Glutens, hydrolyzates, reaction products with lauroyl chloride, sodium salts

Toxicity to reproduction

Administrative data

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:

Between 02 January 2009 and 15 July 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

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)

Remarks:

Date of inspection 19/08/2008. Date of signature 04/03/2009.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Glutens, hydrolyzates, reaction products with lauroyl chloride, sodium salts

EC Number:

500-500-3

EC Name:

Glutens, hydrolyzates, reaction products with lauroyl chloride, sodium salts

Cas Number:

161074-67-5

Molecular formula:

Not applicable

IUPAC Name:

Acylation product between lauroyl chloride and amino acids

Test material form:

other: Extremely pale yellow liquid

Details on test material:

Sponsor’s identification: LCE08082
Description : Extremely pale yellow liquid
Label : LCE08082
Batch : 0818600011
Dry extract : 29,8%
Date received : 05 September 2008
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

Sponsor’s identification: LCE08082
Description : Extremely pale yellow liquid
Label : LCE08082
Batch : 0818600011
Dry extract : 29,8%
Date received : 05 September 2008
Storage conditions: room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source:
Wistar Han™:HsdRccHan™:WIST strain rats from Harlan Laboratories UK Ltd, Oxon, UK

- Age at study initiation:
Approximately 12 weeks old

- Weight at study initiation:
males 298 to 357 g; females weighed 209 to 254 g

- 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 and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet Harlan Laboratories U.K. Ltd., Oxon, UK) was used throughout the study period. Certificates of analysis of the batches used are given in Addendum 2.

- Water:
The animals were allowed free access to water. 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:
For 12 days

- Environmental enrichment:
Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd. Cheshire, UK) except for mated females during gestation and lactation. Mated females were also given softwood flakes as bedding, throughout gestation and lactation.

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:
Males: 43 Days 01/04/2009 - 13/05/2009
Females: up to 56 days 01/04/2009 - 26/05/2009

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of the study, the test material was prepared at the appropriate concentrations as a solution in Arachis oil. The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd, Shardlow, UK. Results are given in Appendix 26 and show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately 4ºC in the dark.
Samples were taken of each test material formulation and were analysed for concentration of LCE08082 at Harlan Laboratories Ltd, Shardlow, UK. The Method used for analysis of formulations and the results obtained are given in Appendix 26. The results indicate that the prepared formulations were within ± 8% of the nominal concentration.

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.:
Not applicable

- Further matings after two unsuccessful attempts:
Not applicable

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

- Any other deviations from standard protocol:
Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of LCE08082 in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.The test material formulations were extracted with methanol to give a final, theoretical test material concentration of approximately 1 mg/ml.Standard solutions of test material were prepared in methanol at a nominal concentration of 1 mg/ml.The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.The test material formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for 14 days.The test material formulations were sampled and analysed within three days of preparation.
For Analytical Method Procedure See Section Any Other Details on Method
For Results See Section Any Other Details Results Section

Duration of treatment / exposure:

The oral administration of the test substance to rats up to fifty-four consecutive days.

Frequency of treatment:

Daily

Details on study schedule:

-Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturation where applicable). The first day of dosing was designated as Day 1 of the study.
- Prior to strat of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
-One day prior to pairing (Day14), blood samples were taken from five males and five females, randomly selected from each dose group and analysed for haematological and blood chemical assessment.
- On day 15, animals were paired on a 1male:1 female basis within each dose group for a maximum of fourteen days.
- Following evidence of mating (designated as Day0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
-On completion of mating (during week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
- Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weigh, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed dirung this period.
-At day 4 post partum, five selected females per dose group were evaluated for functional/sensory reponses to various stimuli.
-Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.
At day 5 post partum, all females and surviving ofsspring were killed and examined macroscopically.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg/day (25 mg/ml)
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg/day (75 mg/ml)
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg/day (250 mg/ml)
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose (including control).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on Preliminary Fourteen Day Repeated Dose Oral (Gavage) Range-Finder in the Rat (see Any Other Details on Method Section)

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

- Other:
Not applicable

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Yes

- Time schedule:
All animals were examined for overt signs of toxicity, ill-health and behavioural change 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, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).
- Cage side observations checked in table [No.2 and 3] are included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
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


BODY WEIGHT: Yes
- 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. Bodyweights were also recorded for all animals on the day of termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (compound intake not applicable to this study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
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.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data:
Yes
- Food efficiency: (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period and for females during maturation and the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.

