Registration Dossier

Administrative data

Description of key information

A NOAEL value of 1000 mg/kg bw/day was obtained in an OECD 422 combined repeated dose toxicity and reproductive effects test.

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
24 May 2010 and 22 September 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Wistar:HsdHan:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon UK
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: males, 322 to 373 g; females, 201 to 244 g
- Fasting period before study: none
- Housing: solid floor polypropylene cages with stainless steel mesh lids with softwood flake bedding (Datesand Ltd., Cheshire, UK); during mating, animals were transferred to polypropylene cages with grid floors suspended over trays of absorbent paper
- Diet (ad libitum): Rodent 2018C, Teklad Global Certified Diet
- Water (ad libitum): mains drinking water
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 55 +/- 15%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 26 May 2010 (first day of treatment) to 10 July 2010 (final necropsy)
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
Arachis Oil BP
Details on oral exposure:
Dosing formulations were prepared twice monthly and stored at +4 deg C in the dark. Stability was previously determined by the test laboratory (Project No.: 2600-0021).

The test item was administered by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated identically with vehicle.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

Chronological Sequence:

i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition, where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weightby sex, clinical observations and landmark developmental signs were also performed during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.
ix) Blood samples were taken from five randomly selected females from each dose group at termination for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test item formulation were taken and analyzed for concentration of undecenyl methoxycrylene at Harlan Laboratories Ltd.

The test item formulations were diluted with acetone to give a final, theoretical test item concentration of approximately 0.1 mg/ml.

The standard and sample solutions were analysed by GC using the following conditions:
GC system : Agilent Technologies 5890, incorporating autosampler and workstation
Column : DB-5 (30 m x 0.53 mm id x 5 μm film)
Oven temperature program : initial 100 ºC for 0 mins; rate 20 ºC/min; final 280 ºC for 8 mins
Injection temperature : 280 ºC
Flame ionisation detector temperature: 280 ºC
Injection volume : 2 μl
Retention time : ~ 4.3 mins

Concentrations ranged for 88 to 104% of nominal.
Duration of treatment / exposure:
see Details on Oral exposure above
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bwt/day
Basis:
other: nominal administered
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Mating:

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.

see Details of oral exposure above.

Positive control:
None
Observations and examinations performed and frequency:
Clinical Observations
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). All observations were recorded.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioural Assessments
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 closureBizarre/Abnormal/Stereotypic behaviour Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.

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, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal wasallowed 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. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.

The following parameters were observed:
Grasp response Touch escape
Vocalisation Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

Food Consumption
During the maturation period, weekly food consumption was recorded for each cage of non-recovery adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum 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 (the ratio of body weight 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
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.

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

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

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Laboratory Investigations
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. Where necessary repeat samples were taken by cardiac puncture at termination.

Animals were not fasted prior to sampling.

Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed.
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Calcium (Ca++)
Glucose Inorganic phosphorus (P)
Total protein (Tot.Prot.) Aspartate aminotransferase (ASAT)
Albumin Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation) Alkaline phosphatase (AP)
Sodium (Na+) Creatinine (Creat)
Potassium (K+) Total cholesterol (Chol)
Chloride (Cl-) Total bilirubin (Bili)
Bile acids (Bile)

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post-fixation).
Sacrifice and pathology:
Pathology
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

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

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

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
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 tissue, Uterus/Cervix, Muscle (skeletal), Vagina.

All tissues were subjected to histology processing Test Site (Harlan Laboratories Ltd., Zelgliweg 1, CH-4452 Itingen, Switzerland) for processing (Principal Investigator: K Weber). The tissues from five selected control and 1000 mg/kg/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a 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 animals 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 liver and thyroid, examination was subsequently extended to include similarly prepared sections of liver and thyroid from five animals per sex from the low and intermediate groups.

Microscopic examination was conducted at the histopathology examination Test Site (AnaPath GmbH, Oberbuchsiten, Switzerland) by the Study Pathologist (K Heider).
Other examinations:
see discussion above
Statistics:
Data for males and females prior to pairing, and functional performance test data, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Bartletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was
found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data for females during gestation and lactation, and offspring 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) were calculated as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
p≥0.05 (not significant)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Adult Responses

Mortality

There were no unscheduled deaths during the treatment period.

Clinical Observations

No clinically observable signs of toxicity were detected during the treatment period.

Increased salivation was detected soon after and also up to one hour after dosing for all animals treated with 1000 mg/kg/day. Increased salivation was also observed for one female treated with 1000 mg/kg/day prior to treatment, although this was only observed on one day. Similar effects were also evident for animals of either sex treated with 300 mg/kg/day and for one male treated with 100 mg/kg/day. Instances of staining around the mouth were also observed in the 1000 and 300 mg/kg/day dose groups and one instance of wet fur was noted for two females treated with 1000 mg/kg/day. Thes eobservations are commonly observed following the oral administration of a slightly irritant or unpalatable test item formulation. In the absence of any associated findings to suggest irritancy, these observations were considered to be unrelated to test item toxicity.

