2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-09-2014 to 23-11-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Test guideline study conducted to GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: 12 weeks
- Weight at study initiation: males weighed 302 to 342g, the females weighed 185 to 224g
- Fasting period before study: none
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum- A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used
- Water (e.g. ad libitum): ad libitum
- Acclimation period: The animals were acclimatized for five days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

IN-LIFE DATES: From: To: 25-09-2014 to 23-11-2014

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

polyethylene glycol

Remarks:

(400)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least fifteen days in the dark at 4 °C. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Samples of the test item formulation were taken and analyzed for concentration of Glycyl
(CAS: 24085-08-3) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study.
- Concentration in vehicle (mg/mL): 0 (control), 25 (low), 75 (intermediate), 250/188/125 (high)
- Amount of vehicle (if gavage): 4 mL/kg of Polyethylene glycol 400

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Results show the formulations to be stable for at least fifteen days in the dark at 4 °C. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.

Samples of the test item formulation were taken and analyzed for concentration of Glycyl (CAS: 24085-08-3) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 25. The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study.

Duration of treatment / exposure:

Males- 43 days
Females- 40-54 days

Frequency of treatment:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000/750/500 mg/kg bw/day
Basis:
other: Initial treatment at 1000 mg/kg bw/day resulted in the termination of one m; dose lowered to 750 mg/kg bw/day from Day 2. Termination of 1 f on day 3 - dose reduced to 500 mg/kg bw/day from day 4.

No. of animals per sex per dose:

12 (twelve)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based upon dose range finding study
- Rationale for selecting satellite groups: The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

- Section schedule rationale (if not random): Samples of tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week, at weekends (except for females during parturition where applicable). All observations
were recorded.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait
Hyper/Hypothermia
Tremors
Skin color
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behavior
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OTHER:

Oestrous cyclicity (parental animals):

A vaginal smear was prepared for each female and the stage of estrus was recorded.

Sperm parameters (parental animals):

Parameters examined in male parental generations: [testis weight, epididymis weight, prostate weight, seminal vesicles weight, sperm count in testes, sperm count in epididymides, other: testes morphology]

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- 5/sex/litter/dose; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring: litter size, sex ratio and viability and offspring growth and development.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Postmortem examinations (parental animals):

SACRIFICE
Surviving adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. Surviving adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.

GROSS NECROPSY
- For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora
lutea were also counted.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown with an * were weighed from all remaining animals:

Adrenals
Prostate*
Brain
Seminal vesicles*
Epididymides
Spleen*
Heart
Testes*
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries*
Uterus (weighed with Cervix)*
Pituitary (post fixation)*

Postmortem examinations (offspring):

SACRIFICE
- Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown with an * were preserved from all remaining animals:

Adrenals
Muscle (skeletal)
Aorta (thoracic)
Ovaries*
Bone & bone marrow (femur including stifle joint)
Pancreas
Bone & bone marrow (sternum)
Pituitary*
Brain (including cerebrum, cerebellum and pons)
Prostate*
Caecum
Rectum
Coagulating gland*
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles*
Epididymides* (preserved in Modified Davidsons fluid)
Skin (hind limb)
Esophagus
Spinal cord (cervical, mid-thoracic and lumbar)
Eyes (fixed in Davidson's fluid)
Gross lesions*
Spleen
Heart
Stomach
Ileum (including peyer’s patches)
Thyroid/parathyroid
Jejunum
Trachea
Kidneys
Testes* (preserved in Modified Davidsons fluid)
Liver
Thymus
Lungs (with bronchi) (lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative)
Urinary bladder
Lymph nodes (mandibular and mesenteric)
Uterus/Cervix*
Mammary gland*
Vagina*


ORGAN WEIGHTS
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown with an * were weighed from all remaining animals:
Adrenals
Prostate*
Brain
Seminal vesicles*
Epididymides*
Spleen
Heart
Testes*
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries*
Uterus (weighed with Cervix)*
Pituitary (post fixation)*