WATER CONSUMPTION:
Yes

- Time schedule for examinations:
Water intake was observed daily by visual inspection of water bottles for any overt changes. Intergroup differences did not indicate any need for more formal gravimetric measurements.


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. 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

Oestrous cyclicity (parental animals):

not applicable

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, epididymis weight, other: histopathological examinations of testes and epididymides

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 post partum:
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 and litter weights on Day 1 and 4 post partum, physical Development and pathology.

GROSS EXAMINATION OF DEAD PUPS:
Dying offspring during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE

- Male animals:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43.

- Maternal animals:
Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

GROSS NECROPSY / ORGAN WEIGHTS

For all females the uterus was examined for signs of implantation and the number of uterine implantations in each born was recorded. This procedurewas enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution. In addition, the corpora lutea of all ovaries from pregnantfemales were counted at necropsy. All adult animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY

The following organs, removed from the five selected males and parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation. Adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroid and liver.

The following reproductive organs were weighed from all animals that were killed at the end of the study:
Adrenals Ovaries
Brain Spleen
Epididymides Testes
Heart Thymus
Kidneys Thyroid
Liver


Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin except where indicated.
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint) Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and pons) Oesophagus
Caecum Rectum
Coagulation gland Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymides• Skin (hind limb)
Eyes* Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart Spleen
Ileum Stomach
Jejunum Thyroid/parathyroid
Kidneys Trachea
Liver Testes•
Lungs (with bronchi) # Thymus
Lymph nodes (cervical and mesenteric) Urinary bladder
Mammary gland Uterus/Cervix
Muscle (skeletal) Vagina

* = eyes fixed in Davidson’s fluid
• = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately
to forty-eight hours later
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative


The following tissues were also removed from the remaining animals:coagulating gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, mammary gland, testes and uterus/cervix.

All tissues were despatched to Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford for processing (Principal Investigator: E Richards). The tissues from five selected control and 1000 mg/kg/day dose group animals, any animal failing to produce a pregnancy and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 1000 mg/kg/day were also processed. In addition, sections of testes and epididymides from all control and 1000 mg/kg/day males were also stained with Periodic Acid Schiff (PAS) stain and examined.
Since there were indications of treatment-related changes in the stomach, examination was subsequently extended to include similarly prepared sections of stomach from five animals per sex from the low and intermediate dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [?] days of age. not applicable for this study.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Surviving offspring were terminated on Day 5 post partum via intracardiac overdose of sodium pentobarbitone.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively. - not applicable for this study

Statistics:

The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Bodyweight and bodyweight change
Food consumption during gestation and lactation
Haematology, blood chemistry, absolute and bodyweight relative organ weights
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
The following statistical procedures were used:
Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Probability values (P) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
p>0.05 (not significant)
In addition, histopathological findings were analysed using the following methods:
Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Probability values (P) were calculated as follows:
P<0.001 +++ --- ***
P<0.01 ++ -- **
P<0.05 + - *
P<0.1 (+) (-) (*)
p>0.01 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs indicate negative differences. Asterisks refer to overall differences between group variation which is non-directional.

Reproductive indices:

Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
Mating Index (%)
=Number of animals mated / Number of animals paired X 100
Pregnancy Index (%)
= Number of pregnant females / Number of animals mated x 100
Gestation / Parturition Data: The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
Parturition Index (%)
= Number of females delivering live offspring / Number of pregnant females x 100

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were
first calculated for each litter and the group mean was calculated using their individual
litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for
each female/litter as follows:
% pre – implantation loss = [(Number of corpora lutea - Number of Corpora Lutea) ÷ Number of implantation sites] x 100
% post – implantation loss =[(Number of implantation sites - Number of implantation sites) ÷ Total number of offspring born] x 100
ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring born ÷Number of offspring alive on Day 1) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 1 ÷ Number of offspring alive on Day 4) x 100
iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinically observable signs of toxicity were detected

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effect on bodyweight development was detected both in males and females

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

no effects observed

Description (incidence and severity):

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

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

no effects observed

Description (incidence and severity):

No intergoup differences were detected.

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects detected in the haematological parameters measured.

