Condensation products of oil shale alkylresorcinols and formaldehyde

Endpoint summary

Description of key information

Dhinsa NK et. al. (2004): 28-day repeated dose toxicity study via the oral route: NOAEL = 150 mg/kg/day based on blood chemistry and liver weights

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 April 2004 - 18 August 2004

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 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: ca. 5 - 6 weeks
- Weight at study initiation: males weighed 144 to 170 g, females weighed 132 to 176 g
- Housing: animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet (e.g. ad libitum): free access to pelleted rodent diet
- Water (e.g. ad libitum): free access to mains drinking water from polycarbonate bottles attached to the cage.
- Acclimation period: 7 days
- Other: Environmental enrichment was provided in the form of wooden chew blocks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 55 ± 15 %
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a suspension in Polyethylene glycol 400. Formulations were prepared weekly and stored at approximately +4ºC in the dark.
- Treatment volume: 4 mL/kg
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD
Summary:
The concentration of SF Resin in the test material formulations was determined spectrophotometrically.

Samples:
The test material formulations were diluted with methanol to give a final, theoretical test material concentration of approximately 0.05 mg/mL.

Standards:
Standard solutions of test material were prepared in methanol at a nominal concentration of 0.05 mg/mL.

Procedure:
The standard and sample solutions were analysed spectrophotometrically using the following conditions:
Spectrophotometer : Perkin-Elmer Lambda 20
Wavelength : λmax at ~ 279 nm
Cell path length : 1 cm
Reference medium : Methanol

Homogeneity Determinations:
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.

Stability Determinations:
The test material formulations were sampled and analysed initially and then after storage at approximately +4 ºC in the dark for fourteen days.

Verification of Test Material Formulation Concentrations:
The test material formulations were sampled and analysed within two days of preparation.

METHOD VALIDATION
Linearity:
A range of standard solutions covering the concentration range 0 to 0.101 mg/mL, were prepared and analysed. The detector response was shown to be linear up to 0.101 mg/mL.

Specificity:
The diluent solvent methanol and a blank Polyethylene glycol 400 (control) were analysed. Analysis of the solvent and a blank Polyethylene glycol 400 (control) produced no signal that interfered with the signal due to the test material.

Accuracy:
Samples of Polyethylene glycol 400 were accurately fortified with known amounts of test material, and analysed. The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.

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

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 15, 150, 750 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
0, 3.75, 37.5, 188 mg/ml
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

5 males and 5 females per dosage group

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends. All observations were recorded.

BODY WEIGHT:
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28. Bodyweights 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.

HAEMATOLOGY
Haematological investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

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) - Cresyl blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Thrombomax HS with calcium’ and Activated partial thromboplastin time (APTT) was assessed by ‘CK Prest’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY:
Blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

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

NEUROBEHAVIOURAL EXAMINATION - FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and on Days 4, 11, 18 and 25, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing.

> BEHAVIOURAL ASSESSMENTS
Detailed individual clinical observations were performed for each animal using a purpose built
arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behaviour
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper/Hypothermia
Skin colour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation

> 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 day, under similar laboratory conditions. The evaluation period was sixteen hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall sixteen hour period and also during the final 20 % of the period (considered to be the asymptotic period).

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. 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:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Startle reflex
Blink reflex

Sacrifice and pathology:

GROSS PATHOLOGY
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone 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
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus

HISTOPATHOLOGY
Samples of the following tissues were removed from all animals and preserved in buffered 10 % formalin:
Adrenals*
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)*
Brain (including cerebrum, cerebellum and pons)*
Caecum*
Colon*
Duodenum*
Epididymides*
Eyes
Gross lesions*
Heart*
Ileum*
Jejunum*
Kidneys*
Liver*
Lungs (with bronchi)* #
Lymph nodes (cervical and mesenteric)*
Muscle (skeletal)
Oesophagus
Ovaries*
Pancreas
Pituitary
Prostate*
Rectum*
Salivary glands (submaxillary)
Sciatic nerve*
Seminal vesicles*
Skin (hind limb)
Spinal cord (cervical)*
Spleen*
Stomach*
Testes*
Thymus*
Thyroid/parathyroid*
Trachea*
Urinary bladder*
Uterus*

# Lungs were inflated to approximately normal inspiratory volume with buffered 10 % formalin before immersion in fixative

The tissues with stars next to them from all control and 750 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 15 and 150 mg/kg/day dose group animals.
Since there were indications of treatment-related changes in the liver, examination was subsequently extended to include similarly prepared sections of this tissue from all animals in the other treatment groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate. Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity 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.
The haematology variable basophils was not analysed since consistently greater than 30 % of the data were recorded as the same value.