Statistics:

quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the Provantis Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed:
homogeneity of variance from mean values : Bartlett’s test
Intergroup variances:ANOVA, or if required, ANCOVA
transformed data were analyzed for significant effect using the Williams Test (parametric) or Shirley Test (nonparametric)
non-homogeneity of means: stepwise Dunnett’s (parametric) or Steel (non-parametric) for significant difference from the control group.
pair-wise tests : Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing.
distribution of the data : Shapiro-Wilk normality test,
assessment of data homogeneity: Bartlett's test.
parametric analysis:(ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett's test.
non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test

Reproductive indices:

Mating Performance and Fertility

The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices
For each group the following were calculated:

Mating Index (%) = [Number of animals mated/Number of animals paired] * 100
Pregnancy Index (%) = [Number of pregnant females/Number of animals mated]* 100

Gestation and Parturition Data

The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = [number of femailes delivering live offspring/Number of pregnant females]*100

Offspring viability indices:

Viability Index (%) = [Number of offspring alive on Day 4/Number of offspring alive on Day 1]*100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day- clonic convulsions (4males) with one early termination.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings considered to be treatment related were observed for the liver, kidneys and thyroid glands.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality
There were four unscheduled deaths on the study.

At 1000 mg/kg bw/day, male number 79 showed clonic convulsions and staining around the mouth on the first day of dosing, the severity of the convulsions observed were considered sufficient for this animal to be terminated for animal welfare considerations. Necropsy and subsequent microscopic evaluation of the tissues did not reveal any obvious underlying reason for the condition of the animal.

At 1000/750 mg/kg bw/day, female number 90 showed clonic convulsions on Day 3; the severity of the convulsions observed were considered sufficient for this animal to be terminated for animal welfare considerations. Necropsy and subsequent microscopic evaluation of the tissues did not reveal any obvious underlying reason for the condition of the animal.

At 300 mg/kg bw/day, male number 55 was found dead on Day 13, no clinical signs had been apparent for this animals prior to its’ death. Necropsy findings included sloughing of the glandular region of the stomach, enlarged liver, reddened adrenals, reddened lungs, reddened mandibular lymph nodes, reddened salivary glands, reddened mammary gland, gaseous distension of the caecum and green discoloration of the skin (hind limb). Microscopic findings included congestion in the lungs, mammary gland, mandibular lymph nodes and salivary glands, slight granulolymphocytic inflammatory infiltrate in the trachea, slight lymphoid hyperplasia for the mesenteric lymph node and autolysis of the stomach but did not reveal any obvious cause for the death of the animal.

At 300 mg/kg bw/day, male number 56 showed piloerection, hunched posture and staining around the mouth on Day 25. These clinical signs persisted to the following morning when lethargy, decreased respiratory rate, pallor of the extremities and disrrhoea were also apparent and the animal was terminated early for animal welfare considerations. Necropsy revealed reddened lungs and black contents in the stomach, jejunum, ileum and caecum but no microscopic findings were apparent for these tissues and histopathological evaluations did not determine the cause of the decline in the clinical condition for this animal.

Clinical Observations
At 1000 mg/kg bw/day, treatment on Day 1 was associated with clonic convulsions for four males, with one of these males having to be terminated early. Two of the males showing convulsion also showed hunched posture. Following the lowering of the dosage to 750 mg/kg bw/day on Day 2 and then to 500 mg/kg bw/day on Day 4, no similar signs were apparent for the high dosage males. For females at 1000 mg/kg bw/day, treatment on Day 1 was associated with clonic convulsions for one animal and hunched posture for five animals. Following the lowering of dosage to 750 mg/kg bw/day, hunched posture persisted for two of these affected females, with one also showing clonic convulsions on Day 2. A further female showed hunched posture and clonic convulsions on Days 2 and 3 and lethargy, hunched posture and piloerection on Day 3. Additionally on Day 3, another female at this dosage showed clonic convulsions and had
to be terminated early. Following the lower of the dosage to 500 mg/kg bw/day, no similar signs were apparent for high dosage females.