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related effect was detected:
STOMACH: Acanthosis, hyperkeratosis, subepithelial inflammatory cell inflitrates, epithelial erosion and ulceration were seen in the forestomach among males only treated with 1000 mg/kg/day were similarly affected. Such changes are typically seen as a direct response to ingestion of an irritant substance and usually resolve on removal of the irritant. While gastric epithelial changes which include ulceration are considered to be adverse, a squamous-lined counterpart to the rodent forestomach is not present in humans.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Functional performance test: There were no treatment-related changes in functional performance.
Sensory reactivity assessments: There were no treatment-related changes in sensory reactivity.
Blood chemistry: There were no treatment-related effects detected in the blood chemical parameters measured.
Necropsy: No toxicologically significant effects were detected.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating: One control female did not show any positive evidence of mating. The were no treatment-related effects on mating or conception rates for animals of either sex treated with 100, 300 or 1000 mg/kg/day.
Gestation length: There were no differences in gestation lenghts. The distribution for treated females was comparable to controls.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No clinically observable signs of toxicity were detected.
Males treated with 1000 mg/kg/day showed episodes of increased salivation immediately after dosing from Day 11 onwards. One male from this treatment group also showed noisy respiration on Days 11 and 13. Two males treated with 300 mg/kg/day showed noisy respiration on Day 6 or Day 35. Observations of this nature are often reported following oral administration of an unpalatable or slightly irritant test material formulation and, in isolation, are considered not to be indicative of systemic toxicity.
Generalised fur loss was evident in two females treated with 1000 mg/kg/day and in one female treated with 300 mg/kg/day. These findings were consistent with low incidence finding for laboratory rats of the strain and age used and were considered not to be of toxicological importance.



BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males
No adverse effect on bodyweight development was detected.
Statistical analysis of the data did not reveal any significant intergroup differences.
Females
No adverse effect on bodyweight development was detected.
Statistical analysis of the data did not reveal any significant intergroup differences.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
oral gavage dosing at 100, 300 and 1000 mg/kg/day


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
not applicable for this study


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
not applicable for this study

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no treatment-related effects on mating performance. The distribution of precoital intervals for treated animals were comparable to controls with the majority of animals showing positive evidence of mating within four days of pairing (i.e. the first oestrus opportunity). One control female did not show any positive evidence of mating and was subsequently non-pregnant. There were no treatment-related effects on fertility.There were no significant intergroup differences in gestation lengths or parturition for treated females, with the majority of females giving birth following 22 ½ and 24 days of gestation. The distribution for treated females was comparable to controls.


ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no treatment-related changes in organ weights from treated animals when compared to controls.
Statistical analysis of the data did not reveal any significant intergroup differences.


GROSS PATHOLOGY (PARENTAL ANIMALS)
Adults
No toxicologically significant macroscopic abnormalities were detected.
One male treated with 1000 mg/kg/day had a mass on the right epididymis. One male treated with 300 mg/kg/day had a small right testis and epididymis and a further male from this treatment group had hydronephrosis in the right kidney. In the absence of any histology correlates the intergroup differences were considered of no toxicological importance.



HISTOPATHOLOGY (PARENTAL ANIMALS)
STOMACH: Acanthosis, hyperkeratosis, subepithelial inflammatory cell infiltrates, epithelial erosion and ulceration were seen in the forestomach among males only treated with 1000 mg/kg/day. Two males treated with 300 mg/kg/day were similarly affected. Such changes are typically seen as a direct response to ingestion of an irritant substance and usually resolve on removal of the irritant. While gastric epithelial changes which include ulceration are considered to be adverse, a squamous-lined counterpart to the rodent forestomach is not present in humans.

OTHER FINDINGS (PARENTAL ANIMALS)
HISTOPATHOLOGY REPRODUCTIVE TRACT AND RELATED ORGANS
PITUITARY: No treatment-related changes were seen. Vacuolation of pars anterior cells is observed commonly as a spontaneous change, more especially among males.
TESTIS/EPIDIDYMIS: No treatment-related changes were seen. Isolated instances of testicular atrophy, suppression of spermatogenesis, and spermatocoel granuloma formation were seen as spontaneous changes.
SEMINAL VESICLES/COAGULATING GLAND: No significant pathology was observed.
PROSTATE: Interstitial chronic inflammatory cell infiltrates are a commonly observed background finding in laboratory maintained rats and there was no relationship to treatment in this investigation.
MAMMARY GLAND: Glandular hyperplasia was observed in the mammary tissue of the majority of females examined. The appearance of the mammary tissue is consistent with pregnancy and lactation.
OVARY: No pathological changes were seen.
UTERUS: Areas of haemorrhage, occasionally with haematoma formation, foam cells and pigment accumulation were seen in the myometrium and adjacent connective tissue of the uterus in the majority of female animals examined from control and high dose groups. These conditions are consistent with normal post partum uterine changes in the rat.
All other morphological changes in the above and remaining tissues were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

food efficiency

water consumption and compound intake

haematology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive performance

other: see 'Remark'