Generalised fur loss was observed for one control female, three females treated with 1000 mg/kg/day and one male treated with 300 mg/kg/day. Scab formation was observed for one female treated with 100 mg/kg/day. These observations are occasionally observed in laboratory maintained animals and are considered to be unrelated to treatment with the test item.

Functional Observations

Behavioural Assessments

No treatment-related observations were detected during the weekly open-field arena observations. Noisy respiration was observed for one male treated with 1000 mg/kg/day during the Week 4 assessments. This was not observed during the daily clinical observations, and as such, was considered to have arisen incidentally and did not represent a true effect of treatment. All remaining inter and intra group differences in urination, defecation and transfer arousal 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.

Functional Performance Tests

No treatment-related effects were detected following the grip strength or motor activity assessments for treated animals when compared to controls.
Statistical analysis of this data did not reveal any significant intergroup differences.

Sensory Reactivity Assessments

No treatment-related effects were evident in sensory reactivity scores for treated animals when compared to controls. 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.

Body Weight

No adverse effects on body weight change were detected for treated males when compared to controls during the treatment period. Females treated with 1000 mg/kg/day showed a statistically significant reduction in body weight change during the first week of treatment, when compared to controls (P<0.05). No such effects were evident thereafter, as such, this reduction was considered not to represent an adverse effect of treatment. No differences in body weight change were evident for females during the gestation or lactation phases of the study.

Food Consumption

No adverse effect on dietary intake or food conversion efficiency (the ratio of body weight gain to dietary intake) was observed for treated males in comparison to controls throughout the treatment period (with the exception of the mating phase, when food consumption was not recorded), or for treated females during the pre-mating phase of the study.

Females treated with 1000 mg/kg/day showed a slight reduction in dietary intake when compared to controls during the first week of treatment. No such reductions were evident thereafter, and as such, this minimal reduction was considered not to represent a true effect of treatment.

Water Consumption

Daily visual inspection of water bottles did not reveal any significant intergroup differences between test and control groups.

Reproductive Performance

Mating

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

Fertility

No treatment-related effects on fertility were detected between treated animals when compared to controls. One male and female pair treated with 100 mg/kg/day which showed evidence of mating, did not achieve a pregnancy. This finding is occasionally observed in reproductive studies of this type, and as such, was considered to be unrelated to treatment.

Gestation Length

No treatment-related effects were detected in the length of gestation between control and treated groups. The length of gestation ranged between 22 to 23½ days for control and test animals. Statistical analysis of the data did not reveal any significant intergroup differences.

Litter Response

In total, all ten females from the control, 300 and 1000 mg/kg/day dose groups and nine females from the 100 mg/kg/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. There was one non-pregnant female in the 100 mg/kg/day dose group. 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

No treatment-related differences in corpora lutea, implantation sites, or pre- and postimplantation losses were detected for females from treated groups when compared to controls. No differences in litter size or viability were evident for offspring from treated litters when compared to those from the controls. Statistical analysis of these data did not result in any significant intergroup differences.

Offspring Growth and Development

No treatment-related effects in litter weights, offspring weights or surface righting were detected for litters from treated groups when compared to control litters. Statistical analysis of these data did not result in any significant intergroup differences. The number of interim offspring found dead or missing (cannabalised by the mother) was essentially similar throughout the control and treatment groups. No treatment-related clinical signs were observed. The clinical signs in the offspring observed in this study were considered to be low incidence findings commonly observed in reproductive studies of this type and unrelated to test item toxicity. Surface righting was not affected at any treatment level and statistical analysis of this data did not reveal any significant intergroup differences.

Laboratory Investigations

Haematology

No toxicologically significant intergroup differences were detected in the haematology parameters investigated. Males treated with 1000 mg/kg/day showed a statistically significant reduction in mean cell haemoglobin when compared to the concurrent controls. The significance achieved was minimal (P<0.05) and all individual values were within the normally expected ranges for this parameter. As such, this reduction was considered to have arisen incidentally. Mean cell haemoglobin concentration (MCHC) was significantly reduced for males treated with 1000 and 300 mg/kg/day when compared to controls (P<0.01 and P<0.05 respectively). All individual values for the treated animals were slightly lower than the normally expected ranges, suggesting that these reductions were attributable to treatment with the test item. However in the absence of any supporting data to suggest that these reductions resulted in a toxicologically significant effect on the haemopoietic system, these reductions in MCHC were considered not to represent an adverse effect. Furthermore, statistically significant increases in neutrophil counts were evident for males treated with 100 mg/kg/day when compared to controls (P<0.01). No such effects were detected for females and there was no evidence of an immune response to the test item. As such, this finding was considered not to represent an adverse effect of treatment.