Probability values (p) are presented 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:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
None of the animals died during the study and no clinically observable signs of toxicity were detected in test or control animals throughout the study period. Increased salivation was detected prior to and up to ten minutes after dosing for animals of either sex treated with 750 mg/kg/day from Day 2 onwards. Isolated incidents were also observed at 150 mg/kg/day. Such observations are often reported following the oral administration of an unpleasant tasting and/or locally irritant test material formulation and, in isolation, are considered not to be indicative of systemic toxicity.
Red/brown staining around the eyes was observed for one male treated with 150 mg/kg/day on Day 21 and one control male on Day 27 only. These were incidental findings and were of no toxicological importance.

BODY WEIGHT AND WEIGHT GAIN
No adverse effect on bodyweight gain was detected. Bodyweight development in test animals was similar to that of controls.

FOOD CONSUMPTION
There was no adverse effect on food consumption during the study. Food efficiency (the ratio of bodyweight gain to dietary intake) in test animals was similar to that of controls.

WATER CONSUMPTION
Daily visual inspection of water bottles revealed no intergroup differences.

HAEMATOLOGY
Females treated with 750 mg/kg/day showed a statistically significant reduction (p < 0.01) in activated partial prothrombin time. This may have been attributed to higher than expected control values, but given the hepatic effects seen, a treatment-related effect cannot be ruled out.
No such effect was detected for males treated with 750 mg/kg/day or for animals of either sex treated with 150 or 15 mg/kg/day.

CLINICAL CHEMISTRY
A statistically significant increase in plasma bilirubin was detected for animals of either sex treated with 750 and 150 mg/kg/day (p < 0.01). Many values were outside the respective normal ranges for rats of the strain and age used. Alanine aminotransferase and aspartate aminotransferase were also statistically significantly elevated for 750 mg/kg/day females, with a number of values outside the normally expected ranges.
No such effects were detected for animals of either sex treated with 15 mg/kg/day.

NEUROBEHAVIOUR
> BEHAVIOURAL ASSESSMENTS
Weekly open field observations revealed no treatment-related effects.
All 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 were of no toxicological importance.

> FUNCTIONAL PERFORMANCE TESTS
No treatment related effects were detected in the functional performance parameters measured.
Statistical analysis of the data revealed no significant intergroup differences.

> SENSORY REACTIVITY ASSESSMENTS
There were no treatment related changes in sensory reactivity.
All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

ORGAN WEIGHTS
A statistically significant increase in liver weight, relative to terminal bodyweight, was detected for 750 mg/kg/day males. Relative liver weight was elevated for 750 mg/kg/day females and extended to 150 mg/kg/day males, but statistical significance was not achieved. Absolute weight was also affected for both 150 and 750 mg/kg/day males, many values were outside the respective normal ranges for rats of the strain and age used.
The remaining intergroup differences were confined to statistically significant reductions in epididymis weight throughout the male dose groups. The dose response relationship was unconvincing and in the absence of histopathological correlates at the high dose, the reduction was considered to be incidental and of no toxicological importance.
No such effects were detected for 150 mg/kg/day females or for animals of either sex treated with 15 mg/kg/day.

GROSS PATHOLOGY
No macroscopic abnormalities were detected at terminal kill.

