Registration Dossier

Administrative data

Description of key information

The oral administration of 1,6-hexanediol reaction product with chloromethyloxirane to rats for a period of ninety consecutive days, to Wistar rats of both sexes at dose levels of 30, 100 or 300 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 300 mg/kg bw/day. A No Observed Effect Level (NOEL) for systemic toxicity was considered to be 100 mg/kg bw/day for male animals; this could not be established in females due to the slight body weight effects noted at 30 mg/kg bw/day. The microscopic liver change evident in animals of either sex treated with 300 mg/kg bw/day was considered to be an adaptive response to treatment and as such is considered to be non-adverse in nature. The stomach findings of ulceration or erosion and hyperplasia including hyperkeratosis (to varying degrees of severity) were noted in animals of either sex treated with 300 mg/kg bw/day and could be considered an adverse effect. However, these findings are likely to reflect an irritant effect of the test item on the non-glandular mucosa and are considered not to be relevant for humans as the unique structure of the rodent’s stomach is considered to have led to a prolonged exposure to the test item and the corresponding anatomical area is not present in man. In terms of risk assessment, the findings observed on this study would suggest that a No Observed Adverse Effect Level (NOAEL) can be established at 300 mg/kg bw/day for animals of either sex because the findings in general do not reflect true systemic toxicity.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han™:RccHan™:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were
examined for signs of ill-health or injury. The animals were acclimatized for nine days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 202 to 228g, the females weighed 149 to 177g, and were approximately six to eight weeks old.

Animal Care and Husbandry
The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Details on route of administration:
Dose Administration
Animals were allocated to treatment groups as follows:

Treatment Dose Level Treatment Concentration Animal Numbers
Group (mg/kg bw/day) Volume (mL/kg) (mg/mL) Male Female
Control 0 4 0 10 (1-10) 10 (11-20)
Low 30 4 7.5 10 (21-30) 10 (31-40)
Intermediate 100 4 25 10 (41-50) 10 (51-60)
High 300 4 75 10 (61-70) 10 (71-80)
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.

The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Vehicle:
arachis oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
see attached
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Serial Observations
General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Behavioral 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 color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior 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 Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour 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 grip strength 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. 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 Behavioral Assessments and Sensory Reactivity Tests.

The following parameters were observed:

Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Ophthalmoscopic Examination
The eyes of all control and treated animals were examined pre-treatment and the eyes of all control and high dose animals were examined before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using an ophthalmoscope was performed.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.

The methods used for hematological and blood chemical investigations are given in Annex 6 and normal ranges are shown in Annex 7.

Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)

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 Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)
Sacrifice and pathology:
Terminal Investigations
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

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 Ovaries
Brain Spleen
Epididymides Testes
Heart Thymus
Kidneys Uterus
Liver

Normal ranges for organ weights are given in Annex 8.

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) Rectum
Caecum Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymides ♦ Skin
Esophagus Spinal cord (cervical, mid-thoracic
Eyes* and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum (including Peyer’s patches) Testes ♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Tongue•
Lungs (with bronchi) # Trachea
Lymph nodes (mandibular and mesenteric) Urinary bladder
Mammary glands Uterus (with cervix)
Muscle (skeletal)• Vagina

• Retained only and not processed
* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative
• Retained only and not processed

All tissues were dispatched to the Test Site (Envigo CRS Limited, Eye Research Centre, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: J Schofield). All tissues from control and 300 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.

Since there were indications of microscopic changes, examination was subsequently extended to include similarly prepared sections of liver, stomach and thyroid from animals in the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by Vasanthi Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS. A complete histopathology phase report is presented in Annex 1 and represents the consensus view of both pathologists.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

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

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test.

Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed 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 shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p>0.05 (not significant)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A summary incidence of daily clinical observations is given in Table 1. Individual data are presented in Appendix 1.

Instances of increased salivation were evident in all animals treated with 300 mg/kg bw/day and in all males treated with 100 mg/kg bw/day throughout the majority of the treatment period. Two females treated with 100 mg/kg bw/day also exhibited increased salivation but only on one occasion each during the study. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are generally considered to be of no toxicological importance. One male treated with 300 mg/kg bw/day exhibited noisy respiration on three separate occasions (Days 20, 22 and 50) during the treatment period, however, these transient clinical signs were considered to reflect occasional difficulties with dosing this individual animal rather than any underlying toxicological effect of the test item. Two male animals treated with 300 mg/kg bw/day exhibited signs of lethargy which persisted for one day only (Day 8). In isolation this was considered incidental and considered to be of no toxicological importance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean weekly body weights and standard deviations are given in Table 6 and are presented graphically in Figure 1 and Figure 2. Group mean weekly body weight gains and standard deviations are given in Table 7 (statistically significant differences are indicated). Individual data are given in Appendix 7 and Appendix 8.

