Fatty acids, C18-unsatd., reaction products with acrylic acid and polyethylenepolyamines

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 12 July 2010 and 17 February 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 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

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-unsatd., reaction products with acrylic acid and polyethylenepolyamines

EC Number:

287-820-4

EC Name:

Fatty acids, C18-unsatd., reaction products with acrylic acid and polyethylenepolyamines

Cas Number:

85586-18-1

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

(Z,9Z)-N-(2-{2-[(8Z)-heptadec-8-en-1-yl]-4,5-dihydro-1H-imidazol-1-yl}ethyl)octadec-9-enimidic acid; 3-[(2-carboxyethyl)({2-[3-(2-carboxyethyl)-2-[(8Z)-heptadec-8-en-1-yl]imidazolidin-1-yl]ethyl})amino]propanoic acid; 3-{2-[(8Z)-heptadec-8-en-1-yl]-3-{2-[(E)-[(9Z)-1-hydroxyoctadec-9-en-1-ylidene]amino]ethyl}imidazolidin-1-yl}propanoic acid

Test animals

Species:

rat

Strain:

other: Wistar Han™:HsdRccHan™:WIST strain rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Source:
Wistar Han™:HsdRccHan™:WIST strain rats were obtained from a reputable supplier. For full details please see full study report.

- Age at study initiation:
Approximately 12 weeks old

- Weight at study initiation:
312 to 378 g (male); 193 to 244 g (female)

- Fasting period before study:
Not applicable

- Housing:
Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating 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:
The animals were allowed free access to food. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used throughout the study period. The diet was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

- Water:
Water intake was measured daily throughout the study (with the exception of the mating phase). Mains drinking water was supplied from polycarbonate bottles attached to the cage. The drinking water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

- Acclimation period:
Seven days

ENVIRONMENTAL CONDITIONS

- Temperature:
(°C): 21 ± 2

- Humidity (%):
55 ± 15

- Air changes (per hr):
At least fifteen air changes per hour

- Photoperiod (hr dark / hrs light):
12 hours continuous light and 12 hours darkness

IN-LIFE DATES:
First day of treatment: 14 July 2010 - Final necropsy day was 29 August 2010.

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

other: Distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in distilled water. The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 3180-0007). Results from the previous study showed the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly during the treatment period and stored at approximately +4ºC in the dark. Samples of each test material formulation were taken and analysed for concentration of test material at Harlan Laboratories Ltd., Shardlow, UK Analytical Services. The method used for analysis of formulations and the results obtained are given in the attached Appendix 26.

The results indicate that the prepared formulations were acceptable for the purpose of this study.

DIET PREPARATION
Not applicable

- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable

- Storage temperature of food:
No data

VEHICLE
Distilled water.

- Justification for use and choice of vehicle (if other than water):
Not applicable

- Concentration in vehicle:
0, 20, 60 and 200 mg/ml

- Amount of vehicle (if gavage):
5 ml/kg

- Lot/batch no. (if required):
Not applicable

- Purity:
Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity, stability and linearity determinations were performed under Harlan Laboratories Ltd project number 3180-0007.

The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

The concentration of the test material in the formulations was determined spectrophotometrically.

The test item formulations were diluted with methanol to give a final, theoretical test item concentration of approximately 0.05 mg/m

Standard solutions of test item were prepared in methanol at a nominal concentration of 0.05 mg/ml.

The test item formulations were sampled and analysed within one day of preparation

The results indicate that the prepared formulations were acceptable for the purpose of this study.

See attached Appendix 26 - Chemical Analysis of Test Material Formulations, Methods and Results

Details on mating procedure:

- M/F ratio per cage:
1/1 (Animals were paired on a 1 male: 1 female basis within each dose group)

- Length of cohabitation:
Up to 14 days

- Proof of pregnancy:
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 the oestrous cycle 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)

- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.:
No data

- Further matings after two unsuccessful attempts:
No data

- After successful mating each pregnant female was individually caged:
Mated females were housed individually during the period of gestation and lactation.

- Any other deviations from standard protocol:
Not applicable

Duration of treatment / exposure:

All males from all treatment groups were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Frequency of treatment:

Daily

Duration of test:

The in-life phase of the study was conducted between 14 July 2010 (first day of treatment) and 29 August 2010 (final necropsy).

