	0.330	0.222
day 119	N	4	4
	Deviation Vs Control [%]		-26.087

Summary Heamatology - End of Recovery		187383- 3
Ascentos™ 1.3		27-Jun-2019 11:03
Sex: Female - Phase: In-life
	C / F	HD / F
WBC                              Mean	1.850 n	2.185
[1E9/L]                           S.d.	0.526	0.565
day 119                         N	5	4
Deviation Vs Control [%]		18.108
RBC                               Mean	8.062 n	8.448
[1E12/L]                         S.d.	0.867	0.352
day 119                         N	5	4
Deviation Vs Control [%]		4.782
HGB                               Mean	14.64 n	15.22
[g/dL]                              S.d.	1.12	0.57
day 119                         N	5	4
Deviation Vs Control [%]		4.00
HCT                               Mean	46.02 n	47.92
[%]                                  S.d.	5.36	1.80
day 119                         N	5	4
Deviation Vs Control [%]		4.14
MCV                               Mean	57.06 n	56.78
[fL]                                  S.d.	2.20	1.90
day 119                         N	5	4
Deviation Vs Control [%]		-0.50
MCH                              Mean	18.24 n	18.00
[pg]                                 S.d.	0.76	0.65
day 119                         N	5	4
Deviation Vs Control [%]		-1.32
MCHC                           Mean	31.92 n	31.72
[g/dL]                              S.d.	1.58	0.10
day 119                         N	5	4
Deviation Vs Control [%]		-0.61
RET%                            Mean	1.574 n	1.555
[%]                                  S.d.	0.211	0.530
day 119                         N	5	4
Deviation Vs Control [%]		-1.207
n=DUNNETT

Summary Heamatology - End of Recovery		187383- 4
Ascentos™ 1.3		27-Jun-2019 11:03
Sex: Female - Phase: In-life
	C / F	HD / F
PLT                                  Mean	610.6 n	660.0
[1E9/L]                             S.d.	79.0	143.7
day 119                           N	5	4
Deviation Vs Control [%]		8.1
EOS%                              Mean	0.640 s	0.600
[%]                                    S.d.	0.483	0.876
day 119                           N	5	4
Deviation Vs Control [%]		-6.250
LYM%                              Mean	79.56 n	79.32
[%]                                    S.d.	6.40	1.27
day 119                           N	5	4
Deviation Vs Control [%]		-0.30
NEUT%                           Mean	17.94 s	17.78
[%]                                    S.d.	6.07	1.14
day 119                           N	5	4
Deviation Vs Control [%]		-0.92
MONO%                          Mean	1.56 n	1.80
[%]                                    S.d.	0.51	0.59
day 119                           N	5	4
Deviation Vs Control [%]		15.38
BASO%                           Mean	0.080 s	0.150
[%]                                    S.d.	0.045	0.058
day 119                           N	5	4
Deviation Vs Control [%]		87.500
LUC%                              Mean	0.280 s	0.375
[%]                                    S.d.	0.084	0.206
day 119                           N	5	4
Deviation Vs Control [%]		33.929
n=DUNNETT; s=DUNN

 

Summary Coagulation - End of Treatment   Ascentos™ 1.3				187383 - 1   16-Jan-2020 10:17
Sex: Male - Phase: In-life
	C / M	LD / M	MD / M	HD / M
PT                   Mean	22.50 a	23.17	23.27	23.01
[sec]               S.d.	1.13	1.16	1.40	1.61
day 91            N	10	10	10	9
Deviation Vs Control [%]		2.98	3.42	2.27
aPTT              Mean	12.47 k	13.42	13.67	13.32
[sec]               S.d.	3.44	3.01	1.91	2.08
day 91            N	10	10	10	9
Deviation Vs Control [%]		7.62	9.62	6.83

Summary Coagulation - End of Treatment

Sex: Female - Phase: In-life

		C / F	LD / F	MD / F	HD / F
PT	Mean	24.16 a	24.44	24.63	25.38
[sec]	S.d.	1.15	2.14	1.60	1.10
day 91	N	10	10	9	10
	Deviation Vs Control [%]		1.16	1.96	5.05
aPTT	Mean	11.71 u	11.81	13.73	15.71 ***
[sec]	S.d.	1.28	1.60	2.55	1.39
day 91	N	10	10	9	10
	Deviation Vs Control [%]		0.85	17.28	34.16

Summary Coagulation - End of Recovery		187383 - 1
Ascentos™ 1.3		16-Jan-2020 10:20
Sex: Male - Phase: In-life
	C / M	HD / M
PT                                 Mean	26.34 n	25.12
[sec]                              S.d.	1.05	0.43
day 119                        N	5	4
Deviation Vs Control [%]		-4.61
aPTT                            Mean	13.98 n	14.02
[sec]                              S.d.	0.81	0.56
day 119                        N	5	5
Deviation Vs Control [%]		0.29
n=DUNNETT

Summary Coagulation - End of Recovery		187383 - 2
Ascentos™ 1.3		16-Jan-2020 10:20
Sex: Female - Phase: In-life
	C / F	HD / F
PT                                 Mean	23.76 s	22.90
[sec]                              S.d.	1.69	1.94
day 119                        N	5	4
Deviation Vs Control [%]		-3.62
aPTT                             Mean	13.50 n	13.80
[sec]                              S.d.	0.72	1.98
day 119                        N	5	4
Deviation Vs Control [%]		2.22

 

Summary Clinical Biochemistry - End of Treatment

Sex: Male - Phase: In-life

		C / M	LD / M	MD / M	HD / M
ALAT	Mean	28.99 u	35.34	27.18	40.58
[U/L]	S.d.	5.26	7.11	3.10	39.41
day 91	N	10	10	10	10
	Deviation Vs Control [%]		21.90	-6.24	39.98
Gluc	Mean	8.257 a	10.312	10.187	9.081
[mmol/L]	S.d.	1.633	2.388	2.086	1.878
day 91	N	10	10	10	10
	Deviation Vs Control [%]		24.888	23.374	9.979
Crea	Mean	24.5 a	22.6	24.3	22.0
[µmol/L]	S.d.	4.9	3.8	6.4	4.2
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-7.8	-0.8	-10.2
ASAT	Mean	90.18 a	103.65	89.90	102.21
[U/L]	S.d.	16.36	32.21	20.63	38.80
day 91	N	10	10	10	10
	Deviation Vs Control [%]		14.94	-0.31	13.34
AP	Mean	72.373 a	75.355	84.315	79.212
[U/L]	S.d.	27.129	18.715	19.758	13.514
day 91	N	10	10	10	10
	Deviation Vs Control [%]		4.120	16.501	9.450
Na	Mean	142.1 u	138.3	136.9 *	137.9
[mmol/L]	S.d.	5.6	2.5	1.6	2.3
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-2.7	-3.7	-3.0
K	Mean	4.833 ad	5.640	6.124 **	6.423 **
[mmol/L]	S.d.	0.783	1.286	0.584	0.815
day 91	N	10	10	10	10
	Deviation Vs Control [%]		16.698	26.712	32.899
TP	Mean	59.41 k	57.87	56.57	56.51
[g/L]	S.d.	3.77	1.99	1.52	2.81
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-2.59	-4.78	-4.88
ALB	Mean	32.889 ad	31.440	31.112 *	31.444
[g/L]	S.d.	2.033	0.906	1.039	1.409
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-4.406	-5.403	-4.394
TBIL	Mean	2.40 k	2.36	2.13	2.28
[µmol/L]	S.d.	0.58	0.20	0.15	0.51
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-1.67	-11.25	-5.00
CHOL	Mean	1.931 a	1.954	2.009	1.813
[mmol/L]	S.d.	0.354	0.209	0.334	0.289
day 91	N	10	10	10	10
	Deviation Vs Control [%]		1.191	4.039	-6.111

 

Sex: Male - Phase: In-life
		C / M	LD / M	MD / M	HD / M
TBA	Mean	34.791 a	40.218	26.144	26.036
[µmol/L]	S.d.	24.915	39.671	11.273	12.741
day 91	N	10	10	10	10
	Deviation Vs Control [%]		15.599	-24.854	-25.165
TG	Mean	0.75 a	0.76	1.00	0.88
[mmol/L]	S.d.	0.36	0.29	0.35	0.25
day 91	N	10	10	10	8
	Deviation Vs Control [%]		1.88	34.32	18.63
Urea	Mean	7.494 a	7.171	7.094	7.158
[mmol/L]	S.d.	0.638	1.029	0.620	1.026
day 91	N	10	10	10	10
	Deviation Vs Control [%]		-4.310	-5.338	-4.484
HDL	Mean	1.366 a	1.376	1.397	1.248
[mmol/L]	S.d.	0.232	0.121	0.214	0.212
day 91	N	10	10	10	10
	Deviation Vs Control [%]		0.732	2.269	-8.638
LDL (calculated)	Mean	0.416 a	0.426	0.412	0.403
[mmol/L]	S.d.	0.146	0.121	0.129	0.129
day 91	N	10	10	10	8
	Deviation Vs Control [%]		2.453	-1.010	-3.078
a=ANOVA

Summary Clinical Biochemistry - End of Treatment

Sex: Female - Phase: In-life

		C / F	LD / F	MD / F	HD / F
ALAT	Mean	25.84 a	26.97	24.65	24.14
[U/L]	S.d.	3.33	5.94	3.25	5.67
day 91	N	10	10	10	9
	Deviation Vs Control [%]		4.37	-4.61	-6.56
Gluc	Mean	7.212 a	7.639	7.711	7.113
[mmol/L]	S.d.	2.122	3.172	2.221	1.730
day 91	N	10	10	10	9
	Deviation Vs Control [%]		5.921	6.919	-1.368
Crea	Mean	29.0 k	28.8	24.9	28.0
[µmol/L]	S.d.	4.7	6.3	4.5	5.2
day 91	N	10	10	10	9
	Deviation Vs Control [%]		-0.7	-14.1	-3.4
ASAT	Mean	83.77 a	75.37	67.62	76.48
[U/L]	S.d.	13.12	15.25	15.34	12.63
day 91	N	10	10	10	9
	Deviation Vs Control [%]		-10.03	-19.28	-8.71
AP	Mean	49.361 k	52.938	42.227	39.657
[U/L]	S.d.	25.848	21.026	14.279	14.598
day 91	N	10	10	10	9
	Deviation Vs Control [%]		7.247	-14.453	-19.660
Na	Mean	140.6 k	140.3	139.5	139.7
[mmol/L]	S.d.	2.7	1.6	3.8	1.0
day 91	N	10	10	10	9
	Deviation Vs Control [%]		-0.2	-0.8	-0.7
K	Mean	3.536 u	3.858	4.141 *	4.859 **
[mmol/L]	S.d.	0.299	0.395	0.610	0.972
day 91	N	10	10	10	9
	Deviation Vs Control [%]		9.106	17.110	37.412
TP	Mean	59.49 k	60.24	62.84	60.38
[g/L]	S.d.	4.95	3.37	4.29	2.91
day 91	N	10	10	10	9
	Deviation Vs Control [%]		1.26	5.63	1.49
ALB	Mean	34.335 a	34.656	35.849	34.416
[g/L]	S.d.	2.459	2.007	2.618	1.476
day 91	N	10	10	10	9
	Deviation Vs Control [%]		0.935	4.409	0.235
TBIL	Mean	2.26 k	2.45	2.70	2.62
[µmol/L]	S.d.	0.34	0.51	0.61	0.48
day 91	N	10	10	10	9
	Deviation Vs Control [%]		8.41	19.47	16.03
CHOL	Mean	1.263 a	1.403	1.593	1.548
[mmol/L]	S.d.	0.245	0.290	0.407	0.357
day 91	N	10	10	10	9
	Deviation Vs Control [%]		11.085	26.128	22.548