Male animals treated with 300 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment which achieved statistical significance (p<0.01). Thereafter, slightly lower body weight gains were noted on some occasions during the treatment period, however, statistical significance was only achieved during Week 11 of the treatment period (p<0.01). Overall body weight gain was found to be approximately 16 % lower than controls. Female animals from all treatment groups exhibited general reductions in body weight gain throughout the treatment period resulting in statistical significance (p<0.05) being noted in the 300 mg/kg bw/day treatment group during weeks 7 and 11. In contrast, a statistically significant (p<0.05) body weight gain was noted in this treatment group during Week 10. Female animals treated with 100 mg/kg bw/day showed a statistically significant (p<0.05) decrease in body weight gain during Week 11 and a statistically significant (p<0.05) increase in body weight gain was noted in this treatment group during Week 10. An increase in body weight/body weight gain is considered not to represent an adverse effect of treatment, therefore, these intergroup differences (body weight gains) were considered to be of no toxicological significance. Overall body weight gains for all female treatment groups were approximately 15 % to 19 % lower than controls but were not in a dose related manner. No such effects were noted in male animals treated with 30 or 100 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Group mean weekly food consumptions are given in Table 8 and are presented graphically in Figure 3 and Figure 4. Weekly food efficiencies are given in Table 9.

No significant effects were detected in food consumption for treated animals of either sex. Any slight reductions noted when compared to control were considered to be due to normal biological variation. Any reductions/fluctuations in food conversion efficiencies in treated animals were considered likely to be due to fluctuations in body weight gains and/or food intake.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not reveal any intergroup differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Individual ophthalmoscopic examination findings are given in Appendix 9.

Ophthalmoscopic examination of animals of either sex from the control and 300 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatmentrelated differences.
Haematological findings:
no effects observed
Description (incidence and severity):
Group mean values and standard deviations for test and control group animals are given in Table 10 (statistically significant differences are indicated). Individual data are given in Appendix 10 and Appendix 11.

There were no toxicologically significant effects detected in the hematological parameters examined.

Male animals treated with 300 mg/kg bw/day showed statistically significant decreases in hemoglobin (p<0.05), mean corpuscular hemoglobin (p<0.05), mean corpuscular hemoglobin concentration (p<0.01), total leukocyte count (p<0.01) and lymphocytes (p<0.01). Eosinophils were statistically significantly reduced (p<0.05) across all male treatment groups, however, a true dose related response was not evident. Male animals treated with 100 mg/kg bw/day exhibited a statistically significant reduction (p<0.05) in activated partial thromboplastin time.

Females from all treatment groups showed a statistically significant reduction (p<0.05) in mean corpuscular hemoglobin. Females treated with 100 and 300 mg/kg bw/day exhibited a statistically significant reduction (p<0.05) in mean corpuscular volume. A true dose related response was not evident in either parameter.

With the exception of the decrease in mean corpuscular hemoglobin concentration in male animals treated with 300 mg/kg bw/day where six animals exhibited values that were very slightly lower than the historical control data range, the majority of other values where statistical significance was noted lay within the historical data ranges for treated animals. In contrast, control male animals (two to five animals per parameter) exhibited increases from the historical control data range in these parameters which was considered to accentuate these apparent reductions and as such these findings are considered to be of no toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Group mean values and standard deviations for test and control group animals are given in Table 11 (statistically significant differences are indicated). Individual data are given in Appendix 12 and Appendix 13.

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

Males treated with 300 mg/kg bw/day showed statistically significant increases in potassium concentration (p<0.05) and albumin/globulin ratio (p<0.01) and a statistically significant decrease (p<0.01) in alanine aminotransferase (ALAT). Animals of either sex treated with 300 mg/kg bw/day also exhibited statistically significant increases in bile acids (p<0.05). Extremely large values were noted in two male animals for potassium which would explain the significant increase when compared to control but were not a reflection of the group as a whole. A very slight increase from the historical control data was noted in albumin/globulin ratio in three treated animals and all values for ALAT were within the historical control data range, however, three control animals exhibited values for ALAT which were actually higher than the historical control data range. As such, the intergroup differences were considered to be of no toxicological importance. The majority of animals treated with 300 mg/kg bw/day exhibited bile acid values which exceeded the historical control data ranges, however, four males and four females from the control group also exhibited values that exceeded these ranges. The increase in bile acids amongst treated animals is considered to be in relation to the adaptive liver changes and as such is considered not to represent an adverse effect of treatment.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
A summary incidence of behavioral assessment observations is given in Table 2 and group mean behavioral assessment scores are given in Table 3. Group mean functional performance test values and standard deviations are given in Table 4 (statistically significant differences are indicated). Individual values are given in Appendix 4 and Appendix 5. Group mean sensory reactivity assessments are given in Table 5. Individual responses are given in Appendix 6.