For surviving animals at 1000/750/500 mg/kg bw/day, treatment was associated with increased post-dosing salivation, with all animals being affected to some extent although the individual incidence showed great variation. Such salivation is frequently observed when a slightly irritant or unpalatable test item is administered via the oral gavage route and is generally considered to reflect distaste of the dosing formulations rather than any systemic effect of treatment. Two
males at 1000 mg/kg bw/day showed noisy respiration on Day 1 and a further high dosage male also showed noisy respiration on Day 30, when receiving 500 mg/kg bw/day. This finding most probably represents accidental inspiration of the test item formulations during the dosing procedure and at the incidence and duration observed on this study was considered not to indicate a systemic effect of treatment.

At 300 mg/kg bw/day, clinical signs were mainly restricted to that previously described for decedent male number 56. One female showed scabbing during the study, otherwise there were no clinical signs apparent for surviving animals at this dosage.

No clinical signs were apparent for animals treated at 100 mg/kg bw/day.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

Body Weight
There was considered to be no effect of treatment on body weight gain for males at 100, 300 or 1000/750/500 mg/kg bw/day. Body weight gains of the treated animals were generally superior to control and the differences in body weight gain that did attain statistical significance were considered to reflect normal biological variation.

There was considered to be no effect of treatment on body weight gain for females during the pre-pairing, gestation and lactation phases of the study at 100, 300 or 1000/750/500 mg/kg bw/day.

There was considered to be no effect of treatment on food consumption or food conversion efficiency for males at 100, 300 or 1000/750/500 mg/kg bw/day. Food intake for treated males appeared higher than control during the post-pairing phase but was consistent with the body weight gain and in the absence of any accompanying effect on food conversion efficiency, this finding was considered to reflect normal biological variation.

There was considered to be no effect of treatment on food consumption or food conversion efficiency for females during the pre-pairing, gestation and lactation phases of the study at 100, 300 or 1000/750/500 mg/kg bw/day.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 500 mg/kg bw/day.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Testes morphology did not reveal any disturbances of spermatogenic staging.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating
There was no effect of treatment on mating performance at 100, 300 or 1000/750/500 mg/kg bw/day.

Fertility
Fertility was unaffected by treatment at 100, 300 or 1000/750/500 mg/kg bw/day.

Gestation Lengths
Gestation length was unaffected by treatment at 100, 300 or 1000/750/500 mg/kg bw/day.

ORGAN WEIGHTS (PARENTAL ANIMALS)
For males at 100 mg/kg bw/day and both sexes at 300 and 1000/750/500 mg/kg bw/day, there was a statistically significant increase in absolute and body weight relative liver weights compared with control.

For females at 100 mg/kg bw/day and both sexes at 300 and 1000/750/500 mg/kg bw/day, there was a statistically significant increase in absolute and body weight relative kidney weights compared with control.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic necropsy of surviving animals revealed enlargement of the liver for the majority of males at 1000/750/500 mg/kg bw/day. Similar enlargement of the liver was apparent for one
female at this dosage, three males and one female at 300 mg/kg bw/day and one male at 100 mg/kg bw/day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic findings, considered to be treatment related but adaptive in nature, were observed for the liver, kidneys and thyroid glands.

For the liver, diffuse midzonal/centrilobular hypertrophy was observed for both sexes at 100, 300 and 1000/750/500 mg/kg bw/day; the incidence and severity tending to increase with dosage.

For the kidneys, an increase incidence of cortical hyaline droplets, compared with control, was apparent for males at 100, 300 and 1000/750/500 mg/kg bw/day, with the highest severity being observed in the high dosage group.

For the thyroid, an increase incidence of diffuse follicular hypertrophy/hyperplasia at minimal/slight degree was observed for both sexes at 300 and 1000/750/500 mg/kg bw/day; the incidence and severity tending to increase with dosage.

OTHER FINDINGS (PARENTAL ANIMALS)

Dose descriptor:

NOAEL

Effect level:

ca. 100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: two deaths observed at 300 mg/ kg bw/day with unknown aetiology.

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At 1000/750/500 mg/kg bw/day, one litter show total litter loss on the day of parturition and another on Day 5 of lactation; the significance of these litter losses in the absence of any increased mortality amongst remaining litters was uncertain.