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg/day (20 mg/ml)
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg/day (60 mg/ml)
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg/day (200 mg/ml)
Basis:
actual ingested

No. of animals per sex per dose:

0 mg/kg/day - Control 10 animals per sex per.
100 mg/kg/day - 10 animals per sex.
300 mg/kg/day - 10 animals per sex.
1000 mg/kg/day - 10 animals per sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on results from a Fourteen day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Harlan Laboratories Ltd. Project Number 3180-0007).

- Rationale for animal assignment (if not random):
Random

- Rationale for selecting satellite groups:
Not applicable

- Post-exposure recovery period in satellite groups:
Not applicable

- Section schedule rationale (if not random):
Random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Yes see attached Table 1 and Appendix 1

- Time schedule:
- Immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, thirty minutes after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes (see above).
- Yes see attached Table 1 and Appendix 1

BODY WEIGHT:
- Yes see attached Tables 4 and 5 and Appendices 7 and 8

- Time schedule for examinations:
Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for
males until termination and weekly for females until mating was evident.

Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
- Yes see attached Table 9 and Appendix 9

- During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing
evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
- Yes see attached Table 10 and Appendix 9

- Food efficiency:
(the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
- Yes see attached Table 11 and Appendix 10

- Time schedule for examinations:
Water intake was measured daily from Day 1 (excluding mating).

OPHTHALMOSCOPIC EXAMINATION: Not applicable

HAEMATOLOGY AND CLINICAL CHEMISTRY:
- Yes see attached Tables 18 and 19 and Appendices 17 to 20

- Time schedule for collection of blood:

Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females).

- Blood samples were obtained from the lateral tail vein or by cardiac puncture at termination, if applicable.

- Anaesthetic used for blood collection: No

- Animals fasted: No

- How many animals:
Five males and 5 females selected from each surviving treatment group on Day 42 and from five randomly selected females from each dose group at termination for haematological and blood chemical assessment on Day 4 post partum.

URINALYSIS: Not applicable

NEUROBEHAVIOURAL EXAMINATION:
- Yes see attached Tables 3 to 6 and Appendices 2 to 6

- Time schedule for examinations:
(Behavioural assessment)
Prior to the start of treatment at weekly intervals thereafter

(Functional Performance Tests)
Final week of treatment.

(Sensory Reactivity)
Final week of treatment.

- Dose groups that were examined:
(Behavioural assessment)
All animals

(Functional Performance Tests)
Five selected males and females per dose level

(Sensory Reactivity)
Five selected males and females per dose level

- Parameters examined:

(Behavioural assessment)
Detailed individual clinical observations were performed for each animal using a purpose-built arena.

The following parameters were observed:
Gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation

(Functional Performance Tests)
Motor Activity.
Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength.
An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

(Sensory Reactivity)
Animals were assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex and finger approach

OTHER:
MATING
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 the oestrous cycle 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.

PREGNANCY AND PARTURITION
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:

i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

LITTER SIZE
On completion of parturition (Day 0 of 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)

PHYSICAL DEVELOPMENT
All live offspring were assessed for surface righting reflex on Day 1 post partum

Ovaries and uterine content:

The ovaries and uterine content was examined after termination:
Yes - Examinations included:
- Gravid uterus weight:

- Number of corpora lutea:
Yes

- Number of implantations:
Yes

- Number of early resorptions:
No

- Number of late resorptions:
No

Fetal examinations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
No

PARAMETERS EXAMINED
The following parameters were examined in offspring: Number of offspring born, number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, individual offspring weights on Day 1 and 4 post partum, physical Development and pathology.

GROSS EXAMINATION OF DEAD PUPS:
Any dead and dying offspring that may have occurred during the study were subjected to a full external and internal examination, and any macroscopic abnormalities recorded.