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

Functional Performance Tests
There were considered to be no toxicologically significant changes in functional performance.

A statistically significant decrease in overall activity (p<0.05) was observed for all treated male animals when compared to control. A statistically significant increase in the final 20% activity (p<0.05) was observed for females treated with 30 and 100 mg/kg bw/day when compared to control. However, as there were no clinical signs to signify a neurotoxic effect of the test item and all intergroup differences weren't occurring in a dose related manner these findings were considered to be of no toxicological relevance.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in Table 13 (statistically significant differences are indicated). Individual data are given in Appendix 15 and Appendix 16.

Males and females treated with 300 mg/kg bw/day exhibited statistically significant increases (p<0.01) in absolute and relative liver weights. Females treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.05) in absolute liver weight but showed a statistically significant increase (p<0.05) in relative liver weight. These findings correlate with the hypertrophic changes noted in the liver during histopathological examination and are considered to be due to an adaptive response to treatment with the test item.

Animals of either sex treated with 300 mg/kg bw/day showed statistically significant increases in absolute and relative kidney weight (p<0.01). Approximately 60% of control male animals, 90% of treated males, 30% of control females and 60% of treated females showed values that exceeded the normal control ranges. As there were no histopathological correlates to account for the effects on the kidneys, these findings are considered to be of no toxicological significance.

No such effects were noted in male animals treated with 100 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A summary incidence of necropsy findings is given in Table 12. Individual data are given inAppendix 14.

The following treatment related effects were noted in the stomachs of animals of either sex treated with 300 mg/kg bw/day: One male animal showed dark colored contents, all males and nine females exhibited raised white patches on the non-glandular region and nine males exhibited signs of sloughing and/or thickening of the non-glandular region or a raised limiting ridge.

The following findings were without histopathological correlates and were considered to be incidental and not related to treatment with the test item:

Three control males, two control females, two males treated with 30 mg/kg bw/day, two males treated with 100 mg/kg bw/day, three males and two females treated with 300 mg/kg bw/day exhibited reddened lungs. One control male, one control female, one female treated with 30 mg/kg bw/day, one male treated with 100 mg/kg bw/day and one male animal treated with 300 mg/kg bw/day exhibited dark patches on the lungs. One male animal treated with 300 mg/kg bw/day exhibited discolored lungs with dark patches. One control male and one control female exhibited an increased pelvic space in the right kidney. One control female exhibited an increased pelvic space in the left kidney with kidneys also being fluid filled. One male animal treated with 100 mg/kg bw/day exhibited an increased pelvic space in the left kidney. One female animal treated with 100 mg/kg bw/day exhibited an increased pelvic space in both kidneys. One male animal treated with 300 mg/kg bw/day exhibited an increased pelvic space in the left kidney which was also fluid filled.

One male animal treated with 100 mg/kg bw/day exhibited a small left horn of the seminal vesicles. One female animal treated with 100 mg/kg bw/day exhibited a dark liver.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A complete histopathology phase report is presented in Annex 1.

The following treatment-related microscopic abnormalities were detected:

Liver
Centrilobular hepatocyte hypertrophy was noted in four males and four females in Group 4 only.

Non Glandular Stomach
Ulceration or erosion was present in seven males and five females in Group 4.

Hyperplasia including hyperkeratosis, of moderate or marked severity, was present in all males in Group 4. Hyperplasia including hyperkeratosis, of mild or moderate severity, was noted in all females in Group 4.

No other findings were present at histopathology which could be attributed to treatment with the test item or correlated with in-life changes noted.

The appearance of the follicular epithelium in the thyroids was variable throughout animals in all groups and no consistent changes were apparent which could be related to treatment with the test item.
Details on results:
The oral administration of 1,6-hexanediol reaction product chloromethyloxirane to rats for a period of ninety consecutive days at dose levels of 30, 100 and 300 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 300 mg/kg bw/day and in female animals treated with 30 and 100 mg/kg bw/day.

