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Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2003 - Oct 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrazolyl-valinesteramid (3-methyl-2-{pentanoyl-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-amino}-butyric acid benzyl ester)
EC Number:
604-047-3
Cas Number:
137863-20-8
Molecular formula:
C31 H35 N5 O3
IUPAC Name:
Tetrazolyl-valinesteramid (3-methyl-2-{pentanoyl-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-amino}-butyric acid benzyl ester)

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat: Wistar Crl:(WI} BR (outbred, SPF-Quality). Untreated animals and virgin females were used at initiation of the study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Number of animals 48 males and 40 females.
Age at Start of Treatment Approximately 6 weeks.
Acclimatisation At least 5 days prior to start of treatment.
Health check A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
Randomisation Prior to commencement of tmatment, by computer-generated random algorithm according 1:0 body weight, with all animals within ± 20% of the sex mean.
Identification Earmark and tattoo.

Accommodation
Group housing of 5 animals per sex in stainless steel suspended cages (55 x 34 x 21.5 cm height) with wire mesh floors. The last and last but one male of each group (males 11 +12, 23+24, 35+36, 47+48) were group housed of 2 males in s·:ainless steel suspended cages (55 x 34 x 21.5 cm height) with wire mesh floors.
During the pre-mating period, males of this 90-day study (Part A) and females of the onegeneration study (Part B) were housed in separate rooms
During the mating procedures, the males of this study were caged together with two females from the same treatment group from the one-generation study (Part B) on a two-to-one-basis (two females with one male) in suspended stainless steel ages with wire mesh floors.
During post-mating, males were housed individually in stainless steel suspended cages (55 x 34 x 21.5 cm height) with wire mesh floors.
During overnight activity monitoring, animals were housed individually in Macrolon plastic cages (MIii type; height 15 cm.) with Woody Clean bedding (Woody-Clean type 3/4; Tecnilab-BMI BV, Someren, The Netherlands).
Results of bedding analyses for contaminants were examined and archived.
Diet
Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1 ), Lage, Germany). Each batch was analysed for nutrients and cortaminants were analysed on a regular basis. Results were examined and archived.
Water
Free access to tap water. Certificates of analysis (performed quarterly) were examined and then retained in the NOTOX archives.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a rubber catheter attached to a plastic disposable syringe.
Vehicle:
propylene glycol
Remarks:
Propylene glycol, specific gravity 1.036
Details on oral exposure:
Oral gavage, using a rubber catheter attached to a plastic disposable syringe.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations was performed according to the following scheme:
Week of Week of 2004 Group Analysis (type of sample)
study**
Week 01 Week 03 2004 1 acc (M)
2 acc + hom + stab(t=o (TMB), stab(t=4. RT (M)
3 acc (M)
4 acc+ hom + stab(t=o (TMB), stab(t=4, RT (M)
Week 06 Week 08 2004 1 acc (M)
2 acc + hom (TM 3)
3 acc
4 acc + hom (TM 3)
Week 12 Week 14 2004 1 acc (M)
2 acc + hom (TM 3)
3 acc
4 acc + hom (TM 3)
Week 14* Week 16 2004 4 hom (TMB)
Week 17 Week 19 2004 1 acc (M)
2 acc + hom (TM 3)
3 acc
4 acc + hom (TM 3)
Duplicate samples were analysed
acc=accuracy, hom=homogeneity, stab=stability {hours),
T=top, M=middle, B=bottom position of container,
RT=room temperature

* These additional analyses were performed as chemical analysis of study week 12 showed the group 4 formulation to be inhomogeneous (low accuracy level of duplicate top samples).
** Samples of week 01 and 06 of study were taken from formulations used during the 90-day study (part A), samples of week 12 and 14 of study were taken from formulations used during the 90-day and one-generation study (Part A+B), and samples of week 17 of study were taken from formulations used during the one-generation study (Part B).
Duration of treatment / exposure:
For at least 90 days continuoJsly (10 weeks of pre-mating, at least 1 day of mating, 3 weeks of i:ost-mating), up to the day prior to necropsy.
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Group Dose level Number of animals
mg/kg/day Males Females
1 Main 0 10 10
1 Additional 0 2 -

2 Main 10 10 10
2 Additional 10 2 -

3 Main 50 10 10
3 Additional 50 2 -

4 Main 250 10 10
4 Additional 250 2 -

Control animals:
yes, concurrent vehicle
Details on study design:
To reduce the number of animals, it was decided to combine a 90-day toxicity study (Part A) with a one-generation study (Part B) performed simultaneously with the same test item and dose levels at NOTOX.
This was done by using the male animals exposed for ten weeks during the 90-day toxicity study for mating with the female animals exposed for two weeks at the same dose level during the one-generation study.
The treatment period for the males and females of the 90-day toxicity study was at least 13 weeks continuously (10 weeks of pre-mating, at least 1 day of mating, 3 weeks of post-mating).
The treatment period for the females of the one-generation study was at least 8 weeks continuously (2 weeks of pre-mating, at least 1 day of mating, 3 weeks of post-coitum, 3 weeks of post-partum).
For the 90-day toxicity study 48 male rats and 40 female r3ts were used; for the one-generation study 96 female rats were used.
The report is divided in two parts:
Part A = 90-day toxicity study
Part B = one-generation reproduction toxicity study