HISTOPATHOLOGY: NON-NEOPLASTIC
LIVER: Centrilobular hepatocyte enlargement was observed in relation to treatment for males treated with 750 mg/kg/day. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature. In this instance the effect was marginal.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Blood chemistry (increase in plasma bilirubin) Histopthology (centrilobular hepatocyte enlargement in liver)

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Blood chemistry (increase in plasma bilirubin) Histopthology (centrilobular hepatocyte enlargement in liver)

Critical effects observed:

not specified

Table 1: Test Material Formulation Concentrations Over the Dosing Period

Nominal Concentration (mg/mL)	Mean Concentration Found		Range
	(mg/mL)	(expressed as % of nominal)	(mg/mL)	(expressed as % of nominal)
3.75	3.78	101	3.42 – 4.00	91 – 107
37.5	38.2	102	37.4 – 38.8	100 – 103
188	196	104	193 – 198	103 – 105

Table 2: Total bilirubin

Dose Level (mg/kg/day)	No. of Males	Total bilirubin (mg/dl) Mean ± Standard deviation	No. of Females	Total bilirubin (mg/dl) Mean ± Standard deviation
0 (control)	5	0.07 ± 0.01	5	0.05 ± 0.02
15	5	0.07 ± 0.01	5	0.06 ± 0.01
150	5	**0.20 ± 0.05	5	**0.19 ± 0.01
750	5	**0.24 ± 0.07	5	**0.36 ± 0.10

** = significantly different from control group p < 0.01

RANGE FINDING TEST

Mortality

No unscheduled deaths occurred during the fourteen day treatment period.

Clinical Observations

No clinically observable signs of toxicity were detected.

Increased salivation was observed prior to and up to ten minutes after dosing for treated animals from Day 1. This observation is often reported following the oral administration of an unpleasant tasting and/or locally irritant test material formulation and, in isolation, is considered not to be indicative of systemic toxicity.

Bodyweight

No adverse effects on bodyweight development were detected for test or control animals.

Necropsy

All animals treated with 750 mg/kg/day showed stomach changes including a raised limiting ridge, thickening of the gastric epithelia and reddened forestomach.

Conclusion

The dose levels for the main twenty-eight day study were chosen, following consultation with the Sponsor, as:

High dose: 750 mg/kg/day

Intermediate dose: 150 mg/kg/day

Low dose: 15 mg/kg/day

- plus a control group treated with vehicle alone.

Conclusions:

Oral administration of the test material, to rats for a period of twenty-eight consecutive days at dose levels of 15, 150 and 750 mg/kg/day, resulted in treatment-related effects at 150 and 750 mg/kg/day. No such effects were demonstrated at 15 mg/kg/day, therefore a ‘No Observed Effect Level’ (NOEL) was considered to be 15 mg/kg/day.
The minor changes detected at 150 mg/kg/day were confined to blood chemical and liver weight changes. These were considered not to represent ‘serious damage’ to health as defined by the criteria given in the EC labelling guide of Commission Directive 2001/59/EC. For this reason, a ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 150 mg/kg/day.

Executive summary:

The repeated dose toxicity of the test material was examined in a sub-actute toxicity test using rats. The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 750 mg/kg/day. A control group of five males and five females was dosed with vehicle alone.

The study was conducted in line with the OECD 407 and EU Method B.7 guidelines. The study was conducted to GLP standard, in a certified laboratory.

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

No effects on mortality, clinical signs, behaviour, functional performance, sensory reactivity, bodyweight, food consumption, water consumption or gross pathology were observed.

Haematology. A statistically significant reduction in activated partial prothrombin time was detected for females treated with 750 mg/kg/day. Males dosed with 750 mg/kg/day or animals of either sex treated at the other dose levels were unaffected.

Blood Chemistry. A statistically significant increase in plasma bilirubin was detected for animals of either sex treated with 750 and 150 mg/kg/day. Females treated with 750 mg/kg/day also showed statistically significant increases in plasma aspartate aminotransferase and alanine aminotransferase compared to controls. No such effects were detected at 15 mg/kg/day.

Organ Weights. Elevated liver weights were detected for animals of either sex treated with 750 mg/kg/day, extending to 150 mg/kg/day males. Females treated with 150 mg/kg/day and animals of either sex dosed at 15 mg/kg/day were unaffected.