Sex: Female - Phase:	In-life
		C / F	LD / F	MD / F	HD / F
TBA	Mean	19.188 ad	27.692	22.572	49.980 *
[µmol/L]	S.d.	13.930	18.318	18.540	28.812
day 91	N	10	10	10	9
	Deviation Vs Control [%]		44.319	17.636	160.475
TG	Mean	0.47 k	0.52	0.55	0.54
[mmol/L]	S.d.	0.24	0.25	0.26	0.21
day 91	N	10	10	10	9
	Deviation Vs Control [%]		10.73	18.03	15.16
Urea	Mean	7.703 a	7.639	6.813	7.324
[mmol/L]	S.d.	1.081	0.989	0.623	1.514
day 91	N	10	10	10	9
	Deviation Vs Control [%]		-0.831	-11.554	-4.914
HDL	Mean	0.975 a	1.049	1.196	1.138
[mmol/L]	S.d.	0.193	0.208	0.259	0.234
day 91	N	10	10	10	9
	Deviation Vs Control [%]		7.590	22.667	16.695
LDL (calculated)	Mean	0.195 a	0.251	0.287	0.303
[mmol/L]	S.d.	0.073	0.095	0.155	0.109
day 91	N	10	10	10	9
	Deviation Vs Control [%]		28.747	47.331	55.373

Summary Clinical Biochemistry - End of Recovery

Sex: Male - Phase: In-life

		C / M	HD / M
ALAT	Mean	30.54 n	22.98
[U/L]	S.d.	7.03	2.34
day 119	N	5	5
	Deviation Vs Control [%]		-24.75
Gluc	Mean	15.464 n	17.524
[mmol/L]	S.d.	2.474	2.721
day 119	N	5	5
	Deviation Vs Control [%]		13.321
Crea	Mean	29.8 s	44.0
[µmol/L]	S.d.	18.6	49.9
day 119	N	5	5
	Deviation Vs Control [%]		47.7
ASAT	Mean	75.34 n	76.06
[U/L]	S.d.	6.70	6.14
day 119	N	5	5
	Deviation Vs Control [%]		0.96
AP	Mean	78.054 s	82.766
[U/L]	S.d.	17.452	14.126
day 119	N	5	5
	Deviation Vs Control [%]		6.037
Na	Mean	137.8 n	134.6
[mmol/L]	S.d.	2.2	3.8
day 119	N	5	5
	Deviation Vs Control [%]		-2.3
K	Mean	6.180 s	6.756
[mmol/L]	S.d.	0.362	1.706
day 119	N	5	5
	Deviation Vs Control [%]		9.320
TP	Mean	52.80 n	54.26
[g/L]	S.d.	3.13	4.72
day 119	N	5	5
	Deviation Vs Control [%]		2.77
ALB	Mean	30.270 n	30.558
[g/L]	S.d.	1.360	2.558
day 119	N	5	5
	Deviation Vs Control [%]		0.951
TBIL	Mean	2.30 n	2.12
[µmol/L]	S.d.	0.56	0.18
day 119	N	5	5
	Deviation Vs Control [%]		-7.83
CHOL	Mean	1.244 n	1.190
[mmol/L]	S.d.	0.157	0.249
day 119	N	5	5
	Deviation Vs Control [%]		-4.341

 

Sex: Male - Phase: In-life
		C / M	HD / M
TBA	Mean	19.458 n	14.650
[µmol/L]	S.d.	13.608	4.785
day 119	N	5	5
	Deviation Vs Control [%]		-24.710
TG	Mean	1.34 s	1.27
[mmol/L]	S.d.	0.33	0.20
day 119	N	4	5
	Deviation Vs Control [%]		-5.10
Urea	Mean	4.856 s	7.556
[mmol/L]	S.d.	0.633	5.152
day 119	N	5	5
	Deviation Vs Control [%]		55.601
HDL	Mean	0.754 n	0.792
[mmol/L]	S.d.	0.130	0.168
day 119	N	5	5
	Deviation Vs Control [%]		5.040
LDL (calculated)	Mean	0.216 n	0.143
[mmol/L]	S.d.	0.092	0.059
day 119	N	4	5
	Deviation Vs Control [%]		-33.857
n=DUNNETT; s=DUNN

 