Reductions in body weight gain were noted in male animals treated with 300 mg/kg bw/day during the first week of treatment. Thereafter, slightly lower body weight gains were noted on some occasions during the treatment period which resulted in an overall body weight gain which was approximately 16 % lower than controls. Female animals from all treatment groups exhibited general reductions in body weight gain throughout the treatment period. Overall body weight gains for all female treatment groups were approximately 15 % to 19 % lower than controls but were not in a dose related manner.

Although there were some statistically significant differences in treated animals from controls for the hematological and blood parameters measured, these differences were considered not to be of toxicological significance as the majority of values lay within the historical control data range and no histopathological correlates were apparent. A significant proportion of control data values also exceeded the historical control data and as such these findings were considered to be due to normal biological variation and not related to treatment of the test item.

Animals of either sex treated with 300 mg/kg bw/day exhibited increases in absolute and relative liver weights. Females treated with 100 mg/kg bw/day showed a reduction in absolute liver weight but showed a statistically significant increase (p<0.05) in relative liver weight. These findings correlate with the hypertrophic changes noted in the liver during histopathological examination and are considered to be due to an adaptive response to treatment with the test item.

Histopathological examination of the stomach revealed ulceration or erosion in the nonglandular region and was present in seven males and five females treated with 300 mg/kg bw/day. Hyperplasia including hyperkeratosis, of moderate or marked severity, was present in all males treated with 300 mg/kg bw/day and was noted at mild or moderate severity in all females from this dose group. These stomach findings could be considered an adverse effect as they point to an irritant effect of the test item on the non-glandular mucosa. However, these stomach findings are considered not to be relevant for humans as the unique structure of the rodent’s stomach is considered to lead to prolonged exposure to the test item and the corresponding anatomical area is not present in man. In terms of risk assessment, the findings observed on this study would suggest that a No Observed Adverse Effect Level (NOAEL) can be established at 300 mg/kg bw/day for animals of either sex because the findings in general do not reflect true systemic toxicity of the test item.

A No Observed Effect Level (NOEL) for systemic toxicity was considered to be 100 mg/kg bw/day for male animals; this could not be established in females due to the equivocal effect on body weight gain at 100 and 30 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: A No Observed Adverse Effect Level (NOAEL) can be established at 300 mg/kg bw/day for animals of either sex because the findings in general do not reflect true systemic toxicity of the test item.
Key result
Critical effects observed:
no
Conclusions:
The oral administration of 1,6-hexanediol reaction product with chloromethyloxirane to rats for a period of ninety consecutive days, to Wistar rats of both sexes at dose levels of 30, 100 or 300 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 300 mg/kg bw/day. A No Observed Effect Level (NOEL) for systemic toxicity was considered to be 100 mg/kg bw/day for male animals; this could not be established in females due to the slight body weight effects noted at 30 mg/kg bw/day. The microscopic liver change evident in animals of either sex treated with 300 mg/kg bw/day was considered to be an adaptive response to treatment and as such is considered to be non-adverse in nature. The stomach findings of ulceration or erosion and hyperplasia including hyperkeratosis (to varying degrees of severity) were noted in animals of either sex treated with 300 mg/kg bw/day and could be considered an adverse effect. However, these findings are likely to reflect an irritant effect of the test item on the non-glandular mucosa and are considered not to be relevant for humans as the unique structure of the rodent’s stomach is considered to have led to a prolonged exposure to the test item and the corresponding anatomical area is not present in man. In terms of risk assessment, the findings observed on this study would suggest that a No Observed Adverse Effect Level (NOAEL) can be established at 300 mg/kg bw/day for animals of either sex because the findings in general do not reflect true systemic toxicity.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:

The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

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

Methods

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 30, 100 and 300 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from all control and high dose animals (Groups 1 and 4) as well as any gross lesions from low and intermediate dose animals (Groups 2 and 3) was performed in the first instance. As there were treatment-related findings in the liver and stomach and equivocal changes in the thyroid, examination of these tissues was subsequently extended to include relevant animals from the low and intermediate dose groups.

Results

Mortality

There were no unscheduled deaths.

Clinical Observations

There were considered to be no clinical signs apparent that could be attributed to systemic toxicity of the test item.

Behavioral Assessment

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

Functional Performance Tests

There were considered to be no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Male animals treated with 300 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Thereafter, slightly lower body weight gains were noted on some occasions during the treatment period, this resulted in an overall reduction in body weight gain when compared to control. Female animals from all treatment groups exhibited general reductions in body weight gain throughout the treatment period, however, true dose related responses were not evident.

