3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)pent-3-en-2-one

Endpoint summary

Description of key information

An OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents) was performed to GLP, a synopsis is given below.

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Between 26 February 2010 and 09 July 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Assigned reliability score of 2 on the basis that the test substance is being used for read-across.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the Law Co

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar Han™:HsdHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source:
Wistar Han™:HsdHan™:WIST strain rats obtained from Harlan Laboratories U.K. Ltd., Oxon, UK.

- Age at study initiation:
six to eight weeks old

- Weight at study initiation:
213 to 248g (male), 166 to 202g (female)

- Fasting period before study:
Not applicable

- Housing:
The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.

- Diet:
A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used (ad libitum). The diet was considered not to contain any level of contaminant.

- Water (e.g. ad libitum):
ad libitum mains water was supplied from polycarbonate bottles attached to the cage. The drinking water was considered not to contain any level of contaminant.

- Acclimation period:
Seven days

ENVIRONMENTAL CONDITIONS
- Temperature:
: (°C): 21 ± 2

- Humidity (%):
: 55 ± 15

- Air changes (per hr):
: At least fifteen air changes per hour

- Photoperiod (hr dark / hrs light):
: 12 hours continuous light and 12 hours darkness

IN-LIFE DATES:
The in-life phase of the study was conducted between 19 March 2010 (Day 1, first day of treatment) and 30 April 2010 (Day 43, Recovery Control and High dose final day of necropsy).

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Method of administration:
Gavage

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Corn oil.

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

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

- Storage temperature of food:
Not applicable.

VEHICLE
- Justification for use and choice of vehicle(if other than water): Most suitable

- Concentration in vehicle:
8.75, 81.3 and 250 mg/ml.

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

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

- Purity:
Unknown

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test material was prepared at the appropriate concentrations as a solution in Corn oil. The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 0766-0168) The analytical method using gas chromatography was satisfactorily validated in terms of linearity, specificity and accuracy. Results from 0766-0168 showed the formulations to be stable for at least twenty one days. Formulations in this study (0766-0169) were therefore prepared twice during the treatment period and stored at approximately 4ºC in the dark.

Samples of each test formulation were taken and analyzed for concentration of test material by Harlan Laboratories Ltd., Shardlow, UK Analytical Services. The results obtained showed that the prepared formulations were acceptable for the purpose of this study.

See Attached Appendix 18 - Chemical Analysis of Test Material Formulations.

Duration of treatment / exposure:

The test material was administered daily, for twenty-eight consecutive days, by oral gavage to all rats. Control animals were treated in an identical manner with 4 ml/kg/day of Corn oil. Recovery control and high dose group animals were maintained for a further fourteen days treatment-free period following termination of treatment.

Frequency of treatment:

Dosing regime: 7 days/week

Remarks:

Doses / Concentrations:
Dose levels of 35, 325 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Male and Female: 10 animals per sex at 0 mg/kg/day (Control)
Male and Female: 5 animals per sex at 35 mg/kg/day
Male and Female: 5 animals per sex at 335 mg/kg/day
Male and Female: 10 animals per sex at 1000 mg/kg/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on a preliminary Seven Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Harlan Laboratories Ltd., Project Number 0766-0168.

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

- Rationale for selecting satellite groups:
For regulatory compliance and to determine potential regression of any detected systemic responses elicited by administration of the test material

- Post-exposure recovery period in satellite groups:
Fourteen days

- Section schedule rationale (if not random):
Random

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes - see attached tables and appendices.

- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes see above

- Time schedule: As above

NEUROBEHAVIOURAL EXAMINATION: Yes see attached tables and appendices

Functional Observations were performed prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity.

Functional performance tests (motor activity, forelimb/hindlimb grip strength and sensory reactivity) were also performed on five selected males during the final week of treatment and five Day 4 post partum females from each dose level.

BODY WEIGHT: Yes - see attached tables and appendices.