Summary Clinical Biochemistry - End of Recovery		187383 - 3
Ascentos™ 1.3		16-Jan-2020 10:24
Sex: Female - Phase: In-life
	C / F	HD / F
ALAT                             Mean	30.78 n	26.78
[U/L]                              S.d.	8.49	3.80
day 119                        N	4	4
Deviation Vs Control [%]		-13.00
Gluc                              Mean	8.505 n	9.868
[mmol/L]                       S.d.	1.091	0.735
day 119                        N	4	4
Deviation Vs Control [%]		16.020
Crea                              Mean	30.2 n	24.8
[µmol/L]                        S.d.	4.5	4.6
day 119                        N	4	4
Deviation Vs Control [%]		-18.2
ASAT                            Mean	85.25 s	78.60
[U/L]                              S.d.	17.99	16.28
day 119                        N	4	4
Deviation Vs Control [%]		-7.80
AP                                 Mean	51.090 n	37.372
[U/L]                              S.d.	14.655	11.663
day 119                        N	4	4
Deviation Vs Control [%]		-26.850
Na                                 Mean	137.0 n	138.8
[mmol/L]                       S.d.	5.2	3.9
day 119                        N	4	4
Deviation Vs Control [%]		1.3
K                                    Mean	3.875 n	3.498
[mmol/L]                       S.d.	0.525	0.319
day 119                        N	4	4
Deviation Vs Control [%]		-9.742
TP                                  Mean	62.20 n	62.30
[g/L]                               S.d.	4.42	4.87
day 119                        N	4	4
Deviation Vs Control [%]		0.16
ALB                               Mean	35.782 n	35.518
[g/L]                               S.d.	2.617	2.778
day 119                        N	4	4
Deviation Vs Control [%]		-0.741
TBIL                              Mean	2.90 n	2.12 **
[µmol/L]                        S.d.	0.16	0.35
day 119                        N	4	4
Deviation Vs Control [%]		-26.72
CHOL                           Mean	1.305 n	1.615
[mmol/L]                       S.d.	0.298	0.277
day 119                        N	4	4
Deviation Vs Control [%]		23.755

		C / F	HD / F
TBA	Mean	26.325 n	34.405
[µmol/L]	S.d.	20.188	21.001
day 119	N	4	4
	Deviation Vs Control [%]		30.693
TG	Mean	0.86 n	0.86
[mmol/L]	S.d.	0.55	0.32
day 119	N	4	4
	Deviation Vs Control [%]		0.00
Urea	Mean	7.638 n	7.552
[mmol/L]	S.d.	1.073	0.392
day 119	N	4	4
	Deviation Vs Control [%]		-1.113
HDL	Mean	0.998 n	1.228
[mmol/L]	S.d.	0.229	0.213
day 119	N	4	4
	Deviation Vs Control [%]		23.058
LDL (calculated)	Mean	0.134 n	0.214
[mmol/L]	S.d.	0.091	0.099
day 119	N	4	4
	Deviation Vs Control [%]		59.480
n=DUNNETT

 

Summary Hormone Analysis – End of Treatment - Males

Group		T3	T4	TSH
	Units	ng/mL	ng/mL	ng/mL
C	Mean	1.83	59.80	1.06
	SD	0.52	16.79	0.49
	N	10	10	10
LD	Mean	1.98	57.39	0.81
	SD	0.47	12.53	0.29
	N	10	9	10
MD	Mean	1.96	59.27	0.77
	SD	0.43	8.91	0.29
	N	10	10	10
HD	Mean	1.95	54.10	0.57**
	SD	0.41	8.09	0.25
	N	9	10	10

Asterisks indicate statistically significant differences to Control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

 

All values are within historical control data- (Historical control Data are summerized in full study report page 869- Annex III)

Summary Hormone Analysis – End of Treatment - Females

Group		T3	T4	TSH
	Units	ng/mL	ng/mL	ng/mL
C	Mean	1.84	34.94	1.66
	SD	0.35	4.16	0.85
	N	10	10	10
LD	Mean	2.13	56.07	1.38
	SD	0.27	46.13	0.68
	N	10	10	9
MD	Mean	2.20	38.50	1.14
	SD	0.63	5.56	0.37
	N	10	10	7
HD	Mean	2.22	42.50	1.53
	SD	0.49	6.01	0.69
	N	10	10	9

 