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significant increase in absolute and body weight relative liver weights compared with control (m). Significant increase in absolute and body weight relative kidney weights compared with control (f).

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings, considered to be treatment related but adaptive in nature, were observed for the liver, kidneys and thyroid glands.

VIABILITY (OFFSPRING)
At 1000/750/500 mg/kg bw/day, offspring survival from Day 1 was slightly inferior to control resulting in lower litter size at Day 4; differences from control did not attain statistical significance and were principally attributable to one litter which showed total litter loss on Day 5 post partum. However, the difference in offspring survival and litter size on Day 4 has to be viewed in the context of an earlier total litter loss on the day of parturition for one female at this dosage.

CLINICAL SIGNS (OFFSPRING)
The clinical sign and necropsy findings apparent for offspring on the study were typical for the age observed and did not indicate any obvious effect of offspring development at 100, 300 or 1000/750/500 mg/kg bw/day. At 1000/750/500 mg/kg bw/day, there was a higher incidence of offspring showing clinical signs; although these were concentrated in only few isolated litters they were suggestive of possible lower maternal care at this dosage.

BODY WEIGHT (OFFSPRING)
There was considered to be no clear effect of maternal treatment on offspring body weights and litter weights at Day 1 or on subsequent offspring body weigh, body weight gain and litter weights on Day 4 post partum at 100, 300 or 1000/750/500 mg/kg bw/day. Offspring performance during assessment of surface righting appeared to be unaffected by maternal treatment at all dosages.

At 1000/750/500 mg/kg bw/day, litter weight and offspring body weight and body weight gain for both sexes on Day 4 was lower than control; differences from control did not attain statistical significance and were principally attributable to one litter which showed total litter loss on Day 5 post partum. When this litter was excluded, offspring body weight and body weight gain on Day 4 was similar to control and did not indicate any adverse effect of maternal treatment on offspring growth and development. Slightly inferior performance during assessment of surface righting, which failed to attain statistical significance, was also attributable to the same litter, and was similar to control when this litter was excluded.

GROSS PATHOLOGY (OFFSPRING)
Necropsy findings apparent for offspring were typical for the age observed and did not indicated any effect of maternal treatment on offspring development at 100, 300 or 1000/750/500 mg/kg bw/day.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

ca. 300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect on growth, survival or development of the offspring

Reproductive effects observed:

not specified

Conclusions:

The oral administration of Glycyl (CAS: 24085-08-3) to rats by gavage, at dose levels of up to 1000 mg/kg bw/day resulted in adverse clinical signs at the high dosage (1000 and 750 mg/kgbw/day). Following the final reduction of the high dosage to 500 mg/kg bw/day treatment was better tolerated, however, there were two deaths at 300 mg/kg bw/day, the aetiology of which was unknown. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity on this study was therefore considered to be 100 mg/kg bw/day. Due to the equivocal nature of findings for isolated litters at the high dosage, it is difficult to state with certainty whether reproductive effects have been observed at this dosage. The No Observed Effect Level (NOEL) for reproduction, including the growth, survival and development of the offspring was therefore considered to be 300 mg/kg bw/day.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 500 mg/kg bw/day. Initial treatment at 1000 mg/kg bw/day resulted in adverse clinical signs on Day 1 resulting in the termination of one male; the high dosage was lowered to 750 mg/kg bw/day from Day 2. Adverse clinical signs remained apparent at 750 mg/kg bw/day resulting in the termination of one female due to animal welfare considerations. Consequently, the high dosage was further lowered to 500 mg/kg bw/day from Day 4 of the study. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on at least five selected males and females from each dose group.

Surviving adult males were terminated on Day 43 or Day 44, followed by the termination of all surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses

Mortality

At 1000 mg/kg bw/day, one male was killed on Day 1 after showing clonic convulsions and staining around the mouth. Following the reduction of this dosage to 750 mg/kg bw/day, one female was killed after showing clonic convulsions on Day 3. Necropsy and subsequent microscopic evaluation of the tissues did not reveal any obvious underlying reason for the condition of these animals.