Blood Clinical Chemistry

No toxicologically significant intergroup differences were detected in the blood chemical parameters investigated. Males treated with 1000 and 300 mg/kg/day showed statistically significant increases in blood calcium levels (P<0.01) and reductions in inorganic phosphorus levels (P<0.05) when compared to controls. With the exception of one control value, all individual values were within the normally expected ranges for these parameters. Furthermore, creatinine levels were significantly higher for males from all treated groups when compared to control values. The significance achieved in each case was minimal (P<0.05). All individual values for the treated animals were within the normally expected ranges, therefore the higher values observed in the treated groups, were considered to be attributable to one lower than expected value from the control group for this parameter. Therefore, this intergroup difference was considered not to represent a true effect of treatment.

Pathology

Organ Weights

Females treated with 1000 mg/kg/day showed statistically significant increases in absolute and body weight-relative liver weights when compared to controls (P<0.001 and P<0.05 respectively). No such effects were evident for males treated with 1000 mg/kg/day. A slight reduction in absolute and body weight-relative liver weights was observed for males treated with 1000 mg/kg/day when compared to controls (P<0.05). Although some individual values were higher than the normally expected ranges for these parameters in the male 1000 mg/kg/day dose group, higher than expected values were also observed in the control groups. In the absence of any convincing histopathological changes to confirm the cause of this slight reduction, these findings were considered to be of minimal toxicological importance. Furthermore, males treated with 300 mg/kg/day showed an increase in liver weights when compared to controls. In the absence of any treatment-related histopathological correlates observed in the liver at this dose level, this
minimal increase (P<0.05) was considered to have arisen incidentally. Finally, males treated with 1000 and 300 mg/kg/day showed a slight reduction in absolute and bodyweight-relative seminal vesicles weights when compared to controls (P<0.05). In the absence of any histopathological correlates, or any indication of an adverse effect of fertility, these minor reductions were considered to have arisen incidentally and were unrelated to treatment with the test item.

Necropsy

Offspring

No treatment-related macroscopic abnormalities were detected in interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to the toxicity of the test item.

Adults

No treatment-related macroscopic abnormalities were detected at terminal kill. Generalised fur loss was observed for two females treated with 1000 mg/kg/day. Macroscopic observations in the treated group were confined to a reddened thymus, which was evident for one female treated with 1000 mg/kg/day. In isolation, and in the absence of any treatment-related histopathological thymic changes, this finding was considered to have arisen incidentally. Isolated incidents of reddened lungs, hydronephrosis and pallor of the right kidneys were observed. In the absence of any histopathological correlates, these findings were considered to be low incidence findings occasionally observed in laboratory maintained animals and unrelated to test item toxicity.

Histopathology

Histopathology examinations revealed the following treatment-related effects:

Liver: Diffuse or periportal hepatocellular hypertrophy was recorded at minimal to slight severity in all 1000 mg/kg/day animals. This hypertrophy was mainly characterized by an increased cytoplasmic density and/or size of periportal liver cells and an increased size of mainly centrilobular liver cells.
Incidence and Mean Severity of Relevant Liver Findings (see Table 1 below)

Thyroid gland: Increased incidence and/or mean severity of diffuse follicular cell hypertrophy in the thyroid were recorded in animals treated with 1000 mg/kg/day when compared with the control group.

Incidence and Mean Severity of Relevant Thyroid Findings (see Table 2 below)

The remaining microscopic findings recorded in this study were within the range of normal background lesions which may be recorded in animals of this strain and age.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity: There were no toxicologically significant effects noted in this study.
Critical effects observed:
not specified

Table 1: Liver findings

Groups 1 2 3 4
Tissues examined 5M 5F 5M 5F 5M 5F 5M 5F
Hepatocellular
Hypertrophy:
Periportal 0 0 0 0 0 0 4 4

Mean severity

0 0 0 0 0 0 1 1
Diffuse 0 0 0 0 0 0 1 4

Mean severity

0 0 0 0 0 0 1 1.3
Total Affected 0 0 0 0 0 0 5 5

Table 2: Thyroid gland findings

Groups 1 2 3 4
Tissues examined 5M 5F 5M 5F 5M 5F 5M 5F
Follicular Cell Hypertrophy:
Diffuse 4 3 4 2 4 2 4 5

Mean Severity

1 1 1 1 1 1 2 1.8
Total Affected 4 3 4 2 4 2 4 5
Conclusions:
The oral administration of undecenyl methoxycrylene to rats by gavage at a maximum dose level of 1000 mg/kg/day did not result in any toxicologically significant effects of treatment. The minor effects detected in this study were not considered to represent an adverse effect of treatment; therefore a ‘No Observed Adverse Effect Level’ (NOAEL) was established at 1000 mg/kg/day for systemic toxicity.
Executive summary:

The oral administration of undecenyl methoxycrylene to rats for a period of forty-two days for males and up to eight weeks for females (including two weeks pre-mating, gestation and early lactation period) at dose levels of 1000, 300 and 100 mg/kg/day resulted in minor treatment-related effects. These were considered of no toxicological significance.