POST-MORTEM EXAMINATION
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Necropsy findings checked in table 21 were included. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Quantitative male data, female data prior to pairing, functional performance test data, blood chemical and haematology data will be analysed by the ProvantisTM Tables and Statistics Module. For each variable, the most suitable transformation of the data will be found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA on ANCOVA and Bartlett’s test. The transformed data will be analysed to find the lowest treatment level that shows a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response is found, but the data shows non-homogeneity of means, the data will be analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Finally, if required, pair-wise tests are performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Female gestation and lactation data and offspring data will be assessed, where appropriate, by one way analysis of variance (ANOVA) incorporating a test for homogeneity of variance will be used. Dose response relationships may be investigated by linear regression. Where variances are shown to be homogeneous, pair-wise comparisons will be conducted using Dunnetts test. Where appropriate, data will be analysed using non-parametic methods: Kruskal-Wallis, ANOVA and Mann-Witney U test.

Reproductive Indices

Where appropriate, the data will be statistically analysed using Levene’s test for homogeneity of variance followed by one way analysis of variance and pair-wise comparison using a choice of versions of multiple comparison tests; or Kruskal-Wallis non parametric one way analysis of variance and Mann Whitney U test/Wilcoxon signed rank test.
Reproductive and viability indices by Fisher's exact test or chi-squared probability test may be used, where applicable (see appended Appendix 1).

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 paired ÷ Number of animals mated) x 100

Pregnancy Index (%) = (Number of animals mated ÷ Number of pregnant females) x 100

Gestation and Parturition Data
The following parameters were calculated for 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 pregnant females ÷ Number of females delivering live offspring) x 100

Historical control data:

Yes – See attached:

Addendum 3: Normal Ranges for Reproductive Parameters in the Pregnant and Lactating Female Wistar Han ™:HsdHan™:WIST Strain Rat
Addendum 4: Normal Ranges for Offspring Parameters in the Wistar Han™: HsdHan™:WIST Strain Rat
Addendum 5: Normal Ranges for Haematological and Blood Chemical Values in the Wistar Han™:HsdHan™:WIST Strain Rat
Addendum 6: Normal Ranges for Functional Performance Assessments in the Wistar Han™:HsdHan™:WIST Strain Rat
Addendum 7: Normal Ranges for Absolute and Body Weight-Relative Organ Weight Values in the Wistar Han™:HsdHan™:WIST Strain Rat

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Episodes of increased salivation were evident in animals of either sex treated with 1000 mg/kg/day f rom Day 9 (males) and Day 14 (females), together with an isolated incident of decreased respiration evident in one male treated with 1000 mg/kg/day on Day 29.
No such effects were detected in animals of either sex treated with 300 or 100 mg/kg/day.Generalised fur loss was evident in one female treated with 1000 mg/kg/day between Days 34 and 4 5. Observations of this nature are commonly observed in group housed animals or during the lactati on phase for parental females and are considered not to be related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths that were related to test item toxicity.
One control male was found dead on Day 40. Macroscopic examination of this animal revealed a perf oration in the oesophagus which may have been due to a misplaced gavage.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See attached Tables 7 and 81 and individual data in Appendices 7 and 8. Also Figure 1 and Figure 2 which depict cumulative bodyweight changes graphically.
Males treated with 1000 mg/kg/day showed a statistically significant reduction (P<0.05) in body weight gain during Week 3 and Week 4 of treatment.
No adverse effects on body weight change were detected for females treated at all dose levels during the pre-mating, gestation or lactation phases of the study when compared to controls.
Males from all treatment groups showed a statistically significant increase (P<0.01) in body weight gain during Week 5 of treatment. Males treated with 100 mg/kg/day also showed a statistically significant increase (P<0.05) in body weight gain during Week 2. An increase in body weight gain is not c onsidered to represent an adverse effect of treatment and the intergroup differences detected durin g Week 5 were considered to be related to the actual body weight loss of one control male that was later found dead during the final week of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See attached Tables 9 and 10, individual cage values during maturation and individual values for fem ales during gestation and lactation in Appendix 9. Food intake is also presented graphically in Figure
3 and Figure 4.
No adverse effect on food consumption was detected for males during the treatment period or for f emales during the pre-mating, gestation or lactation phases of the study.

Food efficiency:

no effects observed

Description (incidence and severity):

Food efficiency (the ratio of body weight gain to dietary intake) was not affected for males throughout the treatment period or for females during the pre-mating phase.