- Time schedule for examinations:

Individual bodyweights were recorded on Day 1 (prior to start of treatment) and on Days 8, 15, 22, 29 and 36. Terminal bodyweights were also recorded at the Day 29 (non-recovery animals) and Day 43 (recovery animals).

FOOD CONSUMPTION: Yes - see attached tables.

- Dietary intake was recorded for each cage group at weekly intervals throughout the study.
FOOD EFFICIENCY: Yes - see attached tables.

- Weekly food efficiency (bodyweight gain/food intake) was calculated retrospectively.

WATER CONSUMPTION: Yes - see attached tables.

- Time schedule for examinations:
Water intake was measured gravimetrically throughout the treatment period (Day 1 to Day 43)

OPHTHALMOSCOPIC EXAMINATION: Not applicable

Not applicable
- Time schedule for examinations:
- Dose groups that were examined:
Not applicable

HAEMATOLOGY: Yes - see attached tables and appendices.

- Time schedule for collection of blood: - At the end of the treatment period (Day 28) and at the end of the treatment-free recovery period (Day 42).
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: - Five males and five females per treatment group.
- Routine haematological parameters were examined.
- Routine blood chemistry parameters were examined.

CLINICAL CHEMISTRY: Yes - see attached tables and appendices.

- Time schedule for collection of blood: - At the end of the treatment period (Day 28) and at the end of the treatment-free recovery period (Day 42).
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: - Five males and five females per treatment group

URINALYSIS: Yes - see attached tables and appendices.

- Time schedule for collection of urine: - Urinalytical investigations were performed on all non-recovery test and control group animals during the final week of treatment (Week 4) and on all recovery group animals during Week 6.

- Metabolism cages used for collection of urine: Yes
- Animals fasted:
Animals were maintained under conditions of normal hydration during collection but without access to food.

THYROID HORMONE ASSESSMENT: Yes
At termination, blood samples were taken from the exsanguination procedure and the plasma from each animal was stored frozen at -20°C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

STAGE OF OESTRUS: Yes - see attached appendix 16.
At termination, a vaginal smear was taken from all females and the stage of oestrus was recorded.

ORGAN WEIGHTS: Yes - see attached tables and appendices.

- Time schedule:
Selected organs were removed from animals that were killed either at the end of the dosing period (Day 29) or at the end of the treatment-free period (Day 43).

How many animals: - Five males and five females per treatment group.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes - see attached tables and appendices.

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

HISTOPATHOLOGY: Yes - see attached tables and appendices.

All control and high dose animals were subjected to a full histological examination and low and intermediate group animals were routinely subjected to examination of liver and spleen.

In addition, since there were indications of treatment-related liver, thyroids and kidney changes, examination was subsequently extended to include similarly prepared sections of thyroid and kidneys from all animals in the other treatment groups and the livers, thyroids and kidneys from recovery control and high dose group animals.

Other examinations:

In addition, sections of testes and epididymides from all Control and high dose males were stained with Periodic Acid-Schiff (PAS) stain and examined.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.

All data were summarised in tabular form. 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 or ANCOVA and Bartlett’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 nonparametric 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 (nonparametric) 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).

Probability values (P) are presented as follows:
P<0.01 **
P<0.05 *
p≥0.05 (not significant)

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

effects observed, treatment-related

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

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:

no effects observed

Details on results:

RESULTS

- Mortality
There were no unscheduled deaths during the study.

- Clinical Observations - See attached Table 1 and Appendix 1.

Clinical observations were confined to increased salivation in animals of either sex treated with 1000 or 325 mg/kg/day throughout the treatment period.

Instances of staining around the mouth were evident in females treated with 1000 mg/kg/day on either Days 3, 11, 22 and 23 and in one female treated with 325 mg/kg/day on Day 17. Lethargy was also detected in one male treated with 1000 mg/kg/day on Day 4 only.

No such effects were evident in animals of either sex treated with 35 mg/kg/day or in recovery animals during the treatment free period.

- Functional Observations - See attached Tables 2 to 5 and Appendices 2 to 6.