No treatment-related effects were evident in the functional performance tests and no adverse effects on body weight change, dietary intake or food conversion efficiency were observed. Haematology and blood chemical investigations did not reveal any toxicologically significant changes. The slight changes detected during these investigations were considered to be of no toxicological importance.

Post-mortem examinations revealed increases in liver weights for females treated at the highest dose level in comparison to controls. These increases were supported by histopathological examinations, which revealed hepatocyte hypertrophy of the liver for animals of either sex treated with 1000 mg/kg/day. Histopathological examinations also revealed changes in the thyroid gland, consisting of increased incidence and/or severity of follicular cell hypertrophy in the 1000 mg/kg/day dose group when compared to the controls. Hepatocellular hypertrophy is commonly observed in the rodent liver following treatment with xenobiotics and, in the absence of degenerative or inflammatory changes, is generally considered to be an adaptive response. Follicular cell hypertrophy of the thyroid glands is a secondary response to the changes seen in the liver as thyroxine is ultimately excreted via the bile, having first been conjugated in the liver. Any induction of the enzymes involved would result in increased thyroxine excretion and compensatory TSH and thyroxine production resulting in the microscopic changes identified. Based on the findings of this study, the treatment-related effects detected in this study were considered not to represent systemic toxicity; therefore a ‘No Observed Adverse Effect Level’ (NOAEL) was established at 1000 mg/kg/day for systemic toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The surrogate material, undecenyl methoxycrylene, was administered to rats for a period of forty-two days for males and up to eight weeks for females (including two weeks pre-mating, gestation and early lactation period) at dose levels of 1000, 300 and 100 mg/kg/day in an OECD 422 Guideline study. No treatment-related effects were evident in functional performance tests and no adverse effects on body weight, dietary intake or food conversion efficiency were observed. Haematology and blood chemical investigations revealed minimal effects at the highest administered dose but these were not considered to be toxicologically significant changes. Post-mortem examinations revealed increases in liver weights for females treated at the highest dose level in comparison to controls. These increases were supported by histopathological examinations, which revealed hepatocyte hypertrophy of the liver for animals of either sex treated with 1000 mg/kg/day. Histopathological examinations also revealed changes in the thyroid gland, consisting of increased incidence and/or severity of follicular cell hypertrophy in the 1000 mg/kg/day dose group when compared to the controls. Hepatocellular hypertrophy is commonly observed in the rodent liver following treatment with xenobiotics and, in the absence of degenerative or inflammatory changes, is generally considered to be an adaptive response. Follicular cell hypertrophy of the thyroid glands is a secondary response to the changes seen in the liver as thyroxine is ultimately excreted via the bile, having first been conjugated in the liver. Any induction of the enzymes involved would result in increased thyroxine excretion and compensatory TSH and thyroxine production resulting in the microscopic changes identified. Based on the findings of this study, the treatment-related effects detected in this study were considered not to represent systemic toxicity; therefore a NOAEL was established at 1000 mg/kg/day for systemic toxicity.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A close structurally analogue has been used to conduct this study as, the substance EHMC is used as a cosmetic ingredient, please see our read across proposal in section 13. The study conducted on UMC is an OECD guidline study which has been conducted to GLP, this would normally be considered a reliability 1 study however as read across has been used it has been doengraded to a reliability of 2 according to the klimisch scale.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: other; digestive: liver; glandular: thyroids

Justification for classification or non-classification

Based on the results for the surrogate material, undecenyl methoxycrylene, the subject chemical would not be rated for repeated dose effects. This is based on a lack of toxicologically significant changes at dose levels up to 1000 mg/kg bw/day. Hepatocellular hypertrophy was considered to be an adaptive response and follicular cell hypertrophy of the thyroid gland a secondary response to the effects seen in the liver. Under EU Directive 67/548/EEC, the test material would not be rated as R48 (Danger of serious damage to health by prolonged exposure). Similarly, the test material would not be rated for “Specific Target Organ Toxicity – Repeated Exposure” according to Directive 67/548/EEC, EU CLP (Regulation (EC) No. 1272/2008, and UN GHS.