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

See attached Table 18 and 17 and individual data in Appendix 17 and Appendix 18.
There were no toxicologically significant effects detected in the haematological parameters measured. Males treated with 1000 mg/kg/day showed a statistically significant reduction (P<0.05) in mean cell haemoglobin concentration. Although all individual values were outside the normal range for rats of the strain and age used, control values were also outside the normal range and in the absence of any associated changes the intergroup difference was considered not to be of toxicological significance.
Females treated with 1000 and 300 mg/kg/day showed a statistically significant reduction (P<0.05) in monocytes. All the individual values were within the normal ranges for rats of the strain and age used and the intergroup difference was considered not to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters measured. Females treated with 1000 mg/kg/day showed a statistically significant reduction in bilirubin. All the individual values were within the normal ranges for rats of the strain and age used and the intergroup difference was considered not to be of toxicological significance.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in functional performance.
Females treated with 1000 and 300 mg/kg/day showed a statistically significant reduction in mean forelimb grip strength. The intergroup differences were confined to one out of the three tests and in the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the finding was considered to be of no toxicological significance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See attached Table 20 and individual data in Appendices 21 and 22.
Males treated with 1000 mg/kg/day showed a statistically significant increase in kidney weight both absolute and relative to terminal body weight (P<0.01).
No toxicologically significant effects were detected in females treated with 1000 mg/kg/day or animals of either sex treated with 300 and 100 mg/kg/day.
Females treated with 1000 mg/kg/day showed a reduction in thyroid/parathyroid and pituitary weigh t both absolute and relative to terminal body weight. The effect on pituitary weight also extended to females treated with 300 mg/kg/day. In the absence of any histology correlates the intergroup differences were considered to be of no toxicological importance. Males from all treatment groups showed a reduction in heart weight both absolute and relative to terminal bodyweight. In the absen ce of a true dose related response or any histology correlates the intergroup differences were considered to be of no toxicological importance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

One male treated with 1000 mg/kg/day had an enlarged left testis and epididymis and a small right te stis and epididymis at necropsy. This male also had a mass on the left epididymis. One female tre ated with 1000 mg/kg/day showed a reddened thymus and a further female from this treatment group had reddened lungs at necropsy. One female treated with 300 mg/kg/day had a small spleen and pale adrenals and one female treated with 100 mg/kg/day also had pale adrenals at necropsy. In the absence of any true dose related responses or any histology correlates the intergroup differences were considered not to be of toxicological importance. One control male had a reddened thymus a nd dark cervical lymph nodes at necropsy. In the absence of treatment, these were considered to be low incidence findings occasionally observed in laboratory maintained rats. One male treated with 300 mg/kg/day and one female treated with 100 mg/kg/day incurred damage to the eyes during the removal procedure. The control male that was found dead on Day 40 had dark coloured contents in the gastro-intestinal tract, a dark liver and spleen, reddened lungs and thymus, a distended stomach and a hole in the oesophagus.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See attached histopathology report in Appendix 25.
The following treatment-related microscopic effects were detected:
Kidneys: Interstitial fibrosis and inflammatory cells, areas of degeneration/necrosis, tubular dilatation and degeneration/necrosis of tubular epithelium, increased severity of tubular basophilia and hyaline droplets, granular and hyaline casts, corticomedullary mineralization, glomerular hyalinization and thickening of the glomerular basement membrane were evident in males treated with 1000 mg/kg/ day.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
ADULT RESPONSES

MORTALITY
There were no unscheduled deaths that were related to test item toxicity.

One control male was found dead on Day 40. Macroscopic examination of this animal revealed a perforation in the oesophagus which may have been
due to a misplaced gavage.

CLINICAL OBSERVATIONS
See attached Table2 and individual data in Appendix 1.

Episodes of increased salivation were evident in animals of either sex treated with 1000 mg/kg/day from Day 9 (males) and Day 14 (females), together with an isolated incident of decreased respiration evident in one male treated with 1000 mg/kg/day on Day 29.

No such effects were detected in animals of either sex treated with 300 or 100 mg/kg/day.