Summary Hormone Analysis – End of Recovery - Males

Group		T3	T4	TSH
	Units	ng/mL	ng/mL	ng/mL
C	Mean	1.55	44.34	1.00
	SD	0.54	9.20	0.33
	N	4	4	5
HD	Mean	1.86	48.59	0.58*
	SD	0.99	9.22	0.22
	N	5	5	5

Summary Hormone Analysis – End of Recovery - Females

Group		T3	T4	TSH
	Units	ng/mL	ng/mL	ng/mL
C	Mean	1.92	38.75	0.67
	SD	0.58	7.89	0.20
	N	5	5	5
HD	Mean	1.78	39.21	1.29
	SD	0.19	4.20	1.35
	N	4	4	4

Mean Testis Weight – End of Treatment

Group		Weight of Testes [g]	Weight of Tunica Albuginea [g]	Calculated Weight of Parenchyma [g]
C	Mean	1.897	0.1075	1.7897
	SD	0.134	0.024	0.153
	N	10	10	10
LD	Mean	1.806	0.120	1.686
	SD	0.117	0.046	0.127
	N	10	10	10
MD	Mean	1.852	0.125	1.727
	SD	0.148	0.045	0.128
	N	10	10	10
HD	Mean	1.755	0.137	1.618*
	SD	0.084	0.057	0.087
	N	10	10	10

 

 

Mean Sperm Motility – End of Treatment

 

Group		Motile Count [%]	Static Count [%]	Rapid [%]
C	Mean	84.0	16	65.6
	SD	5.9	5.9	11.8
	N	10	10	10
LD	Mean	86.0	14	69.3
	SD	3.6	3.6	4.1
	N	10	10	10
MD	Mean	85.4	14.6	69.7
	SD	4.1	4.1	6.7
	N	10	10	10
HD	Mean	76.8	23.2	54.7
	SD	15.1	15.1	20.3
	N	10	10	10

 

Mean Testicular Sperm Count – End of Treatment

Group		Million Sperms/g mean
C	Mean	105.7
	SD	10.8
	N	9
LD	Mean	108.9
	SD	12.9
	N	8
MD	Mean	106.2
	SD	17.2
	N	8
HD	Mean	114.4
	SD	22.5
	N	7

 

Mean Sperm Morphology – End of Treatment

Group	Animal No.	Head and Neck
		Amorphous Head (AH)	Banana Shaped head (BH)	Hookless sperm (HkS)	Pin Head (PH)	Head only(HO)	Bent neck
C	Mean	0.00	0.00	0.00	0.00	0.80	0.00
	SD	0.00	0.00	0.00	0.00	0.79	0.00
	N	10	10	10	10	10	10
HD	Mean	0.00	0.00	0.00	0.00	1.60	0.00
	SD	0.00	0.00	0.00	0.00	0.84	0.00
	N	10	10	10	10	10	10

Group	Animal No.	Tail
		Bent Tail (BT)	Broken Tail (BrT)	Coiled tail (CT)	Tail Only (TO)
C	Mean	2.90	0.10	0.00	1.90
	SD	1.29	0.32	0.00	0.99
	N	10	10	10	10
HD	Mean	3.00	0.70	0.00	2.00
	SD	1.70	1.06	0.00	1.49
	N	10	10	10	10

Group	Animal No.	Number of sperms evaluated	Total Number of abnormal sperms/findings*	Total Number of normal sperms/findings	% normal	% abnormal
C	Mean	200	5.70	194.30	97.15	2.85
	SD	0.00	1.70	1.70	0.85	0.85
	N	10	10	10	10	10
HD	Mean	200	7.30*	192.70*	96.35*	3.65*
	SD	0	1.34	1.34	0.67	0.67
	N	10	10	10	10	10

* = one sperm may have multiple abnormalities/findings

Mean Testis Weight – End of Recovery

Group		Weight of Testes [g]	Weight of Tunica Albuginea [g]	Calculated Weight of Parenchyma [g]
C	Mean	1.859	0.106	1.754
	SD	0.131	0.024	0.145
	N	5	5	5
HD	Mean	1.808	0.098	1.710
	SD	0.111	0.027	0.107
	N	5	5	5

Mean Testicular Sperm Count – End of Recovery

Group		Million Sperms/g mean
C	Mean	134.2
	SD	21.2
	N	5
HD	Mean	137.6
	SD	14.2
	N	5

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 treatmentrelated 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.

