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

Endpoint summary

Description of key information

Test material: the liquid marketed product "Proteol OAT", batch LCE08082 which contains ca.27.5% of the solid substance "Glutens, hydrozylates, reaction products with lauroyl chloride, sodium salts".

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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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.

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

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

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

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral 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.


DIET PREPARATION
A pelleted diet Rodent 2018C Teklad Global Certified Diet Harlan UK Ltd, Oxon, UK was used throughout the study period.

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

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

- Storage temperature of food:
No data

VEHICLE
Arachis oil

- Justification for use and choice of vehicle (if other than water):
considered suitable following vehicle determination

- Concentration in vehicle:
0, 25, 75, 250 mg/ml

- Amount of vehicle (if gavage):
4 mg/kg

- Lot/batch no. (if required):
0818600011

- Purity:
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 for a period of up to fifty-four consecutive days.

Frequency of treatment:

Daily

Remarks:

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

Remarks:

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

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

- Rationale for selecting satellite groups:
Not applicable

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

- Section schedule rationale (if not random):
Random

Positive control:

Not applicable

Observations and examinations performed and frequency:

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.

BODY WEIGHT:
Yes

- Time schedule:
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:
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.



FOOD EFFICIENCY:
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.

OPHTHALMOSCOPIC EXAMINATION:
No
- Time schedule for examinations:
Not applicable

- Dose groups that were examined:
Not applicable

HAEMATOLOGY AND CLINICAL CHEMISTRY:
Yes

- 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 on Day 14 (day prior to pairing). Blood samples were obtained from the lateral tail vein at Day 14. Animals were not fasted prior to sampling.

- Anaesthetic used for blood collection:
No

- Animals fasted:
No

- How many animals:
Five males and 5 females selected from each test and control group on Day 14.


- For a list of Parameters investigated, see Any Other Deatails on Method Section

URINALYSIS: No
- Time schedule for collection of urine:
Not applicable

- Metabolism cages used for collection of urine:
Not applicable

- Animals fasted:
Not applicable

- Parameters examined: Not applicable

NEUROBEHAVIOURAL EXAMINATION:
Yes

- 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. 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 Day 1 and 4 post partum
iii) Sex of offspring on Day 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring and litter weights on Day 1 and 4 post partum


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

Sacrifice and pathology:

GROSS PATHOLOGY:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

All adult animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.


HISTOPATHOLOGY:
Samples of tissues were preserved from five males and five females from each dose group, in buffered 10% formalin except where indicated. The tissues shown in bold were also removed from the remaining animals. (see Any Other Details on Methods Section for list of tissues)
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.

Other examinations:

The following organs were removed from all adult animals killed at the end of the treatment period, dissected free from fat and weighed before fixation: Adrenals, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Thyroid, Liver

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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

ADULT RESPONSES

CLINICAL SIGNS
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.



MORTALITY
There were no unscheduled deaths during the study.

BODY WEIGHT AND WEIGHT GAIN
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.


FOOD CONSUMPTION AND FOOD EFFICIENCY
Males
No adverse effect on food consumption or food efficiency was detected for males throughout the treatment period.
Females
No adverse effect on food consumption or food efficiency was detected for females throughout the two week maturation period or during gestation or lactation phase of the study.

WATER CONSUMPTION
Daily visual inspection of water bottles did not reveal any significant intergroup differences in water consumption for treated animals when compared to controls.


HAEMATOLOGY
No treatment-related effects were detected in the haematological parameters measured.
Statistical analysis of the data did not reveal any significant intergroup differences

CLINICAL CHEMISTRY
No treatment-related effects were detected in the blood chemical parameters measured.
Statistical analysis of the data did not reveal any significant intergroup differences.


NEUROBEHAVIOUR
(Behavioural assessment)
There were no treatment-related changes in behavioural parameters measured.

(Functional Performance Tests)
There were no treatment-related changes in functional performance.


(Sensory Reactivity)
There were no treatment-related changes in sensory reactivity.
Any 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 were of no toxicological importance.