Histopathology. Histopathological investigation revealed treatment-related liver changes, identified as centrilobular hepatocyte enlargement for males treated with 750 mg/kg/day. Females treated with 750 mg/kg/day and animals of either sex treated at the other dose levels were unaffected.

Oral administration of the test material, to rats for a period of twenty-eight consecutive days at dose levels of 15, 150 and 750 mg/kg/day, resulted in treatment-related effects at 150 and 750 mg/kg/day. No such effects were demonstrated at 15 mg/kg/day, therefore a ‘No Observed Effect Level’ (NOEL) was considered to be 15 mg/kg/day.

The minor changes detected at 150 mg/kg/day were confined to blood chemical and liver weight changes. These were considered not to represent ‘serious damage’ to health as defined by the criteria given in the EC labelling guide of Commission Directive 2001/59/EC. For this reason, a ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 150 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The sub-acute toxicity of the substance is adequately addressed by the key study. This study has been assaigned a reliability score of 1 in accordance with the scoring system of Klimisch et. al. (1997). No longer term studies are available. The quality of the database is good.

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

ORAL

The repeated dose toxicity of the test material was examined in a sub-acute toxicity test using rats. The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 750 mg/kg/day. A control group of five males and five females was dosed with vehicle alone.

The study was conducted in line with the OECD 407 and EU Method B.7 guidelines. The study was conducted to GLP standard, in a certified laboratory.

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

No effects on mortality, clinical signs, behaviour, functional performance, sensory reactivity, bodyweight, food consumption, water consumption or gross pathology were observed.

Haematology. A statistically significant reduction in activated partial prothrombin time was detected for females treated with 750 mg/kg/day. Males dosed with 750 mg/kg/day or animals of either sex treated at the other dose levels were unaffected.

Blood Chemistry. A statistically significant increase in plasma bilirubin was detected for animals of either sex treated with 750 and 150 mg/kg/day. Females treated with 750 mg/kg/day also showed statistically significant increases in plasma aspartate aminotransferase and alanine aminotransferase compared to controls. No such effects were detected at 15 mg/kg/day.

Organ Weights. Elevated liver weights were detected for animals of either sex treated with 750 mg/kg/day, extending to 150 mg/kg/day males. Females treated with 150 mg/kg/day and animals of either sex dosed at 15 mg/kg/day were unaffected.

Histopathology. Histopathological investigation revealed treatment-related liver changes, identified as centrilobular hepatocyte enlargement for males treated with 750 mg/kg/day. Females treated with 750 mg/kg/day and animals of either sex treated at the other dose levels were unaffected.

Oral administration of the test material, to rats for a period of twenty-eight consecutive days at dose levels of 15, 150 and 750 mg/kg/day, resulted in treatment-related effects at 150 and 750 mg/kg/day. No such effects were demonstrated at 15 mg/kg/day, therefore a ‘No Observed Effect Level’ (NOEL) was considered to be 15 mg/kg/day.

The minor changes detected at 150 mg/kg/day were confined to blood chemical and liver weight changes. These were considered not to represent ‘serious damage’ to health as defined by the criteria given in the EC labelling guide of Commission Directive 2001/59/EC. For this reason, a ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 150 mg/kg/day.

INHALATION

An adaptation to the standard data requirements has been provided to fulfil this data requirement, as it is not considered to be the most appropriate route of exposure, and since a more appropriate study is available, testing for this endpoint was omitted.

DERMAL

An adaptation to the standard data requirements has been provided to fulfil this data requirement, as it is not considered to be the most appropriate route of exposure, and since a more appropriate study is available, testing for this endpoint was omitted.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study was available to address this endpoint. The study was considered to be reliable enough to address this endpoint alone, and is therefore is selected as the key study.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

With respect to the repeated dose toxicity information available for this substance, no classification is required in accordance with Regulation (EC) No. 1272/2008 or Directive 67/548/EEC. The minor changes detected at 150 mg/kg/day were confined to blood chemical and liver weight changes. These were considered not to represent ‘serious damage’ to health as defined by the criteria given in the EC labelling guide of Commission Directive 2001/59/EC.