Remarks on result:

other: Generation: Parents and offsprings (migrated information)

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinically observed signs of toxicity were detected for offspring from all treatment groups.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Offspring litter size and viability: Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum were comparable to control.
Offspring growth and development: Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4 post partum were comparable to control.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Histopathological findings:

not examined

Other effects:

not specified

Description (incidence and severity):

In total nine females from the control group and all ten females treated with 100, 300 and 1000 mg/kg/day 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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No effects

CLINICAL SIGNS (OFFSPRING)
No clinically observable signs of toxicity were detected for treated litters when compared to controls. The clinical signs observed throughout the control and treated groups are commonly observed in reproductive toxicity studies and not considered to represent toxicity. There were no interim offspring deaths observed in the control groups, however, the number of interim deaths observed in the treated litters were low, and in the absence of a dose-related response, were not indicative of toxicity.

BODY WEIGHT (OFFSPRING)
There were no differences in litter weights or mean offspring bodyweights between control and treated animals. Statistical analysis of the data did not reveal any significant differences between control and treated groups.

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)
In total nine females from the control group and all ten females treated with 100, 300 and 1000 mg/kg/day 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.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

viability

clinical signs

body weight and weight gain

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

 RESULTS

1.1                      Homogeneity of Test Material Formulations

Nominal Concentration (mg/ml)	Sampling Location	Concentration Found (mg/ml)
		1	2	3	Mean
25	Top	23.8	23.9	24.0	23.9
	Middle	23.9	24.1	24.1	24.0
	Bottom	24.0	24.0	24.0	24.0
250	Top	252	251	252	252
	Middle	251	250	251	251
	Bottom	251	251	251	251

1.2                      Stability of Test Material Formulations

Nominal Concentration (mg/ml)	Concentration Found Initially (mg/ml)	Concentration Found After Storage for 14 Days
		(mg/ml)	(expressed as % of initial)
25	24.0	24.1	100
250	251	250	100

1.3                      Verification of Concentration of Weekly Test Material Formulation

Week Number	Nominal Concentration (mg/ml)	Concentration Found
		(mg/ml)	(expressed as % of nominal)
1	0	ND	-
	25	24.9	100
	75	74.8	100
	250	253	101
2	0	ND	-
	25	23.3	93
	75	71.5	95
	250	249	100
3	0	ND	-
	25	24.4	97
	75	75.6	101
	250	252	101
4	0	ND	-
	25	27.0	108
	75	80.0	107
	250	259	104
5	0	ND	-
	25	23.0	92
	75	70.4	94
	250	243	97
6	0	ND	-
	25	23.3	93
	75	73.4	98
	250	250	100
7	0	ND	-
	25	23.7	95
	75	75.2	100
	250	251	100

ND= none detected

-= not applicable

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

Observations		Dose Level (mg/kg/day)
		0 (Control)	100	300	1000
Mated pairs	n	10	10	10	10
Females showing evidence of copulation	n	9	10	10	10
Pregnant females	n	9	10	10	10
Conception Days 1-5	n	9	10	10	10
Gestation = 22 ½ Days	n	4	6	4	7
Gestation = 23 Days	n	3	1	2	1
Gestation = 23 ½ Days	n	2	3	3	2
Gestation = 24 Days	n	0	0	1	0
Dams with live young born	n	9	10	10	10
Dams with live young at Day 4post partum	n	9	10	10	10
Corpora lutea/dam	n	14.9	13.9	14.0	12.4
Implants/dam		14.2	13.2	12.5	11.4
Live offspring/dam Day 1post partum		13.4	12.9	11.8	11.1
Live offspring/dam at Day 4post partum		13.3	12.9	11.7	10.9
Sex ratio: % males Day 1post partum		47.8	52.3	48.9	54.1
Sex ratio: %males at Day 4post partum		47.4	52.3	48.7	53.8
Litter weight (g) at Day 1post partum		78.8	77.1	72.2	65.8
Litter weight (g) at Day 4post partum		110.9	114.5	104.4	92.8
Male offspring weight (g) at Day 1post partum		6.1	6.1	6.3	6.2
Female offspring weight (g) at Day 1post partum		5.8	5.9	6.0	6.0
Male offspring weight (g) at Day 4post partum		8.6	8.9	9.2	9.1
Female offspring weight (g) at Day 4post partum		8.2	8.9	8.9	8.7
LOSS OF OFFSPRING/DAM
Pre-implantation (corpora lutea minus implantations)
0	n	4	4	3	4
1	n	3	5	4	4
2	n	1	1	1	1
3	n	0	0	1	0
4	n	0	0	0	1
6	n	0	0	1	0
Pre-natal (implantations minus live births)
0	n	3	7	6	8
1	n	3	3	2	1
2	n	1	0	1	1
3	n	1	0	1	0
Post natal (live births minus offspring alive on Day 4post partum)
0	n	8	10	9	9
1	n	1	0	1	0
2	n	0	0	0	1