ORGAN WEIGHTS
No treatment-related effects were detected in the organ weights measured.
Statistical analysis of the data did not reveal any significant intergroup differences.


GROSS PATHOLOGY
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: NON-NEOPLASTIC
The following treatment-related changes were observed:
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 HISTOPATHOLOGY
Remaining histopathological changes seen among surviving control and intermediate dose animals were all considered to be spontaneous in origin and unrelated to treatment. The following conditions warrant specific mention:

ADRENAL GLANDS: Cortical vacuolation was seen in a few control and treated male rats and was of no toxicological significance in this investigation.

BONE MARROW: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study there was no difference between control and treated groups.

HEART: Focal myocarditis was observed in a few control and treated rats and is a common background lesion in laboratory maintained rats. The severity of the condition was never greater than minimal, or one or two foci, and should not be interpreted as being indicative of any ongoing myocardial disease.

KIDNEYS: Isolated groups of basophilic tubules are frequently encountered in the renal cortex of laboratory maintained rats and have no pathological significance at the severities or frequencies reported in this study. Similarly focal corticomedullary mineralisation is a commonly observed background condition among female rats and the slight disparity between control and treatment groups in this investigation is of no toxicological significance. Hydronephrosis was reported for a two rats and is widely considered to be a condition of congenital origin. Globular accumulations of eosinophilic material, as a consequence of excessive accumulation of 2-microglobulin in renal proximal tubular epithelial cells, are occasionally encountered as a spontaneous change in male rats.

LIVER: Scattered mononuclear cell foci were observed in the majority of control and treated rats examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered. Isolated instances of generalised hepatocyte enlargement, periportal pigment accumulation and lipid-type vacuolation were also seen.

LUNGS: A minimal severity of bronchus associated lymphoid tissue was reported for the majority of control and high dose animals examined in the study and is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis and accumulations of alveolar macrophages are commonly observed pulmonary changes in laboratory maintained rats of this age and are not suggestive of significant respiratory disease.

OESOPHAGUS: Inflammatory cell infiltrates in the peripheral musculature is a commonly observed change that is considered to be related to the physical trauma of gavage dosing.

SKELETAL MUSCLE: Mononuclear cell foci are commonly observed in the skeletal muscle of laboratory maintained rats, especially in post-partum females, and are of no toxicological significance at the incidences seen in this investigation.

SPLEEN: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits for males and previously pregnant females.

THYROID: Follicular cell hypertrophy is commonly seen among untreated rats of either sex and there was no indication of a relationship to treatment in this study.

THYMUS: Lymphoid atrophy is frequently seen among pregnant and lactating female rats and there was no evidence of a treatment related group distribution of incidence or severity in this study.


REPRODUCTIVE TRACT AND RELATED ORGANS

PITUITARY: No treatment-related changes were seen. Vacuolation of pars anterior cells is commonly seen as a spontaneous change, more especially among male rats

TESTIS/EPIDIDYMIS: No treatment-related changes were seen. Epididymal spermatocoel granuloma formation and testicular atrophy are observed occasionally as a spontaneous conditions.

SEMINAL VESICLES/COAGULATING GLAND: No treatment-related changes were seen. Reduced secretory content was observed occasionally.

PROSTATE: Interstitial chronic inflammatory cell infiltrates are a commonly observed background finding in laboratory maintained rats.

MAMMARY GLAND: Glandular hyperplasia was observed in the mammary tissue of the majority of female rats examined. The appearance of the mammary tissue is consistent with pregnancy and lactation.

OVARY: No pathological changes were seen.

UTERUS: Areas of haemorrhage and fibrosis 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.

VAGINA: Keratinisation of the epithelium is a normal cyclical change among female rats.


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.

Dose descriptor:

NOEL

Remarks:

systemic toxicity

Effect level:

1 000 other: mg/kg/day

Sex:

female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

1 000 other: mg/kg/day

Sex:

male

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

1.                          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

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 4 post partum.

Males 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.

Results

Adult Responses:

Mortality.There were no unscheduled deaths during the study.

Clinical Signs.No toxicologically significant clinical signs were detected.

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

Functional Performance Tests.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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed