L-Valine, N-(1-oxopentyl)-N-[[2'-(2H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-, phenylmethyl ester

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Effects on fertility

Description of key information

The NOAELs in a 90-day one-generation reproductive toxicity study in rats were 50 mg/kg/day (maternal toxicity), 250 mg/kg/day (maternal fertility), and 10 mg/kg/day (breeding and developmental effects). For general toxicity endpoints, the NOAELs in repeated dose animal studies were 50 mg/kg/day (90-day toxicity study in rats) and 100 mg/kg/day (28-day study in rats, dietary).

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2013 - Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

GLP compliance:

yes

Limit test:

no

Justification for study design:

90-day toxicity study combined with one-generation reproduction toxicity study with PBS 859 OS administered by oral gavage in Wistar rats.
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 simultanecusly with the same test item and dose levels at NOTOX.
The nature and purpose of the one-generation study was to assess the effect of the test substance on male and female reproductive performance, such as gonadal function, oestrus cycle, mating behaviour, conception, parturition, lactation and weaning, when administered by gavage during one complete reproductive cycle. The study might also provide preliminary information of the test substance about developmental toxic effects, such as neonatal morbidity, mortality, behaviour and teratogenesis.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat: Wistar Crl:(WI} BR (outbred, SPF-Quality). Untreated animals and virgin females were useci at initiation of the study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number of animals Fo 96 females
Age at Start of Treatment Fo 13 -14 weeks.
Acclimatisation Fo At least 5 days prior to start of treatment.
Health check Fo A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
Randomisation Fo At least 5 days before study Btart, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification Earmark and tattoo.

Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21 ± 3°C (actual range: 17.8- 25.5°C), a relative humidity of 30 - 70% (actual range: 28 - 85%) and 12 hours artificial light and
12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal level of
70% for relative humidity.
Based on laboratory historical data these conditions were considered not to have affected the study integrity.
Accommodation
Upon arrival, Fo-females were housed in groups of 4 females/cage in suspended stainless steel cages.
During the mating procedures, parental females were caged together with males from the same treatment group from the 90-day toxicity study (Part A) on a two-to-one-basis (two females with one male) in suspended stainless steel cages with wire mesh floors.
Females that mated were individually housed in labelled polcarbonate cages containing Woody Clean bedding (Woody-Clean type 3/4; Tecnilab-BMI BV, Someren, The Netherlands).
Certificates of analysis were examined and then retained in the NOTOX archives.
Offspring was kept with the dam until weaning.
In order to reduce environmental influences as much as possible, cages were arranged in a latin square design over the cage rack during the study period. D Jring mating and from 04 May 2004 onwards, cages were arranged per dose group. Each cage was identified with a colour-coded label according to dose group, showing the study number, animal identifications, and other experimental details. From arrival until mating, males of the 90-day toxicity study (Part A) and females of this one-generation study were housed in separate rooms. During the final stage of the pregnancy period (from day 16 of gestation onwards) and during lactation, paper (Enviro-dri,BMI, Someren, The Netherlands) was supplied to each dam for incorporation in the nest. The paper was analysed for contaminants. This was replaced when soiled.
Diet
Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1 ), Lage, Germany). Each batch was analysed for nutrients and contaminants were analysed on a regular basis. Results were examined and then retained in the NOTOX archives.
Water
Free access to tap water. Certificates of analysis (performed quarterly) were examined and then retained in the NOTOX archives.
Analysis of bedding, paper, diet and water did not reveal an)' findings that were considered to have affected study integrity.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

Oral gavage, using a rubber catheter attached to a plastic disposable syringe.
Formulations were placed on a magnetic stirrer during dosing.

Details on mating procedure:

Females were paired on a two-to-one-basis (two females with one male) with males from the same treatment group from the 90-day
toxicity study (Part A). See schedule next page. Each morning following pairirtg, a vaginal smear was prepared daily from each female and examined for the presence of spermatozoa. The day on which evidence of mating was found was designated day 0 of gestatiort (=day 0 post-coitum). Once mating occurred, the female was housed individually and vaginal smearing was discontinued. When both females paired with one male were
found to be pregnant, this mc1le was placed back in the 90-day toxicity study (Part A).

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:

The females were exposed for 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).

Frequency of treatment:

PBS 859 OS was administered by daily oral gavage to fermale Wistar rats at dose levels of 10, 50 or 250 mg/kg body weight/day.

Details on study schedule:

Acclimatisation: 03 March 2004
Start treatment: 08 March 2004
Start mating: 22 March 2004
Delivery of litters 13 to 27 April 2004
Culling of pups 19 April to 03 May 2004
Necropsy 23 March to 17 May 2004

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:

24 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The test substance was administered daily for at least 8 weeks by oral gavage to SPF-bred female Wistar rats. One control group and three treated groups were tested, each consisting of 24 females.

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 rats were used; for the one-generation study 96 female rats were used.

Positive control:

no

Parental animals: Observations and examinations:

The following parameters were evaluated: clinical signs daily; body weight and food consumption weekly; reproduction processes; breeding parameters; pup development; macroscopy at termination; organ weights on a selection of tissues.