n= Number   x = Mean

Applicant's summary and conclusion

Conclusions:

The oral administration of LCE08082 to rats for a period of up to fifty five days at dose levels of up to 1000 mg/kg/day resulted in treatment related changes in males treated with 1000 and 300 mg/kg/day. For systemic toxicity the 'No Observed Effect Level' (NOEL) for males was therefore, considered to be 100 mg/kg/day. No such effect was detected in females treated with 1000 or 300 mg/kg/day and for systemic toxicity the ‘No Observed Effect Level’ (NOEL) for females was therefore, considered to be 1000 mg/kg/day.
The gastric changes detected in 1000 and 300 mg/kg/day males may be considered to be adverse, however because a squamous-lined counterpart to the rodent forestomach is not present in human stomachs, these effects in this study, are not considered to be indicative of a hazard to human health and, for the purposes of hazard evaluation, the “No Observed Adverse Effect Level” (NOAEL) for males, should be regarded as 1000 mg/kg/day.
No treatment-related effects were detected in the reproductive parameters measured, therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny 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 material on reproduction (including offspring development) and complies with the recommendations 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 comply 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 material was administered by gavage to three groups each of ten male and ten femaleWistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-five consecutive days (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 (Arachis oil BP).

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected males and females from each dose group.

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.

Males were terminated on Day 43, followed by the termination of all females and 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 during the study.

Clinical Observations.

No toxicologically significant clinical signs were detected.

Behavioural Assessment.

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

Functional Observations.

There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments.

There were no treatment-related changes in sensory reactivity.

Bodyweights.

No adverse effects on bodyweight change were detected for treated animals when compared to controls.

Food Consumption and Food Efficiency.No adverse effects on food consumption or food efficiency were detected for treated animals when compared to controls.

Water Consumptions.No intergroup differences were detected.

Haematology.

There were no treatment-related effects detected in the haematological parameters measured.

Blood Chemistry. There were no treatment-related effects detected in the blood chemical parameters measured.

Pathology:

Necropsy.No toxicologically significant effects were detected.

Organ Weights.No toxicologically significant effects were detected in the organ weights measured.

Histopathology.The following treatment-related effect was detected:

STOMACH:

Acanthosis, hyperkeratosis, subepithelial inflammatory cell infiltrates, epithelial erosion and ulceration were seen in the forestomach among males only treated with 1000 mg/kg/day. Two males treated with 300 mg/kg/day were similarly affected. Such changes are typically seen as a direct response to ingestion of an irritant substance and usually resolve on removal of the irritant. While gastric epithelial changes which include ulceration are considered to be adverse, a squamous-lined counterpart to the rodent forestomach is not present in humans.

Conclusion.

The oral administration of LCE08082 to rats for a period of up to fifty five days at dose levels of up to 1000 mg/kg/day resulted in treatment related changes in males treated with 1000 and 300 mg/kg/day. The 'No Observed Effect Level' (NOEL) for males was therefore, considered to be 100 mg/kg/day. No such effects were detected in females treated with 1000 or 300 mg/kg/day and the 'No Observed Effect Level' (NOEL) for females was therefore, considered to be 1000 mg/kg/day. The gastric changes detected in 1000 and 300 mg/kg/day males may be considered to be adverse, however because a squamous-lined counterpart to the rodent forestomach is not present in human stomachs, these effects in this study, are not considered to be indicative of a hazard to human health and, for the purposes of hazard evaluation, the “No Observed Adverse Effect Level” (NOAEL) for males, should be regarded as 1000 mg/kg/day.

No treatment-related effects were detected in the reproductive parameters measured, therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny was considered to be 1000 mg/kg/day.