- Behavioural Assessments
There were no treatment-related changes detected in parameters measured.

- Functional Performance Tests
There were no toxicologically significant changes in the functional performance parameters measured.

The statistically significant differences in hind/fore limb grip strength, overall activity and the final 25% activity detected were considered to be of no toxicological importance (see attached Form 1, Justification of No Observed Effect Level).

- Sensory Reactivity Assessments
There were no treatment-related changes detected in sensory reactivity. All inter 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.

- Bodyweight - See attached Table 6 and 7, Figure 1 and Figure 2 and Appendices 7 and 8.

No adverse effects on bodyweight development were detected in animals from all treatment groups or recovery animals during the treatment free period when compared to controls.

- Food Consumption - See attached Table 8 and 9 and Figure 3 and Figure
No adverse effects on food intake or food efficiency were detected.

- Water Consumption - See attached Table 10.

Water consumption was increased in animals of either sex treated with 1000 mg/kg/day throughout the treatment period. No such effects were evident in the remaining treatment groups or in recovery animals during the treatment free period.

- Haematology - See attached Table 11 and Appendices 9 to 11.

There were no toxicologically significant effects detected in the haematological parameters examined.
The intergroup differences detected in haemoglobin, erythrocyte count, haematocrit, clotting time, platelet count and neutrophil count were considered not to be of toxicological importance (see attached Form 1, Justification of No Observed Effect Level).

Blood Chemistry - See attached Table 12 and Appendix 12.

There were no toxicologically significant effects detected in the blood chemical parameters examined. The intergroup differences detected in total protein, albumin, calcium, phosphorus, creatinine, triglycerides and bilirubin were considered not to be of toxicological importance (see attached Form 1, Justification of No Observed Effect Level).

- Urinalysis - See attached Table 13 and Appendix 13.

No treatment-related effects were detected in urinalytical values in all treated animals and in recovery animals following fourteen days without treatment.

- Organ Weights - See attached Table 14 and Appendices 14 and 15.

Males treated with 1000 or 325 mg/kg/day and females from all treatment groups showed statistically significant increases in liver weight, both absolute and relative to terminal bodyweight. The effect on liver weight continued in recovery animals following fourteen days without treatment. Males treated with 1000 or 325 mg/kg/day also showed statistically significant increases in kidney weight, both absolute and relative to terminal bodyweight.
The remaining intergroup differences in heart and thyroid weight were considered not to be of toxicological significance (see attached Form 1, Justification of No Observed Effect Level).

- Necropsy - See attached Table 115 and Appendix 16.

No toxicologically significant macroscopic abnormalities were detected. The incidental finding recorded at necropsy was considered to be of no toxicological importance (see attached Form 1, Justification of No Observed Effect Level).

- Stage of Oestrus - See attached Table 15 and Appendix 16.
Assessment of the stage of oestrus for females at termination showed no significant differences between treated and control groups.

- Histopathology - See attached Appendix 17.

The following treatment-related changes were identified:

Liver: Centrilobular hepatocellular hypertrophy was evident in animals of either sex treated with 1000 mg/kg/day and in males treated with 325 mg/kg/day. Following the completion of the treatment free period complete regression of centrilobular hepatocellular hypertrophy was evident in recovery animals.

Kidneys: Increased severity of hyaline droplets/granules in the proximal tubules was evident in males treated with 1000 or 325 mg/kg/day. Following the completion of the treatment free period hyaline droplets had regressed to normal control background levels in recovery animals.

Thyroid: Follicular cell hypertrophy was noted in males from all treatment groups, in females treated with 1000 and 325 mg/kg/day and also one female treated with 35 mg/kg/day. Following the completion of the treatment free period follicular cell hypertrophy had regressed to normal control background levels in recovery animals.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOEL

Effect level:

other: Not established

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

See attached (0766-0169) Form 1, Keys to Tables, Tables, Figures, Appendices and Addenda.

 

The following information relates to resulst from the Seven Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study.

 

- Mortality.

There were no unscheduled deaths during the treatment period.