At 300 mg/kg bw/day, one male was found dead on Day 13 without any clinical signs being apparent. Necropsy revealed sloughing of the glandular region of the stomach, enlarged liver, reddened adrenals, lungs, mandibular lymph nodes, salivary glands and mammary gland, gaseous distension of the caecum and green discoloration of the skin (hindlimb). Microscopic findings included congestion in the lungs, mammary gland, mandibular lymph nodes and salivary glands, slight granulolymphocytic inflammatory infiltrate in the trachea, slight lymphoid hyperplasia for the mesenteric lymph node and autolysis of the stomach but did not reveal any obvious cause for the death of the animal.

At 300 mg/kg bw/day, one male was killed on Day 26 after showing piloerection, hunched posture, staining around the mouth, lethargy, decreased respiratory rate, pallor of the extremities and diarrhoea. Necropsy revealed reddened lungs and black contents in the stomach, jejunum, ileum and caecum although no microscopic findings were apparent for these tissues and histopathological evaluations did not reveal any obvious underlying reason for the condition of this animal.

Clinical Observations

At 1000 mg/kg bw/day, treatment was associated with clonic convulsions and hunched posture for some animals on Day 1. Following the lowering of the dosage to 750 mg/kg bw/day on Day 2, no similar signs were apparent for males but clonic convulsions and hunched posture was still observed for some females. Following the lowering of the dosage to 500 mg/kg bw/day, no similar signs were apparent for either sex.

For surviving animals at 1000/750/500 mg/kg bw/day, treatment was associated with increased post-dosing salivation, with all animals being affected to some extent.

There were no clinical signs considered to be of toxicological importance at 100 or 300 mg/kg bw/day.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters at 100, 300 or 1000/750/500 mg/kg bw/day.

Functional Performance Tests

There were no changes in functional performance considered to be related to treatment at 100, 300 or 1000/750/500 mg/kg bw/day.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores.

Body Weight

There was considered to be no effect of treatment on body weight gain for either sex at 100, 300 or 1000/750/500 mg/kg bw/day.

Food Consumption and Food Conversion Efficiency

There was considered to be no effect of treatment on food consumption or food conversion efficiency for either sex at 100, 300 or 1000/750/500 mg/kg bw/day.

Water Consumption

There was no obvious effect on water intake for either sex at 100, 300 or 1000/750/500 mg/kg bw/day.

Reproductive Performance

Mating

There was no effect of treatment on mating performance at 100, 300 or 1000/750/500 mg/kg bw/day.

Fertility

Fertility was unaffected by treatment at 100, 300 or 1000/750/500 mg/kg bw/day.

Gestation Lengths

Gestation length was unaffected by treatment at 100, 300 or 1000/750/500 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was considered to be no clear effect of treatment on the numbers of corpora lutea or implantations, pre- and post-implantation losses, litter size or sex ratio at birth/Day 1 or subsequent offspring survival to Day 4 of age at 100, 300 or 1000/750/500 mg/kg bw/day. However at 1000/750/500 mg/kg bw/day, one litter show total litter loss on the day of parturition and another on Day 5 of lactation; the significance of these litter losses in the absence of any increased mortality amongst remaining litters was uncertain.

Offspring Growth and Development

There was considered to be no clear effect of maternal treatment on offspring body weights, litter weights and surface righting ability at Day 1 or subsequently offspring body weight, body weight gain and litter weights on 4 post partum at 100, 300 or 1000/750/500 mg/kg bw/day.

The clinical sign and necropsy findings did not indicate any obvious effect on offspring development at 100, 300 or 1000/750/500 mg/kg bw/day. At 1000/750/500 mg/kg bw/day there was a higher incidence of offspring showing clinical signs although these were concentrated in only a few isolated litters.

Laboratory Investigations

Hematology

There were no obvious effects of treatment on hematology parameters for either sex at 100, 300 or 1000/750/500 mg/kg bw/day.

Blood Chemistry

There were no adverse effects of treatment on the blood chemical parameters for either sex at 100, 300 or 1000/750/500 mg/kg bw/day.