Generalised fur loss was evident in one female treated with 1000 mg/kg/day between Days 34 and 45. Observations of this nature are commonly observed in group housed animals or during the lactation phase for parental females and are considered not to be related to treatment.

FUNCTIONAL OBSERVATIONS
See attached Tables 3 to 6 and individual data in Appendices 23 to 26.

Behavioural Assessments

Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.

Functional Performance Tests

There were no toxicologically significant changes in functional performance.

Females treated with 1000 and 300 mg/kg/day showed a statistically significant reduction in mean forelimb grip strength. The intergroup differences were confined to one out of the three tests and in the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the finding was considered to be of no toxicological significance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and the differences were of no toxicological importance.

BODYWEIGHT
See attached Tables 7 and 81 and individual data in Appendices 7 and 8. Also Figure 1 and Figure 2 which depict cumulative bodyweight changes graphically.

Males treated with 1000 mg/kg/day showed a statistically significant reduction (P<0.05) in body weight gain during Week 3 and Week 4 of treatment.

No adverse effects on body weight change were detected for females treated at all dose levels during the pre-mating, gestation or lactation phases of the study when compared to controls.

Males from all treatment groups showed a statistically significant increase (P<0.01) in body weight gain during Week 5 of treatment. Males treated with 100 mg/kg/day also showed a statistically significant increase (P<0.05) in body weight gain during Week 2. An increase in body weight gain is not considered to represent an adverse effect of treatment and the intergroup differences detected during Week 5 were considered to be related to the actual body weight loss of one control male that was later found dead during the final week of treatment.

FOOD CONSUMPTION
See attached Tables 9 and 10, individual cage values during maturation and individual values for females during gestation and lactation in Appendix 9. Food intake is also presented graphically in Figure 3 and Figure 4

No adverse effect on food consumption was detected for males during the treatment period or for females during the pre-mating, gestation or lactation phases of the study. Food efficiency (the ratio of body weight gain to dietary intake) was not affected for males throughout the treatment period or for females during the pre-mating phase.

WATER CONSUMPTION
See attached Table 11 and individual and group mean water consumptions for females in Appendix 10.

Males treated with 1000 and 300 mg/kg/day showed an increase in water consumption throughout the treatment period. Females treated with 1000 mg/kg/day also showed an increase in water consumption during maturation, gestation and lactation.
Statistical significance was achieved during the first week of gestation.
No such effect was detected in females treated with 300 mg/kg/day or animals of either sex treated with 100 mg/kg/day.

HAEMATOLOGY
See attached Table 18 and 17 and individual data in Appendix 17 and Appendix 18.

There were no toxicologically significant effects detected in the haematological parameters measured.
Males treated with 1000 mg/kg/day showed a statistically significant reduction (P<0.05) in mean cell haemoglobin concentration. Although all individual values were outside the normal range for rats of the strain and age used, control values were also outside the normal range and in the absence of any associated changes the intergroup difference was considered not to be of toxicological significance.
Females treated with 1000 and 300 mg/kg/day showed a statistically significant reduction (P<0.05) in monocytes. All the individual values were within the normal ranges for rats of the strain and age used and the intergroup difference was considered not to be of toxicological significance.

BLOOD CHEMISTRY
See attached Table 19 and individual data in Appendix 19 and Appendix 20.

There were no toxicologically significant effects detected in the blood chemical parameters measured.
Females treated with 1000 mg/kg/day showed a statistically significant reduction in bilirubin. All the individual values were within the normal ranges for rats of the strain and age used and the intergroup difference was considered not to be of toxicological significance.

REPRODUCTIVE PERFORMANCE

Mating
A summary incidence of adult performance is given in Table 1. Group values for mating performance are presented in Table 12. Individual data are given in Appendix 11.

No treatment-related effects were detected in mating performance. All paired animals mated within the first four days of pairing.
Statistical analysis of the pre-coital interval data did not reveal any significant intergroup differences.

Fertility
A summary incidence of adult performance is given in Table 1. Group values for fertility, litter data and implantation losses are given in Tables 12, 13 and 14. Individual data are given in Appendices 11 to 13.

No treatment-related effects were detected on fertility for treated animals when compared to controls.