 

- Clinical Observations.

Incidents of increased salivation were evident post dosing from Day 4 or 5 onwards for animals of either sex treated with 1000 and 500 mg/kg/day. Observations of this nature are commonly observed following oral administration of an unpalatable test material formulation and in isolation are considered not to be of any toxicological significance.

 

- Bodyweight.

No adverse effect on bodyweight development was detected for test animals when compared to controls.

 

- Food Consumption.

No adverse effect on dietary intake or food efficiency was detected in treated animals when compared to controls throughout the treatment period. 

 

- Water Consumption.

An increase in overall water consumption was detected in animals of either sex from all treatment groups throughout the treatment period.

 

- Necropsy.

No treatment-related macroscopic abnormalities were detected.

 

- Discussion.

No clinically observable signs of toxicity were detected during the study. Increased salivation was evident in males at 500 mg/kg/day on Day 7 only and in females from this treatment group on Days 5 and 7. At 1000 mg/kg/day increased salivation was evident in males from Day 4 and in females from Day 5 onwards. Daily measurement of water consumption showed a slight increase in water intake for treated animals throughout the treatment period. These findings are often observed following the oral administration of an unpalatable or slightly irritant test material formulation, and in the absence of any macroscopic observations suggesting irritancy are considered not to be indicative of systemic toxicity.

 

- Conclusion.

The oral administration of Ebanol to rats for a period of seven consecutive days at dose levels of 100, 500 and 1000 mg/kg/day produced no toxicologically significant effects in parameters measured. The “No Observed Adverse Effect Level” (NOAEL) and a suitable high dose level for use on future toxicity studies was, therefore, considered to be 1000 mg/kg/day.

 

Conclusions:

Conclusion. The oral administration of Ebanol to rats by gavage, at dose levels of 1000, 325 and 35 mg/kg/day, resulted in non-adverse treatment-related effects in animals of either sex from all treatment groups.

The liver and thyroid changes together with associated organ weight changes detected were considered to be adaptive in nature and do not represent an adverse effect of treatment. The kidney changes identified in males at 1000 mg/kg/day were consistent with well documented changes and are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. A No Observed Effect Level (NOEL) has therefore not been established. However, the No Observed Adverse Effect Level (NOAEL) for either sex was considered to be 1000 mg/kg/day.

Executive summary:

Results.

 

- Mortality.

There were no unscheduled deaths during the study.

 

- Clinical Observations.

Clinical observations were confined to increased salivation in animals of either sex treated with 1000 or 325 mg/kg/day throughout the treatment period. Instances of staining around the mouth were evident in females from these treatment groups from Day 3 onwards and lethargy was also detected in one male treated with 1000 mg/kg/day on Day 4.

No such effects were evident in animals of either sex treated with 35 mg/kg/day or in recovery animals during the treatment free period.

 

Functional Observations.

 

- Behavioural Assessment.

There were no treatment-related changes detected in parameters measured.

 

- Functional Performance Tests.

There were no toxicologically significant changes in functional performance.

 

- Sensory Reactivity Assessments.

There were no treatment-related changes detected in sensory reactivity.

 

- Bodyweight.

No adverse effects on bodyweight development were detected for all treated animals or recovery animals during the treatment free period when compared to controls.

 

- Food Consumption.

No adverse effects on food intake or food efficiency were detected.

 

- Water Consumption.

Water consumption was increased in animals of either sex treated with 1000 mg/kg/day throughout the treatment period.

No such effects were evident in the remaining treatment groups or in recovery animals during the treatment free period.

 

- Haematology.

There were no toxicologically significant effects detected in the haematological parameters measured.

 

- Blood Chemistry.

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

 

- Urinalysis.

No treatment-related effects were detected in the urinalytical parameters examined in any treated animals or in recovery animals following fourteen days without treatment.

 

- Organ Weights.