 

All values are within historical control data- (Historical control Data are summerized in full study report page 869- Annex III)

Individual Oestrus Cyclicity – End of Treatment Period

Group	Female No.	Last Day of the Treatment Period
C	51	P
	52	P
	53	E/M
	54	P
	55	E
	56	D
	57	D
	58	M/D
	59	D
	60	M
LD	61	E
	62	D
	63	P/E
	64	P
	65	M/D
	66	P
	67	D
	68	E
	69	D
	70	D
MD	71	D
	72	D
	73	M/D
	74	M
	75	M/D
	76	D
	77	D
	78	M/D
	79	P
	80	P
HD	81	D
	82	M/D
	83	E
	84	M
	85	P
	86	P
	87	D
	88	D
	89	D
	90	P

P = proestrous, E = estrous, M = metestrous, D = diestrous

 

 

*Tables regarding detailed description of all histopathological findings can not be included in this part due to some unexpected formatting issues (probably related to our IUCLID version). Please find all histopathological findings in tabular form in the attached Annex I.

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.

 

*Due to the large data volumes the tables regarding ophthalmological examination, sensory activity, grip strength, toxic response data, terminal body weight, organ weight and organ/body weight ratios vaginal parameters, necropsy data (with statistical treatment) are included in the attached annex II. 

 

*Historical control data are summarized in full study report, starting on page 860 - Annex III.

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.

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:

1 (reliable without restriction)

Additional information

On the basis of an OECD-conform 90-Day Repeated Dose Oral Toxicity study (OECD TG 408) 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.

On the basis of an OECD-conform combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test (according to OECD 422) 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:

No mortality and no clinical signs of systemic toxicity were observed in the animals of this study. Daily oral treatment with Methyl 4-hydroxybenzoate had no effect on body weight and food consumption. At the end of the respective treatment periods of male and female animals there were no changes in clinical pathology results up to 1000 mg/kg bw/day and organ weights were inconspicuous. No relevant macroscopic and no histopathological abnormalities were observed in treated animals. Thus, the NOAEL for general toxicity is determined to be 1000 mg/kg bw/day.No test item-related effects on the reproduction and developmental parameters analysed in this study, i.e. oestrous cycle,  copulation, fertility and delivery indices, number of corpora lutea, implantation sites and live pups, pre- and post-implantation loss, number of male and female pups, sex ratio, still births, runts, litter weight data, anogenital distance, nipple retention and external abnormalities. Thus, the NOAEL for reproduction/developmental toxicity and systemic toxicity is determined to be 1000 mg/kg bw/day.

A 28 day oral toxicity study was performed in rats (according to OECD 407). The animals were treated with 50, 250 and 1000 mg/kg bw/d. No spontaneous mortality occurred during the study. However, one male and one female at 1000 mg/kg bw/d were sacrificed for ethical reasons on Day 14 and 24 respectively, showing clinical signs indicative of ill health. Microscopic examination revealed minimal/slight erosions in the stomach, correlating to the irregular surface recorded at necropsy, slight red pulp atrophy of the spleen and slight/moderate lymphoid atrophy of the thymus, correlating to the reduced size recorded at necropsy. Piloerection and/or hunched posture were noted among the surviving animals at 1000 mg/kg bw/d during the observation period. Laboured respiration, rales and gasping were noted in most animals at 1000 mg/kg bw/d and one female at 250 mg/kg bw/d during the observation period. These clinical signs could be considered related to treatment with the test substance. However the signle incidance of mortality and the absence of further toxicological sings in other animals does not reveal a susbtance related effect.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. hematology, clinical chemistry, urinalysis, functional observations, ophthalmoscopy, body weight, food consumption, estrus cycle, spermatogenesis, macroscopic and microscopic examination).Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for the test item, Methylparaben, was considered to be 250 mg/kg body weight/day.