One female treated with 1000 mg/kg/day did not achieve pregnancy following evidence of mating. In the absence of any histopathological correlates in the reproductive organs to elucidate the cause of the non-pregnancy in either the paired female or male partner which did not produce a pregnancy, the intergroup difference was considered to be incidental and of no toxicological importance.

Gestation Length
Group values for gestation length are given in Table 12. Individual lengths are given in Appendix 11.

No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths between 22 to 24 days.
Statistical analysis of the data did not reveal any significant intergroup differences.

Litter Responses

In total, all females from the control and 100 mg/kg/day dose groups, eight females from the 300 mg/kg/day dose group and nine females from the 1000 mg/kg/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size and Viability
Group mean corpora lutea and implantation counts, litter size, implantation losses and survival indices and sex ratio are given in Tables 13 to 15. Individual data are given in Appendices 12 to 14.

No significant differences were detected for corpora lutea for treated animals when compared to controls. Litter sizes and viability for treated groups were also comparable to controls. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Females treated with 300 mg/kg/day showed a statistically significant reduction in implantation sites (P<0.05). In the absence of a dose related effect or a subsequent effect on litter size at birth the intergroup difference was considered to be of no toxicological significance.

NECROPSY (ADULTS)
See attached Tables 21 to 22 and individual data in Appendices 23 and 24.

There were no toxicologically significant macroscopic abnormalities detected.

One male treated with 1000 mg/kg/day had an enlarged left testis and epididymis and a small right testis and epididymis at necropsy. This male also had a mass on the left epididymis. One female treated with 1000 mg/kg/day showed a reddened thymus and a further female from this treatment group had reddened lungs at necropsy. One female treated with 300 mg/kg/day had a small spleen and pale adrenals and one female treated with 100 mg/kg/day also had pale adrenals at necropsy. In the absence of any true dose related responses or any histology correlates the intergroup differences were considered not to be of toxicological importance. One control male had a reddened thymus and dark cervical lymph nodes at necropsy. In the absence of treatment, these were considered to be low incidence findings occasionally observed in laboratory maintained rats. One male treated with 300 mg/kg/day and one female treated with 100 mg/kg/day incurred damage to the eyes during the removal procedure. The control male that was found dead on Day 40 had dark coloured contents in the gastro-intestinal tract, a dark liver and spleen, reddened lungs and thymus, a distended stomach and a hole in the oesophagus.

ORGAN WEIGHTS
See attached Table 20 and individual data in Appendices 21 and 22.

Males treated with 1000 mg/kg/day showed a statistically significant increase in kidney weight both absolute and relative to terminal body weight (P<0.01).

No toxicologically significant effects were detected in females treated with 1000 mg/kg/day or animals of either sex treated with 300 and 100 mg/kg/day.

Females treated with 1000 mg/kg/day showed a reduction in thyroid/parathyroid and pituitary weight both absolute and relative to terminal body weight. The effect on pituitary weight also extended to females treated with 300 mg/kg/day. In the absence of any histology correlates the intergroup differences were considered to be of no toxicological importance. Males from all treatment groups showed a reduction in heart weight both absolute and relative to terminal bodyweight. In the absence of a true dose related response or any histology correlates the intergroup differences were considered to be of no toxicological importance.

HISTOPATHOLOGY
See attached histopathology report in Appendix 25.

The following treatment-related microscopic effects were detected:

Kidneys: Interstitial fibrosis and inflammatory cells, areas of degeneration/necrosis, tubular dilatation and degeneration/necrosis of tubular epithelium, increased severity of tubular basophilia and hyaline droplets, granular and hyaline casts, corticomedullary mineralization, glomerular hyalinization and thickening of the glomerular basement membrane were evident in males treated with 1000 mg/kg/day.

Liver: Centrilobular hepatocyte hypertrophy was seen in all males treated with 1000 mg/kg/day.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no differences in litter weights or mean offspring body weights between control and treated animals.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: under the conditions of the test, number of corpora lutea, implantation rate, postimplantation loss, litter size at first littercheck, body weights of pups or results of external examination gave no indication of embryotoxic or teratogenic effects.