No such effects were noted in male animals treated with 30 or 100 mg/kg bw/day.

Food Consumption

No significant effects were detected in food consumption for treated animals of either sex.

Water Consumption

Visual inspection of water bottles did not reveal any intergroup differences.

Ophthalmoscopy

Ophthalmoscopic examination of animals of both sexes from the control and 300 mg/kg bw/day dose group during Week 12 of the treatment period did not indicate any treatmentrelated differences.

Hematology

There were no toxicologically significant effects detected in the hematological parameters examined.

Blood Chemistry

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

Necropsy

The following treatment related effects were noted in the stomachs of animals of either sex treated with 300 mg/kg bw/day: One male animal showed dark colored contents, all males and nine females exhibited raised white patches on the non-glandular region and seven males exhibited signs of sloughing and/or thickening of the non-glandular region or a raised limiting ridge.

All other findings were considered to be incidental and not related to treatment with the test item.

Organ Weights

Males and females treated with 300 mg/kg bw/day exhibited increases in absolute and relative liver weights. Females treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.05) in absolute liver weight but showed a statistically significant increase (p<0.05) in relative liver weight.

No such effects were noted in male animals treated with 100 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day.

Histopathology

Liver

Centrilobular hepatocyte hypertrophy was noted in four males and four females treated with 300 mg/kg bw/day only.

Non Glandular Stomach

Ulceration or erosion was present in seven males and five females treated with 300 mg/kg bw/day.

Hyperplasia including hyperkeratosis, of moderate or marked severity, was present in all males treated with 300 mg/kg bw/day. Hyperplasia including hyperkeratosis, of mild or moderate severity, was noted in all females treated with 300 mg/kg bw/day.

No other findings were present at histopathology which could be attributed to treatment with the test item or correlated with in-life changes noted.

Conclusion

The oral administration of 1,6-hexanediol reaction product chloromethyloxirane to rats for a period of ninety consecutive days, to Wistar rats of both sexes at dose levels of 30, 100 or 300 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 300 mg/kg bw/day. A No Observed Effect Level (NOEL) for systemic toxicity was considered to be 100 mg/kg bw/day for male animals; this could not be established in females due to the slight body weight effects noted at 30 mg/kg bw/day. The microscopic liver change evident in animals of either sex treated with 300 mg/kg bw/day was considered to be an adaptive response to treatment and as such is considered to be non-adverse in nature. The stomach findings of ulceration or erosion and hyperplasia including hyperkeratosis (to varying degrees of severity) were noted in animals of either sex treated with 300 mg/kg bw/day and could be considered an adverse effect. However, these findings are likely to reflect an irritant effect of the test item on the non-glandular mucosa and are considered not to be relevant for humans as the unique structure of the rodent’s stomach is considered to have led to a prolonged exposure to the test item and the corresponding anatomical area is not present in man. In terms of risk assessment, the findings observed on this study would suggest that a No Observed Adverse Effect Level (NOAEL) can be established at 300 mg/kg bw/day for animals of either sex because the findings in general do not reflect true systemic toxicity.

Endpoint conclusion
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEC
16 mg/m³
Study duration:
subacute
Species:
rat

Additional information

No systemic adverse findings were made in the oral gavage OECD 408 90 -day subchronic repeat dose toxicity study. Therefore, the NOAEL from this study is 300 mg/kg/day. The results from this study are considered to be most appropriate for derivation of both worker and general population long-term systemic oral and dermal exposure DNELs. Following E.C.H.A. guidance (E.C.H.A. RIP Guidance on information requirements and chemical safety assessment. Chapter R.8) and using the the rat oral repeated-dose NOAEL of 300 mg/kg/day the rsulting DNELs are:

Oral DNEL General Population long-term systemic = 0.83 mg/kg/day.

Dermal DNEL General Population long-term systemic = 1.7 mg/kg/day

Inhalation DNEL General Population long-term systemic = 2.9 mg/m3 (0.31 ppm)

Dermal DNEL Worker long-term systemic = 2.8 mg/kg/day

Inhalation DNEL Worker long-term systemic = 4.9 mg/m3 (0.52 ppm)

Justification for classification or non-classification

No significant adverse target organ effects were observed in rats exposed to 1,6-hexanediol reaction product with chloromethyloxirane

by either whole body inhalation or oral gavage. Therefore, no Classification and Labeling for systemic target organ effects is required.