As reported in the OECD 442 Study males and female wistar rats were treated with methyl paraben for ta time period of 28 and 63 days respectivels. Based on the longer exposure period in females and due to the fact that no toxicologically relevant effects werer observed up to the highest dose (1000 mg/kg bw/d) in both genders the NOAEL for repeated dose toxicity was set as 1000 mg/kg bw/d. Furthermore perliminary results of the requested 90 day oral toxicity study confired a NOAEL of 1000 mg/kg bw/d.

 

In a chronic dietary study rats were fed diets containing 2 and 8% Methylparaben for 96 weeks. Animals receiving diets containing 8% Methylparaben (equivalent to a Methylparaben intake of 5.5 - 5.9 g/kg bw/d) showed a slower rate of weight gain than control animals. This difference was much more striking in the early part of the study. At the end of the study these differences disappeared. This effect was more striking in the case of males than females. On animals receiving 2% of Methylparaben (equivalent to an Methylparaben intake of 0.9 - 1.2 g/kg bw/d), however, it was impossible to differentiate treated animals from untreated. Food intake and hence Methylparaben intake remained constant throughout the study. Tissues from all rats surviving the chronic study were entirely normal. The only significant findings were seen in the case of the rats dying during the study; in all such animals there was extensive consolidation of the lung and pneumonia, and in this respect there was no difference between the control and treated animals.

 

Mongrel dogs were treated with Methylparaben by daily application of a capsule for up to 422 days. No toxic symptoms were observed in any of the animals. The weights of the test animals before and after treatment showed respectable increases. At termination of study all animals appeared to be in excellent condition. One of the females receiving 0.5 g/kg bw/d of Methylparaben was mated toward the end of the study and delivered a litter of healthy pups. At study termination complete blood count examination was made on all dogs. No effect was observed. Complete urinalysis at the end of the study showed no changes. Pathology revealed no changes.

 

Rabbits were treated with 2.5 mg Methylparaben/animal/day for 120 days. All animals survived. There were no clinical signs or effects on body weight, body weight gain, temperature or gastro-intestinal tract observed.

 

In another study guinea pigs were treated with up to 100 mg Methylparaben/animal/day for 120 days daily. All animals survived. There were no clinical signs or effects on body weight, food consumption or haematology.

 

Rats treated with 0.5 to 5 mg Methylparaben/animal/day for 80 days daily showed no effects on body weight (gain), gastro-intestinal activity and haematology. All animals survived and no clinical signs were noted.

Repeated dose toxicity: via oral route - systemic effects (target organ) respiratory: other

Justification for classification or non-classification

On the basis of a combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test (according to OECD 422) and a sub chronic oral toxicity study (accordign to OECD 408) with Methyl 4-hydroxybenzoate in male and female Wistar rats the NOAEL for repeated dose toxicity is determined to be 1000 mg/kg bw/day.

It can reasonably be deduced that Methylparaben does not exert systemic toxic effects after dermal application and thus does not have to be classified, because the LD50 of Methylparaben after the administration of a single oral dose was set at 2,100 mg/kg bw in rats. The NOAEL for repeated dose toxicity was set as 1000 mg/kg bw/day based on the results of the OECD 408 and OECD 422 Study. Furthermore the substance does not have to be classified as skin irritating and it is unlikely that higher amounts than tested in the acute oral toxicity study will be systemically available via the intact skin barrier. Therefore, testing is not scientifically necessary.

 

In accordance with Section 1.2 of REACH Annex XI, there is sufficient weight of evidence from several independent sources of information leading to the conclusion that Methylparaben does not exert systemic toxic effects after acute inhalation exposure and thus does not have to be classified, because

- the LD50 value for acute oral toxicity of Methylparaben is >2000 mg/kg bw,

- Methylparaben does not have to be classified as skin irritating, and

- inhalation to consumer is very unlikely to occur, since the substance is embedded in cosmetical matrices for application by the consumer.

- risk characterisation calculations show that there is no concern for workers arising from the Methylparaben production (for calculation details please refer to “Waiver_Acute toxicity: inhalation”).

Therefore, it is concluded that testing of inhalation toxicity of Methylparaben is not scientifically necessary.