Oestrous cyclicity (parental animals):

Mortality / Viability, Clinical signs, Body weights, Food consumption, Water consumption, Reproduction processes

Litter observations:

Each litter was examined to determine the following if practically possible:
- The numbers of live and dead pups at the First Litter Chuck(= check at day 1 of lactation) and daily thereafter (if possible, defects or cause of death were evaluated)
- The individual weight of all live pups on days 1, 4, 7, 14 and 21 of lactation
- Sex of all pups (by assessment of the ano-genital distance)
- The number of pups with physical or behavioural abnormalities daily

Postmortem examinations (parental animals):

Macroscopic examination, Organ weights

Postmortem examinations (offspring):

Macroscopic examination

Statistics:

For each dose group reproduction parameters were expre:;sed in two ways:
- As a mean (with standard deviation) of the number observed for each litter
- Relative to a second parameter and calculated on a total group basis

The following statistical methods were used to analyse the data:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-toone t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

An increased incidence of salivation was observed for females treated at 50 and 250 mg/kg. At 50 mg/kg salivation was observed during the first three we:eks of treatment and at 250 mg/kg salivation was observed during the complete treatment period.
Salivation was observed at a much lower incidence in animals of the control and 10 mg/kg groups.
Incidental findings that were noted included alopecia of several body parts, scabs, a wound, hunched posture, rales, piloerection, lean appearance, broken upper incisors, lethargy, uncoordinated movements, laboured respiration, pale appearance, and diarrhoea. 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:

mortality observed, treatment-related

Description (incidence):

Two females were killed in extremis.
Female 50 treated at 50 mg/kg was killed in extremis on day 16 of treatment (day 2 of mating).
This female showed lethargy, uncoordinated movements and laboured respiration just before necropsy. At necropsy, many dark red foci on the lungs was noted.
Female 90 treated at 250 mg/kg was killed in extremis on day 51 of treatment (a day after delivery). Sixteen pups were dead at first litter check and one pup was killed in extremis. The female showed hunched posture, piloerection, diarrhoea and lean appearance just before necropsy. No macroscopic finding was observed at necropsy.
No other unscheduled deaths 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 tmatment up to 250 mg/kg body weight/day.
Minor statistically significant differences arising between controls and animals receiving 50 and 250 mg/kg/day were considered to have arisen by chance as they were inconsistent over time, and were thus not considered to represent a change of biological significance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption (absolute and/or relative) of females tmated at 250 mg/kg was statistically significantly decreased during days 7-21 of lactation. In acdition, the mean over means for the lactation period was decreased at 250 mg/kg.
Minor statistically significant decreases in relative food consumption during post-coitum for group 3 and 4 females were very slight and were thus considered to be of no toxicological relevance.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Reproduction parameters were unaffected by treatment up to 250 mg/kg body weighday.
Mating performance, duration of gestation, fertility parame,ters, number of corpora lutea, and number of dead and living pups at first litter check were similar for the control and treated groups.
The decreases in duration of gestation for group 3 and 4 when compared to controls were considered to have arisen as a result of slightly high control values and in the absence of a dose response relationship considered to be of no toxicological significance.

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Breeding parameters were affected by treatment at 50 and 250 mg/kg body weight/day. These findings consisted of an increased number of postnatal loss and breeding loss, and thus decreased viability and weaning indices at these doses.
The increased postnatal loss and decreased viability index at 10 mg/kg was mainly due to the loss of 12 pups in one litter (number 34 ), and was thus comdered not to be toxicological relevant.
The number of living pups on day 4 and 21 post partum were similar for the control and treated groups.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental clinical symptoms consisted of (very) small, pale or cold appearance, blue discolouration and alopecia of several parts of the body, little or no milk, less hair, umbilical hernia, purple appearance, cannibalism, lean and lethargy. Macroscopic examination of the pups revealed small appearance, no milk, pelvic dilation of the right kidney, diaphragmatic hernia, constricted and enlarged spleen, and cannibalism.
No relationship with treatment was
established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of living pups on day 4 and 21 post partum were similar for the control and treated groups.
The increased postnatal loss and decreased viability index at 10 mg/kg was mainly due to the loss of 12 pups in one litter (number 34 ), and was thus cosidered not to be toxicological relevant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 50 and 250 mg/kg, body weights of pups was decreased when compared to the control group. This reached statistical significance on day 7 with treatment at 50 mg/kg, and during the complete lactation period with treatment at 250 mg/kg.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

One pup of the 250 mg/kg dose group (male 3 of dam 89) showed the heart enlarged and a ventricle-septum defect. This finding was considered incidental and was not considered to be caused by treatment.

Gross pathological findings:

not specified

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Incidental clinical symptoms consisted of (very) small, pale or cold appearance, blue discolouration and alopecia of several parts of the body, little or no milk, less hair, umbilical hernia, purple appearance, cannibalism, lean and lethargy. Macroscopic examination of the pups revealed small appearance, no milk, pelvic dilation of the right kidney, diaphragmatic hernia, constricted and enlarged spleen, and cannibalism.
No relationship with treatment was
established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

other: body weight/day, a delay in pup devHlopment

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

250 mg/kg bw/day

Treatment related:

no

Conclusions:

Based on the results in this one-generation study with only female parental animals, the definitive parental No Observed Adverse Effect Level (NOAEL) was established as being 50 mg/kg body weight/day.
The definitive reproductive NOAEL was established as being 250 mg/kg body weight/day.
The definitive breeding and developmental NOAEL was ef,tablished as being 10 mg/kg body weight/day

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The NOAELs in a 90-day one-generation reproductive toxicity study in rats were 50 mg/kg/day (maternal toxicity), 250 mg/kg/day (maternal fertility), and 10 mg/kg/day (breeding and developmental effects). For general toxicity endpoints, the NOAELs in repeated dose animal studies were 50 mg/kg/day (90-day toxicity study in rats) and 100 mg/kg/day (28-day study in rats, dietary).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Mortality was unaffected by the treatment.

250 mg/kg: decreased food consumption during lactation

Reproduction parameters were unaffected.

50 and 250 mg/kg: Increased postnatal loss, increased breeding loss and decreased body weights of pups during lactation were observed at 50 mg/kg/day and 250 mg/kg/day.

90 days; the females were exposed for at least 8 weeks continuously ( 2 weeks of pre-mating, at least one day of mating, 3 weeks of post-coitum, 3 weeks of post-partum)