Males treated with 1000 or 325 mg/kg/day and females from all treatment groups showed statistically significant increases in liver weight, both absolute and relative to terminal bodyweight. The effect on liver weight continued in the recovery animals following fourteen days without treatment. Males treated with 1000 or 325 mg/kg/day also showed statistically significant increases in absolute and relative kidney weight. No such effects were detected in animals of either sex treated with 35 mg/kg/day.

 

- Necropsy.

No toxicologically significant macroscopic abnormalities were detected.

 

- Stage of Oestrus.

Assessment of the stage of oestrus for females at termination showed no significant differences between treated and control groups.

 

- Histopathology.

The following treatment-related changes were identified:

 

Liver: Centrilobular hepatocellular hypertrophy was evident in animals of either sex treated with 1000 mg/kg/day and in males treated with 325 mg/kg/day. Following the completion of the treatment free period complete regression of centrilobular hepatocellular hypertrophy was evident in recovery animals.

 

Kidneys: Increased severity of hyaline droplets/granules in the proximal tubules was evident in males treated with 1000 or 325 mg/kg/day. Following the completion of the treatment free period hyaline droplets had regressed to normal control background levels in recovery animals.

 

Thyroid: Follicular cell hypertrophy was noted in males from all treatment groups, females treated with 1000 and 325 mg/kg/day and one female treated with 35 mg/kg/day. Following the completion of the treatment free period follicular cell hypertrophy had regressed to normal control background levels in recovery animals.

 

Conclusion. The oral administration of Ebanol to rats by gavage, at dose levels of 1000, 325 and 35 mg/kg/day, resulted in non-adverse treatment-related effects in animals of either sex from all treatment groups.

 

The liver and thyroid changes together with associated organ weight changes detected were considered to be adaptive in nature and do not represent an adverse effect of treatment. The kidney changes identified in males at 1000 mg/kg/day were consistent with well documented changes and are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. A No Observed Effect Level (NOEL) has therefore not been established. However, the No Observed Adverse Effect Level (NOAEL) for either sex was considered to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

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

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

Conclusion.

The oral administration of Ebanol to rats by gavage, at dose levels of 1000, 325 and 35 mg/kg/day, resulted in non-adverse treatment-related effects in animals of either sex from all treatment groups.

 

Some liver and thyroid changes together with associated organ weight changes detected were considered to be adaptive in nature and do not represent an adverse effect of treatment. The kidney changes identified in males at 1000 mg/kg/day were consistent with well documented changes and are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health.

A NOEL was not absolutely established. However, the clear NOAEL for either sex was considered to be 1000 mg/kg/day.

Waiver for inhalation toxicity:

In line with Column 2, point 8.6.1, Annex VIII of Regulation 1907/2006, a repeat-dose inhalation study does not need to be performed as the substance has low vapour pressure and high melting point, so the potential for the generation of inhalable forms is low. The use of this substance should not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be unlikely to occur, and a repeat-dose inhalation study is not required. As an objective of Regulation 1907/2006 is to reduce, replace or refine animal testing, based on the above information and information in this dossier, it can be reasonably expected that inhalation exposure is not expected and as such, it is not warranted to test the hypothesis in animals.

Waiver for dermal toxicity:

The physicochemical and toxicological properties suggest low potential for significant rate of absorption through the skin. Furthermore the results of laboratory animal studies show low acute dermal toxicity. The 28 day repeated dose study via dietary administration does not exacerbate systemic toxicity effects which suggest bioavailability is low, thereby there is low toxicity potential. This intrinsic property/toxicity potential can be extrapolated to repeated dermal route administration.

On the basis that the test substance (Ebanol) is being used to support MTCP Crude on the basis of read-across, the NOAEL for MTCP Crude is also considered to be 1000 mg/kg/day and that data for repeat-dose dermal and inhalation toxicity are not required.

Justification for classification or non-classification

According to Directive 67/548/EEC and Regulation (EC) 1272/2008, no classification is warranted.

On the basis that the test substance (Ebanol) is being used to support MTCP Crude on the basis of read-across, the classification for repeat-dose toxicity for MTCP Crude is also considered to be unclassified.