Pathology

Necropsy

Macroscopic necropsy of surviving animals revealed enlargement of the liver for the majority of males at 1000/750/500 mg/kg bw/day. Similar enlargement of the liver was apparent for one female at this dosage, three males and one female at 300 mg/kg bw/day and one male at 100 mg/kg bw/day.

Organ Weights

For males at 100 mg/kg bw/day and both sexes at 300 and 1000/750/500 mg/kg bw/day, there was a statistically significant increase in absolute and body weight relative liver weights compared with control.

For females at 100 mg/kg bw/day and both sexes at 300 and 1000/750/500 mg/kg bw/day, there was a statistically significant increase in absolute and body weight relative kidney weights compared with control.

Histopathology

Microscopic findings, considered to be treatment related but adaptive in nature, were observed for the liver, kidneys and thyroid glands.

For the liver, diffuse midzonal/centrilobular hypertrophy was observed for both sexes at 100, 300 and 1000/750/500 mg/kg bw/day; the incidence and severity tending to increase with dosage.

For the kidneys, an increase incidence of cortical hyaline droplets, compared with control, was apparent for males at 100, 300 and 1000/750/500 mg/kg bw/day, with the highest severity being observed in the high dosage group.

For the thyroid, an increase incidence of diffuse follicular hypertrophy/hyperplasia at minimal/slight degree was observed for both sexes at 300 and 1000/750/500 mg/kg bw/day; the incidence and severity tending to increase with dosage.

Conclusion

The oral administration of Glycyl (CAS: 24085-08-3) to rats by gavage, at dose levels of up to 1000 mg/kg bw/day resulted in adverse clinical signs at the high dosage (1000 and 750 mg/kg bw/day). Following the final reduction of the high dosage to 500 mg/kg bw/day treatment was better tolerated, however, there were two deaths at 300 mg/kg bw/day, the aetiology of which was unknown. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity on this study was therefore considered to be 100 mg/kg bw/day. Due to the equivocal nature of findings for isolated litters at the high dosage, it is difficult to state with certainty whether reproductive effects have been observed at this dosage. The No Observed Effect Level (NOEL) for reproduction, including the growth, survival and development of the offspring was therefore considered to be 300 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

There is a single screening study addressing reproductive performance. THis study was performed according to OECD guidelines and GLP and as such the qulity is deemed appropriate.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Treatment of the parental animals with up to the adjusted top dose of 500 mg/kg bw/day had no effect on mating performance, Fertility or Gestation Length at the top dose tested.

There was considered to be no clear effect of treatment on the numbers of corpora lutea or implantations, pre- and post-implantation losses, litter size or sex ratio at birth/Day 1 or subsequent offspring survival to Day 4 of age at up to the adjusted top dose of 500 mg/kg bw/day. However at the maximum dose tested, one litter show total litter loss on the day of parturition and another on Day 5 of lactation; the significance of these litter losses in the absence of any increased mortality amongst remaining litters was uncertain.

There was considered to be no clear effect of maternal treatment on offspring body weights, litter weights and surface righting ability at Day 1 or subsequently offspring body weight, body weight gain and litter weights on 4 post partum at up to the adjusted top dose of 500 mg/kg bw/day.

The clinical sign and necropsy findings did not indicate any obvious effect on offspring development at 100, 300 or 500 mg/kg bw/day. At 500 mg/kg bw/day there was a higher incidence of offspring showing clinical signs although these were concentrated in only a few isolated litters.

Short description of key information:

Reproductive Toxicity Screening: Subacute Reproductive Test (oral gavage), rat (Wistar-Han) m/f (OECD guideline 422, GLP): NOAEL: 100 mg/kg bw/day (parent); NOEL 300 mg/kg bw/day (offspring).

Justification for selection of Effect on fertility via oral route:

single screening study conducted according to OECD guideline 422 and GLP

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

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

At the maximum concentration tested, there was no evidence that the test substance had an adverse effect upon gonad (histo)pathology, reproductive performance or fetal/pup development. The total loss of two litters was observed at the highest dose tested, however this could not be directly attributed to the substance given a lack of effects observed in the surviving litters. Based upon the results of the screening study the classification criteria for reproductive toxicity or development set out in Commission Regulation 1272/2008/EC are not met.