Details on embryotoxic / teratogenic effects:
Group values for offspring body weight and body weight change, surface righting reflex and the incidence of clinical signs are given in Tables 13, 16 and 17. Individual values and observations are given in Appendices 12, 15 and 16. Please also see Table 21 and Appendix 23 for summary and individual offspring necropsy findings

VIABILITY (OFFSPRING)
Of the litters born, litter size at birth and subsequently on Day 1 and Day 4 post partum were comparable to controls.

CLINICAL SIGNS (OFFSPRING) –
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, bruising, no milk in stomach, cold, weak, dehydrated and physical injuries were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test item toxicity.

BODY WEIGHT (OFFSPRING)
Offspring body weight gain and litter weights at birth and subsequently on Day 1 and Day 4 post partum were comparable to controls. No adverse effects were detected in surface righting reflex on Day 1.

SEXUAL MATURATION (OFFSPRING)
Not applicable

ORGAN WEIGHTS (OFFSPRING)
Not applicable

GROSS PATHOLOGY (OFFSPRING)
Neither the incidence, type or distribution of macroscopic findings observed at necropsy of decedent offspring nor offspring killed at scheduled termination (Day 5 of age) indicated any adverse effect of maternal treatment.

HISTOPATHOLOGY (OFFSPRING)
Not applicable

OTHER FINDINGS (OFFSPRING)
No treatment-related effects were detected for surface righting reflex for offspring from treated animals when compared to offspring from control females.

Statistical analysis of the data did not reveal any significant intergroup differences.

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

(3180 -0008)- See attached Tabulated Summary of Reproduction and Development, Keys to Tables, Figures, Appendices and Addenda.

Study Findings for the Fourteen day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Harlan Laboratories Ltd. Project Number 3180-0007).

 

Results.

 

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

No clinically observable signs of toxicity were detected.

Incidents of increased salivation and noisy respiration were evident in one female treated with 1000 mg/kg/day between Days 2 and 13.

No such effects were evident in males treated with 1000 mg/kg/day or in animals of either sex treated with 500 or 250 mg/kg/day.

Body Weight

Overall body weight gains were slightly reduced in animals of either sex treated with 1000 mg/kg/day when compared to controls.

No such effects were evident in animals of either sex treated with 500 or 250 mg/kg/day.

Food Consumption

No adverse effect on dietary intake or food efficiency was detected in treated animals when compared to controls throughout the treatment period.

Water Consumption

An increase in overall water consumption was detected in males treated with1000 mg/kg/day.

No such effects were evident in females treated with 1000 mg/kg/day or in animals of either sex treated with 500 or 250 mg/kg/day.

Necropsy

No toxicologically significant macroscopic abnormalities were detected.

One female treated with 1000 mg/kg/day had hydronephrosis in the right kidney at necropsy. This is considered to be a congenital abnormality and of no toxicological importance.

DISCUSSION

The oral administration of PR-4758 - Technical Grade, to rats for a period of fourteen consecutive days at dose levels of 250, 500 and 1000 mg/kg/day resulted in treatment related effects in animals of either sex treated with 100 mg/kg/day.

Incidents of increased salivation and noisy respiration were evident in one female treated with 1000 mg/kg/day between Days 2 and 13. An increase in overall water consumption was also detected in males treated with 1000 mg/kg/day. Observations of this nature are commonly observed following oral administration of an unpalatable or slightly irritant test material formulation and in isolation are not considered to be of toxicological significance.

Overall body weight gains were slightly reduced in animals of either sex treated with 1000 mg/kg/day when compared to controls. In the absence of any associated clinical signs of toxicity or any adverse effect on food consumption the slight reduction in body weight gain was considered not to represent a true toxicological effect.

CONCLUSION

The oral administration of PR-4758 - Technical Grade to rats for a period of fourteen consecutive days at dose levels of 250, 500 and 1000 mg/kg/day produced no toxicologically significant effects in the parameters measured. The “No Observed Adverse Effect Level” (NOAEL) and a suitable high dose level for use on future toxicity studies was, therefore, considered to be 1000 mg/kg/day. 

Applicant's summary and conclusion

Conclusions:

The oral administration of PR-4758 - Technical Grade to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg/day, resulted in treatment-related effects in males treated with 1000 mg/kg/day. No toxicologically significant effects were detected in females treated with 1000 mg/kg/day or animals of either sex treated with 300 or 100 mg/kg/day, hence the 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day for females and 300 mg/kg/day for males.