Examinations

Observations and examinations performed and frequency:
- Mortality/ Viability At least twice daily.
- Clinical signs At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals,
this was also performed outsidei the home cage in a standard arena. The time of onset, degree and duration were recorded. All symptoms were recorded ar d graded according to fixed scales: Maximum grade 1: grade 0 = absent, grade 1 = present Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe
- Functional Observations After the mating period, the following tests were performed on the MAIN males and MAIN females:
hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
- Body weights Weekly
- Food consumption Weekly
- Water consumption Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
- Ophthal moscopic
Sacrifice and pathology:
When almost all females of the one-generation study (Part B) delivered their litter, and there was no need to consider additional mating, all males and tamales of this 90-day toxicity study (Part A) were sacrificed. The animals were fasted overnight (with a maximum of 20 hours) before necropsy. All animals surviving to the end of the ob:;ervation period were deeply anaesthetised using iso-flurane and subsequently exsanguinated.
The additional males were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs. Descriptions of all macroscopic abnormalities were recorded.
Samples of the following tissues and organs were collectej and fixed in neutral phosphate buffered 4% formaldehyde solution (v/v):
All gross lesions
Coagulation gland
Epididymides*
Pituitary gland
Prostate gland
Seminal Vesicles
Testes*
The main males and main females were necropsied and descriptions of all macroscopic abnormalities were recorded.

Examination of reproductive organs of males
Organ weights
Histotechnology
Histopathology

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No mortality occurred during the study period.
An increased incidence of salivation was observed for males treated at 250 mg/kg and females treated at 50 and 250 mg/kg.
Salivation was observed at a much lower incidence in animals of the control and 10 mg/kg groups.
Incidental findings that were noted included muscle twitchi 19, alopecia and scabs of several body parts, swelling of the penis, broken lower incisors, lethargy, clonic spasms, rales, chromodacryorrhoea of the left eye, exophthalmos of the r ght eye, a wound on the tail, thickened area of the nose, and brown staining of the back. These findings are commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were unaffected by treatment up to 250 mg/kg body weight/day.
The increased body weights observed for several days for males of all treated groups were considered unrelated to treatment as no clear dose-response relationship was apparent.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects were observed for ophthalmoscopy with treatment of PBS 859 DS up to 250 mg/kg bw/day.
At pretest, no ophthalmoscopic abnormalities were observed.
At the end of treatment, one male of the control group showod focal lens opacity of the right eye and one female of the 250 mg/kg dose group showed focal Iens opacity of both eyes.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Several haematological parameters were affected by treatmi~nt at 250 mg/kg. These changes
(not always statistical significant) were observed for males and/or females and consisted of
effects on white blood cells (decreased levels of neutrophils and increased levels of
lymphocytes), effects on the haematopoetic system (decreaBed levels of erythrocytes count,
haemoglobin, haematocrit, mean corpuscular volume, and increased levels of platelet count and
red cell distribution width), and effects on clotting parameter:, (decreased prothrombin time and
partial thromboplastin time).
Statistically significant differences arising between controls c1nd animals receiving 10 and 50
mg/kg/day were considered to have arisen by chance as no dose response relationship was
apparent, and thus not to represent a change of biological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Some clinical biochemistry parameters were affected by treatment at 250 mg/kg. For males, statistical significant increased levels of bilirubin, cholesterol and potassiuni were observed. And for females, statistical significant decreased levels of aspartate aminotransferase and increased levels of bilirubin, cholesterol and phosphorus were observed.
At 10 and 50 mg/kg, statistical significant increased levels of bilirubin were observed for females.
The statistical significant decreased level of potassium for females treated at 10 mg/kg was considered to be of no toxicological significance in the absence of a treatment-related distribution.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No changes were observed in hearing ability, pupillary refle)(, static righting reflex and grip strength in the animals treated with PBS 859 DS, when com Jared to control animals.
The variation in motor activity did not indicate a relation with treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased liver weight (absolute and relative) was observed for males and females treated at 250 mg/kg. This was considered to be treatment related.
Increased liver weight (absolute) was observed for males treated at 50 mg/kg. Decreased heart weight (absolute and/or relative) was observed for males at 10, 50 and 250 mg/kg and for females at 250 mg/kg. Increased kidneys weight (absolute) was observed for males at 50 and 250 mg/kg and females at 250 mg/kg. Increased testes wnight (absolute) was observed for males at all treatment groups, and increased pituitary weight (absolute) was observed for females at 250 mg/kg. These changes were probably due to the higher terminal body weight and in the absence of any organ dysfunction and/or dose l'esponse relationship considered to be of no toxicological significance.
All other statistical significant changes were considered unrelated to treatment with PBS 859 OS, as no dose-response relationship was apparent.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlargement of the liver was noted in two group 4 (250 mq/kg/day) females. This was confirmed by microscopic examination.
Incidental findings included (dark) red discolouration of thn caecum, mandibular lymph nodes and thymus, tan foci on the preputial glands, enlarged ma ridibular lymph nodes and spleen, dark red foci on the thymus and liver, seminal vesicles rec uced in size, pelvic dilation of the kidneys, greenish soft nodule on the epididymides, alopecia, liver grown together with the diaphragm, watery-clear cyst on the right uterus horn, thickened uterus, and adrenal glands grown together with the kidneys. These findings are occaf,ionally seen among rats used in these types of studies. In the absence of a treatment-related dis:ribution they were considered changes of no toxicological significance.
Fluid in the uterus, found in females of groups 1, 3 and 4, is related to a stage in the oestrous cycle and is a normal finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sperm Examination
No treatment related effects on sperm count, motility and morphology were observed.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
gross pathology
mortality
ophthalmological examination
organ weights and organ / body weight ratios

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
blood
erythrocyte development
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
endocrine system
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for PBS 859 OS of 50 mg/kg/day was established.