No treatment-related effects were observed for reproduction, therefore, a ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/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 meets 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 meet the requirements of 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 ten male and ten female Wistar Han™:HsdHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Distilled water).

 

Clinical signs, behavioural 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 mating phase, and for five selected parental females from each dose group on Day 4post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

 

Surviving males were terminated on Day 43, followed by the termination of all females and surviving offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results

 

Adult Responses:

 

Mortality.

There were no unscheduled deaths that were considered to be related to test item toxicity.

 

Clinical Signs.

Episodes of increased salivation were evident in animals of either sex treated with 1000 mg/kg/day together with an isolated incident of decreased respiration evident in one male from this treatment group. No such effects were detected in animals of either sex treated with 300 or 100 mg/kg/day.

 

Behavioural Assessment.

There were no treatment-related changes in the behavioural parameters measured.

 

Functional Performance Tests.

There were no toxicologically significant changes in functional performance.

 

Sensory Reactivity Assessments.

There were no treatment-related changes in sensory reactivity. 

 

Bodyweights.

Males treated with 1000 mg/kg/day showed a statistically significant reduction in body weight gain during Week 3 and Week 4 of treatment.

 

No such effects were detected in females treated with 1000 mg/kg/day or animals of either sex treated with 300 or 100 mg/kg/day.

 

Food Consumption and Food Efficiency.

No adverse effects on food consumption or food efficiency were detected for treated animals when compared with controls.

 

Water Consumptions.

Males treated with 1000 and 300 mg/kg/day showed an increase in water consumption throughout the treatment period. Females treated with 1000 mg/kg/day also showed an increase in water consumption during maturation, gestation and lactation. No such effect was detected in females treated with 300 mg/kg/day or animals of either sex treated with 100 mg/kg/day.

 

Haematology.

There were no toxicologically significant effects detected in the haematological parameters measured.

 

Blood Chemistry.

There were no toxicologically significant effects detected in the blood chemical parameters measured.

 

Reproductive Performance:

Mating.

There were no treatment-related effects on mating in animals of either sex treated with 1000, 300 or 100 mg/kg/day.

 

Fertility.

There were no treatment-related effects on conception rates for animals of either sex treated with 1000, 300 or 100 mg/kg/day.

 

Gestation Length.

There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

 

Litter Responses.

 

Offspring Litter Size and Viability.

Of the litters born, litter size at birth and subsequently on Day 1 and Day 4post partumwere comparable to controls.

 

Offspring Growth and Development.

Offspring body weight gain and litter weights at birth and subsequently on Day 1 and Day 4post partumwere comparable to controls. No adverse effects were detected in surface righting reflex on Day 1.

 

Pathology.

 

Necropsy.

There were no toxicologically significant macroscopic abnormalities detected.

 

Organ Weights.

Males treated with 1000 mg/kg/day showed an increase in kidney weight both absolute and relative to terminal body weight. No toxicologically significant effects were detected in females treated with 1000 mg/kg/day or animals of either sex treated with 300 and 100 mg/kg/day.

 

Histopathology.

The following treatment related microscopic effects were detected:

 

Kidneys:Interstitial fibrosis and inflammatory cells, areas of degeneration/necrosis, tubular dilatation and degeneration/necrosis of tubular epithelium, increased severity of tubular basophilia and hyaline droplets, granular and hyaline casts, corticomedullary mineralization, glomerular hyalinization and thickening of the glomerular basement membrane were evident in males treated with 1000 mg/kg/day.

 

Liver:Centrilobular hepatocyte hypertrophy was seen in all males treated with 1000 mg/kg/day.

 

Conclusion.

The oral administration of PR-4758 - Technical Grade to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg/day, resulted in treatment-related effects in males treated with 1000 mg/kg/day. No toxicologically significant effects were detected in females treated with 1000 mg/kg/day or animals of either sex treated with 300 or 100 mg/kg/day, hence the 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day for females and 300 mg/kg/day for males.

 

No treatment-related effects were observed for reproduction, therefore, a ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.